DOES BIOTURBATION BY THE TADPOLE SHRIMP LEPIDURUS PACKARDI
PROMOTE CRUSTACEAN ABUNDANCE AND TAXONOMIC RICHNESS IN
CALIFORNIA VERNAL POOLS?
A Thesis
Presented to the faculty of the Department of Biological Sciences
California State University, Sacramento
Submitted in partial satisfaction of
the requirements for the degree of
MASTER OF SCIENCE
In
Biological Sciences
by
Russell Croel
SPRING
2014
DOES BIOTURBATION BY THE TADPOLE SHRIMP LEPIDURUS PACKARDI
PROMOTE CRUSTACEAN ABUNDANCE AND TAXONOMIC RICHNESS IN
CALIFORNIA VERNAL POOLS?
A Thesis
by
Russell Croel
Approved by:
__________________________________, Committee Chair
Jamie Kneitel, Ph.D.
__________________________________, Second Reader
Ronald M. Coleman, Ph.D.
__________________________________, Third Reader
James W. Baxter, Ph.D.
____________________________
Date
ii
Student: Russell Croel
I certify that this student has met the requirements for format contained in the University
format manual, and that this thesis is suitable for shelving in the Library and credit is to
be awarded for the thesis.
_________________________, Graduate Coordinator _________________
Jamie Kneitel, Ph.D.
Date
Department of Biological Sciences
iii
Abstract
of
DOES BIOTURBATION BY THE TADPOLE SHRIMP LEPIDURUS PACKARDI
PROMOTE CRUSTACEAN ABUNDANCE AND TAXONOMIC RICHNESS IN
CALIFORNIA VERNAL POOLS?
by
Russell Croel
Ecosystem engineers are increasingly recognized for their potential in facilitating
habitat restoration efforts. An example of ecosystem engineering in aquatic habitats is
bioturbation, the disruption of sediment at the water-sediment interface by animal
activity. Among the varying effects they have on aquatic communities, bioturbating
animals can facilitate zooplankton recruitment by digging up buried, resting eggs and
returning them to the sediment surface, where they have a higher probability of hatching.
Such facilitation has been demonstrated in studies involving lake and permanent-pond
ecosystems, but the effects of bioturbation in temporary ponds, such as California vernal
pools, have largely been overlooked. Vernal pools are home to a strong bioturbator, the
endemic notostracan Lepidurus packardi. I hypothesized that bioturbation by L. packardi
facilitates the hatching of buried, resting eggs by returning them to the sediment surface. I
tested this hypothesis by removing L. packardi from mesocosms filled with natural vernal
pool soil and comparing the resulting crustacean communities to those in unmanipulated
iv
mesocosms. I predicted that mesocosms with fewer L. packardi would have fewer
crustacean individuals and/or taxa in the active community, because fewer buried eggs
would be returned to the sediment surface. I directly tested L. packardi’s digging abilities
by conducting complementary microcosm experiments where I buried propagules
(resting eggs and plant seeds) at different depths and added freely roaming or caged L.
packardi. These experiments also allowed me to determine whether L. packardi can
influence the hatching of resting eggs through kairomones (chemical signals).
I found no support for my hypothesis. In the mesocosm experiment, four taxa were
actually more abundant, not less, in mesocosms with fewer L. packardi. This indicates
that L. packardi was suppressing these taxa in the Control mesocosms, most likely
through predation. In the microcosm experiments, I found that L. packardi did not
translocate propagules buried ≥ 0.5 cm deep, and that it also consumed eggs (but not
seeds) lying on the sediment surface. I further found no evidence for kairomones. Results
from the microcosm experiments additionally suggest that i) egg translocation was not
masked by egg predation; and ii) propagule translocation simply did not occur. I conclude
that bioturbation by L. packardi does not facilitate crustacean recruitment in California
vernal pools, and that this taxon influences other crustacean taxa primarily through
predation on both resting and active stages.
___________________________, Committee Chair
Jamie Kneitel, Ph.D.
___________________________
Date
v
ACKNOWLEDGEMENTS
I thank the outstanding faculty and staff of the Department of Biology for providing
me with support and encouragement throughout my academic career at CSUS.
I especially thank Dr. Ronald Coleman and Dr. James Baxter for their constructive
comments and criticisms of this research. Their guidance and coursework have made me
a better scientist, teacher, and writer, and I am grateful that they were on my committee.
Dr. Jamie Kneitel, my advisor, deserves singular recognition. He exemplifies what it
means to be a great mentor and ecologist. I feel honored to know him and to be part of his
scientific pedigree.
None of this would have been possible without the unending support of my wife. Not
only did she encourage me and provide me with uninterrupted study time over the years,
but she also helped me collect data for this project when I was sidelined by a leg injury.
In a very literal sense, I could not have completed this project without her.
I dedicate this to my father, Philip Miles Croel.
vi
TABLE OF CONTENTS
Page
Acknowledgements ............................................................................................................ vi
List of Tables ................................................................................................................... viii
List of Figures .................................................................................................................... ix
INTRODUCTION .............................................................................................................. 1
METHODS ......................................................................................................................... 8
Mesocosm Experiment.................................................................................................... 8
Microcosm Experiments ............................................................................................... 15
RESULTS ......................................................................................................................... 21
Mesocosm Experiment.................................................................................................. 21
Microcosm Experiments ............................................................................................... 28
DISCUSSION ................................................................................................................... 30
Literature Cited ................................................................................................................. 43
vii
LIST OF TABLES
Tables
Page
1. Summary of crustacean taxa abundance (excluding L. packardi)
observed in each treatment group ................................................................................. 22
2. Independent-samples t-test results for evenness, total abundance,
and per-taxon abundances in mesocosm experiment .................................................... 25
viii
LIST OF FIGURES
Figures
Page
1. Number of L. packardi captured at each treatment application .................................... 11
2. NMDS ordination of mesocosms .................................................................................. 23
3. Comparison of Bosmina sp., Cypris sp., and Limnocythere
ceriotuberosa abundances in Control (unmanipulated) mesocosms
vs. mesocosms from which L. packardi was removed weekly..................................... 26
4. ANCOVA comparison of Eucypris sp. abundance in Control (unmanipulated)
mesocosms vs. mesocosms from which L. packardi was removed
weekly ........................................................................................................................... 27
5. Comparison of nauplii and seedling abundances in microcosm
experiments .................................................................................................................. 29
ix
1
INTRODUCTION
Ecosystem engineers are organisms that modify their physical habitat in ways that
affect resource availability to other organisms (Jones et al., 1994). Such habitat
modification can promote biodiversity by creating habitat space or ameliorating abiotic
stresses. For example, beaver dams can increase habitat complexity in riparian zones,
thereby increasing plant species richness (Bartel et al., 2010), and seaweed canopies can
reduce thermal and desiccation stresses in intertidal zones, enhancing recruitment and
survival of intertidal organisms (Bertness et al., 1999). Because of the positive impacts
ecosystem engineers can have on biodiversity, they are increasingly being recognized for
their potential in facilitating habitat restoration efforts (Byers et al., 2006).
A key goal of habitat restoration is re-establishing biodiversity in ecosystems
degraded by human activity (Palmer et al., 1997). An obvious first step in using
ecosystem engineers in restoration efforts is identifying engineer species and assessing
how their activities affect the community. If an engineer species is found to positively
influence the establishment or persistence of other species, its focused use in restoration
efforts could contribute to the success of those efforts. For example, lakes that are subject
to nutrient loading from human activity can shift from a pristine, clear-water state to a
turbid, algae-dominated state. Planting macrophytes in such lakes can facilitate a return to
the clear-water state (Byers et al., 2006), in part because macrophytes provide
zooplankton, which consume algae, refuge from predators.
One example of ecosystem engineering is bioturbation. In aquatic habitats,
bioturbation is the disruption of sediment at or below the water-sediment interface due to
2
burrowing and foraging by animals. Species that disrupt the sediment can be large in size,
such as sting rays and manatees, but small benthic invertebrates are considered the
dominant bioturbators in aquatic ecosystems, due largely to their sheer numbers
(Meysman et al., 2006). Such taxa include annelids (Mermillod-Blondin and Lemoine,
2010), echinoderms (Lohrer et al., 2004), and crustaceans (Gyllström et al., 2008).
Bioturbation can have both positive and negative effects on other aquatic species. On
the negative side, suspended sediment resulting from bioturbation can reduce macrophyte
cover by creating turbid conditions that occlude light (Croel and Kneitel, 2011).
Suspended sediment can also clog the feeding apparati of suspension feeders, such as
bivalves (Pillay et al., 2007) and cladocerans (Kirk, 1991). Further, bioturbation by
relatively large invertebrates can bury or displace smaller invertebrates, limiting their
access to food resources (Brenchley, 1981; Pillay et al., 2007). On the positive side,
bioturbation has been shown to enhance nutrient cycling (Mermillod-Blondin and
Rosenberg, 2006), contribute benthic food resources to food webs (Creed et al., 2010),
and increase the densities of epibenthic invertebrates by changing sediment topography
(Sun and Fleeger, 1994). Bioturbation can also stimulate macrophyte growth by mixing
relatively oxygen-rich water into anoxic sediments (Mermillod-Blondin and Lemoine,
2010).
One way that bioturbating animals can positively affect zooplanktonic taxa is by
digging up their buried, resting eggs and returning them to the sediment surface (Marcus
and Schmidt-Gengenbach, 1986). Resting eggs are embryos that undergo a period of
obligate dormancy called diapause. They are produced in large numbers by many
3
zooplanktonic taxa, such as small crustaceans in highly variable habitats. Resting eggs
accumulate in the sediment and form an egg bank, a process analogous to the formation
of seed banks in terrestrial habitats (De Stasio, 1989; Cáceres, 1998; Brendonck and De
Meester, 2003). Crustacean eggs in diapause can remain viable for years, and even
centuries (Hairston et al., 1995; Cáceres, 1998; Frisch et al., 2014). The eggs hatch, or
break diapause, when abiotic cues such as photoperiod, temperature, and salinity
collectively signal favorable environmental conditions (Brendonck, 1996; Brendonck and
De Meester, 2003; Vandekerkhove et al., 2005). Eggs must be at or near the sediment
surface to experience the full effects of these cues (Cáceres, 1998). Eggs covered by as
little as 0.5 cm of sediment become insulated from hatching cues and generally will not
hatch (Brendonck and De Meester, 2003; Gleason et al., 2003). Bioturbation can
positively affect crustacean abundance by translocating these buried-but-viable resting
eggs to the sediment surface, where they have a much higher probability of hatching
(Marcus and Schmidt-Gengenbach, 1986; Brendonck and De Meester; 2003; Gyllström
et al., 2008).
In addition to potentially increasing crustacean abundance, bioturbation might also
increase taxonomic richness in the active community (Brendonck and De Meester, 2003).
Some sediments exhibit greater richness, in the form of diapausing eggs, than the active
zooplankton community in any given season (Hairston and Kearns, 2002; Brendonck and
De Meester, 2003; Vandekerkhove et al., 2005). This mismatch can be attributed to a lifehistory strategy called bet hedging, which posits that the hatching of a relatively small
fraction of eggs prevents all offspring from dying should abiotic conditions lead to
4
abortive hatchings (i.e., hatched individuals do not survive long enough to reproduce;
Hildrew, 1985; Simovich and Hathaway, 1997; Belk, 1998; Vanschoenwinkel et al.,
2010). Cumulative hatching fractions for some temporary-pond crustaceans, for example,
have been found to be as low as 3% (Hildrew, 1985; Belk, 1998; Simovich and
Hathaway, 1997). Although hatching fractions vary widely (Brendonck, 1996), in general
resting eggs accumulate faster than they hatch (De Stasio, 1989; Cáceres, 1998).
Bioturbation might contribute to taxonomic richness of the active aquatic community by
promoting recruitment of species that would otherwise remain in diapause.
Several studies have demonstrated that bioturbation can return buried, resting eggs
back to the sediment surface. Marcus and Schmidt-Gengenbach (1986), for example,
buried copepod eggs and found that a polychaete bioturbator returned a sizeable fraction
(~15%) of them to the sediment surface. The eggs hatched and were recovered from the
water as nauplii. Consequently, Marcus and Schmidt-Gengenbach (1986) suggested that
bioturbation may be an important recruitment mechanism for zooplanktonic taxa. Kearns
et al. (1996), using small, plastic beads as proxies for eggs, reported similar upward
translocation, primarily due to bioturbation by oligochaetes. More recently, Gyllström et
al. (2008) showed that bioturbation by an amphipod increased the taxonomic richness of
zooplankton in the active community. They attributed this result to the amphipod digging
up buried, resting eggs, which facilitated their hatching. Other studies have shown that
the translocation of resting stages is not limited to crustaceans. Ståhl-Delbanco and
Hansson (2003), for instance, showed that recruitment of dormant algae cells in the
sediment was dramatically higher in the presence of an amphipod bioturbator.
5
The effects of bioturbation on crustacean communities have been examined primarily
in marine, lake, and permanent-pond ecosystems. Few studies have examined the effects
of bioturbation in temporary ponds, let alone the effects specifically on crustacean
communities in these unique ecosystems. Temporary ponds occur where seasonally
variable climatic conditions produce annual cycles of inundation and desiccation. These
cycles result in distinct aquatic and terrestrial communities in pond basins at different
times of year. Temporary pond ecosystems tend to be characterized by high levels of
species richness and endemism and are generally considered biodiversity hotspots
(Holland and Jain, 1981; King et al., 1996; Semlitsch and Bodie, 1998; Simovich, 1998).
In Mediterranean climate regions, such as the Central Valley of California, temporary
ponds are known as vernal pools. California vernal pools contain many plant and
invertebrate species that are listed as threatened or endangered (Federal Register, 2003).
Despite their global ubiquity (Keeley and Zedler, 1998), temporary pond systems
worldwide are in steady decline due to habitat loss and invasive species (Holland, 1978;
Blaustein and Schwartz, 2001; Brinson and Malvárez, 2002; De Meester et al., 2005). In
California, for example, only one-tenth of the historical expanse of vernal pool habitat in
the Central Valley remains due to agriculture and urbanization (Holland, 1978; Holland,
1998). The loss of vernal pools combined with their high levels of endemism makes this
habitat a focus of conservation efforts in the state. Understanding the factors that
contribute to and maintain biodiversity in vernal pools is a pre-requisite for their
successful management and restoration (Simovich, 1998). For example, knowing whether
6
one species was particularly important in maintaining crustacean diversity in California
vernal pools could inform and help guide management and restoration strategies.
The tadpole shrimp Lepidurus packardi (Crustacea: Branchiopoda: Notostraca:
Triopsidae) is a benthic omnivore that grows up to 8 cm long. It is endemic to California
vernal pools and, like other members of the family Triopsidae (Yee et al., 2005;
Waterkeyn et al., 2011b), is a strong bioturbator (Croel and Kneitel, 2011). It occurs as
far north as Shasta County and as far south as Tulare County, with the most known
occurrences in Sacramento County (U.S. Fish and Wildlife Service, 2007). It is also one
of the four large branchiopods federally listed as endangered (Federal Register, 2003).
Lepidurus packardi forages by sifting the sediment surface and underlying layers for food
items, often digging shallow depressions (~0.5 cm deep; personal observation) in the
process. Because of this bioturbation, L. packardi is recognized as an ecosystem engineer
(Croel and Kneitel, 2011). Although the presence of L. packardi is associated with higher
crustacean taxonomic richness compared to pools without L. packardi (King et al., 1996;
Croel and Kneitel, 2011), the impact of its bioturbation on the crustacean community in
California vernal pools has not been experimentally tested.
The goal of this study was to experimentally test the hypothesis that bioturbation by
Lepidurus packardi positively affects other crustacean taxa in California vernal pools. I
tested this hypothesis by reducing the density of L. packardi in vernal pool mesocosms
and comparing the resulting crustacean communities to those in unmanipulated
mesocosms. I predicted that mesocosms with reduced L. packardi densities would have
lower total crustacean abundance, taxonomic richness, and per-taxon abundances,
7
because fewer buried eggs would be returned to the sediment surface in these
mesocosms. I also compared water physicochemisty between treatments to assess how
bioturbation by L. packardi affected general abiotic conditions in the mesocosms. I
conducted complementary microcosm experiments in which I buried fairy shrimp eggs at
different depths. These microcosm experiments allowed me to directly test L. packardi’s
digging and egg-translocation abilities. They also allowed me to assess whether L.
packardi influenced resting-egg hatching via kairomones (chemical signals released by
predators; Lass and Spaak, 2003), which otherwise might be a confounding factor in this
study. Combined, these microcosm experiments would help me interpret the results of the
mesocosm experiment and shed greater light on how L. packardi affects other crustacean
taxa in California vernal pools.
8
METHODS
Mesocosm experiment
In December 2011, I arranged 20 mesocosms in a 4 × 5 array in an outdoor area on
the campus of California State University, Sacramento. A white, canvas drop cloth (3.7 ×
4.6 m) was placed beneath the array for weed control. The mesocosms were black,
circular, plastic pond liners (diameter = 0.6 m, depth = 0.18 m, volume = 51 L). I filled
each mesocosm with 10 kg of dry soil collected from vernal-pool basins in the Elder
Creek watershed of Sacramento County (Kneitel and Lessin, 2010). This amount of soil
yielded a soil layer about 4 cm deep. The soil surface in each mesocosm was smoothed
by hand to ensure an even depth throughout. Before being added to the mesocosms, the
soil was mixed in a cement mixer to homogenize the egg bank. Previous studies using
this soil (Kneitel and Lessin, 2010; Croel and Kneitel, 2011) demonstrated that it
contained eggs for many of the crustacean taxa typical of California vernal pools, e.g.,
cladocerans (water fleas), ostracods (seed shrimp), copepods, anostracans (fairy shrimp),
and notostracans (tadpole shrimp).
Due to lack of natural rainfall, I inundated the mesocosms with well water from a
hose on 1 January, 2012, to initiate the experiment. I filled each mesocosm to the top
using a gentle spray. To prevent the spray from disrupting the soil, I covered the surface
of the soil with bubble wrap prior to filling (Roast et al., 2004). After this initial filling,
water levels in the mesocosms were regulated solely by natural weather conditions. The
mesocosms always contained ample water during the experiment, never dropping below
9
about two-thirds full. Throughout the experiment, floating leaves from nearby trees were
removed by hand when observed in the mesocosms. Submerged leaves were left in place.
The experiment included two treatment groups: an unmanipulated group (hereafter,
the Control group) and an experimental group in which I reduced the density of L.
packardi (hereafter, the Removal group). Two replicates of each group were placed in an
alternating arrangement in each row of four mesocosms. I removed L. packardi from the
Removal group by gently sweeping the water column with a 15 × 20 cm, 500-μm mesh
aquarium net. The sweeping procedure consisted of one circular sweep counterclockwise,
one circular sweep clockwise, and then one linear sweep through the diameter, keeping
the bottom of the net as close as possible to the sediment surface without touching it. The
contents of the net were transferred to a shallow pan, in which I located and removed L.
packardi individuals via forceps or pipette. The remaining contents of the pan were
returned to the source mesocosm. The Control group was not manipulated, except that I
applied the sweeping procedure described above to these mesocosms as a disturbance
control. The contents of the net underwent the same handling as in the Removal group,
but all individuals, including L. packardi, were returned to the mesocosms. Treatments
were applied once per week beginning on 27 January, which was when I first observed
nauplii in the mesocosms. The final treatment was applied on 18 March, for a total of
eight treatments. I performed the sweeping procedure once in each mesocosm for the first
four treatments. Thereafter, I repeated the procedure twice in each mesocosm to increase
capture efficiency.
10
Croel and Kneitel (2011) used the same sweeping procedure and were able to greatly
reduce L. packardi densities in their mesocosms, in most cases to zero. Although I did not
necessarily expect to achieve complete removal of L. packardi in the present study, I
anticipated removal efficiencies comparable to Croel and Kneitel (2011). This was not
the case, however. During most treatment applications, the number of tadpole shrimp
removed from the Removal mesocosms approximated the number captured in the Control
mesocosms (Figure 1). This indicates that tadpole shrimp were hatching continuously
from resting eggs in the Removal mesocosms, which was unexpected. Although L.
packardi abundance was similar in both treatment groups for most of the experiment, this
does not necessarily mean that the two groups were identical with respect to L. packardi.
Compared to the Control group, individuals in the Removal group were generally smaller
because most were ~7 days old or less. In any event, not being able to achieve complete
removal of tadpole shrimp makes the experiment more conservative, as it lessens any
differences between the groups.
On 9 January, seven of the 20 mesocosms (three Control replicates and four Removal
replicates) were visited by waterfowl. I excluded these seven mesocosms from the
experiment because their sediments had been substantially disrupted, which could have
confounded my results. To maintain a balanced design, I randomly chose one of the
remaining Control replicates and also excluded it from the experiment. As a result, all
analyses in this study are based on six replicates per group rather than 10. To prevent
further visitation by waterfowl, I covered the mesocosm array with fine-filament bird
netting (1.2-cm aperture mesh), suspended 1.8 m above the array. The netting was left in
11
Figure 1. Number of L. packardi captured at each treatment application. After being
counted, L. packardi individuals were returned to mesocosms in the Control group, but
were not returned to mesocosms in the Removal group. In the Control group, the number
captured reflects the population size on that date. In the Removal group, the number
captured is the number of L. packardi permanently removed from the mesocosms on that
date. The first treatment was applied on 27 January, but this date is not shown because no
L. packardi were captured. The black arrow at 18 March indicates the final treatment
application. The gray arrow at 24 March indicates the end of the experiment, when all
mesocosms were destructively sampled. Error bars show ± 1 SD.
12
place for the remainder of the experiment. On two other occasions I observed evidence of
animal visitation in the form of paw prints on mesocosm rims. However, there was no
evidence that the sediment in the visited mesocosms had been disturbed, and so these
mesocosms were retained in the study.
On 23 March, one day prior to terminating the experiment, I collected water
physicochemistry data. I used an Oakton pH/CON 300 Meter to measure water
temperature (oC), conductivity (μS), and pH, and an Oakton pH/DO 300 Meter to
measure dissolved oxygen (mg/L). These measurements were taken in situ in the early
afternoon. I also collected 50-mL water samples for laboratory analyses of turbidity
(NTU), PO43- (as orthophosphate, mg/L), and NO3- (mg/L). Lab analyses were completed
within 8 h of sample collection. All water samples were first filtered through a 243-μm
sieve to remove coarse particulates. I then used a LaMotte 2020i Turbidity Meter to
quantify the turbidity of the filtered samples. For the PO43- and NO3- analyses, I further
processed the water samples by centrifuging them at 4000 rpm for 10 minutes to pellet
the suspended sediment, which can interfere with testing reagents. The supernatant was
then syringe-filtered through a 0.45-μm nylon membrane. I used a Hach DR2800
spectrophotometer to quantify PO43- (via Method 8048) and NO3- (via Method 8171) in
the filtered supernatant.
I destructively sampled the mesocosms for crustaceans on 24 March by performing
the previously described sweeping procedure four times in each mesocosm. I counted L.
packardi individuals and returned them to the mesocosms. All other crustaceans were
preserved in 95% ethanol. I sorted and counted the preserved crustaceans under a 25x
13
dissection microscope. I identified crustaceans to the lowest taxonomic level possible
according to Thorp and Covich (2010). For ostracods, I also used a 100x light microscope
as well as the identification key in Pennak (1989). Due to the large number of crustaceans
captured (~20,000 individuals), I did not key-out each individual. Rather, I used the
identification keys to identify three to five individuals per taxon per mesocosm, and then
I sorted the remaining individuals based on morphological similarity to the identified
taxa. Most taxa were identified to genus; two were identified to species. I did not collect
data for non-crustacean invertebrates, but the abundance of such taxa in the preserved
samples was low (~5 or fewer total individuals per mesocosm).
I conducted an Analysis of Similarities (ANOSIM) to test for a treatment effect on
crustacean-community similarity among treatments. This analysis used 5000
permutations and was based on Bray-Curtis dissimilarities calculated from a matrix of
taxa abundances in each mesocosm. I interpreted a significant result with a Similarity
Percentage (SIMPER) analysis to reveal which taxa were contributing most to the
dissimilarity between treatments. To visualize the communities in ordination space, I
used a 2D non-metric multidimensional scaling (NMDS) plot, which was based on the
same data matrix as the ANOSIM. I saved the dimension scores from this plot and used
Pearson’s correlations to examine relationships between these scores and water
physicochemistry variables.
I conducted additional tests to determine whether specific aspects of community
structure were affected by L. packardi removal. I used independent-samples t-tests to
compare evenness and total crustacean abundance between treatments. The measure of
14
evenness used was Pielou’s evenness (E), calculated as E = H/lnS, where H is Shannon’s
Diversity Index (−Σpi[lnpi], where pi is the proportion of individuals of taxon i relative to
the total number of individuals in all taxa) and S is taxonomic richness (Pielou, 1966).
Total abundance was the total number of all crustacean individuals in each mesocosm. I
did not compare taxonomic richness because both treatments had a mean richness of 10.
To determine how individual taxa responded to reduced L. packardi density, I conducted
an independent-samples t-test on the abundance of each taxon, with the exceptions of
Cypris sp. and Eucypris sp. Data for Cypris sp. did not satisfy parametric assumptions,
even after transformation, and so were analyzed non-parametrically via the MannWhitney U-test. Eucypris sp. abundance was compared with Analysis of Covariance
(ANCOVA), using water temperature as the covariate, because preliminary analyses
showed that this taxon’s abundance was strongly correlated with water temperature in the
mesocosms (Pearson’s r = −0.85, P = 0.001). Prior to this analysis, I confirmed that
ANCOVA assumptions were satisfied (i.e., homogeneity of regression slopes and
covariate independence of treatment effect).
I used Multivariate Analysis of Variance (MANOVA) to compare abiotic conditions
(i.e., water physicochemistry) between treatments. Dependent variables were turbidity,
pH, conductivity, and water temperature. Data for PO43- and NO3- concentrations were
invariable and thus excluded from this analysis. Dissolved oxygen was strongly
correlated with water temperature (Pearson’s r = 0.84, P = 0.001) and so was also
excluded to preserve degrees of freedom.
15
Lepidurus packardi was excluded from all analyses. Two other taxa, Moina sp. and
Pseudocandona sp., were also excluded from all analyses because they were extremely
rare in the mesocosms in both abundance and occurrence. Prior to the parametric
analyses, I used Shapiro-Wilk’s test and Levene’s test to check data for normality and
equal variance, respectively. Data were square-root or log transformed as needed to
satisfy parametric assumptions. All figures show untransformed data for clarity. I used
PAST 1.94b (Hammer et al., 2001) for the analyses based on Bray-Curtis dissimilarities.
All other analyses were conducted in SPSS 21.0.
Microcosm experiments
I conducted three microcosm experiments that directly tested whether L. packardi
could dig up buried resting eggs. I also considered whether plant seeds could be
translocated, as an added measure. This allowed me to assess whether propagules (i.e.,
both eggs and seeds) in general could be translocated by L. packardi. These experiments
also tested for any kairomone-related influences exerted by L. packardi. Accounting for
the potential influence of kairomones was necessary because they might otherwise
confound the mesocosm results.
All three microcosm experiments were set up at the same time and conducted
simultaneously. These experiments used clear, plastic, food-storage containers (14 cm
square × 8 cm tall, volume = 1.5 L) as microcosms. Each was filled with 350 mL (~ 440
g) of dry soil collected from vernal-pool basins in the Gill Ranch Conservation Bank in
Sacramento County. This amount of soil yielded a soil layer about 2 cm deep. I glided a
16
straight edge over the soil surface to make it smooth and uniform. I then sprinkled 7 mg
of fairy shrimp resting eggs (~2000 eggs) as evenly as possible on the soil surface. The
eggs belonged to the freshwater taxon Streptocephalus sp. and were obtained from a
commercial supplier (www.arizonafairyshrimp.com). Preliminary studies showed that
these eggs hatched quickly upon rehydration (within 24 h) under laboratory conditions.
Because of time constraints, using this quick-hatching taxon as an indicator of egg
translocation was more practical than relying on the soil’s natural egg bank, as crustacean
eggs in California vernal pools generally take at least 2 – 4 weeks to hatch after
inundation (Ahl, 1991; Gallagher, 1996; personal observation). This constraint did not
apply to seeds because germination of some vernal-pool plant species is rapid (≤ 3 days;
Bliss and Zedler, 1998; personal observation). Also, preliminary studies confirmed that
seeds in the soil used in these experiments reliably germinated within a few days
following inundation. It was thus more practical to leverage the seeds naturally present in
the soil rather than add them.
In one microcosm experiment, the propagules were allowed to remain at the soil
surface (the 0 cm-burial experiment). In the other two experiments, I buried the
propagules under 0.5 cm- and 1 cm-deep layers (respectively) of vernal pool soil that had
been autoclaved for 90 min. The autoclaved soil acted as a barrier to hatching and
germination once the microcosms were inundated (Gleason et al., 2003; personal
observation). The 0.5 cm-burial and 1 cm-burial experiments allowed me to directly test
whether L. packardi could dig through the soil barrier and translocate propagules to the
sediment surface. To create the 0.5-cm and 1-cm deep layers, I added 100 mL (~114 g)
17
and 200 mL (~228 g), respectively, of autoclaved soil atop the propagules. The
autoclaved soil layer was smoothed with a straight edge to achieve as uniform a depth as
possible.
After adding the eggs and autoclaved soil, I laid a piece of bubble wrap atop the soil
(Roast et al., 2004) and then gently inundated each microcosm with 1 L of deionized tap
water. The bubble wrap prevented the spray from disrupting the soil. Once the
microcosms were inundated, I added the treatment, which was the influence of L.
packardi. Levels for this treatment were L. packardi present, absent, and caged. Each
treatment was replicated four times, for a total of 12 replicates per experiment. For the L.
packardi-present treatment, I added one L. packardi to each replicate. The movement of
L. packardi in these replicates was unrestricted; individuals were allowed to freely roam
and bioturbate. The L. packardi-absent treatment had no L. packardi added. For the L.
packardi-caged treatment, one L. packardi was added to each replicate but was
sequestered in a 6 × 6.5 cm, 53-μm mesh cage constructed from an aquarium net. The
cage was suspended above the sediment in one corner of the microcosm and prevented L.
packardi from contacting the sediment. The handle of the cage was bent over the corner
of the microcosm and taped to the outer sidewall. The cage’s mesh was fine enough to
prevent nauplii from swimming into the cage, where they might be eaten by L. packardi.
Sequestering L. packardi in cages allowed me to test for kairomone-related influences
this taxon may have on propagules (Waterkeyn et al., 2012). The L. packardi used for
these experiments had a mean (± SD) carapace length of 1.19 ± 0.13 cm and were
obtained from surplus mesocosms immediately before being added to the microcosms.
18
I prepared two additional groups of microcosms as controls. I included an autoclavecontrol group to ensure that the autoclaved soil added atop the propagules contained no
viable eggs or seeds of its own. This group contained two replicates, each filled with 350
mL of autoclaved soil only. A cage-control group, containing four replicates, was also
included. The microcosms in this group were identical to the L. packardi-caged replicates
in the 0 cm-burial experiment, except that no L. packardi were added to the cages. This
controlled for any effect of the cage itself on hatching or germination.
Once all 42 replicates (12 replicates in each of three experiments, plus six control
replicates) were filled and all L. packardi added, I placed the microcosms in an
environmental chamber and maintained them under a 24oC/14 h light:19 oC/10 h dark
regime. Nine replicates (one replicate of each L. packardi treatment from each
experiment) were placed on each of four shelves. The six cage- and autoclave-control
replicates were placed on a fifth shelf.
I removed all L. packardi from the microcosms after 24 h. To remove them from the
L. packardi-present replicates, I plucked them out with forceps, taking care not to touch
the sediment. I briefly dipped the forceps in all other microcosms as a control. For the L.
packardi-caged replicates, I detached and removed the cages from the microcosms. I
removed the cages from the cage-control replicates at the same time. I held each cage
over a separate container and rinsed the mesh with water to dislodge any nauplii that may
have been stuck to the mesh. I visually inspected the rinse water for nauplii, and if any
were observed I added them to the total number of nauplii captured at the end of the
experiment (I observed nauplii in the rinse water for only one microcosm). Seven of the
19
12 L. packardi individuals in the replicates with cages did not survive the 24-h period,
presumably due to starvation. One individual from an L. packardi-present replicate also
did not survive. I returned the microcosms to the environmental chamber once all L.
packardi and cages had been removed.
Three days after removing L. packardi, I siphoned the water of each microcosm
through a 53-μm sieve to capture fairy shrimp nauplii. The siphon used 5-mm (internal
diameter) plastic tubing. Nauplii were immediately preserved in 95% ethanol and counted
under a 25x dissection microscope. The siphoning removed all standing water, but the
soil remained saturated and was conducive to seed germination. Accordingly, I returned
the microcosms to the environmental chamber for an additional eight days, after which I
counted the number of seedlings in each microcosm.
In the 0-cm burial experiment, I predicted that nauplii and seedlings would be similar
in abundance across L. packardi treatments, because the propagules would be fully
exposed to hatching and germination cues. In the 0.5-cm burial experiment, I predicted
that nauplii and seedlings would be most abundant in microcosms containing freely
roaming L. packardi. In these microcosms, I expected L. packardi to dig up many of the
buried propagules and translocate them to the sediment surface, where they would
hatch/germinate. I expected no nauplii or seedlings in the L. packardi-caged or L.
packardi-absent treatments, because the propagules would stay buried and hence remain
insulated from emergence cues (Gleason et al., 2003; personal observation). Similarly, I
predicted that no nauplii or seedlings would be found in the 1-cm burial experiment,
reasoning that this is too deep for L. packardi to dig (Gleason et al., 2003; personal
20
observation). I further expected to find no evidence of kairomones in these experiments
(Waterkeyn et al., 2012).
I used one-way ANOVAs to analyze abundance data for each experiment. These tests
used L. packardi treatment (present, absent, or caged) as the fixed factor and nauplii
abundance or seedling abundance as the dependent variable. Data for the 0.5 cm-burial
experiment were square-root transformed to meet parametric assumptions. These
analyses were conducted in SPSS 21.0.
21
RESULTS
Mesocosm experiment
The preserved samples contained 19,690 crustaceans belonging to 12 taxa (Table 1).
Most taxa were found in all six mesocosms of each treatment group, with three
exceptions. Moina sp. occurred in just one mesocosm, Pseudocandona sp. occurred in
three mesocosms (two Control replicates and one Removal replicate), and Simocephalus
sp. occurred in four mesocosms of each treatment group. The number of crustacean taxa
in each mesocosm ranged from 9 to 11, with a mean richness of 10 in both treatment
groups.
Weekly removal of L. packardi from the Removal group had no significant effect on
crustacean-community similarity between treatments (ANOSIM: R = 0.35, P = 0.12).
Because this result was not significant, I did not proceed with the SIMPER analysis.
Consistent with the ANOSIM result, the NMDS plot showed no discernable patterns in
community composition related to L. packardi removal (Figure 2). The stress value for
this ordination was low (stress = 0.091).
I found a strong positive relationship between Dimension 1 scores of the NMDS plot
and water temperature (Pearson’s r = 0.88, P < 0.001). Dimension 1 scores were also
strongly negatively related to the abundance of Eucypris sp. (Pearson’s r = −0.97, P <
0.001), which was the numerically dominant taxon in all mesocosms (Table 1). This
indicates that variation in Eucypris sp. abundance was responsible for most of the
dissimilarity observed along Dimension 1. No other taxon’s abundance was significantly
related to Dimension 1 scores. Dissolved oxygen was also positively related to
22
Table 1. Summary of crustacean taxa abundance (excluding L. packardi) observed in
each treatment group. For each group, n = 6. Removal group = mesocosms from which L.
packardi was removed weekly. Control group = unmanipulated mesocosms. Total = total
number of individuals captured.
Mean ± SE abundance
Taxonomic group
Taxon
Removal group
Control group
Total
Anostraca
Linderiella occidentalis
25.7 ± 4.9
28.0 ± 4.3
322
Cladocera
Bosmina sp.
15.5 ± 1.3
6.2 ± 1.6
130
Daphnia sp.
264.0 ± 89.0
408.7 ± 177.6
4036
Moina sp.
1
0
1
Simocephalus sp.
17.2 ± 9.8
15.7 ± 8.1
197
Hesperodiaptomus sp.
24.2 ± 4.8
32.5 ± 2.9
340
Leptodiaptomus sp.
276.2 ± 47.1
288.0 ± 31.9
3385
Candona sp.
21.2 ± 5.6
15.8 ± 3.6
222
Cypris sp.
74.7 ± 9.3
34.5 ± 6.2
655
Eucypris sp.
941.8 ± 237.3
775.0 ± 181.0
10,301
Limnocythere ceriotuberosa
13.0 ± 4.4
3.3 ± 1.0
98
Pseudocandona sp.
1
2
3
12 taxa
1674 ± 270
1608 ± 171
19,690
Copepoda
Ostracoda
Total
23
Figure 2. Non-metric multidimensional scaling ordination of mesocosms. The stress
value for this ordination was 0.091.
24
Dimension 1 scores (Pearson’s r = 0.66, P = 0.020). Dimension 2 scores were related to
turbidity (Pearson’s r = 0.74, P = 0.006) and Daphnia sp. abundance (Pearson’s r = 0.78,
P = 0.003).
Reducing L. packardi density had no significant effect on evenness or total crustacean
abundance, but it did significantly affect the abundances of four taxa (Table 2). Contrary
to my hypothesis, however, all four affected taxa were more abundant in mesocosms
from which L. packardi had been removed. The affected taxa were the cladoceran
Bosmina sp. (t = 4.56, df = 10, P = 0.001; Figure 3A) and the ostracods Cypris sp. (U =
1.00, P = 0.004; Figure 3B), Limnocythere ceriotuberosa (square-root transformed, t =
2.37, df = 10, P = 0.039; Figure 3C) (Table 2), and Eucypris sp. (square-root
transformed, F1,9 = 5.70, P = 0.041; Figure 4). Water temperature also had a significant
effect on Eucypris sp. abundance (F1,9 = 45.00, P < 0.001). Adjusted for covariation with
water temperature, Eucypris sp. abundance in the Control treatment was (backtransformed, estimated marginal mean ± 95% CI) 645.0 ± 182.8 individuals, compared to
949.8 ± 221.8 individuals in the Removal treatment.
Overall abiotic conditions in the mesocosms, as measured by the water
physicochemistry variables, were not significantly different between treatments (Pillai’s
Trace = 0.57; df = 4,7; F = 2.32; P = 0.16).
25
Table 2. Independent-samples t-test results for evenness, total abundance, and per-taxon
abundances in mesocosm experiment. Eucypris sp. abundance was analyzed separately
because it covaried with water temperature. Taxonomic richness was not analyzed
because both groups had the same mean richness. df = 10.
Variable
t
P-value
Evenness
0.55
0.60
Total abundance
0.21
0.84
Linderiella occidentalis
0.36
0.73
Bosmina sp.
4.56
0.001
Daphnia sp.
0.73
0.48
Simocephalus sp. a
0.05
0.97
Hesperodiaptomus sp.
1.48
0.17
Leptodiaptomus sp.
0.21
0.84
Candona sp. b
0.67
0.52
−
0.004
2.37
0.039
Cypris sp. c
Limnocythere ceriotuberosa a
a
square-root transformed
b
log transformed
c
Mann-Whitney U-test; U = 1.00
26
Figure 3. Comparison of Bosmina sp., Cypris sp., and Limnocythere ceriotuberosa
abundances in Control (unmanipulated) mesocosms vs. mesocosms from which L.
packardi was removed weekly. (A) Comparison of Bosmina sp. abundance. (B)
Comparison of Cypris sp. abundance (analyzed non-parametrically). (C) Comparison of
L. ceriotuberosa abundance. Panel C shows untransformed data for clarity. Error bars
show ± 1 SE.
27
Figure 4. ANCOVA comparison of Eucypris sp. abundance in Control (unmanipulated)
mesocosms vs. mesocosms from which L. packardi was removed weekly. This figure
shows untransformed data for clarity.
28
Microcosm experiments
In the 0 cm-burial experiment, nauplii abundance was significantly lower in the L.
packardi-present treatment compared to the L. packardi-caged treatment (F2,9 = 6.55, P =
0.018; Figure 5A). There was no significant difference in seedling abundance between
treatments (F2,9 = 0.41, P = 0.68; Figure 5B).
In the 0.5 cm-burial experiment, burying propagules sharply reduced their hatching
and germination. There was no significant difference between L. packardi treatments in
either nauplii abundance (square-root transformed, F2,9 = 0.15, P = 0.87; Figure 5C) or
seedling abundance (square-root transformed, F2,9 = 3.95, P = 0.059; Figure 5D),
although the latter difference approached significance, trending toward more seedlings in
the L. packardi-present treatment compared to the other two treatments.
I did not analyze data from the 1 cm-burial experiment because only 3 nauplii and 2
seedlings were observed among all 12 replicates.
No nauplii or seedlings were found in the control group containing autoclaved soil
only, indicating that this soil contained no viable eggs or seeds. Also, in the cage-control
group, there was no significant difference in either nauplii abundance (ANOVA: F1,6 =
0.015, P = 0.91) or seedling abundance (ANOVA: F1,6 = 4.31, P = 0.083) compared to
the L. packardi-caged replicates in the 0 cm-burial experiment. This indicates that the
cage itself did not affect hatching or germination.
29
Figure 5. Comparison of nauplii and seedling abundances in microcosm experiments. (A,
B) 0 cm-burial experiment. (C, D) 0.5 cm-burial experiment. Lepidurus packardi
treatment: Present = freely roaming L. packardi, Absent = no L. packardi added, Caged =
L. packardi present but sequestered in cage. Nauplii and seedlings were counted 3 days
and 11 days (respectively) after removing L. packardi from the microcosms. In A,
different letters above bars indicate a significant difference between treatments (P =
0.018). All other differences are non-significant (P ≥ 0.059). C and D show
untransformed data for clarity. Error bars show ± 1 SE.
30
DISCUSSION
I found no support for my hypothesis that bioturbation by L. packardi returns buried,
resting eggs to the sediment surface and facilitates their hatching. In the mesocosm
experiment, I predicted that removing L. packardi (or at least reducing its density) would
reduce crustacean taxonomic richness, total abundance, and per-taxon abundances.
However, none of these variables decreased in the Removal treatment. Contrary to my
hypothesis, in fact, four taxa were significantly more abundant in mesocosms where L.
packardi density had been reduced. This shows that L. packardi was suppressing these
taxa in the Control mesocosms. In the microcosm experiments, which were in part
designed to directly test L. packardi’s digging ability, I found that L. packardi did not dig
up propagules buried ≥ 0.5 cm deep. Overall, my results suggest that bioturbation by L.
packardi does not facilitate the hatching of buried propagules. They also indicate that L.
packardi’s influence on other crustacean taxa is generally negative.
There are at least five possible, non-mutually exclusive explanations for L. packardi’s
suppression of the four taxa in the Control mesocosms. The first and most likely
explanation is predation on these taxa by L. packardi. Although I know of no studies that
have examined the feeding habits of L. packardi specifically, several studies have
demonstrated that notostracans consume adult crustaceans (Christoffersen, 2001; Boix et
al., 2006; Waterkeyn et al., 2011b) and resting eggs in the sediment (Waterkeyn et al.,
2011a). My own observations of L. packardi in aquaria verify that it too consumes adult
crustaceans. The results of my 0-cm burial microcosm experiment show that L. packardi
also consumes resting eggs. Given that tadpole shrimp are known predators of both active
31
and resting crustacean stages, it is likely that predation played a large role in suppressing
the four affected taxa in the Control mesocosms. I cannot determine from my data
whether resting or active stages were predominantly consumed, but it was probably the
latter. If resting stages had mainly been consumed, I would expect all taxa to decline,
because resting-egg consumption by tadpole shrimp appears to be indiscriminate
(Waterkeyn et al., 2011a). Further, three of the four affected taxa were ostracods, which,
similar to L. packardi, are benthic and substrate feeders. It seems logical that L. packardi
would preferentially consume active-stage ostracods, because it would encounter them
more often than it would open-water taxa (but see Boix et al., 2006). Both Waterkeyn et
al. (2011b) and Yee et al. (2005) showed that notostracans in the genus Triops negatively
affected ostracod taxa, with the former study directly showing consumption of adult
ostracods as the main cause. The decline of the cladoceran Bosmina sp. is more difficult
to explain in terms of predation on active stages. Notostracans are able to capture and
consume cladocerans (Christoffersen, 2001; Waterkeyn et al., 2011b), but why this
cladoceran taxon alone was affected is unclear. It could be that this cladoceran simply
spent more time near the sediment than other cladoceran taxa, and thus was
disproportionately vulnerable to predation by L. packardi.
The second possible explanation for the reduced taxa abundances in the Control
mesocosms is competition with L. packardi. Tadpole shrimp and ostracods share the
same general feeding habit, i.e., foraging along the sediment surface. The larger size of L.
packardi may have allowed it to more effectively compete for benthic food resources. In
addition, L. packardi’s near-constant movement along and within the sediment could
32
have resulted in interference competition by displacing food items or ostracods directly
(sensu Brenchley, 1981). However, competitive interactions would probably not explain
why a cladoceran taxon also declined in abundance, as cladocerans are pelagic
suspension-feeders.
A third possible explanation is that the suspended sediment caused by L. packardi’s
bioturbation may have clogged the feeding apparati of the affected taxa in the Control
mesocosms (Newcombe and MacDonald, 1991). Such clogging has been shown to
negatively affect certain suspension feeders, including cladocerans (Kirk, 1991).
However, in the present study only one suspension feeder (Bosmina sp.) out of six
(excluding Moina sp.) was negatively affected. If clogging were an issue, more
suspension feeders would likely have been affected. The other taxa affected in this study
were ostracods, which are not suspension feeders, although they do filter/strain food
particles to some extent (Pennak, 1989). Several observational studies have reported a
negative relationship between ostracods and suspended sediment (e.g., Cohen et al.,
1993; Ruiz et al., 2013), but it is unclear whether this was due to clogging or some other
factor associated with suspended sediment (Donohue and Molinos, 2009).
The fourth possible explanation also involves suspended sediment, which may have
inhibited the growth of planktonic and periphytic algae through light limitation. These
algae are a major food resource for temporary-pond crustaceans, including notostracans
(Boix et al., 2006). This explanation is doubtful, however. Although the Control
mesocosms had noticeably higher turbidity than the Removal mesocosms (Control-group
turbidity [mean ± SD]: 268 ± 67 NTU; Removal-group turbidity: 196 ± 29 NTU; data not
33
shown), turbidity in the Control mesocosms was probably not high enough to have
caused light limitation. The results of Croel and Kneitel (2011) support this idea. Using
the same mesocosm containers, they found that it took over four times the turbidity
observed in the present study (~1150 NTU vs. ~270 NTU, respectively) to cause only a
relatively small (25%) reduction in macrophyte cover. Thus, light limitation in the
Control mesocosms was probably nominal compared to the Removal mesocosms.
Finally, although I found no evidence of kairomone-mediated hatching suppression in
the microcosm experiments, strictly speaking this finding applies just to the anostracan
eggs used in those experiments. Therefore, it is still possible that the eggs of the affected
taxa were able to detect the presence of L. packardi via kairomones (Lass and Spaak,
2003) and subsequently remain in diapause (Waterkeyn et al., 2012). This is an unlikely
explanation for my mesocosm results, however. Kairomones would have been present in
all mesocosms, albeit at different concentrations depending on treatment, because tadpole
shrimp were never completely absent from the Removal group. And while there is some
evidence that the eggs of certain crustacean taxa can detect kairomones and delay their
hatching, the evidence so far is limited to vertebrate predators that target only adult
crustaceans (Blaustein, 1997; Spencer and Blaustein, 2001; Bozelli et al., 2008).
Waterkeyn et al. (2012) also found no evidence that crustacean eggs can detect
notostracan kairomones. They further note that a strategy of delayed hatching would not
benefit crustaceans in the presence of notostracan predators, because their eggs would
still be vulnerable to predation in the sediment.
34
Assuming that predation was a factor in my results, it could be posited that egg
translocation occurred in the mesocosm experiment but was offset by egg consumption,
such that the net effect on crustacean abundance was neutral or negative. The merits of
this idea depend on L. packardi’s rate of predation on active and resting stages relative to
the number of eggs in the mesocosms’ soil. To my knowledge, there are no published
studies reporting predation rates for L. packardi in either natural pools or artificial
containers. However, studies have quantified predation rates for the notostracans Triops
cancriformis (Waterkeyn et al., 2011a; Waterkeyn et al., 2011b) and Lepidurus arcticus
(Christoffersen, 2001). These studies show that notostracans can exert considerable
predation pressure on crustacean adults and eggs, at least in small, experimental
containers that offer no other food options. To illustrate, Waterkeyn et al. (2011a)
extrapolated their laboratory results to estimate that a dense population of Triops sp. (300
individuals/m2) could completely strip an egg bank of resting eggs in 100 days or fewer.
If bioturbation by tadpole shrimp returned eggs to the sediment surface and facilitated
their hatching, this could very well be undetectable if more eggs were being eaten than
were being translocated. However, these studies likely overestimate predation rates, for
two reasons. First, the researchers imposed artificially high encounter rates on predator
and prey by using small containers (200 mL – 10 L) from which the prey could not
escape. Second, these studies did not include the broad array of food items normally
consumed by notostracans in their natural habitats. Rather, the researchers experimentally
limited their subjects’ food options to one or a few prey items. Under such controlled
conditions, a high rate of predation is not surprising. Notostracans indeed consume
35
animal prey, but under natural conditions they also rely heavily on detritus and plant
matter (Boix et al., 2006). If notostracans were as voracious a predator (of either resting
or active stages) in natural temporary ponds as these studies suggest, they would quickly
become the only crustacean taxon found in these water bodies. Yet, L. packardi co-occurs
with as many as 26 other crustacean taxa in California vernal pools (King et al., 1996).
Therefore, the predation pressure exerted by notostracans in habitats containing their
natural assortment of food items is arguably much less than in small, laboratory
containers with restricted food options. Under realistic field densities, for instance,
Christoffersen (2001) estimated that one adult Lepidurus sp. would consume a maximum
of just six cladocerans per 24 h period. In contrast to putatively low predation rates in
natural temporary ponds, the sediments in such ponds can contain a huge number of
resting eggs, e.g., 105 – 107 eggs/m2, in their uppermost layers (Hairston and Kearns,
2002; Brendonck and De Meester, 2003). The soil I used in the mesocosms was probably
no exception. In principle, these eggs represent a massive potential food resource for L.
packardi. In practice, however, these eggs were probably just a fraction of L. packardi’s
diet in the mesocosms because plants, algae, and detritus were also available (because
natural vernal pool soil was used). Therefore, any signal of egg translocation should have
been only marginally muted by egg predation in the mesocosm experiment.
The results of the microcosm experiments can also shed light on the potentially
masking effect of egg predation. In the 0-cm burial experiment, L. packardi clearly ate
eggs (Figure 2A) but not seeds (Figure 2B). In the 0.5-cm burial experiment, freely
roaming L. packardi could have translocated eggs but then eaten them (which might
36
explain why I found no significant difference in nauplii abundance between groups;
Figure 2C). However, if freely roaming L. packardi had translocated eggs in this
experiment, it almost surely would have also translocated seeds. Since L. packardi was
not eating seeds (i.e., Figure 2B), I should have found a significant difference between
treatments in seedling abundance in the 0.5-cm burial experiment, if propagule
translocation had in fact occurred. There was no such difference, however (Figure 2D,
although this difference approached significance). This indicates that seeds had not been
translocated. If seeds had not been translocated, it is unlikely that eggs were. From the
microcosm experiments alone, therefore, I infer that egg translocation was not masked by
consumption because it did not occur in the first place. This argument rests on the
assumption that eggs and seeds have the same potential to be translocated. The
anostracan eggs used in the microcosm experiments each had an estimated mass of ~4 μg.
This is less than seeds of vernal-pool plants, which can range from ~20 μg (Linhart,
1974) to ~200 μg (Zammit and Zedler, 1990). Even so, both types of propagule are very
small relative to adult or juvenile notostracans. It is reasonable to expect that eggs and
seeds in vernal pools have a similar potential for translocation. My microcosm
experiments therefore provide evidence that propagule translocation simply did not
occur. It is possible that the propagules were buried too deep for L. packardi to reach, but
my own observations refute this. I observed freely roaming L. packardi digging ~0.5 cm
deep in the microcosm containers. Waterkeyn et al. (2011a) additionally showed that the
notostracan Triops cancriformis, similar in size to my L. packardi, can dig as deep as 1.5
cm. It is therefore doubtful that the propagules were out of reach.
37
One weakness with the mesocosm experiment was that I was unable to achieve a
sizeable difference in tadpole shrimp density between treatments until the end of the
experiment. This indicates that tadpole shrimp were hatching continuously from resting
eggs in the Removal mesocosms. This was not observed in a prior study using the same
mesocosms, soil, and netting procedure (Croel and Kneitel, 2011), and so was
unexpected. Although the continuous presence of juveniles throughout a season has been
previously reported (Ahl, 1991; Gallagher, 1996; Alexander and Schlising, 1998), it was
thought that these juveniles probably hatched from subitaneous eggs (i.e., eggs that hatch
in the same season they are produced; Ahl, 1991). Lepidurus packardi needs a minimum
of 41 days, and an average of 54 days, to produce eggs of either type (subitaneous or
resting; Helm, 1998). Moreover, I did not observe ovisacs on any individuals until the 18
March treatment. These observations mean that all L. packardi captured in the Removal
mesocosms prior to and likely including the 10 March treatment could only have come
from resting eggs (assuming that some of the nauplii observed on 27 January were L.
packardi). The divergence in L. packardi abundance beginning with the 18 March
treatment (Figure 1) probably reflects the hatching of the first cohort of subitaneous eggs
in the Control mesocosms. Continuous hatching from resting eggs presumably also
occurred in the Control mesocosms, but L. packardi density in these mesocosms may
have been kept in check by intraspecific competition or cannibalism from larger, older
individuals (Golzari et al., 2009; Waterkeyn et al., 2011b). This might explain why the
weekly abundances of L. packardi in the two treatment groups were similar, despite the
consistent removal of this taxon from one group. While this issue would have lessened
38
any differences between the two groups of mesocosms, it did not necessarily compromise
my ability to detect egg translocation. The L. packardi individuals removed from the
Removal mesocosms were typically smaller than those observed in the Control
mesocosms (although I did not collect size data) because they were younger, most having
hatched only within the previous ~7 days. Assuming that larger L. packardi individuals
disrupt sediment more extensively than smaller ones, more eggs would potentially have
been translocated in the Control mesocosms than in the Removal mesocosms. The fact
that the Control treatment had 37% greater turbidity than the Removal treatment (data not
shown) supports the idea that larger individuals in the former group were causing more
sediment disruption. Although this quantitative difference in turbidity may have
manifested only within the final week or two of the experiment, when subitaneous eggs
began to hatch, my qualitative, weekly observations of turbidity indicate that sediment
disruption was greater in the Control mesocosms for most of the experiment.
Water temperature appeared to contribute strongly to community dissimilarity
between mesocosms, as suggested by the correlation between temperature and Dimension
1 scores of the NMDS plot. However, dissimilarity along Dimension 1 was driven almost
entirely by variation in Eucypris sp. abundance. The strong negative association between
Dimension 1 scores and Eucypris sp. abundance shows that these variables are basically
one and the same. Therefore, the positive correlation between water temperature and
Dimension 1 can be interpreted as simply reflecting the negative correlation between
water temperature and Eucypris sp. abundance. The temperature gradient itself probably
originated from a large tree to the west of the study area that partially shaded the
39
mesocosm array in the morning hours for much of the experiment. It is curious that
Eucypris sp. was the only taxon to exhibit a relationship with water temperature, given
that this abiotic variable is a reliable indicator of habitat suitability for temporary-pond
crustaceans (Brendonck and De Meester, 2003). It may be that there was not enough
variation in the abundances of the other taxa to allow their relationships with water
temperature to be detectable. The positive relationship between dissolved oxygen and
Dimension 1 scores was probably a by-product of the positive relationship between
dissolved oxygen and temperature; warmer mesocosms likely had higher rates of
photosynthesis and lower rates of respiration (because Eucypris sp. was less abundant in
warmer mesocosms), hence they had more dissolved oxygen. Turbidity was the only
abiotic correlate of Dimension 2, and the only taxon related to this dimension was
Daphnia sp. Taken together, these results indicate that the two most abundant taxa in this
study, along with temperature, dissolved oxygen, and turbidity, were mainly responsible
for the dissimilarity in crustacean communities between mesocosms.
Based on an analysis of data published in King et al. (1996), Croel and Kneitel (2011)
reported that vernal pools containing L. packardi had over twice as many crustacean taxa
as pools without L. packardi. Croel and Kneitel (2011) suggested that this might be due
to bioturbation by this taxon, but the results of the present study cast doubt on this
suggestion. Indeed, my results suggest that L. packardi suppresses crustacean taxa via
predation rather than facilitates them via bioturbation. The lack of support for my
hypothesis was somewhat surprising. Lepidurus packardi clearly digs within and disrupts
the sediment as it forages (Croel and Kneitel, 2011; personal observation). It is difficult
40
to imagine that buried eggs (and seeds) are not returned to the sediment surface in L.
packardi’s ejecta. It could be that the L. packardi individuals observed in this study were
not large enough to dig up buried eggs. Individuals in the Control mesocosms were
smaller than normal for their age, reaching maximum carapace lengths of only ~1.2 cm
despite being as old as 7 weeks. Adults this age are usually about twice as long as this
(personal observation). The relatively small size of adults, as well as their continuous
hatching from resting eggs, were perhaps both tied to the unusually dry early-winter
conditions of 2012, when the mesocosm study was initiated. In any event, I observed
these ~1.2 cm-long individuals actively digging in the sediment in the microcosm
experiments, so size (or lack thereof) probably had little to do with my results. The short
timeline of the microcosm experiments, on the other hand, may have been a factor.
Tadpole shrimp were in the microcosms for just 24 hours, and although I observed the
freely roaming L. packardi digging ~0.5 cm deep soon after being added, they perhaps
were not in the microcosms long enough to measurably translocate eggs. Hatching
phenology could be another reason why I found no evidence of egg translocation, at least
in the mesocosm experiment. By the time L. packardi individuals in that experiment had
grown large enough to extensively bioturbate and dig up eggs, it may be that the window
of opportunity for the hatching of other crustacean taxa was closed because the requisite
abiotic cues were no longer optimal (Brendonck, 1996; Brendonck and De Meester,
2003). Rather than hatching, therefore, eggs translocated to the sediment surface might
stay in diapause and hatch in a future season. Their return to the sediment surface would
be undetectable in a single-season experiment unless the eggs were collected and directly
41
counted. This was beyond the scope of this study, but directly counting eggs in the
sediment post-bioturbation (e.g., Marcus and Schmidt-Gengenbach, 1986) might be a
more effective way of assessing translocation than counting individuals in the active
community. These temporal considerations could be addressed in a longer-term
experiment that tracked the abundances of crustaceans in the active community, as well
as eggs in the sediment, following exposure to L. packardi. Ideally, such an experiment
would include treatments where L. packardi individuals of different sizes were added to
mesocosms immediately after inundation and left in for varying amounts of time over the
course of multiple seasons. It might then be possible to determine if an egg-translocation
signal emerges over a longer time scale, and/or if a size threshold for L. packardi needs to
first be attained.
Sediment disruption in California vernal pools occurs from other animals besides L.
packardi. For example, cattle frequently wade into vernal pools for forage and water
(Brendonck and De Meester, 2003; Marty, 2005; personal observation) and humans wade
into them to sample invertebrates for research purposes. Cattle and humans would
obviously mix the sediment much more extensively than L. packardi owing to their larger
mass. Such mixing would undoubtedly involve bidirectional transport of eggs and seeds;
each step down would plunge propagules on the surface deeper into the sediment, while
each step up would bring buried propagules to the sediment surface. Not only could
bioturbation by large animals affect recruitment within a single pool, it could also affect
recruitment in other pools as well. Waterkeyn et al. (2010), for instance, showed that
humans may be important, albeit inadvertent, vectors of local dispersal for temporary-
42
pond crustaceans by transporting their eggs in mud stuck to boots and vehicles.
Vanschoenwinkel et al. (2008, 2011) demonstrated the potential for similar egg transport
in mud stuck to the fur and skin of large mammals that wallowed in temporary ponds.
Clearly, eggs in the sediment can potentially be translocated over different spatial scales
by the activity of various animals.
The results of the present study indicate that L. packardi is not one of these animals,
however. I found no evidence that it translocates crustacean eggs and facilitates their
hatching, despite this taxon being a strong bioturbator in California vernal pools. How L.
packardi can dig so vigorously through the sediment and yet not translocate buried eggs
is unclear, but investigating the mechanism behind this presents opportunities for future
research. It could be that L. packardi eats eggs as it encounters them while bioturbating,
but my results suggest that consumption did not mask translocation in this study.
Although my hypothesis was not supported, my results do shed light on L. packardi’s
role as a predator of crustaceans in vernal pools. This study joins the growing body of
research showing that notostracans in general have negative effects on other crustacean
taxa in temporary ponds, due mainly to their role as predators in these unique bodies of
water.
43
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