draft - tspace repository: home · 2017-04-06 · draft 2 18 l. k. freidenburg 19 environmental...

Draft

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

https://mc06.manuscriptcentral.com/cjz-pubs

Canadian Journal of Zoology

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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: [email protected] 13

1Current address: School of Forestry and Environmental Studies, Yale University, 370 Prospect 14

Street, New Haven, Connecticut 06511; email: [email protected] 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


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


Average m

ortality/egg m

ass

(b)

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7.6

8.0

8.4

8.8


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)


(a)

0.0

1.0

2.0

3.0


Growth rate (mg/day)

(b)

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21

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25

27


Final Gosner stage

(a)

0

4

8

12

16


Total length (mm)

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