1309
Herbivory by an invasive snail increases nitrogen
fixation in a nitrogen-limited stream
Clay Porter Arango, Leslie Anne Riley, Jennifer Leah Tank, and
Robert Ogden Hall, Jr.
Abstract: Despite anthropogenic nitrogen contributions, nitrogen fixation contributes half of biosphere inputs but has
rarely been quantified in streams. Herbivory controls algal biomass and productivity in streams, and we hypothesized that
herbivory could also control nitrogen fixation. We released periphyton from herbivory in nitrogen-limited Polecat Creek,
Wyoming, where heavy grazing by the invasive New Zealand mudsnail (Potamopyrgus antipodarum) dominates nitrogen
cycling. One and two weeks after releasing periphyton, we found higher rates of nitrogen fixation on heavily grazed rocks
(two-way analysis of variance (ANOVA), p = 0.012). Time elapsed after snail manipulation had no effect (two-way ANOVA, p = 0.24). Grazing changed periphyton composition by reducing the proportion of green algae and increasing the
proportion of nitrogen-fixing diatoms (multivariate ANOVA, p = 0.001). Nitrogen fixation rates increased disproportionately to nitrogen-fixing algal cells, indicating that snails increased nitrogenase efficiency, probably by improving light and
(or) nutrient availability to nitrogen fixers. We incorporated our nitrogen fixation rates into a published nitrogen budget
for Polecat Creek and found that nitrogen flux into the periphyton was 50% higher when we included nitrogen fixation.
Herbivory can increase nitrogen fixation in streams, and future studies should measure nitrogen fixation for a more thorough understanding of stream nitrogen cycling.
Résumé : Malgré les contributions anthropiques d’azote, la fixation d’azote contribue à la moitié des apports de la biosphère; cependant, la fixation d’azote a rarement été mesurée dans les cours d’eau. L’herbivorisme contrôle la biomasse et
la productivité des algues dans les cours d’eau et nous émettons l’hypothèse selon laquelle l’herbivorisme pourrait aussi
contrôler la fixation d’azote. Nous avons protégé du périphyton de l’herbivorisme dans Polecat Creek, Wyoming, où le
broutage intensif par un gastéropode envahissant de Nouvelle-Zélande (Potamopyrgus antipodarum) domine le recyclage
de l’azote. Une et deux semaines après la manipulation, les taux de fixation de l’azote sont plus élevés sur les pierres fortement broutées (analyse de variance à deux critères de classification, p = 0,012). Le temps écoulé depuis la manipulation
des gastéropodes reste sans effet (analyse de variance à deux critères de classification, p = 0,24). Le broutage change la
composition du périphyton en réduisant la proportion d’algues vertes et en augmentant la proportion de diatomées fixatrices d’azote (analyse de variance multidimensionnelle, p = 0,001). Les taux de fixation d’azote augmentent de façon disproportionnée au nombre de cellules d’algues fixatrices d’azote, ce qui indique que les gastéropodes augmentent l’efficacité
de la nitrogénase, probablement en améliorant la disponibilité de la lumière et (ou) des nutriments aux fixateurs d’azote.
Nous avons incorporé nos taux de fixation d’azote dans un bilan d’azote publié pour Polecat Creek et avons découvert que
le flux d’azote vers le périphyton est 50 % plus élevé quand nous incluons la fixation d’azote. L’herbivorisme peut augmenter la fixation d’azote dans les cours d’eau et les études futures devraient mesurer la fixation d’azote afin d’obtenir
une compréhension plus complète du recyclage de l’azote dans les cours d’eau.
[Traduit par la Rédaction]
Introduction
Industrial nitrogen (N) fixation has perturbed the global N
cycle by doubling N flux into the biosphere (Vitousek et al.
1997), but biological N fixation still accounts for half the
global N flux. Among aquatic ecosystems (lakes, estuaries,
and oceans), N fixation contributes the largest input to eutrophic lakes (Howarth et al. 1988), but N fixation has re-
ceived little attention in streams, where conditions often
inhibit N fixation. For example, streams with high N concentration from anthropogenic activities put N-fixing algae
at a competitive disadvantage (Howarth et al. 1988). Also,
many well-studied stream ecosystems are forested, and low
light levels limit algae (Horne and Carmiggelt 1975; Grimm
and Petrone 1997). However, in streams in arid regions such
as the western United States, N fixation could be a substan-
Received 4 August 2008. Accepted 22 May 2009. Published on the NRC Research Press Web site at cjfas.nrc.ca on 6 August 2009.
J20705
Paper handled by Associate Editor John Richardson.
C.P. Arango1,2 and J.L. Tank. Department of Biological Sciences, University of Notre Dame, Notre Dame, IN 46556, USA.
L.A. Riley. School of Biological Sciences, Washington State University, Pullman, WA 99163, USA.
R.O. Hall, Jr. Department of Zoology and Physiology, University of Wyoming, Laramie, WY 82071, USA.
1Corresponding
2Present
author (e-mail: [email protected]).
address: Department of Biological Sciences, Central Washington University, Ellensburg, WA 98926-7537, USA.
Can. J. Fish. Aquat. Sci. 66: 1309–1317 (2009)
doi:10.1139/F09-079
Published by NRC Research Press
1310
tial source of bioavailable N due to higher light levels (Minshall 1978) and the frequency of lower water-column N-to-P
ratios (Grimm and Fisher 1986).
Primary controls on N fixation in streams include temperature (Marcarelli and Wurtsbaugh 2006), light (Horne and
Carmiggelt 1975; Grimm and Petrone 1997), and phosphorus (P) availability (Marcarelli and Wurtsbaugh 2006). In
marine systems, herbivory can increase N fixation (Wilkinson and Sammarco 1983; Williams and Carpenter 1997),
whereas in freshwater ponds, herbivory can decrease N fixation (Gettel et al. 2007). However, despite many studies of
herbivory in stream ecosystems, none has examined how
herbivory could influence N fixation.
In streams, herbivores alter periphyton structure and function (i.e., rate of primary production) (summarized by Feminella and Hawkins 1995), and it follows that herbivores
could also influence N fixation rates (Fig. 1). For example,
indiscriminant herbivory can dramatically reduce total periphyton biomass (Lamberti and Resh 1983; Mulholland et
al. 1991), which could reduce N-fixing taxa and N fixation
rates. Herbivory could increase or decrease N fixation by altering the physical structure of the periphyton, depending on
the morphology of the N-fixing taxa. If herbivores ingest filamentous N-fixing taxa in the periphyton overstory, N fixation rates could decrease. Alternatively, if overstory
herbivory favors adnate, prostrate, or crustose N-fixing taxa
in the periphyton understory (Stevenson 1997; Wellnitz and
Ward 1998), N fixation rates could increase. Further, herbivory could increase N fixation rates by altering the composition of the periphyton through selective grazing that avoids
unpalatable N-fixing cyanobacteria (Dodds et al. 1995) or
by allowing inferior competitors (i.e., N fixers) to thrive
(Power et al. 1988). Finally, herbivory could increase N fixation by improving resource availability (i.e., light and nutrients) to N-fixing taxa and increasing nitrogenase
efficiency (Williams and Carpenter 1997).
Plant–herbivore interactions emphasize how consumers
regulate resource availability at the ecosystem level (Carpenter et al. 1987; Power 1990; Vanni 2002), particularly in
freshwater habitats (Gruner et al. 2008). Because they fundamentally alter ecosystem dynamics by rerouting nutrient
fluxes through the food web (Strayer et al. 1999; Carlsson
et al. 2004), invasive species highlight the importance of
plant–animal interactions. For example, invasive zebra mussels (Dreissena polymorpha) consume the majority of phytoplankton in large water bodies through exceptionally high
filtration rates (Strayer et al. 1999), and the golden apple
snail (Pomacea bridgesii) mineralizes enough phosphorus to
initiate phytoplankton blooms in wetlands (Carlsson et al.
2004). In Polecat Creek, located in the Greater Yellowstone
Ecosystem in northwest Wyoming, the exotic New Zealand
mudsnail (Potamopyrgus antipodarum) reaches very high biomass, consumes nearly all net primary production, and controls stream N cycling through high N excretion rates (Hall
et al. 2003).
We tested how snail herbivory affected N fixation in
Polecat Creek, an optimal system, by reducing ambient biomass of Potamopyrgus, which are routinely found at extremely high densities (20 000 – 500 000 snailsm–2; Hall et
al. 2006). Pilot studies identified that Epithemia spp., a prostrate diatom with N-fixing endosymbionts, dominated the N-
Can. J. Fish. Aquat. Sci. Vol. 66, 2009
fixing component of the algal assemblage and that filamentous N-fixing cyanobacteria were relatively rare. We hypothesized that releasing periphyton from herbivory would
increase N fixation by one of two mechanisms. First, reducing herbivory could increase N fixation if filamentous Nfixing cyanobacteria became more abundant. Alternatively,
because prostrate diatoms can increase in abundance when
grazers remove overstory algae (Steinman et al. 1987), we
hypothesized that releasing periphyton from herbivory
would increase filamentous green algae, which would reduce Epithemia spp. and decrease N fixation during the 2week experiment. To quantify the importance of N fixation
in the context of whole-stream N dynamics, we incorporated our measured N fixation rates into a detailed N
budget reported in a previous study of Polecat Creek (Hall
et al. 2003).
Materials and methods
Study site
Polecat Creek, a geothermal, spring-fed stream located
south of Yellowstone National Park, USA, has optimum
conditions for N fixation due to low stream water N-to-P ratio (during this study, molar N:P = 0.93 ± 0.05 standard error (SE), n = 8), year-round warm temperatures and stable
flows, and high primary production due to high light levels
(Hall et al. 2003).
Experimental design
In July 2005, we manipulated snail densities in four levels:
ambient biomass (33.2 g ash-free dry mass (AFDM)m–2),
40% of ambient biomass (13.3 g AFDMm–2), 20% of ambient biomass (6.6 g AFDMm–2), and no snails (0.0 g
AFDMm–2). We used length–mass regressions to estimate
the number of 2.5 mm long snails required to achieve target biomass levels. Each treatment was replicated five
times for a total of 40 experimental units (one to three
rocks in each unit, with total rock area from 81 to
114 cm2). Experimental rocks were placed in plastic cages
(~90 cm2 each) with 1 mm mesh screens on all sides to
promote water exchange. Cages were placed in holes cut
in floating rafts, which covered the experimental rocks
with approximately 8 cm of stream water (Lamberti et al.
1987b). Throughout the experiment, we anchored the rafts
to a stream bank in water approximately 0.6 m deep. Because Hall et al. (2003) measured extremely high primary
production in late July at Polecat Creek (average 10.6 g
O2m–2day–1), we reasoned that a short-term manipulation
of snail biomass would elicit an algal response and minimize cage effects. Therefore, we randomly assigned each
cage a sampling time of one or two weeks postmanipulation. We sampled 20 experimental units in week 1 and the
other 20 in week 2. After N fixation assays, we recovered
snails from each cage to quantify final snail biomass for
use in analyses as a continuous variable.
In situ N fixation assay
We used in situ chambers to measure N fixation rates
with an acetylene (C2H2) reduction assay (Flett et al. 1976;
Marcarelli and Wurtsbaugh 2006). In this assay, nitrogenase
reduces C2H2 to ethylene (C2H4), providing a measure of niPublished by NRC Research Press
Arango et al.
1311
Fig. 1. Possible effects of herbivory on nitrogen (N) fixation. Herbivory can reduce N fixation by directly consuming N-fixing algal biomass. Herbivory can alter the physical structure of the periphyton to decrease N fixation by directly grazing filamentous N fixers or to
increase N fixation by grazing other filamentous algae and benefiting prostrate N fixers. Through selective avoidance of unpalatable N fixers, herbivory can increase N fixation by reducing other algae. Finally, grazing can mineralize nutrients and increase light, which could
benefit N fixation.
trogenase activity. First, we saturated unfiltered stream
water with C2H2 bubbled through a 10% HCl solution to remove impurities. After placing the experimental rocks into
2.2 L clear polyvinyl chloride (PVC) chambers, we amended
1.5 L of unfiltered stream water with 150 mL of C2H2saturated water to achieve a 10% partial pressure of C2H2.
Each chamber was sealed with a septum-equipped lid for
headspace sampling and incubated in the stream for 3–4 h.
At the end of the assay, we equilibrated gases between the
water and headspace and took replicate 5 mL headspace
samples and injected them into pre-evacuated 3 mL glass
vials. In control samples, we found no background C2H4
production in rocks incubated without C2H2-saturated
water, and we found no C2H4 contamination of C2H2.
Within 48 h, we injected 1 mL of the C2H4 samples into a
Shimadzu GC-14A gas chromatograph (Columbia, Maryland, USA) with Hayesep T column and flame ionization
detector (injector = 100 8C, column = 35 8C, detector =
180 8C, ultra high purity helium carrier gas at 50 mLmin–1).
Using temperature-dependent Bunsen coefficients, we calculated total C2H4 production, which we divided by assay
length to calculate C2H4 production rate. Using the theoretical conversion factor of 1 mol of N2 fixed for every
3 mol of C2H4 produced (Grimm and Petrone 1997; Marcarelli and Wurtsbaugh 2006), we calculated N fixation
rate (as mg Nh–1). Areal N fixation rate (mg Nm–2h–1)
was calculated based on the rock surface area of the experimental unit, which we quantified using gravimetric methods.
Response metrics for the algal assemblage
In week 1, we measured metabolism on all experimental
units after collecting gas samples from the N fixation assays,
but our results varied widely and may have been confounded
by the C2H2 incubation. Therefore, in week 2, we incubated
one replicate from each treatment in sealed, recirculating
chambers to measure metabolism without measuring N fixation on that replicate. To quantify metabolism, we used a
dissolved oxygen meter (YSI-85; YSI Incorporated, Yellow
Springs, Ohio) to measure oxygen increase during a light in-
cubation and oxygen decrease (community respiration, CR)
during a dark incubation (Bott 1996). Subsequently, we calculated gross primary production (GPP) as the sum of net
oxygen flux and the absolute value of CR, and we scaled
both metrics by the assayed rock area (mg O2m–2h–1).
Periphyton was removed from assayed rocks using wire
brushes, and we filtered a subsample of the periphyton
slurry onto pre-ashed Pall A/E filters (1 mm pore size; East
Hills, New York), which we froze for chl a analysis. The remaining slurry was preserved in 1% Lugol’s solution for
later algae identification. We extracted chl a from filters using the hot ethanol method (Sartory and Grobbelaar 1984)
and measured chl a with the fluorometric method (Welschmeyer 1994) on a Turner Designs TD-700 fluorometer (Sunnyvale, California). We subsampled 0.1 L of preserved
slurry into Palmer–Maloney chambers and used a Nikon Diaphot TMD inverted microscope (Melville, New York) at
400 to identify algae according to Prescott (1978). Sampling effort curves indicated that 500 cells per sample adequately characterized the algal assemblage. Algae were
identified to genus or until we could classify them into one
of five functional groups: green algae, diatoms, N-fixing diatoms (Epithemia spp.), cyanobacteria, and N-fixing cyanobacteria, which we differentiated by presence or absence of
heterocysts. We calculated biovolume of Epithemia spp. according to Hillebrand et al. (1999).
Water chemistry
On the days that we measured N fixation, we collected
filtered (Pall A/E, 1 mm pore size; East Hills, New York)
water samples in acid-washed HDPE bottles, prerinsed with
filtered site water, stored them on ice in the field, and froze
them upon return to the laboratory. We used a Shimadzu
UV-VIS spectrophotometer (Columbia, Maryland) and
10 cm path to measure ammonium (NH4+) with the phenate
method (American Public Health Association, American
Water Works Association, and Water Pollution Control Federation (APHA) 1995), and we quantified soluble reactive
phosphorus (SRP) with the molybdate method (APHA
1995). Nitrate (NO3–) was measured on a DIONEX 600 ion
Published by NRC Research Press
1312
chromatograph (Sunnyvale, California) with ED-50 electrochemical detector (US Environmental Protection Agency
(USEPA) 1993). Dissolved inorganic N (DIN) was calculated as the sum of NH4+-N and NO3–-N concentrations.
Statistical analyses
We log-transformed data that failed a one-sample
Kolmogorov–Smirnoff test for normality to meet the assumptions of parametric statistical analysis. We used twoway analysis of variance (ANOVA) to test our a priori hypothesis that treatments with lower snail biomass would
have less N fixation and to identify significant differences
in chl a standing crop, final snail biomass, and total algal
cells. We analysed algal cells and percent composition of
the algal assemblage with multivariate ANOVA
(MANOVA), using two-way ANOVA (week with two levels
and snail biomass with four levels) with a Bonferroni-adjusted p value of 0.05/5 algal categories = 0.01 to determine
significant differences in post-hoc tests.
After testing our a priori hypothesis, we used regression
analyses to explore the predictive power of the explanatory
variables that we collected. Based on the specific hypotheses
presented in Fig. 1, we used the following variables: chl a,
final snail biomass, percent N fixers (N-fixing diatoms + Nfixing cyanobacteria), percent N-fixing diatoms, percent Nfixing cyanobacteria, and week coded as a categorical variable. We used forward stepwise multiple linear regression
analysis to identify functional relationships between N fixation rates and the explanatory variables. Finally, we used
simple linear regression to evaluate relationships between
snail biomass and ecosystem metabolism and between ecosystem metabolism and N fixation. All analyses were performed in SYSTAT 12.0 (Systat Software Inc., Chicago,
Illinois).
Results
Grazing effects on N fixation and the algal assemblage
We found highest chl a standing crop at lowest snail biomass and lowest chl a standing crop at highest snail biomass
(two-way ANOVA, F[3,26] = 3.08, p = 0.045; Fig. 2a). Chl a
biomass among treatments fell within the ambient range normally found in this region (0.77–6.35 mg chl acm–2; L.A.
Riley, unpublished data). Standing crop of chl a did not
vary between weeks (two-way ANOVA, F[1,26] = 0.27, p =
0.61), and there was no significant interaction (two-way
ANOVA, F[3,26] = 0.55, p = 0.65).
Snail biomass measured at the end of the experiment was
somewhat lower than the target biomass estimated with
length–mass regressions, probably due to mortality or to adding smaller snails than intended. Nevertheless, when we analysed snail biomass as a dependent variable, treatment levels
remained distinct (two-way ANOVA, F[3,26] = 91.55,
p < 0.00001, data not shown) and did not differ among
weeks (two-way ANOVA, F[1,26] = 0.36, p < 0.55, data not
shown), allowing us to analyse N fixation rates categorically.
We predicted that rocks incubated at highest (ambient)
snail biomass (33.2 g AFDMm–2) would have higher N fixation. N fixation rates were highest in the 13.3 g AFDMm–2
treatment and lowest in the 6.6 g AFDMm–2 treatment
(two-way ANOVA, F[3,26] = 4.03, p = 0.012; Fig. 2b), but
Can. J. Fish. Aquat. Sci. Vol. 66, 2009
Fig. 2. Chlorophyll a standing crops and nitrogen (N) fixation
(±1 standard error, SE) among experimental snail standing stocks in
weeks 1 (solid bars) and 2 (shaded bars). Letters above bars indicate significant differences among treatments. (a) Chl a standing
crop declined with increasing snail biomass. (b) Areal N fixation
was highest in the 13.3 g ash-free dry mass (AFDM)m–2 treatment.
they did not vary by week (two-way ANOVA, F[1,26] =
1.43, p = 0.24), and there was no significant interaction
(two-way ANOVA, F[3,26] = 2.83, p = 0.058). Total algal
cell density (cellscm–2) did not vary between weeks (twoway ANOVA, F[1,26] = 0.00, p = 0.99) or among treatments
(two-way ANOVA, F[3,26] = 2.44, p = 0.090), and there was
no interaction (two-way ANOVA, F[3,26] = 1.07, p = 0.38).
We predicted that snail grazing would increase N fixation
rates by altering the algal assemblage. Cell density (i.e.,
cellscm–2) among the components of the algal assemblage
differed between weeks 1 and 2 (Wilks’ L, F[20,73] = 3.04,
p < 0.0001; Figs. 3a and 3b), apparently due to changes in
total abundance of N-fixing diatoms rather than changes in
N-fixing cyanobacteria. However, post-hoc analyses did not
meet the Bonferroni-corrected p value to allow us to interpret further. The relative assemblage composition (i.e., percent abundance) changed between weeks 1 and 2 (Wilks’
L, F[20,73] = 2.27, p = 0.0014; Figs. 3c and 3d) due to an
increase in N-fixing diatoms (two-way ANOVA, F[1,23] =
10.51, p = 0.0036) and a decrease in green algae (two-way
ANOVA, F[1,23] = 10.11, p = 0.0042). We found no differences in Epithemia spp. biovolume (mm3cm–2) among treatments (two-way ANOVA, F[3,20] = 0.83, p = 0.49) or
between weeks (two-way ANOVA, F[1,20] = 1.77, p = 0.20).
Thus, grazing by Potamopyrgus had a borderline effect on
Published by NRC Research Press
Arango et al.
1313
Fig. 3. Total algal cells (±1 standard error, SE) by functional group between (a) week 1 and (b) week 2, and algae composition by functional group between (c) week 1 and (d) week 2. Algae capable of fixing nitrogen are indicated by hatched bars. Algal cells differed between weeks 1 and 2, but post-hoc tests did not meet the Bonferroni-adjusted criterion for significance. Algae composition differed
significantly in week 2 compared with week 1 (Wilks’ L, p = 0.001), with a significant decrease in the relative abundance of green algae
(two-way analysis of variance (ANOVA), p = 0.004) and a significant increase in the relative abundance of N-fixing diatoms (two-way
ANOVA, p = 0.008). AFDM, ash-free dry mass.
cell density, and grazing significantly increased the relative
abundance of N-fixing diatoms and decreased the relative
abundance of green algae. Relative abundance of N-fixing
cyanobacteria did not change between weeks or among
treatments.
We compared the magnitude of change in N-fixing algal
cells with the magnitude of change in N fixation rates in
the snail exclusion and the 33.2 g AFDMm–2 treatment in
week 2. Average number of N-fixing cells was greater in
the 33.2 g AFDMm–2 treatment by a factor of 1.3
(9930 cellscm–2 compared with 7878 cellscm–2). By contrast, N fixation rate was greater in the 33.2 g AFDMm–2
treatment by a factor of 2.8 (1.1 mg Nm–2h–1 compared
with 0.4 mg Nm–2h–1). Therefore, two weeks after manipulating snail biomass, N fixation rates increased disproportionately to changes in abundance of N-fixing cells.
chl a and snail biomass, both of which were positively related to N fixation rates. The MLR did not include percentage of N-fixing diatoms or N-fixing cyanobacteria in the
model.
Nitrogen fixation is energetically expensive and requires
abundant reduced carbon; we measured ecosystem metabolism (i.e., GPP and CR) to relate carbon dynamics to N fixation. However, because we were only able to measure
metabolism on one treatment replicate (due to equipment
limitation), we could not include these metrics in MLR analysis. Further, our low sample size (n = 4) made it difficult to
detect significant relationships, and GPP was not significantly related to snail biomass (r2 = 0.88, p = 0.06;
Fig. 4a). Although GPP was positively related to N fixation
(r2 = 0.95, p = 0.03; Fig. 4b), CR was not (r2 = 0.80, p =
0.11, data not shown).
Predicting N fixation rates
We used forward stepwise multiple linear regression
(MLR) analysis to test the predictive ability of a number of
key variables that we hypothesized would affect N fixation
rates (Fig. 1). The MLR explained two-thirds of the overall
variability in the data set (R2 = 0.67, p < 0.0001), and four
of the six variables that we tested were included in the
model (Table 1). The most important predictor, percentage
of N fixers (N-fixing diatoms + N-fixing cyanobacteria),
was positively related to N fixation rates. The second predictor, week, was negatively related to N fixation, indicating
higher N fixation one week after the biomass manipulation.
The model included two more variables, standing crop of
Discussion
Comparing N fixation among aquatic habitats
N fixation has not been well studied in streams, so we
compared our results with the few published rates from
streams and with reports from other aquatic habitats. N fixation rates from our ambient snail biomass treatments
(1.1 mg Nm–2h–1) were similar to the average rate
(0.72 mg Nm–2h–1) reported in a review by Marcarelli et
al. (2008). Our average rate is higher than N fixation rates
reported for oceans (0.004 mg Nm–2h–1), estuaries
(0.06 mg Nm–2h–1), and eutrophic lakes (0.2 mg Nm–2h–1)
(Howarth et al. 1988). In common with most other studies
Published by NRC Research Press
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Can. J. Fish. Aquat. Sci. Vol. 66, 2009
Table 1. Multiple linear regression of variables expected to predict N fixation
rates.
Predictor
Chlorophyll a (mgm–2)
Final snail biomass (g AFDMm–2)
N-fixing diatoms (%)
N-fixing cyanobacteria (%)
Total N fixers (%)
Week (coded categorical variable)
Overall model
p
0.0012
0.0073
>0.05
>0.05
0.0001
0.0015
<0.0001
Coefficient
0.13
0.02
R2
0.10
0.12
1.12
–0.19
0.23
0.22
0.67
Note: AFDM, ash-free dry mass.
Fig. 4. Relationships between (a) gross primary production (GPP)
and snail biomass and (b) GPP and nitrogen (N) fixation. In week
2, we measured metabolism on one of five replicates from each
treatment, but we did not measure N fixation on the same replicate.
Therefore, in (b), we use this metabolism estimate as an index of
GPP for the other four replicates for which we measured N fixation. AFDM, ash-free dry mass.
of N fixation in streams, Polecat Creek had optimum conditions for N fixation (high light levels, low DIN, and low
N:P). However, the maximum rate that we measured was
much lower than that reported from a Sonoran desert
stream (51 mg Nm–2h–1; Grimm and Petrone 1997) and a
California mountain stream (11 mg Nm–2h–1; Horne and
Carmiggelt 1975) where N fixation occurred in homoge-
nous patches of N-fixing algae. In contrast, we measured
N fixation in mixed algal assemblages caused by heavy
grazing by Potamopyrgus. The heavily grazed algal assemblage may have reduced spatial variability (Hillebrand
2008), contributing to lower areal N fixation rates due to
interspecific competition for resources. Although we do
not know what a more representative ‘‘average’’ N fixation
rate might be for streams, our comparison with other
aquatic habitats indicates that N fixation rates in streams
may be much higher than in other aquatic habitats when
conditions allow.
Relationship between herbivory and N fixation
In our experiments, the N fixation response in week 2
clearly followed our prediction, implying that it took greater
than one week for the algal assemblage to recover after releasing periphyton from herbivory. We hypothesized several
mechanisms by which herbivores could change N fixation
rates, and in our system, we can reject the first, that herbivory would reduce N fixation by consuming biomass of N
fixers. Potamopyrgus may avoid basal cells despite heavy
grazing pressure (Holomuzki et al. 2006) given that adnate
diatoms dominated the N-fixing component of the algal assemblage in Polecat Creek. Additionally, Polecat Creek has
very high primary production compared with other streams
(Hall et al. 2003), which may have allowed Potamopyrgus
to selectively consume highly productive green algae, as
seen by their reduced relative abundance in week 2.
Although streams with high rates of primary production
may generally sustain N fixation rates under heavy grazing
pressure, our results may have differed if our experiment
had run longer.
We also hypothesized that herbivores could either increase or decrease N fixation by altering the physical structure of the periphyton (i.e., removing filamentous taxa) or
through selective grazing (i.e., avoiding unpalatable taxa).
Herbivory restricts filament length and N fixation efficiency
in lakes (Chan et al. 2004), and benthic grazing by snails
(Lymnaea elodes) in oligotrophic arctic lakes decreases N
fixation of filamentous cyanobacteria (Gettel et al. 2007).
Filamentous N-fixing cyanobacteria were relatively rare in
Polecat Creek, but in combination with N-fixing diatoms,
they were an important predictor of N fixation. Filamentous
cyanobacteria did not increase after removing snails, suggesting that snails were not an important control over cyanobacterial abundance or that green algae outcompeted
cyanobacteria after release from grazing. Green algae dominated filamentous algal forms, and the proportion of green
Published by NRC Research Press
Arango et al.
algae decreased under high grazing pressure, whereas N-fixing
diatoms increased. Typically, grazing removes erect and
filamentous rather than prostrate algae, which snails only
consume under experimentally
induced starvation
(Steinman 1991) or heavy grazing pressure (Muller 1999).
Removal of green algae may have been due to its filamentous form or its greater palatability, but we cannot make
this distinction with our data set. In either case, we observed that snails altered the physical structure and composition of the periphyton with consequent increases in N
fixation. However, cell density (i.e., cellscm–2) did not
change, so the composition change cannot account for
higher rates of N fixation.
We also hypothesized that herbivory could increase N fixation by improving resource availability to N-fixing taxa.
The disproportionate increase in N fixation compared with
the increase in N-fixing cells indicates that snails increased
N fixation by improving nutrient and (or) light availability
to N-fixing diatoms in basal periphyton layers. Although N
fixation has not been examined in this context in streams,
grazing can increase photosynthesis efficiency by increasing
light penetration and nutrient diffusion from the water column (Lamberti and Resh 1983; Steinman 1996). We examined the N fixation response over a two-week snail
manipulation rather than in the stable algal assemblage that
would develop in the absence of snails. Epithemia is an epiphytic diatom in other streams (Bergey et al. 1995), which
leaves open the possibility that if our experiment had continued for longer, N fixation rates might have increased in the
absence of snail grazing if filamentous algae became a dominant component of the periphyton.
Because N fixation requires abundant energy, it is frequently related to photosynthetic rates in terrestrial environments (Bormann and Gordon 1984; Giraud and Fleischman
2004) and to light availability in streams (Horne and Carmiggelt 1975, Grimm and Petrone 1997) and lakes (Higgins
et al. 2001). We observed higher N fixation with higher
GPP, supporting a link between photosynthesis and N fixation. Although we did not observe higher GPP per unit
chl a, which is often seen with herbivory in systems with
high periphyton biomass (Feminella and Hawkins 1995), we
did see a trend toward increased areal GPP with higher snail
biomass, despite a small sample size. Increased areal GPP
may provide another mechanism by which grazing increases
N fixation rates, but higher areal GPP is not a universal response in grazing studies, though it has been observed in
other snail herbivory studies (e.g., Lamberti et al. 1987a).
Incorporating N fixation into the whole-stream N budget
We compared N fixation rates measured in the highest
snail biomass treatment (i.e., ambient density) with wholestream N fluxes measured by Hall et al. (2003) to understand the relative role of N fixation to the daytime N budget
in Polecat Creek. Because we used the theoretical conversion factor of 1 mol of N2 fixed for every 3 mol of C2H4
produced, this comparison comes with the caveat that actual
N fixation rates may be higher or lower than our estimates.
Our measured N fixation rate was about half of wholestream NH4+ uptake (2.1 mg Nm–2h–1; Hall et al. 2003,
their Table 2). Incorporating N fixation into the wholestream N budget indicates that total N flux into the periphy-
1315
ton is about 50% greater than measured using short-term releases of NH4+ alone. However, Polecat Creek has high
assimilatory N demand because geothermal P inputs and
warm temperatures stimulate primary production. In systems
with lower assimilatory N demand, the contribution of similar rates of N fixation to stream N budgets could be much
higher. Further, the N fixation rates that we measured were
not as high as maximum rates reported from other stream
studies (51 mg Nm–2h–1 (Grimm and Petrone 1997);
11 mg Nm–2h–1 (Horne and Carmiggelt 1975)), but those
studies do not report assimilatory rates for context. Placing
our N fixation rates in the context of a more complete N
budget (Hall et al. 2003) indicates that streams with Nfixing taxa should include N fixation to gain a more complete understanding of whole-stream N fluxes and periphyton N demand.
Although N fixation constitutes a relatively large N flux
compared with whole-stream NH4+ uptake in Polecat Creek,
excretion from Potamopyrgus contributes 7.8 mg Nm–2h–1
(Hall et al. 2003, their table 1). Therefore, N fixation is
only 14% of the NH4+-N flux driven by herbivory, and an
even smaller fraction in comparison with total N flux by Potamopyrgus (i.e., excretion + egestion = 15.7 mg Nm–2h–1).
Including N fixation in the Polecat Creek N budget only reinforces the conclusion that invasive snails dominate the N
cycle of Polecat Creek by dominating consumer biomass.
Animals can affect whole-system nutrient cycling via direct effects such as increasing nutrient availability via waste
excretion or by indirect effects such as altering nutrient dynamics by changing the assemblage structure (Vanni 2002).
Hall et al. (2003) documented that the invasive Potamopyrgus directly affected the N cycle because its excretion accounted for nearly two-thirds of whole-stream NH4+
demand. Regenerated nutrients can contribute substantially
to food web productivity and can have many indirect and
positive food web interactions (Pfister 2007). For example,
our data show that an invasive snail can indirectly affect
the N cycle over the short term by altering the composition
of the algal assemblage and increasing the rate and efficiency of N fixation. From a longer-term perspective, the
postinvasion algal assemblage of Polecat Creek probably
differs from the preinvasion assemblage, though we know
of no earlier studies for comparison. A recent meta-analysis
suggests that freshwater herbivores generally reduce species
richness but that species richness generally increases with
herbivory in productive ecosystems (Hillebrand et al. 2007).
Polecat Creek is a highly productive ecosystem (Hall et al.
2003), which may suggest that Potamopyrgus induced an algal assemblage with higher richness after invasion, though
the consequences for N fixation are unknown. Our study
demonstrates that herbivory may influence N fixation along
multiple pathways, with structural changes in the algal assemblage leading to improved resource availability and
higher periphyton N fixation. However, increased N fixation
may not be a universal response to herbivory if heavy grazing strongly reduces periphyton biomass through indiscriminant grazing.
Acknowledgements
Gretchen Gettel shared her nitrogen fixation protocols and
her chambers during a trial run for this study. Scott TarbutPublished by NRC Research Press
1316
ton helped set up the experiment, and Lisa Kunza shared her
equipment for acetylene reduction assays. Gary Lamberti
provided access to a microscope, Konrad Kulacki and Michelle Evans-White helped with algae identification, and
conversations with Amy Marcarelli improved the data analysis. Hank Harlow provided logistical support at the University of Wyoming – National Park Service Research Station.
We thank two anonymous reviewers and John Richardson
for critical comments that improved this manuscript. C.P.A.
received funding from the Arthur J. Schmitt Presidential Fellowship and the Bayer Predoctoral Fellowship while conducting this research. Additional funding was provided by
NSF-DEB 0111410, USGS – Wyoming Water Development
Commission.
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