Methods
General approach: a conservative test of
contingency
Our overall approach is to compare the NCE of fish on the abundance of
different zooplankton taxa across four experiments performed in
different years, but with similar conditions, objectives, measurables
and implementation. We are unaware of any prior studies that have
examined context dependence of risk effects on prey abundance. The
experiments all shared treatments that allow us to examine NCEs of fish
on zooplankton abundance by comparing caged-fish and no-fish treatments.
The NCE on the abundance of given zooplankton taxa could thus be
compared across experiments to identify context dependence of risk
effects.
The venue was constructed to capture much of the complexity of natural
systems. Each experiment used the same large mesocosms set outdoors
(subject to natural environmental variation) and used a natural resource
base (phytoplankton) supported by nutrient addition, and a speciose
(>10 taxa) zooplankton prey assemblage. Each experiment ran
for 3-5 generations of the prey species with the longest generation
times (see Peacor et al. 2012), allowing the potential for ecological
feedbacks to affect abundance. Although the venue is obviously less
complex than a natural system (e.g., lacking in other predator taxa), it
incorporates much more of the complexity of a natural system than a
typical laboratory experiment that has few prey species, a simple and
controlled resource base, and controlled abiotic conditions.
Our approach is thus different than most studies that have identified
context dependency in the effect of ecological factors on fitness
components over short time scales. Typically, a particular biotic
factor, such as prey density, or abiotic factor, such as physical
complexity from plants, is systematically varied to investigate the
influence of that factor. We ask, rather, could factors that are
typically ignored or unmeasured (because they are deemed inconsequential
to the study of the risk effect) influence the results of a study of
risk effects on abundance in a natural system?
Differences that developed among the complex experimental communities
among years are likely caused by factors such as variation in starting
abundances that could lead to priority effects (Drake 1991, Chase et al.
2009), small differences in start dates across years, nutrient addition,
annual temperature variation, etc. These differences naturally caused
variation across experiments, such as the amount of periphyton in the
tanks and the absolute and relative abundance of a given taxa. It is
these incidental differences that we hypothesize could underlie NCE
contingencies. They are small relative to differences among natural
systems (e.g. different ponds and lakes), because natural systems have
e.g. higher spatial-temporal variation and multiple predators.
Subsequently, our test of contingency among experiments is a
conservative test for the contingencies relative to those expected in
natural systems. Note that by terming the differences “incidental,” we
do not mean they were solely due to stochastic environmental
differences. Rather, the differences were incidental to the experimental
test of NCEs on prey abundance; i.e. they would not be considered
important to the outcomes of a study of risk effects of fish on
zooplankton abundance.
We provide a description of the experimental methodology that was common
to all experiments. Where there were minor differences among
experiments, such as small differences in the average size of fish used,
a range is provided. Wherever a range is provided in the methods the
specific details are provided in a table in the Supporting Information.
Other differences among experiments and methodological differences are
summarized after the general description and described in more detail in
Supporting Information. The timing of manipulations and samplings are
given relative to the day experimental treatments were initiated (as
provided in Supporting Information), defined as day 0.
Methodology shared among experiments
Outdoor mesocosm experiments were conducted at the E.S. George Reserve
(ESGR) of the University of Michigan near Pinckney, Michigan, USA
(42°28’N, 84°00’W). Cylindrical cattle watering tanks were employed and
contained approximately 1100 L of well water. Tanks were 1.9 m in
diameter and 0.75 m tall, and were filled to a depth of 45 cm. Washed
sand was added to each mesocosm as a bottom substrate. Each mesocosm was
covered with a fiberglass window screen lid to deter colonization by
insects. On particularly sunny days, 60% green shade cloth lids were
used on top of the window screen lids to reduce heating.
The design of each experiment included the presence/absence of fish
kairomone (i.e. chemicals) and additional treatments to explore
different questions in each experiment (Supporting Information). A
randomized block design was used for each experiment, and replicates
ranged from 6-16 depending on the treatment and experiment. The
non-consumptive effect of fish was created by maintaining one
zooplanktivorous bluegill sunfish, Lepomis macrochirus (standard
lengths given in Supporting information) in each of two or three
floating cages within each kairomone treatment mesocosom. No-fish
treatments consisted of mesocosms with the same number of empty floating
cages. Large holes in the sides and bottom of the boxes were covered
with fine netting to allow for diffusion of fish kairomones without
permitting zooplankton to pass through. In Exps. 1 and 2, three large
snails (Planorbella cf. trivolvis >11.2 mm in
diameter) were kept inside each cage to graze on periphyton that could
grow on the mesh windows. Adding snails was deemed extraneous and not
implemented in Exps. 3 and 4.
Fish originated from Patterson Lake, Livingston County, Michigan. In
order to ensure fish health and to equalize fish cue (e.g. in case fish
differed in cue production) we rotated the fish from the experimental
mesocosms to a culture tank once a week. Culture tanks consisted of
outdoor mesocosms of ~50 fish of similar size fed
zooplankton three times per week. Fish in culture tanks were not fed for
24 hours before being rotated back into the experiment. While in the
experimental cages, each fish was fed twice a week, including the day
they were added to the cages. To feed fish, an average of 200Daphnia were added per cage. The no-fish cages received an equal
amount of Daphnia that were killed by microwaving to ensure a
population did not build in the cages.
Calculations of nutrient inputs indicate that nutrients from fish
excretion were inconsequential, as they were overwhelmed by other
sources, including external supply and internal recycling by zooplankton
(See Supporting Information in Peacor et al. 2012). Two experiments also
provide evidence that nutrient inputs from fish had no ecological
effects. In one, caged fish had no effect on phytoplankton growth in
chambers placed outside, but near, the cages and performed soon after
(8-10 days) treatments were initiated. In a second in-situ experiment,
fish excretion had no effect on phytoplankton growth in chambers placed
within the fish cages (Rafalski and Peacor, unpublished).
An initial pulse of nutrients (see below) and a phytoplankton inoculum
were added to each tank between 35 and 56 days before the start of the
experiment. The phytoplankton inoculum consisted of water collected from
ponds in the ESGR and filtered through 35 µm Nitex mesh. A community of
zooplankton was added to each tank between day -49 and -27 (Exp. 3
received two inoculums). This initial inoculum came from a single lake
(Exps. 2-4) or multiple lakes (Exp. 1, see Supporting Information for
details on source lakes). We collected zooplankton using a 64µm
zooplankton net, and undesirable animals such as insects (e.g.Chaoborus ) and Hydra were removed prior to adding the
inoculum to the mesocosms. To decrease zooplankton heterogeneity among
mesocosms, on day -24 to -6 we collected samples of zooplankton from
each mesocosm using a 64 µm zooplankton net, mixed all samples, and
redelivered subsamples of this mixed community to all mesocosms. The
procedure was done twice for Exps. 3 and 4. Although this procedure
reduces variation in initial densities among tanks, there will clearly
be more variation at treatment initiation than in a more controlled
experiment that would start with constant densities.
Inorganic nutrients were supplied to the mesocosms to support
phytoplankton growth as a resource for zooplankton at an N:P ratio of
15:1 or 20:1 (Supporting Information). An initial pulse of
NH4NO3 and
KH2PO4 was added to each mesocosm when
phytoplankton were added, and thereafter we supplied maintenance doses
weekly. The rate and overall amount of nutrients added were similar
among experiments (Supporting Information), though there was some
variation in rates and the frequency at which nutrients were added,
varying from twice per week to continuous (Supporting Information). In
some cases, the weekly rate was reduced to reduce phytoplankton and
filamentous algal growth when deemed excessive (Supporting Information).
To reduce periphyton growth and to cycle nutrients back to the water
column, we collected visible filamentous algae by hand each week,
clumped it, allowed it to dry and returned it to the mesocosms. In Exps.
1 and 2, we also added Planorbella cf. trivolvis snails to each
mesocosm to reduce periphyton growth.
Zooplankton were sampled on day 31-71. Samples were passed through a
53-64 µm mesh sieve and preserved. Zooplankton were identified and
enumerated. Zooplankton identification was resolved to species or genus
for Cladocera, adult cyclopoid and adult calanoid for Copepoda (referred
to as “cyclopoid” and “calanoid”, exception described below), and
ostracods for Ostracoda.
There were a number of methodological differences among experiments due
to the specific questions addressed which we summarize here, and
describe in more detail in Supporting Information. (1) The zooplankton
collection method differed; in Exps. 1 and 2 we subsampled different
positions in the tank to determine both zooplankton position in the
tanks and overall density, while in Exps. 3 and 4 we measured overall
density from combined samples of the entire water column. This
difference should not affect our assessment of an NCE of fish on
zooplankton abundance. (2) The experiments examined different aspects of
risk effects with additional treatments. In three experiments, the
additional treatments were included in the no-fish or caged-fish
treatment, as they had no influence and their inclusion increased the
power to identify treatment effects (Supporting Information). (3) The
origin of the zooplankton was the same (a single lake) for Exps. 2-4,
but differed for Exp. 1 in which collection was from multiple lakes. (4)Hydra , which are predators of some zooplankton species, were
present in Exp. 1, but not Exps. 2-4. (5) Because the size and
ecological role of the juvenile stage of cyclopoids can be variable
(Burns and Gilbert 1993), we counted only adult cyclopoids. However, in
Exp. 4 the counting resources were more limited and thus the cyclopoid
counts include both juveniles and adults.
All procedures involving animals were approved by University of
Michigan’s Committee on the Use and Care of Animals under approval
number 07765 (for experiments 1 and 2) and Michigan State University’s
Institutional Animal Care and Use Committee approval no. 02/12-025-00
(for experiments 3 and 4).