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Environmental drivers of carry-over effects in a pond-
breeding amphibian
Journal: Canadian Journal of Zoology
Manuscript ID cjz-2016-0080.R1
Manuscript Type: Article
Date Submitted by the Author: 07-Nov-2016
Complete List of Authors: Freidenburg, L; Yale University, School of Forestry & Environmental Stuides
Keyword: amphibian, oviposition site, carry-over effects, light environment, TEMPERATURE < Discipline, <i>Rana sylvatica</i>, wood frog
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Environmental drivers of carry-over effects in a pond-breeding amphibian 1
L. K. Freidenburg 2
Department of Ecology and Evolutionary Biology, University of Connecticut, 75 N. Eagleville 3
Road, Storrs, Connecticut 06269 4
Corresponding author: 5
L. K. Freidenburg 6
School of Forestry and Environmental Studies 7
Yale University 8
370 Prospect Street 9
New Haven, CT 06511 10
Phone: 203-432-5321 11
Fax: 203-432-3929 12
Email: kealoha.freidenburg@yale.edu 13
1Current address: School of Forestry and Environmental Studies, Yale University, 370 Prospect 14
Street, New Haven, Connecticut 06511; email: kealoha.freidenburg@yale.edu 15
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L. K. Freidenburg 18
Environmental drivers of carry-over effects in a pond-breeding amphibian 19
Abstract 20
Breeding animals confront a complex environment when deciding where to oviposit, and 21
this decision may depend on fine-scale variation in environmental conditions that have the 22
potential to affect not only embryos but also subsequent larvae. I evaluated the influences of two 23
variables, light and temperature, at wood frog (Rana sylvatica (LeConte, 1825)) oviposition sites. 24
First, in four ponds varying in canopy cover, I moved a subset of egg masses from the original 25
oviposition site to an alternative site in the same pond and monitored embryos until hatching 26
commenced. I found that embryos in the alternative site experienced delays in hatching an 27
average of 2.5 days. Second, in each of the four ponds, I placed hatchlings from the two sites in 28
enclosures throughout the pond. After two weeks, larval performance was assessed with respect 29
to development and growth. Larvae from the alternative oviposition site gained less mass (on 30
average 15% less) and developed more slowly (up to two Gosner stages) than larvae from the 31
original oviposition site. Collectively, these results show that in selecting oviposition sites, wood 32
frogs can use local cues to support high performance of their offspring and that those positive 33
effects can carry over well into the larval period. 34
Key words: amphibian, oviposition site, temperature, carry-over effects, light environment, Rana 35
sylvatica, wood frog 36
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Introduction 38
Parental investment in offspring varies tremendously, with some parents investing many 39
resources and years in their offspring while others simply join gametes and abandon the resulting 40
zygote to its fate. For many species, parental investment ends at egg deposition. Under these 41
circumstances, the only parental behavior that can influence offspring performance is oviposition 42
site choice. Among amphibians it is well documented that adults can and do choose oviposition 43
sites with specific characteristics (Seale 1982; Petranka et al. 1994; Spieler and Linsenmair 1997; 44
Dillon and Fiaño 2000; von May et al. 2009). The spatial scale of most studies has typically been 45
at the pond level, with a particular focus on how adult responses to predators or competitors 46
shape species distributions across different habitats (Resetarits and Wilbur 1989; Magnusson and 47
Hero 1991; Resetarits 2005). More recently, research directed at the environmental variables of 48
oviposition sites provides a within pond perspective of adult choices (e.g., Skidds et al. 2007; 49
Pereyra et al. 2011). 50
Environmental heterogeneity exists at most amphibian breeding sites, and placement of 51
offspring within such environments could determine the reproductive success of breeding adults. 52
Within breeding ponds, the decision on where to oviposit may depend on fine-scale variation in 53
environmental conditions. These conditions have the potential to affect not only the embryos but 54
also may have carry-over effects on the subsequent larvae. My research has focused on exploring 55
the effect of oviposition site choice on the performance of wood frog (Rana sylvatica (LeConte, 56
1825)) embryos and hatchlings. 57
Wood frogs are explosive breeders that typically lay their eggs in communal oviposition 58
sites (Howard 1980; Seale 1982; Waldman 1982). These sites often remain in the same location 59
year after year (Herreid and Kinney 1967; Seale 1982; L. K. Freidenburg personal observation). 60
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While the adaptive advantage to laying eggs communally has been well studied (Waldman 1982; 61
Waldman and Ryan 1983; Petranka and Thomas 1995), the choice of the site itself has not (but 62
see Dougherty et al. 2011). Given that wood frogs are one of the earliest amphibians to breed in 63
the spring, temperature may have an important influence on oviposition site. By laying eggs 64
communally, wood frogs can raise the ambient temperature surrounding the egg masses 65
(Waldman 1982; Waldman and Ryan 1983). It is also possible that females select sites within a 66
particular range of temperatures (Seale 1982). Light and temperature are correlated in bodies of 67
water (Wetzel 1983), and high temperature has been shown to accelerate growth and 68
development in amphibian larvae (Duellman and Trueb 1986; McDiarmid and Altig 1999). 69
Additionally, incubation temperature of eggs can affect the phenotypes of developing embryos, 70
with impacts on swimming performance and morphology (Watkins and Vraspir 2006). 71
Wood frogs tend to breed in temporary, fish-free ponds, and within this pond type, 72
environmental conditions (e.g., canopy cover, water temperature, water chemistry, vegetation, 73
etc.) can vary tremendously. In particular, their distribution among ponds spans a range in 74
canopy cover, from ponds lacking any canopy cover to ponds completely shaded by the 75
surrounding vegetation. Halverson et al. (2003) found predictable changes in larval 76
characteristics linked to a pond’s location along the canopy gradient. Specifically, across 17 77
wetlands, larval wood frog developmental stage and body size were linked to the light 78
environment. Here, I address two questions: (1) Do abiotic conditions at the oviposition site, 79
such as those characterized by light and temperature, influence embryo performance (survival, 80
growth, development) or lead to carry-over effects in larvae? And (2) Do these influences vary 81
among ponds? 82
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To these questions, I selected four ponds spanning a range of canopy cover and 83
conducted egg mass transplants within each pond, comparing the performance of embryos left at 84
the original oviposition site to those in an alternative site. In order to determine if carry-over 85
effects existed, I placed larvae from the two locations in field enclosures and documented their 86
subsequent performance. 87
Material and Methods 88
This experiment took place at the Yale-Myers Forest in northeastern Connecticut, USA. 89
The forest covers 3213 hectares and encompasses a diversity of habitats; the property is 90
dominated by second growth oak, maple, and hemlock forests interspersed with marshes, beaver 91
ponds, lakes, vernal ponds, old fields, and clearcuts. Four ponds were chosen in which to conduct 92
my experiment: Morse Bog, Dentist Pond, Clearcut Pond, and Blacksmith Pond. These ponds 93
spanned a range of canopy cover and size and were used by breeding wood frogs for at least the 94
previous four years (D. K. Skelly and L. K. Freidenburg, unpublished data). In addition, the light 95
environment within each pond has been mapped (Halverson et al. 2003). These light 96
measurements were obtained by taking hemispherical photographs along a Cartesian grid set up 97
in each pond (sampling points were 5 m apart). Photos were taken twice during the year (leaves 98
on and off the trees). The photos were digitized, and a measure of incident light at each sampling 99
point was calculated (Global Site Factor, henceforth GSF). 100
I designed the experiment in two phases. The first phase (Embryo Experiment) examined 101
embryo performance at two different pond locations, and the second phase (Hatchling 102
Experiment) determined what effect, if any, embryonic environment had on the resulting larvae. 103
Natural wood frog breeding locations within each of the ponds remained unchanged during the 104
four years leading up to this experiment (D. K. Skelly and L. K. Freidenburg, unpublished data), 105
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and the four ponds in which the experiment took place varied with respect to light and 106
temperature gradients (Freidenburg 2003). All experiments were approved by the University of 107
Connecticut’s Institutional Animal Care and Use Committee protocols. 108
Embryo Experiment. In March 2000, ponds were monitored to determine when wood frog 109
breeding began. As soon as eggs appeared in the ponds, I collected 50 egg masses (except from 110
Clearcut where only 10 egg masses were laid) from the original oviposition site; the masses were 111
haphazardly chosen from the center and all sides of the oviposition site (N, S, E, W) in order to 112
ensure a representative sample of the masses present. Twenty-five masses were placed on a 113
cradle and left in the original oviposition site (Original Site) while the other 25 masses were 114
moved to an alternative site and placed on an identical cradle (Relocated Site). The cradles were 115
square (43 cm x 43 cm) PVC pipe frames holding a plastic mesh center (mesh size = 1.3 cm) 116
with floats on each corner. The design of the cradle allowed the egg masses to be placed closely 117
together, mimicking their natural placement, and to remain suspended in the water column at the 118
same height as the naturally deposited masses. Thus, outside of their placement on the cradles, 119
the egg masses were exposed to the same aspects of the pond environment (e.g., predators, 120
water) as the un-manipulated egg masses. 121
In each pond I chose one alternative site in which to place a cradle. I chose a site with a 122
lower light level than the original site on the expectation that light level affects water 123
temperature and therefore site quality. When choosing the alternative site, I attempted to find 124
sites that were similar to the original site in characteristics except light level including distance 125
from shore, substrate, and depth (Table 1). 126
I monitored the egg masses throughout the embryonic period, visiting each pond at least 127
three times before hatching began. During each visit, water chemistry, egg mortality, and embryo 128
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developmental stage were recorded. I recorded the water temperature and dissolved oxygen at 129
each site using a YSI meter. On each cradle, the total number of dead embryos was recorded. 130
The developmental stage of the embryos was determined by staging (Gosner 1960) 50 embryos 131
for each egg mass, which allowed me to obtain an average developmental stage for each egg 132
mass and an overall average for each cradle. Since the egg masses were all together on their 133
‘cradle’, it’s not possible to determine which hatchlings came from which egg masses. What I 134
could and did do is monitor hatching on each cradle and get an estimate when half of the total 135
number of embryos had hatched. All measurements were done in situ. 136
The length of the embryonic period was defined as the amount of time it took at least 137
50% of the embryos to hatch. As the embryos hatched, I collected the hatchlings and held them 138
in enclosures at their respective sites in order to use them in the second part of the experiment 139
(see below). Developmental rate for each egg mass was calculated (end Gosner stage-beginning 140
Gosner stage/# days of experiment), and the initial size (total length, TL), mass (mg), and 141
developmental stage were determined for a subsample of the animals (n >10 per cradle). 142
Statistical analyses focused on performance differences between Original Site and 143
Relocated Site as well as among the four ponds. Paired t-tests were used to compare light and 144
water temperature differences between the Original Site and Relocated Site. I used two-way 145
ANOVA with Tukey post-hoc tests to analyze the effects of pond and Site on egg mass 146
developmental rate, hatchling initial stage, and hatchling initial size. 147
Hatchling Experiment. The second phase of the experiment was designed to assess any 148
effects the embryonic environment may have had on hatchling performance. In each pond, 16 149
enclosures were placed in four sites (four enclosures per site) for a total of 64 enclosures across 150
ponds. Within each enclosure I placed four hatchlings; two enclosures at a site received 151
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hatchlings from the Original Site and the other two enclosures received hatchlings from the 152
Relocated Site. The larvae remained in the field enclosures for two weeks before I terminated the 153
experiment. This design allowed me to estimate the effect embryonic environment had on the 154
early life history of the larvae. The four sites within each pond were chosen to represent the 155
range in water depth (shallow, deep) and light environment (closed, open) within each pond. 156
Thus, each pond had a shallow/closed, shallow/open, deep/closed, and deep/open site. The 157
cylindrical enclosures measured 61 cm in height and 10 cm in diameter. These enclosures 158
consisted of an internal open-ended cylinder constructed of black plastic mesh (mesh size = 1.3 159
cm) and then covered with a cylinder of black fiberglass window screening (mesh size = 1.5 160
mm) closed at the top and bottom. At each site, four enclosures were attached to a stake and 161
placed at similar depths within a 0.4 m2 area in order to maximize the similarity of their 162
microhabitat. I collected substrate at each site within each pond, allowed the substrate to dry at 163
least 24 hours, and then placed 15 g of substrate in each enclosure. Before stocking enclosures, I 164
weighed, measured (snout-vent length (SVL) and TL), and staged an initial sample of 15 165
hatchlings from each cradle. Length measurements were done using a dissecting scope fitted 166
with an ocular micrometer. At the end of the experiment, all surviving larvae within the field 167
enclosures were weighed, measured (SVL and TL), and staged. 168
Statistical analyses estimated the effect of embryonic environment, as defined by site, and 169
pond on hatchling performance. Two-way ANOVAs were used with pond and site as factors and 170
survival, developmental rate, growth rate, or final size as the response variable. In the case of 171
non-normally distributed data, transformations (arcsine square root or log) were performed 172
(arcsine square root: survival, developmental rate; log: growth rate, final size). 173
Results 174
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Embryo Experiment. As expected, light levels were, in most ponds, higher at the original site 175
than at the alternative site. This difference varied from large (55% more light at original site, 176
Blacksmith Pond) to slightly negative (-0.4% less light at original site, Clearcut Pond). 177
Temperature loggers placed at original and alternative sites reflected inter-site differences in 178
light levels. For three of the ponds, temperature differences averaged 2.1 C (two-tailed paired t-179
test: Blacksmith Pond df = 118, p = 0.007; Dentist Pond df = 117, p = 0.000; Morse Bog df = 180
119, p = 0.000), while in the fourth pond the difference between the two sites averaged 0.2 C 181
(two-tailed paired t-test: Clearcut Pond df = 116, p = 0.002). 182
Embryonic conditions influenced the duration of the embryonic period. At both the 183
original oviposition site and the Original Site, it took an average of 13 days for 50% of the egg 184
masses to hatch, indicating that the presence of the cradle did not affect the developmental time 185
of the embryos. In contrast, Relocated Site egg masses took an average of 2.5 days longer to 186
reach 50% hatching (Figure 1). These differences in hatching time can be linked to differences in 187
developmental rate. Overall, there was a Site effect; developmental rates were 16% slower in the 188
darker, colder Relocated Site than in the lighter, warmer Original Site (ANOVA: MS = 0.013, 189
F1,142 = 18.74, p < 0.001; Figure 2a). A significant Pond effect was also present (ANOVA: MS = 190
0.013, F3,142 = 188.884, p < 0.001; Figure 2b). The interaction between Site and Pond was not 191
quite significant (ANOVA: MS = 0.002, F3,142 = 2.505, p = 0.062). Egg masses in the most open 192
canopy pond, Morse Bog, developed 1.5 times faster than those in Clearcut Pond and Blacksmith 193
Pond and 0.25 times faster than those in Dentist Pond (Figure 2b). At one extreme, rates tended 194
to be 15.5% slower at the Relocated Site in Blacksmith Pond. At the other extreme, rates tended 195
to be 5% faster at the Relocated Site in Clearcut Pond. 196
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Embryo mortality differed among ponds ((ANOVA: MS = 1.622, F3,142 = 22.094, p < 197
0.001) but not between sites (Figure 3), with Morse Bog suffering the least mortality. An 198
interaction between pond and site indicated that in some ponds, site did affect mortality 199
(ANOVA: MS = 0.744, F3,142 = 10.132, p < 0.001). Blacksmith Pond exhibited the largest 200
difference between sites (Original Site: mean deaths/mass = 22 ± 3.4; Relocated Site: mean 201
deaths/mass = 70 ± 10.6). 202
Size at hatching differed between sites, with Relocated Site hatchlings averaging lengths 203
6% smaller than Original Site hatchlings (1–way ANOVA: F1,6 = 10.501, p = 0.018, r2 = 0.636, 204
Figure 4). Pond did not significantly affect either developmental stage (1-way ANOVA: F3,4 = 205
1.248, p = 0.403, r2 = 0.483) or size (1-way ANOVA: F3,4 = 0.540, p = 0.680, r
2 = 0.288). 206
Overall, embryos placed in a low light/low temperature environment developed more 207
slowly and hatched at smaller sizes than embryos in a higher light/higher temperature 208
environment. 209
Hatchling Experiment. Hatchling survival was linked to embryonic environment; on average, 210
hatchlings from the Original Site were 24% more likely to survive for two weeks than were 211
hatchlings from Relocated Site (ANOVA: MS = 0.238, F1,56 = 10.348, p = 0.002). There was no 212
effect of pond on survivorship (ANOVA: MS = 0.106, F3,56 = 0.883, p = 0.455). 213
The developmental rate of hatchlings stocked into the field enclosures varied between 214
sites (ANOVA: MS = 0.023, F1,50 = 21.304, p < 0.001) and among ponds (ANOVA: MS = 0.074, 215
F3,50 = 68.813, p < 0.001). At the time of stocking, Relocated Site larvae were less developed 216
(mean Gosner stage ± se: 21 ± 0.26) and smaller (mean TL ± se: 8.65 ± 0.27 mm) than Original 217
Site larvae (mean Gosner stage ± SE: 22 ± 0.28; mean TL ± se: 9.26 ± 0.20 mm). Among the 218
four ponds, initial Gosner stage ranged from 20 (Dentist Pond) to 23 (Clearcut Pond), and initial 219
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length ranged from 7.5 mm (Dentist Pond) to 10.8 mm (Clearcut Pond). While Relocated Site 220
larvae started out less developed and smaller than their Original Site counterparts, compensatory 221
developmental rates minimized these differences over time in some of the ponds. Overall, larvae 222
from Relocated Sites developed more quickly than those from Original Sites. However, the range 223
in developmental rates varied two-fold among ponds, from 0.21 stages/day in Clearcut Pond to 224
0.45 stages/day in Dentist Pond. Additionally, a pond by site interaction was detected (ANOVA: 225
MS = 0.118, F3,50 = 36.556, p < 0.001). This interaction appeared to be driven largely by one 226
pond; in Morse Bog, Original Site larvae developed at a rate of 0.21 stages/day while Relocated 227
Site larvae developed at a rate of 0.42 stages/day. 228
I measured growth rate as a function of both size and mass. The reason to use both final 229
size and growth rate is because final size shows the cumulative influence of both embryonic and 230
hatchling environments while growth rate applies specifically to the hatchling environment. 231
When measured as SVL, growth rate differed among ponds but not between sites, and there was 232
no interaction between the two factors (Figure 5a). Post-hoc pairwise comparisons indicated that 233
Morse Bog and Dentist Pond hatchlings had the highest growth rates while hatchlings from 234
Clearcut Pond experienced negative growth (Figure 5). However, when measured as a function 235
of mass, growth rate was influenced by both pond and site, and the interaction term was nearly 236
significant. Original Site larvae put on weight faster than Relocated Site hatchlings, and larvae 237
from Morse Bog and Dentist Pond outgrew those from Clearcut and Blacksmith Ponds (Figure 238
5b). 239
After two weeks in field enclosures, the effect of the embryonic environment could still 240
be detected through measures of stage and size. Larvae from Relocated Sites were less developed 241
(Figure 6a) and were smaller than hatchlings from Original Sites (Figure 6b). Developmentally, 242
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Blacksmith Pond larvae lagged behind larvae from the other three ponds by 6%. With respect to 243
size, Morse Bog and Dentist Pond had the largest larvae (mean TL ± se: 13.7 ± 0.1) compared to 244
Clearcut and Blacksmith Ponds (mean TL ± se: 10.6 ± 0.3 mm). 245
Discussion 246
It is well understood that amphibians and many other animal taxa are able to discriminate 247
among potential breeding sites and make choices based on the presence of potential predators or 248
competitors (Spieler and Linsenmair 1997; Anbutsu and Togashi 2002; Andrews et al. 2000; 249
Camara 1997; Kern et al. 2013; Pereya et al. 2010; Refsnider and Janzen 2010). In this study, I 250
have asked whether oviposition decisions have consequences for wood frog embryos and 251
whether carry-over effects can be detected in the performance of larvae from different embryonic 252
sites. Additionally, the generalist nature of wood frog breeding habitats allowed me to assess 253
how an abiotic gradient (canopy cover) in concert with other embryonic conditions can influence 254
the early life history of an amphibian. While most prior studies have focused on how biotic 255
interactions such as competition and predation affect larval amphibian ecology, a growing body 256
of work suggests that abiotic gradients may play a significant role (Anzalone et al. 1998; Smith 257
et al. 2000; Belden and Blaustein 2002; Skelly et al. 2002; Halverson et al. 2003; Skelly et al. 258
2014). 259
Embryos. Overall, I found that oviposition decisions made by wood frogs could have a sizable 260
influence on the length of the embryonic period, developmental rate of embryos, and size at 261
hatching. Embryos moved to an alternative site (Relocated Site) characterized by cooler 262
temperatures and more shade, took an average of 19% longer to hatch than did embryos from the 263
original site (Figure 2a). Sih and Maurer (1992) report qualitatively similar results in an embryo 264
transplant experiment conducted with Ambystoma barbouri (Kraus and Petranka, 1989). They 265
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found that development of embryos transplanted to exposed rock surfaces (alternative location) 266
lagged behind embryos located under rocks (original location). Conflicting results have been 267
reported for two previous wood frog embryo transplants. Howard (1980) transplanted wood frog 268
egg masses and concluded that oviposition location was determined by the presence of other egg 269
masses (e.g., not an abiotic factor). Seale (1982) selected numerous transplant locations with 270
lower temperatures than the documented oviposition locations. She found that with the exception 271
of one site, wood frogs did not oviposit in any of the transplant locations. These results were as 272
striking as those of Howard (1980), yet exactly opposite in character. Neither of these studies did 273
more than report the breeding activity in response to transplanted egg masses, so it is not 274
possible to determine the performance of the transplanted embryos. 275
Much has been made of the fact that wood frogs exhibit communal egg mass deposition. 276
Adaptive advantages to this behavior have been well-established; egg masses deposited in 277
communal clumps have higher temperatures than those deposited singly, and even within the 278
communal site, egg masses in the center have higher temperatures than those at the periphery 279
(Waldman 1982; Waldman and Ryan 1983). This thermal advantage typically results in 280
increased hatching success for embryos located within clumps versus those located singly or at 281
the periphery of clumps (Waldman 1982). The findings of this study show that the thermal 282
advantages derived from communal aggregations can be overridden by the thermal properties of 283
the specific site selected. I transplanted egg masses in such a way as to mimic the natural 284
situation of clumped masses and yet still observed large differences in performance between 285
embryos at the original site and the alternative site. Several authors have noted that wood frogs 286
do not always breed in the same place from year to year, leading to the conclusion that as long as 287
they communally oviposit, pond location does not matter (e.g., Howard 1980). However, I found 288
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that location within a pond does, in fact, matter. It may be that many sites within a pond offer 289
acceptable conditions for the embryos. The choice of oviposition site must take into account 290
myriad factors of which light and temperature are only two. Water depth, emergent vegetation, 291
and woody debris are abiotic factors that can influence oviposition site decisions (Herreid and 292
Kinney 1967; Seale 1982; Waldman and Ryan 1983; L. K. Freidenburg unpublished data). 293
Indeed, using the GSF information from the ponds in this study, higher light levels existed in 294
some locations away from where the wood frogs chose to breed. In some cases, a higher light 295
environment was located in either relatively deep water or where substrate upon which to attach 296
eggs was absent. 297
Hatchlings. Hatchling performance after two weeks in field enclosures could be traced to carry-298
over effects from embryonic conditions. Survival rates of larvae from the alternative site 299
(Relocated Site) were lower than those of larvae from the original site (Original Site). The 300
reasons for this are unclear. It could be that the smaller initial size and younger developmental 301
stage of the Relocated Site hatchlings put them at a physiological disadvantage in the field 302
enclosures, perhaps linked to lower tolerance of temperature extremes. Another reason for poor 303
survival could be reduced ability to exploit available food resources. Predators were excluded 304
from the enclosures, removing direct predation as a source of mortality. However, the non-lethal 305
presence of predators is known to have indirect effects on amphibian larvae (Skelly and Werner 306
1990; Skelly 1992; Anholt and Werner 1996). These indirect effects include changes in behavior 307
and morphology that can lead to reduced growth in prey species (Werner 1991; Skelly 1992; 308
Mathis et al. 2008; McCollum and Leimberger 1997; Relyea 2001; Relyea 2007; Wilson et al. 309
2005; Orizaola and Braña 2005). Predators were observed in the ponds containing field 310
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enclosures, so it is possible that the already stunted Relocated Site hatchlings responded to 311
predator cues by reduced feeding, leading to the observed increase in mortality. 312
Larvae from the Original and Relocated Sites exhibited differences in size and stage up to 313
two weeks after hatching. Hatchlings from the cooler, more shaded site were typically smaller 314
and less developmentally advanced than hatchlings from the original site. Reptile embryos 315
incubated at varying temperatures have been shown to produce phenotypically different 316
hatchlings. In snakes, embryos incubated at cold temperatures resulted in hatchlings less 317
responsive to predator cues and less mobile than hatchlings incubated at higher temperatures 318
(Burger 1998). In lizards and snakes, incubation temperatures affect hatchling size, body 319
morphology, activity levels, thermoregulatory behavior, and locomotion (Van Damme et al. 320
1992; Shine and Harlow 1996; Elphick and Shine 1998; Downes and Shine 1999; Andrews et al. 321
2000; Lin et al. 2005; Ji et al. 2006). 322
I found striking differences in growth rates among wood frog hatchlings. Embryonic 323
environment did not appear to affect growth rate when measured as a function of length. 324
However, when measured as a function of mass, growth rates differed between Original Site and 325
Relocated Site hatchlings, with Original Site hatchlings growing faster than Relocated Site 326
hatchlings. Body length and mass measure different facets of growth and do not necessarily 327
increase at the same rate. Indeed, for small individuals there may be selection to increase length 328
at the expense of weight in order to escape from predators. Predators of larval amphibians are 329
often gape-limited, and thus an increase in length may allow prey to escape from susceptible 330
sizes. Longer tadpoles may also be faster swimmers (Wilson et al. 2005). In fishes, a clear link 331
exists between prey size and susceptibility to predation (Werner and Gilliam 1984; Harvey 1991; 332
Olson 1996). Another factor selecting for an increase in length may be susceptibility to 333
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cannibalism. Many amphibian species are known to be cannibalistic, and intraspecific size 334
differences can lead to the cannibalistic interactions (e.g., Crump 1983; Petranka and Thomas 335
1995). 336
Competitive interactions also can be affected by size differences. The differences I 337
observed between hatchlings from the two sites could result in competitive asymmetries among 338
individuals within a pond, a result observed in other amphibian species (e.g., Brunkow and 339
Collins 1996). Individuals that lag behind with respect to size and developmental stage may 340
metamorphose at a smaller size. Size at metamorphosis is a critical life history trait as it is 341
associated with adult fitness (Smith 1987; Berven 1990). Thus, embryonic conditions that lead to 342
smaller larvae may initiate a chain of interactions that can have repercussions throughout the 343
entire life history of an organism. 344
Environmental heterogeneity. Wood frogs are habitat generalists, breeding in a wide variety of 345
pond types. In this study, a comparison among different pond types revealed that pond type alone 346
could affect performance. I found that temperature differences between the alternative and 347
original sites were most extreme in the closed canopy pond (Blacksmith Pond). Surprisingly, 348
however, even the two most open canopy ponds (Dentist Pond and Morse Bog) exhibited 349
substantial differences in water temperature between the alternative and original sites. Clearly, 350
even a relatively small vernal pond provides a heterogeneous environment for its inhabitants, and 351
it is likely that the inhabitants have evolved mechanisms by which they can detect and respond to 352
environmental gradients. Wood frog adults, however, may not necessarily be choosing the best 353
oviposition sites available. I compared only two locations within a pond; the original site chosen 354
by the adults and an alternative site chosen to represent less favorable habitat based on light and 355
temperature. That clear differences exist in both embryo and hatchling performance suggests that 356
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pond location, as defined by abiotic conditions, has the potential to profoundly influence early 357
life history performance and to have carry-over effects in later life history stages. 358
Previous research has indicated that adult amphibians avoid ovipositing in the presence of 359
predators and competitors (e.g., Resetarits and Wilbur 1989; Crump 1991). While avoidance of 360
competitors and predators is useful for explaining large-scale distributional patterns, it may not 361
explain within-pond oviposition behavior. My results indicate that habitat quality, as defined by 362
light and temperature, may play an important role in oviposition site choice. All of my ponds 363
contained non-fish amphibian predators, and monitoring of the original and alternative 364
oviposition sites indicated that predators were widely distributed throughout a given pond. In 365
temperate regions, clear distributional patterns of amphibians can be linked to the presence of 366
fish. However, a variety of predators exist in virtually all aquatic systems in which amphibians 367
breed. Likewise, many breeding habitats contain potential competitors in the form of other 368
anurans as well as invertebrates (e.g., Petranka and Thomas 1995). Given these conditions, the 369
question may not be just how competitors and predators affect oviposition site choice, but also 370
how local environmental conditions within pond influence this choice. While predators and 371
competitors certainly play a role in these finer scale decisions, habitat quality offers an 372
alternative cue for breeding adults. While suites of predators can change from year to year in 373
ponds, the angle at which the sun reaches the pond does not change (barring habitat modification 374
and successional effects). Light, therefore, can serve as a reliable cue to indicate optimal 375
oviposition sites. 376
Acknowledgments 377
I would like to thank A. Halverson, M. Urban, S. Bolden, and D. Skelly for help in the field. 378
Partial funding for this was came from the Francis Trainor Fund at the University of Connecticut. 379
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Previous versions of this manuscript benefited from the comments of M. Holgerson, M. Lambert, 380
D. Skelly, L. Swierk, and K. Wells. 381
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Table 1. Oviposition site characteristics for both the original site (Original Site = 1) and the 516
alternative site (Relocated Site = 2). Measures for depth (cm), temperature (°C), and dissolved 517
oxygen (DO; mg/L) are given as averages. Global site factor (GSF) is the average value for a 518
given location during the spring months. 519
520
521
Pond Site Depth Temp DO GSF 522
______________________________________________________________________________ 523
524
Morse Bog 1 15.5 16.5 8.6 0.933 525
526
2 14.0 14.6 7.1 0.909 527
528
Dentist 1 14.2 14.9 8.3 0.934 529
530
2 16.5 12.7 8.1 0.774 531
532
Clearcut 1 24.0 9.6 6.0 0.858 533
534
2 28.5 9.4 7.5 0.862 535
536
Blacksmith 1 42.0 9.4 8.5 0.707 537
538
2 31.0 7.2 7.4 0.157 539
______________________________________________________________________________ 540
541
542
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Figure 1. Median days to hatching as determined by the number of days it took 50% of the 543
embryos to hatch out. Note: I used all the egg masses in Clearcut Pond (n = 10) in the 544
experiment, so there are no unmanipulated egg masses with which to compare the two sites. 545
Open bars = original site, stippled bars = Original Site, hatched bars = Relocated Site. 546
Figure 2. Developmental rate of egg masses [(final stage-initial stage)/days]. (a) Developmental 547
rate (stages/day) of egg masses from Original Site and Relocated Site. (b) Developmental rate 548
(stages/day) of pooled egg masses (Original Site and Relocated Site) according to pond. Ponds 549
are arranged in order from most open to most closed canopy. Error bars = se. 550
Figure 3. Average embryo mortality as a function of (a) site, Original Site = open bar and 551
Relocated Site = hatched bar, and (b) pond type, where ponds are arranged in order from most 552
open to most closed canopy. Error bars = se. Mortality was calculated by determining the 553
number of dead embryos per egg mass on the last sampling day. The number of egg masses per 554
cradle ranged from 17-25 with the exception of Clearcut Pond that had 5 egg masses per cradle. 555
Figure 4. Initial size (total length in mm) of hatchlings from all ponds. Original Site = open 556
bar, Relocated Site = hatched bar. Error bars = se. 557
Figure 5. Growth rate measured as a function of (a) size (mm/day) and (b) mass (mg/day). 558
Ponds are arranged in order from open to closed canopy. Original Site = open bars, Relocated 559
Site = hatched bars. Error bars = se. 560
Figure 6. The effect of the embryonic environment (Site) on larval (a) stage (Gosner) and (b) 561
size (mm) after two weeks in field enclosures. Note truncated vertical axes. Original Site = 562
open bar, Relocated Site = hatched bar. Error bars = se. 563
564
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7
14
21
28
Morse Bog Dentist Clearcut Blacksmith
Number of days for 50% hatching
Figure 1
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0.0
0.5
1.0
1.5
Original Site Relocated Site
Gosner stages/day
(a)
0.0
0.6
1.2
1.8
Morse Bog Dentist Clearcut Blacksmith
Gosner stages/day
(b)
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5
10
15
20
25
30
35
40
45
50
Original Site Relocated
Average m
ortality/egg m
ass
(a)
0
12
24
36
48
60
72
Morse Bog Dentist Clearcut Blacksmith
Average m
ortality/egg m
ass
(b)
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7.6
8.0
8.4
8.8
Original Site Relocated Site
Initial size (mm)
Figure 4
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-0.4
-0.2
0.0
0.2
0.4
0.6
1 2 3 4
Growth rate (mm/day)
Morse Bog Dentist Clearcut Blacksmith
(a)
0.0
1.0
2.0
3.0
Morse Bog Dentist Clearcut Blacksmith
Growth rate (mg/day)
(b)
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21
23
25
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Original Site Relocated Site
Final Gosner stage
(a)
0
4
8
12
16
Original Site Relocated Site
Total length (mm)
(b)
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