Amphibian Ecology and Conservation

Techniques in Ecology and Conservation Series

Series Editor: William J. Sutherland

Bird Ecology and Conservation: A Handbook of Techniques

William J. Sutherland, Ian Newton, and Rhys E. Green

Conservation Education and Outreach Techniques

Susan K. Jacobson, Mallory D. McDuff, and Martha C. Monroe

Forest Ecology and Conservation: A Handbook of Techniques

Adrian C. Newton

Habitat Management for Conservation: A Handbook of Techniques

Malcolm Ausden

Conservation and Sustainable Use: A Handbook of Techniques

E.J. Milner-Gulland and J. Marcus Rowcliffe

Invasive Species Management: A Handbook of Techniques

Mick N. Clout and Peter A. Williams

Amphibian Ecology and Conservation: A Handbook of Techniques

C. Kenneth Dodd, Jr
Amphibian Ecology and
Conservation
A Handbook of Techniques

Edited by
C. Kenneth Dodd, Jr

1
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Preface

As this volume is completed, more than 6400 amphibian species have been
recognized, with new taxa being described nearly every day. The last few dec-
ades have seen an explosion in systematic research, particularly in the tropics.
Long-recognized centers of diversity have been explored using increasingly
sophisticated sampling techniques, yielding many new taxa. At the same time,
new centers of speciation, such as Sri Lanka and the Western Ghats of India,
have been discovered, while molecular techniques have yielded previously
unsuspected diversity within some well-known taxa, such as the plethodontid
salamanders of southeastern North America and the green toad (Bufo viridis)
complex of Eurasia. For amphibian systematists, these are exciting times.
Unfortunately, amphibians are now at greater peril than at any time in recent
geologic history, a situation chronicled in two recent data-rich books (Lannoo
2005; Stuart et al. 2008). Habitats are being lost at alarming rates because of
expanding human populations and generally favorable economic conditions
fostering development; emerging infectious diseases, particularly amphibian
chytrid fungus (Batrachochytrium dendrobatidis), threaten worldwide impacts;
non-indigenous species proliferate, affecting amphibians and their habitats; and
amphibians, with their permeable skins, diverse life histories, and often biphasic
life cycles requiring both terrestrial and aquatic habitats, are being saturated by
a host of lethal and sublethal toxic substances. New threats, such as the effects
of global climate change, further imperil amphibians, especially those with
limited distributions and dispersal capabilities. Fully one-third of all amphib-
ians are now considered threatened (Stuart et al. 2004), and 168 species have
become extinct within the last two decades. Clearly, these are treacherous times
for many frogs, salamanders, and caecilians.
Amphibians are, quite frankly, engaging animals. Despite Linnaeus’ early
characterization of amphibians in the context of “Terrible are thy works, O
God”, biologists have come to appreciate that their diverse life histories and
shear numbers offer a wealth of material for research on basic ecological princi-
ples, such as trophic interactions, phenotypic plasticity, predator–prey interac-
tions, community structure, mate choice and recognition, water balance, and
many others. In response to threats, conservation biologists have probed these
and other questions in hopes of understanding amphibian biology in order to
prevent declines and extinctions. The basic and applied themes of biology merge
vi | Preface

in these disciplines: understanding ecology leads to conservation options (see

Gascon et al. 2007), and conservation-based research leads to a better appreci-
ation of ecological principles.
To say that there are a great many techniques available in ecological and
conservation-based research on amphibians is an understatement. The pages
of journals such as Herpetological Review and Applied Herpetology contain tech-
niques papers with every issue. Specialized books, such Heyer et al. (1994),
Henle and Veith (1997), and Gent and Gibson (1998), offer additional summar-
ies that are as applicable today as when they were published. No one volume can
include all techniques. The current volume is meant not to supplant these earlier
works, but to supplement them and add new areas not previously summarized,
such as occupancy modeling, landscape ecology, genetics, telemetry, and dis-
ease biosecurity. Our objectives have been to delineate important new develop-
ments, to give an idea as to what the techniques tell or do not tell a researcher,
to focus attention on biases and data inference, and to get readers to appreciate
sampling as an integral part of their science, rather than just a means of captur-
ing animals. The techniques used will set the boundaries within which results
can or should be interpreted.
As noted earlier, amphibian systematics is a flourishing field, with many
new opportunities made available by combining large datasets using molecular
and morphological data with powerful computer analysis. The phylogeny of
amphibians is undergoing increasingly sophisticated analysis. Some analyses,
such as those of Frost et al. (2006), suggest relationships that differ substantially
from “traditional” concepts. If accepted, extensive nomenclatural changes will
be warranted. Although Frost et al. (2006) have advocated substantive changes
in nomenclature, many of which will likely be accepted with further study, other
amphibian biologists disagree with automatically accepting every change pro-
posed by these authors. In this book, I have decided to retain the older nomen-
clature rather than make a taxonomic decision each time a name is mentioned.
The intended audience of this volume (biologists starting their careers in ecology
and conservation) likely will be more familiar with the older generic names of
Bufo, Rana, and Hyla, and initially may be confused by the unfamiliar replace-
ment names. Readers should be aware, however, that names such as Lithobates
( Rana, in part), Anaxyrus ( Bufo, in part), and others currently unfamiliar
may soon be more commonplace.
I wish to thank the following for taking their valuable time to review manu-
scripts and offer suggestions for improving this volume: James Austin, Larissa
Bailey, Bruce Bury, Dan Cogălniceanu, Sarah Converse, Steve Corn, Rafael
Ernst, Alisa Gallant, Marian Griffey, Kerry Griffis-Kyle, Richard Griffiths,
 Preface | vii

Margaret Gunzburger, Tibor Hartel, Robert Jehle, Steve Johnson, Y.-C. Kam,
Sarah Kupferberg, Frank Lemckert, Harvey Lillywhite, John Maerz, Joseph
Mitchell, Clinton Moore, Erin Muths, James Petranka, Benedikt Schmidt,
Ulrich Sinsch, Kevin Smith, Lora Smith, Joseph Travis, Susan Walls, and
Matthew Whiles. I greatly appreciate the support from Ian Sherman and Helen
Eaton at Oxford University Press and editorial help from freelance copy-editor
Nik Prowse, and thank series editor, Bill Sutherland, for inviting me to edit the
amphibian volume. This volume is dedicated to all the biologists who take up
the challenge of amphibian ecology and conservation.
C. Kenneth Dodd, Jr

References
Frost, D. R., Grant, T., Faivovich, J., Bain, R. H., Haas, A., Haddad, C. F. B., de Sá, R. O.,
Channing, A.,Wilkinson, M., Donnellan, S. C. et al. (2006). The amphibian tree of
life. Bulletin of the American Museum of Natural History, 297, 1–370.
Gascon, C., Collins, J. P., Moore, R. D., Church, D. R., McKay, J. E., and Mendelson,
III, J. R. (eds) (2007). Amphibian Conservation Action Plan. IUCN/SSC Amphibian
Specialist Group, Gland and Cambridge.
Gent, T. and Gibson, S. (eds) (1998). Herpetofauna Worker’s Manual. Joint Nature
Conservation Committee, Peterborough.
Henle, K. and Veith, M. (eds) (1997). Naturschutzrelevante Methoden der Feldherpetologie.
Mertensiella 7.
Heyer, W. R., Donnelly, M. A., McDiarmid, R. W., Hayek, L.-A., and Foster, M. S.
(eds). (1994). Measuring and Monitoring Biological Diversity. Standard Methods for
Amphibians. Smithsonian Institution Press, Washington DC.
Lannoo, M. J. (ed) (2005). Amphibian Decline. The Conservation Status of United States
Species. University of California Press, Berkeley, CA.
Stuart, S., Chanson, J. S., Cox, N. A., Young, B. E., Rodrigues, A. S. L., Fishman, D. L.,
and Waller, R. W. (2004). Status and trends of amphibian declines and extinctions
worldwide. Science, 306, 1783–6.
Stuart, S., Hoffman, M., Chanson, J. S., Cox, N. A., Berridge, R. J., Ramani, P., and
Young, B. E. (eds) (2008). Threatened Amphibians of the World. Lynx Edicions,
Barcelona; IUCN, Gland; Conservation International, Arlington, VA.
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Contents

List of contributors xxv

Part 1 Introduction 1
1 Amphibian diversity and life history 3
Martha L. Crump
1.1 Introduction 3
1.2 Amphibian species richness and distribution 3
1.3 Amphibian lifestyles and life history diversity 5
1.3.1 Caecilians 6
1.3.1.1 Aquatic 7
1.3.1.2 Combination aquatic and terrestrial 7
1.3.1.3 Terrestrial and/or fossorial 7
1.3.2 Salamanders 7
1.3.2.1 Aquatic 8
1.3.2.2 Combination of aquatic and terrestrial 8
1.3.2.3 Terrestrial 9
1.3.2.4 Fossorial 10
1.3.2.5 Arboreal 10
1.3.3 Anurans 10
1.3.3.1 Aquatic/aquatic 10
1.3.3.2 Terrestrial/aquatic 11
1.3.3.3 Arboreal/aquatic 13
1.3.3.4 Fossorial/aquatic 14
1.3.3.5 Terrestrial/non-aquatic 14
1.3.3.6 Arboreal/non-aquatic 14
1.3.3.7 Fossorial/non-aquatic 15
1.4 Amphibian declines and why they matter 15
1.4.1 Economics 16
1.4.2 Ecosystem function 16
1.4.3 Esthetics 17
1.4.4 Ethics 17
1.5 References 17

2 Setting objectives in field studies 21

Dan Cogălniceanu and Claude Miaud
2.1 Basic concepts for a good start 21
x | Contents

2.2 Steps required for a successful study 24

2.2.1 Temporal and spatial scales 24
2.2.2 Choosing the model species 25
2.2.3 Pilot/desk study 26
2.2.4 Elaborate a conceptual model 26
2.2.5 The SMART approach 27
2.2.6 Applying the SMART approach to plan an
amphibian inventory 28
2.2.7 Experimental versus field studies 29
2.2.8 Methods for sampling, data storage, and analysis 29
2.3 Trade-offs and pitfalls 30
2.4 Ethical issues 31
2.5 Acknowledgments 32
2.6 References 32

Part 2 Larvae 37
3 Morphology of amphibian larvae 39
Roy W. McDiarmid and Ronald Altig
3.1 Background 39
3.2 Larval caecilians 40
3.2.1 Morphology and ontogeny 40
3.2.2 Coloration 42
3.2.3 Diversity 42
3.3 Larval and larviform salamanders 42
3.3.1 Morphology and ontogeny 42
3.3.2 Coloration 44
3.3.3 Diversity 44
3.4 Anuran tadpoles 45
3.4.1 Morphology and ontogeny 45
3.4.2 Coloration 49
3.4.3 Diversity 49
3.5 Summary 50
3.6 References 51

4 Larval sampling 55
David K. Skelly and Jonathan L. Richardson
4.1 Introduction 55
4.1.1 Why sample larvae? 55
4.1.2 Target responses 56
4.1.3 Timing 57
4.1.4 Sampling effort 57
4.2 Sampling techniques 58
4.2.1 Box/pipe sampler 58
 Contents | xi

4.2.1.1 Description 58
4.2.1.2 Application 60
4.2.1.3 Considerations 60
4.2.2 Dip net 60
4.2.2.1 Description 60
4.2.2.2 Application 61
4.2.2.3 Considerations 61
4.2.3 Seine 61
4.2.3.1 Description 61
4.2.3.2 Application 62
4.2.3.3 Considerations 62
4.2.4 Leaf litterbags 62
4.2.4.1 Description 62
4.2.4.2 Application 63
4.2.4.3 Considerations 63
4.2.5 Trapping 64
4.2.5.1 Description 64
4.2.5.2 Application 64
4.2.5.3 Considerations 64
4.2.6 Mark–recapture 65
4.2.6.1 Description 65
4.2.6.2 Application 66
4.2.6.3 Considerations 66
4.3 Other techniques 66
4.3.1 Bottom net 67
4.3.2 Electroshocking 67
4.3.3 Visual encounter survey 67
4.4 Conclusions 67
4.5 Acknowledgments 68
4.6 References 68

5 Dietary assessments of larval amphibians 71

Matt R. Whiles and Ronald Altig
5.1 Background 71
5.2 Larval caecilians and salamanders 72
5.3 Anuran tadpoles 72
5.4 Assessing food sources and diets 73
5.4.1 Category I: preparatory studies 73
5.4.2 Category II: gut contents 74
5.4.3 Procedures: anurans and small predators 74
5.4.4 Processing and analysis of gut contents 75
5.5 Category III: assimilatory diet 78
5.5.1 Stable-isotope analysis 78
5.5.2 Procedures 80
5.5.3 Fatty acid analyses 81
xii | Contents

5.6 Summary 82
5.7 References 83

6 Aquatic mesocosms 87
Raymond D. Semlitsch and Michelle D. Boone
6.1 Introduction 87
6.2 Historical background 88
6.3 Why use mesocosms? 90
6.4 Types of mesocosm 93
6.5 Setting up mesocosms 95
6.6 Common experimental designs 96
6.7 Case studies 98
6.7.1 Community ecology 98
6.7.2 Evolutionary ecology 99
6.7.3 Ecotoxicology 100
6.7.4 Land use and management 100
6.8 Conclusion 101
6.9 References 102

7 Water-quality criteria for amphibians 105

Donald W. Sparling
7.1 Introduction 105
7.2 Dissolved oxygen 105
7.3 Temperature 108
7.4 pH 110
7.5 Conductivity, hardness, and salinity 112
7.6 Total and dissolved organic carbon 113
7.7 Pollutants 114
7.7.1 Fertilizers and nitrogenous compounds 114
7.7.2 Pesticides 114
7.7.3 Metals 115
7.7.4 Organic pollutants and halogenated hydrocarbons 115
7.7.5 Pharmaceuticals 116
7.8 Summary and conclusions 116
7.9 References 117

Part 3 Juveniles and adults 121

8 Measuring and marking post-metamorphic
amphibians 123
John W. Ferner
8.1 Introduction 123
8.2 Toe-clipping 125
 Contents | xiii

8.2.1 Anurans 125

8.2.2 Salamanders 129
8.2.3 Ethical issues related to toe-clipping of amphibians 129
8.3 Branding 130
8.3.1 Anurans 130
8.3.2 Salamanders 131
8.4 Tagging and banding 131
8.4.1 Anurans 131
8.4.2 Salamanders 133
8.4.3 Caecilians 133
8.5 Trailing devices 133
8.6 Pattern mapping 134
8.6.1 Anurans 134
8.6.2 Salamanders 135
8.7 Passive integrated transponder (PIT) tags 136
8.7.1 Anurans 137
8.7.2 Salamanders 137
8.7.3 Caecilians 137
8.8 Taking measurements 138
8.9 Recommendations 138
8.10 References 139

9 Egg mass and nest counts 143

Peter W. C. Paton and Reid N. Harris
9.1 Background: using egg mass and nest counts
to monitor populations 143
9.2 Oviposition strategies 144
9.3 Egg-mass counts 145
9.4 Amphibian nests and nest counts 147
9.5 Clutch characteristics 150
9.6 Spatial distribution of eggs 152
9.7 Breeding phenology 152
9.8 Number of surveys needed 153
9.9 Estimating egg-mass detection probabilities 154
9.10 Variation in counts among observers 154
9.11 Marking eggs 155
9.12 Situations in which nest counts are not practical 155
9.12.1 Nest destruction 155
9.12.2 Desertion of attendant 155
9.13 How to count eggs in a nest 156
9.14 Estimating hatching success 156
9.15 Analysis of egg-mass count data 157
9.16 Summary 157
9.17 References 158
xiv | Contents

10 Dietary assessments of adult amphibians 167

Mirco Solé and Dennis Rödder
10.1 Introduction 167
10.1.1 Adult anurans 167
10.1.2 Adult caudata 168
10.1.3 Adult gymnophiona 169
10.2 Methods to obtain prey items: historical overview 169
10.2.1 Stomach flushing: materials 170
10.2.2 When should sampling be conducted? 171
10.2.3 How to perform the flush 171
10.3 Data analysis 172
10.3.1 Measuring prey availability and electivity 174
10.3.2 Comparing trophic niche structure 176
10.3.3 Fatty acid and stable-isotope analyses 177
10.3.4 Further biochemical analyses 178
10.4 General considerations 178
10.4.1 Ontogenetic changes 179
10.4.2 Seasonal changes 179
10.5 Conclusions 180
10.6 Acknowledgments 180
10.7 References 180

11 Movement patterns and radiotelemetry 185

Dale M. Madison, Valorie R. Titus, and Victor S. Lamoureux
11.1 Introduction 185
11.2 Equipment 186
11.2.1 Receivers and antennas 186
11.2.2 Transmitters 186
11.2.2.1 External transmitters 187
11.2.2.2 Internal transmitters 188
11.3 Surgical techniques 189
11.3.1 Surgery 189
11.3.2 Recovery and healing 191
11.4 Tracking procedures 191
11.4.1 Animal release 191
11.4.2 Locating signal source 192
11.4.3 Data collection 193
11.5 Analysis of movement data 193
11.6 Validation of telemetry procedures 197
11.6.1 Internal condition and mass 197
11.6.2 Movements 198
11.6.3 Reproduction 198
11.6.4 Injury and survivorship 199
 Contents | xv

11.7 Conclusions 199

11.8 References 200

12 Field enclosures and terrestrial cages 203

Elizabeth B. Harper, Joseph H.K. Pechmann, and
James W. Petranka
12.1 Introduction: amphibians in the terrestrial environment 203
12.2 What are the purposes of terrestrial enclosures? 204
12.3 Defining the research question 205
12.3.1 Questions related to habitat types 205
12.3.2 Questions with treatments that can be assigned
within enclosures 207
12.4 Constructing enclosures 208
12.4.1 Location of enclosures 208
12.4.2 Size and number of enclosures 209
12.4.3 Building enclosures that minimize escapes and
trespasses 216
12.4.4 “Standardizing” conditions among enclosures 218
12.5 Study species 218
12.5.1 Choice of species 218
12.5.2 Source and age of animals 219
12.6 Census techniques 219
12.6.1 Individual marks 219
12.6.2 Methods of capture 220
12.6.3 Frequency of censuses 220
12.7 Response metrics 221
12.7.1 Vital rates: survival, growth, age at reproductive
maturity, fecundity 221
12.7.2 Physiological responses 222
12.7.3 Behavioral responses 222
12.8 Thinking outside the box 223
12.9 References 223

Part 4 Amphibian populations 227

13 Drift fences, coverboards, and other traps 229
John D. Willson and J. Whitfield Gibbons
13.1 Introduction 229
13.2 Drift fences, funnel traps, and other passive
capture methods 229
13.2.1 What are passive traps? 229
13.2.2 How are passive traps constructed, aligned,
and monitored? 231
xvi | Contents

13.2.3 What can passive traps tell you?

What can they not tell you? 235
13.3 Coverboards and other traps that require active capture 236
13.3.1 What are coverboards and other active traps? 236
13.3.2 How are active traps constructed, aligned, and
monitored? 237
13.3.3 What can active traps tell you? What can they
not tell you? 240
13.4 References 241

14 Area-based surveys 247

David M. Marsh and Lillian M.B. Haywood
14.1 Introduction: what are area-based surveys? 247
14.1.2 Why use area-based surveys? 247
14.2 Kinds of area-based survey 249
14.3 Specific examples of area-based surveys 249
14.3.1 Leaf-litter plots 249
14.3.2 Natural-cover surveys for terrestrial salamanders 250
14.3.3 Nocturnal transects 251
14.3.4 Quadrats for stream amphibians 252
14.3.5 Soil quadrats for caecilians 252
14.4 Modifications 253
14.4.1 Distance sampling 253
14.4.2 Adaptive cluster sampling 253
14.5 Design issues: choice of sampling unit 254
14.5.1 Design issues: how many replicates? 255
14.5.2 Reducing variation among replicates 255
14.5.3 How many times to survey each replicate? 256
14.6 An example of study design 257
14.7 Assumptions of area-based surveys 259
14.8 Summary and recommendations 260
14.9 References 260

15 Rapid assessments of amphibian diversity 263

James R. Vonesh, Joseph C. Mitchell, Kim Howell, and
Andrew J. Crawford
15.1 Background: rapid assessment of amphibian diversity 263
15.1.1 When is an RA needed? 264
15.2 Planning an RA 265
15.2.1 Developing objectives 265
15.2.2 Costs and funding 266
15.2.3 Team selection and training 266
15.2.4 Permits 267
 Contents | xvii

15.2.5 Data management 267

15.2.6 Developing the sampling plan 268
15.2.6.1 Selecting sampling sites 268
15.2.6.2 Selecting sampling time 270
15.2.6.3 Selecting sampling techniques 270
15.2.6.3.1 Visual encounter surveys (VESs) 270
15.2.6.3.2 Assumptions and limitations of VESs 272
15.2.6.4 Determining what data to collect 273
15.2.6.5 Field logistics 274
15.3 In the field 274
15.4 Compiling data and interpreting results 274
15.5 Recommendations and reporting 275
15.6 Summary 275
15.7 References 276

16 Auditory monitoring of anuran populations 281

Michael E. Dorcas, Steven J. Price, Susan C. Walls, and
William J. Barichivich
16.1 Introduction 281
16.2 MCS 281
16.2.1 History and current status of MCS 282
16.2.2 Study objectives: what can MCS tell us? 283
16.2.3 Survey design 283
16.2.4 Other survey-design issues to consider:
the efficiency of MCS 284
16.2.5 Limitations of MCS data 287
16.3 ARS 288
16.3.1 Sources for ARS 289
16.3.2 Construction, deployment, and retrieval of data 289
16.3.3 Monitoring environmental data 292
16.3.4 Answering questions using ARS 292
16.3.5 Limitation of ARS 294
16.4 Conclusions 294
16.5 Acknowledgments 295
16.6 References 295

17 Measuring habitat 299

Kimberly J. Babbitt, Jessica S. Veysey, and George W. Tanner
17.1 Introduction 299
17.2 Habitat selection 299
17.3 Spatial and temporal scale 300
17.4 Approaches for examining habitat selection 300
17.5 Determining availability 301
xviii | Contents

17.6 What to measure 302

17.7 Weather variables 302
17.8 Aquatic habitat 304
17.9 Physical habitat variables 304
17.10 Chemical variables 307
17.11 Measuring vegetation 309
17.11.1 Tree (overstory) measurements 310
17.11.2 Shrub (midstory) measurements 312
17.11.3 Ground (understory) measurements 313
17.12 Edaphic features 314
17.13 Conclusion 314
17.14 References 315

Part 5 Amphibian communities 319

18 Diversity and similarity 321
C. Kenneth Dodd, Jr
18.1 Introduction 321
18.2 Data transformation 322
18.3 Species diversity 323
18.3.1 Sampling considerations 323
18.3.2 Species richness 324
18.3.3 Species accumulation curves 325
18.3.4 Heterogeneity 327
18.3.5 Evenness and dominance 329
18.4 Similarity 329
18.5 Software 331
18.6 Summary 334
18.7 References 334

19 Landscape ecology and GIS methods 339

Viorel D. Popescu and James P. Gibbs
19.1 Introduction 339
19.1.1 Relevance of landscape ecology to amphibian
biology and conservation 339
19.1.2 Defining a “landscape” from an amphibian’s
perspective 341
19.1.3 GIS 342
19.2 Applications of spatial data for amphibian conservation 346
19.2.1 Multiscale predictors of species occurrence and
abundance 346
19.2.2 Landscape thresholds 347
19.2.3 Connectivity/isolation in amphibian populations 349
 Contents | xix

19.2.4 Landscape permeability 351

19.2.5 Landscape genetics 352
19.3 Spatial statistics 353
19.4 Limitations and future directions 354
19.5 References 356

Part 6 Physiological ecology and genetics 361

20 Physiological ecology: field methods and
perspective 363
Harvey B. Lillywhite
20.1 Introduction 363
20.1.1 Important features of amphibians 364
20.2 Heat exchange, body temperature, and thermoregulation 365
20.2.1 Thermal acclimation of physiological function 365
20.2.2 Early methods in field studies of amphibian
thermal relations 366
20.2.3 Precautions for temperature measurements 367
20.2.4 Telemetry 371
20.3 Water relations 372
20.3.1 Cutaneous water exchange: terrestrial environments 372
20.3.2 Cutaneous water exchange: aquatic environments 374
20.4 Measuring water exchange 374
20.5 Energetics 375
20.6 Modeling amphibian–environment interactions 376
20.6.1 Mathematical models 376
20.6.2 Physical models 377
20.7 Other issues and future research directions 379
20.8 References 382

21 Models in field studies of temperature and

moisture 387
Jodi J. L. Rowley and Ross A. Alford
21.1 Introduction 387
21.1.1 The importance of understanding the temperature
and moisture environments of amphibians 387
21.1.2 Physical models of amphibians 389
21.2 Field models for investigating temperature and moisture 390
21.2.1 Models with zero resistance to EWL 390
21.2.2 A system of models that allows for variable EWL 390
21.2.2.1 Rationale 390
21.2.2.2 Model design and construction 391
xx | Contents

21.2.2.3 Example of model construction and

validation in the laboratory 392
21.2.2.4 Field validation of use of models to
characterize available temperatures 395
21.2.2.5 The importance of matching the spectral
properties of models to living animals 399
21.2.2.6 Models as indicators of relative moisture
availability of microenvironments 399
21.3 Summary and future developments 402
21.4 References 403

22 Genetics in field ecology and conservation 407

Trevor J. C. Beebee
22.1 Background: the importance of genetics in ecology
and conservation 407
22.2 Molecular methods for investigating amphibian
populations 408
22.2.1 Laboratory facilities 408
22.2.2 Sampling 409
22.2.3 DNA extraction 409
22.2.3.1 Proteinase K/phenol/chloroform method 410
22.2.3.2 Kit-based DNA extraction 410
22.2.3.3 Chelex method 410
22.2.3.4 FTA cards 410
22.2.3.5 Summary of DNA-extraction methods 410
22.2.4 Quantifying DNA recoveries 411
22.2.5 The basis of PCR analysis 411
22.2.6 Choice of analytical methods 412
22.2.6.1 Mitochondrial DNA (mtDNA) 412
22.2.6.2 Random amplification of polymorphic
DNA (RAPD) and amplified fragment
length polymorphism (AFLP) analyses 413
22.2.6.2.1 RAPD analysis 413
22.2.6.2.2 AFLP analysis 414
22.2.6.2.3 Microsatellite analysis 414
22.3 Analysis of genetic data 417
22.3.1 Cryptic species or life-stage identification 417
22.3.2 Genetic diversity, inbreeding, and bottlenecks 417
22.3.3 Identification of barriers to movement 419
22.3.4 Defining populations and population size 420
22.3.5 Historical issues 421
22.3.6 Behavior and sexual selection 423
22.4 Future developments 423
22.5 References 425
 Contents | xxi

Part 7 Monitoring, status, and trends 429

23 Selection of species and sampling areas:
the importance to inference 431
Paul Stephen Corn
23.1 Introduction 431
23.2 Sampling 433
23.3 Study sites and consequences of convenience sampling 435
23.4 Abundance and inference 439
23.5 Conclusions 442
23.6 Acknowledgments 443
23.7 References 443

24 Capture–mark–recapture, removal sampling,

and occupancy models 447
Larissa L. Bailey and James D. Nichols
24.1 Introduction 447
24.2 Estimating amphibian population size and vital rates 448
24.2.1 Marking: tag type and subsequent encounter 448
24.2.2 Capture–mark–recapture 449
24.2.3 Removal sampling 455
24.3 Estimating amphibian occupancy and vital rates 455
24.3.1 Occupancy estimation 456
24.3.2 Estimation of occupancy vital rates:
extinction and colonization 457
24.4 Summary and general recommendations 458
24.5 Disclaimer 459
24.6 References 459

25 Quantifying abundance: counts, detection

probabilities, and estimates 465
Benedikt R. Schmidt and Jérôme Pellet
25.1 Background: imperfect detection in amphibian
ecology and conservation 465
25.2 Imperfect detection 466
25.2.1 Counts underestimate abundance 467
25.2.2 Per-visit and cumulative detection probabilities 468
25.2.3 Temporal and spatial variation in
detection probabilities 469
25.3 Components of imperfect detection 470
25.4 How to deal with imperfect detection 471
xxii | Contents

25.4.1 Estimation of abundance 471

25.4.2 Other approaches to dealing with
imperfect detection 473
25.5 Designing a sampling protocol 476
25.6 Software 476
25.7 Outlook 477
25.8 References 477

26 Disease monitoring and biosecurity 481

D. Earl Green, Matthew J. Gray, and Debra L. Miller
26.1 Introduction 481
26.2 Amphibian diseases of concern 482
26.2.1 Infectious diseases 482
26.2.2 Parasitic diseases 487
26.2.3 Toxins 487
26.3 Disease monitoring: detection and diagnosis 487
26.3.1 Disease surveillance 487
26.3.2 Sample size 490
26.3.3 Sample collection and shipment 490
26.3.4 Diagnostics 494
26.4 Biosecurity: preventing disease transmission 497
26.4.1 Human and animal safety 497
26.4.2 Washing and disinfecting equipment 498
26.4.3 Movement of animals and disease management 499
26.5 Conclusions 500
26.6 References 501

27 Conservation and management 507

C. Kenneth Dodd, Jr
27.1 Introduction 507
27.1.1 Statutory protection 508
27.1.2 Protecting habitats 508
27.2 Managing amphibian populations 509
27.3 Wetland breeding sites 510
27.3.1 Wetland integrity and hydroperiod 510
27.3.2 Wetland creation and restoration 512
27.3.3 Core habitat and buffer zones 514
27.3.4 Vegetation structure, composition, and
canopy cover 516
27.3.5 Water quality 516
27.4 Terrestrial habitats 517
27.4.1 Contiguous habitats and edge effects 517
27.4.2 Silviculture 517
 Contents | xxiii

27.4.3 Restoring degraded lands 518

27.5 Migratory and dispersal routes 519
27.5.1 Corridors between habitat fragments 519
27.5.2 Crossing transportation corridors 519
27.6 Intensive manipulation of individuals 521
27.6.1 Captive breeding 521
27.6.2 Relocation, repatriation, translocation (RRT) 521
27.6.3 Disease and biosecurity 522
27.7 Conclusion 523
27.8 References 523

Index 529
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Contributors

Ross A. Alford School of Marine and Tropical Biology, James Cook University,
Townsville, Queensland 4811, Australia. E-mail: [email protected]
Ronald Altig Department of Biological Sciences, Mississippi State University,
Mississippi State, Mississippi 39762-5759, USA. E-mail: [email protected]
Kimberly J. Babbitt Department of Natural Resources and the Environment, University
of New Hampshire, Durham, NH 03824, USA. E-mail: [email protected]
Larissa L. Bailey Department of Fish, Wildlife and Conservation Biology, Colorado
State University, Fort Collins, CO 80523, USA. E-mail: [email protected]
William J. Barichivich Florida Integrated Science Center, US Geological Survey, 7920
NW 71st Street, Gainesville, FL 32653, USA. E-mail: [email protected]
Trevor J. C. Beebee School of Life Sciences, University of Sussex, Falmer, Brighton
BN1 9QG, UK. E-mail: [email protected]
Michelle D. Boone Department of Zoology, 212 Pearson Hall, Miami University,
Oxford, Ohio 45056, USA. E-mail: [email protected]
Andrew J. Crawford Smithsonian Tropical Research Institute, Apartado Postal
0843-03092, Balboa, Ancón, Republic of Panama. E-mail: [email protected]
Dan Cogălniceanu University Ovidius Constant‚a, Faculty of Natural Sciences, Bvd.
Mamaia 124, Constant‚a, Romania. E-mail: [email protected]
Paul Stephen Corn U.S. Geological Survey, Aldo Leopold Wilderness Research
Institute, 790 E. Beckwith Ave., Missoula, MT 59801, USA. E-mail: [email protected]
Martha L. Crump Department of Biological Sciences, Northern Arizona University,
Flagstaff, Arizona 86011, USA. E-mail: marty.crump@ nau.edu
C. Kenneth Dodd, Jr Department of Wildlife Ecology and Conservation, University
of Florida, Gainesville, FL 32611, USA. E-mail: [email protected]
Michael E. Dorcas Department of Biology, Davidson College, Davidson, NC 28035,
USA. E-mail: [email protected]
John W. Ferner Department of Biology, Thomas More College, Crestview Hills,
Kentucky 41017, USA. E-mail: [email protected]
J. Whitfield Gibbons Savannah River Ecology Laboratory, P.O. Drawer E, Aiken,
South Carolina 29802, USA. E-mail: [email protected]
James P. Gibbs Department of Environmental and Forest Biology, State University
of New York, Syracuse, NY 13210, USA. E-mail: [email protected]
Matthew J. Gray Center for Wildlife Health, Department of Forestry, Wildlife and
Fisheries, University of Tennessee, 274 Ellington Plant Sciences Building, Knoxville,
TN 37996, USA. E-mail: [email protected]
xxvi | Contributors

D. Earl Green U.S. Geological Survey, National Wildlife Health Center, 6006
Schroeder Drive, Madison, WI 53711, USA. E-mail: [email protected]
Elizabeth B. Harper State University of New York, College of Environmental
Sciences and Forestry, 1 Forestry Drive, Syracuse, New York 13210, USA. E-mail:
[email protected]
Reid N. Harris Department of Biology, James Madison University, Harrisonburg,
Virginia 22807, USA. E-mail: [email protected]
Lillian M. B. Haywood Department of Biology, Washington and Lee University,
Lexington, VA 24450, USA. E-mail: [email protected]
Kim Howell Department of Zoology and Wildlife Conservation, PO Box 35064,
University of Dar es Salaam, Dar es Salaam, Tanzania. E-mail: [email protected]
Victor S. Lamoureux Binghamton University, PO Box 6000, Binghamton, New
York 13902, USA. E-mail: [email protected]
Harvey B. Lillywhite Department of Zoology, University of Florida, Gainesville, FL
32611, USA. E-mail: [email protected]fl.edu
Dale M. Madison Binghamton University, PO Box 6000, Binghamton, New York
13902, USA. E-mail: [email protected]
David M. Marsh Department of Biology, Washington and Lee University, Lexington,
VA 24450, USA. E-mail: [email protected]
Roy W. McDiarmid Patuxent Wildlife Research Center, US Geological Survey, National
Museum of Natural History, Washington DC 20036, USA. E-mail: [email protected]
Claude Miaud Laboratoire d’ Alpine UMR CNRS 5553, Université de Savoie, 73376
Le Bourget-du-Lac, France. E-mail: [email protected]
Debra L. Miller Veterinary Diagnostic and Investigational Laboratory, The University
of Georgia, College of Veterinary Medicine, 43 Brighton Road, Tifton, GA 31793,
USA. E-mail: [email protected]
Joseph C. Mitchell Mitchell Ecological Research Service, LLC, PO Box 5638,
Gainesville, FL 32627–5638, USA. E-mail: [email protected]
James D. Nichols U.S. Geological Survey, Patuxent Wildlife Research Center, 12100
Beech Forest Road, Laurel, MD 20708, USA. E-mail: [email protected]
Peter W. C. Paton Department of Natural Resources, University of Rhode Island,
Kingston, Rhode Island 02881, USA. E-mail: [email protected]
Joseph H. K. Pechmann Department of Biology, 132 Natural Sciences Building,
Western Carolina University, Cullowhee, North Carolina 28723, USA. E-mail:
[email protected]
Jérôme Pellet A. Mailbach Sàrl, CP 99, Ch. de la Poya 10, CH-1610 Oron-la-Ville,
Switzerland. E-mail : [email protected]
James W. Petranka Department of Biology, University of North Carolina at Asheville,
Asheville, North Carolina 28804, USA. E-mail: [email protected]
 Contributors | xxvii

Viorel D. Popescu Department of Wildlife Ecology, University of Maine, Orono,

ME 04469, USA. E-mail: [email protected]
Steven J. Price Department of Biology, Wake Forest University, Winston-Salem, NC
27109, USA. E-mail: [email protected]
Jonathan L. Richardson School of Forestry and Environmental Studies, Yale
University, 370 Prospect Street, New Haven, Connecticut 06511, USA. E-mail:
[email protected]
Dennis Rödder Zoological Research Museum Alexander Koenig, Adenauerallee 160,
D-53113, Bonn, Germany and Faculty of Geography/Geosciences, Trier University,
Wissenschaftspark Trier-Petrisberg, Am Wissenschaftspark 25–27, D-54286 Trier,
Germany. E-mail: [email protected]
Jodi J. L. Rowley School of Marine and Tropical Biology, James Cook University,
Townsville, Queensland 4811, Australia. E-mail: [email protected]
Benedikt R. Schmidt Zoologisches Institut, Universität Zürich, Winterthurerstrasse
190, CH-8057 Zürich, Switzerland. E-mail: [email protected]
Raymond D. Semlitsch Division of Biological Sciences, 105 Tucker Hall, University
of Missouri, Columbia, Missouri 65211, USA. E-mail: [email protected]
David K. Skelly School of Forestry and Environmental Studies, Yale University, 370
Prospect Street, New Haven, Connecticut 06511, USA. E-mail: [email protected]
Mirco Solé Department of Biology, Universidade Estadual de Santa Cruz, Rodovia
Ilhéus – Itabuna, km 16, Ilhéus, Bahia, Brazil. E-mail: [email protected]
Donald W. Sparling Cooperative Wildlife Research Laboratory, Life Science II,
MS6504, Southern Illinois University, Carbondale, Illinois 62901, USA. E-mail:
[email protected]
George W. Tanner Department of Wildlife Ecology and Conservation, University of
Florida, Gainesville, FL 32611, USA. E-mail: tannerg@ufl.edu
Valorie R. Titus Binghamton University, PO Box 6000, Binghamton, New York
13902, USA. E-mail: [email protected]
Jessica S. Veysey Department of Natural Resources and the Environment, University
of New Hampshire, Durham, NH 03824, USA. E-mail: [email protected]
James R. Vonesh Department of Biology, Virginia Commonwealth University, 1000
West Cary Street, Richmond, VA 23284, USA. E-mail: [email protected]
Susan C. Walls Florida Integrated Science Center, US Geological Survey, 7920 NW
71st Street, Gainesville, FL 32653, USA. E-mail: [email protected]
Matt R. Whiles Department of Zoology and Center for Ecology, Southern Illinois
University, Carbondale, Illinois 62901-6501, USA. E-mail: [email protected]
John D. Willson Savannah River Ecology Laboratory, P.O. Drawer E, Aiken, South
Carolina 29802, USA. E-mail: [email protected]
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 Part 1
Introduction
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 1
Amphibian diversity and life history
Martha L. Crump

When the first crossopterygian crawled out of the rich Devonian waters and cast the
first envious vertebrate gaze at the terrestrial world, a boundless empire awaited colon-
ization. Although the change from an ungainly lobe-finned locomotion to a terrestrial
walking gait . . . was agonizingly slow, generations succeeded generations, archotypes [sic]
gave way to new evolutionary experiments, and the land became the home for the first
quadrupeds—the amphibians.
William E. Duellman (1970)

1.1 Introduction
Over the past 350 million years, amphibian descendents of lobe-finned fishes
have radiated into most habitats on Earth. In doing so, they have acquired spec-
tacular and sometimes bizarre physiological, morphological, behavioral, and
ecological attributes that mold their innovative life histories. Amphibians have
highly permeable skin, which makes them both vulnerable to losing water and
able to absorb water. Their eggs, covered with jelly capsules rather than hard
shells, lose water rapidly. For these reasons, amphibians require relatively moist
environments.
Many sampling techniques have been developed in North America or Europe where
most amphibians exhibit the complex life cycle of aquatic eggs, aquatic larvae, and
metamorphosis into terrestrial adults that return to water to breed. Not all amphib-
ians fit this stereotype. The spectacular diversity of amphibian life histories provides a
focus for studying their natural history, as well as presents a challenge since researchers
must ensure that field methods are appropriate for the target species.

1.2 Amphibian species richness and distribution

Scientists have named approximately 1.75 million species of living organisms
(Groom et al. 2006). About 72% of all named animals are insects; about 5%
are vertebrates. Approximately 0.5% of all animal species—6347 species (see
AmphibiaWeb; http://amphibiaweb.org)—belong to the class Amphibia. The
4 | Amphibian ecology and conservation

class gets it name from the Greek words amphi meaning “two” and bios mean-
ing “mode of life” because many species have a diphasic life history: they spend
part of their lives in water and part on land. Biologists divide the class into three
orders: Gymnophiona (caecilians), Urodela (salamanders), and Anura (frogs)
(Figure 1.1).
Gymnophiona, from the Greek words gymnos and ophis meaning “naked ser-
pent,” encompasses 174 species. Caecilians, which resemble large earthworms,
are long, skinny animals with no legs and reduced eyes. Annuli (grooves) encir-
cle their bodies. Their tails are either greatly reduced or absent, and a sensory
tentacle sits between each eye and nostril. Some caecilians have small dermal
scales beneath the surface of their mucus-covered skin. These scales, composed
mainly of collagen fibers and minerals, are not found in salamanders or anurans.
Adult caecilians range in length from a little more than 10 cm to about 1.5 m.
Most are highly specialized for burrowing. Others live on the ground but are
cryptic and secretive. Some are aquatic. Caecilians occur in tropical habitats
around the world except for Madagascar and the Papuan–Australian region
(Pough et al. 2004).

(a) (b)

(c) (d)

Fig. 1.1 Representatives of the three orders of amphibians. (a) Anura: Rana palmipes,
from Ecuador, (b) Anura: Bufo arenarum, from Argentina, (c) Urodela: Phaeognathus
hubrichti, from Alabama, USA, (d) Gymnophiona: Hypogeophis rostratus, Seychelles.
Photographs (a) and (b) by Martha L. Crump; photographs (c) and (d) by C. Kenneth
Dodd, Jr.
 1 Amphibian diversity and life history | 5

Five hundred and seventy-one species belong to Urodela, from the Greek
words uro and delos meaning “tail evident.” All salamanders have tails, and adults
have elongate bodies. Most have front and back legs of about the same length;
the limbs of a few aquatic species are greatly reduced or absent. Salamanders
are completely aquatic, terrestrial, combined aquatic and terrestrial, fossor-
ial, or arboreal. Adults range in size from about 30 mm to nearly 2 m. Most
salamander species occur in eastern and western North America and temper-
ate Eurasia, although plethodontids have radiated extensively in Central and
South America. There are no salamanders in sub-Saharan Africa, Australasia,
Australia, or much of tropical Asia, and they are missing from most islands
(Pough et al. 2004).
Frogs, which include toads, make up the order Anura from the Greek words
an and oura, meaning “without tail.” Although tadpoles have tails, adults do
not. Anurans live in the water, on the ground, underground, and in the trees.
Most have long, strong back legs well suited for jumping. Males of most species
call to attract females for mating. Adults of the 5602 recognized species range
in size from about 13 mm to 30 cm. Anurans live almost everywhere except
where restricted by cold temperatures or extremely dry conditions, and except
for many oceanic islands (Pough et al. 2004).
Duellman identified 43 areas worldwide with exceptionally high numbers
of amphibian species, endemic species (those found nowhere else), or both
(Duellman 1999). Nineteen of these high diversity areas are in the western hemi-
sphere; the others are in Eurasia, Africa, and the Papuan–Australian region.
The neotropical region houses 54% of the world’s amphibian species.
Amphibians live in nearly every habitat except for open oceans, most oceanic
islands, polar regions, and some extremely dry deserts (Wells 2007). These
restrictions are imposed on them because of their highly permeable skin that
loses water, and because they are ectothermic: the energy needed to raise their
body temperatures comes from the sun. Thus, amphibians become inactive at
low temperatures. These characteristics, however, work to their advantage as
well. In dry areas and those with seasonal rainfall, amphibians absorb water
through their skin by contacting moist soil. Their low metabolic rates translate
into low energy requirements and allow them to estivate, often underground,
during unfavorable conditions.

1.3 Amphibian lifestyles and life history diversity

Textbooks, management guides, and monitoring manuals, especially those ori-
ginating in Europe and North America, often give an oversimplified impression
6 | Amphibian ecology and conservation

of amphibian life histories. In fact, amphibian life histories are often complex,
and many are still poorly understood. Not all species have an aquatic larval stage
and a terrestrial adult stage.
Reproductive mode, a central aspect of amphibian life history, refers to the
site of egg deposition, egg and clutch characteristics, type and duration of
embryonic and larval development, and type of parental care if any (Duellman
and Trueb 1986). Many amphibians are not tied to aquatic habitats for repro-
duction. Instead, they reproduce on land, underground, or in trees, even in
the temperate zones. The following brief discussion of selected lifestyles and
life histories reveals that similar behaviors have evolved in diverse taxonomic
groups and geographical areas: amphibian “experiments” toward greater inde-
pendence from standing or flowing water and perhaps from lower predation
pressure as well.
Readers wishing more information concerning amphibian life histories
should consult Duellman (2007) and Wells (2007). For reviews of reproduct-
ive modes, see Salthe (1969), Salthe and Duellman (1973), Wake (1982, 1992),
Haddad and Prado (2005), and Duellman (2007). For reviews of parental care
see Crump (1995, 1996).

1.3.1 Caecilians
Caecilian lifestyles include aquatic, combined aquatic and terrestrial, terrestrial,
and fossorial. Fully aquatic caecilians generally have compressed bodies with
well-developed dorsal fins on the posterior portion. Fossorial species generally
have blunt heads, used for pushing and compacting the soil while burrowing.
Although details of reproductive biology are unknown for many species, cae-
cilians display two basic modes: oviparous (egg-laying) and viviparous (bear-
ing live young) (Wake 1977, 1992). Oviparous caecilians lay eggs on land. In
some species the eggs hatch into larvae that wriggle to water. Caecilian larvae
exhibit less dramatic metamorphosis than do salamanders or frogs. They hatch
almost fully developed, and the larval period is short. The eggs of some ovip-
arous species undergo direct development. That is, development occurs within
the egg capsule; there is no free-living larval stage. Some oviparous females stay
with their eggs, which probably protects them from predators and from drying
out. Viviparity evolved independently several times in caecilians. Females retain
eggs in their oviducts until development is complete (Wake 1993; Wilkinson
and Nussbaum 1998). After the developing young exhaust their yolk reserves,
they scrape lipid-rich secretions from their mothers’ oviductal epithelium with
their fetal teeth. Gestation lasts for many months, and the newborn are large
relative to their mothers.
 1 Amphibian diversity and life history | 7

1.3.1.1 Aquatic
All species in the South American family Typhlonectidae are either fully aquatic
or semi-aquatic. Some in the latter group spend the day in burrows they con-
struct next to water, then emerge at night to feed in shallow water. All typhlonec-
tids are assumed to be viviparous. Soon after the young are born, they shed their
gills and quickly acquire adult morphology.

1.3.1.2 Combination aquatic and terrestrial

Caecilians of the two most primitive families—Ichthyophiidae and
Rhinotrematidae—all appear to have complex life cycles encompassing both
water and land. As far as is known, ichthyophiids from southeastern Asia lay
their eggs in burrows or under vegetation at the edge of water. The female coils
around her eggs. The hatchling larvae wriggle to water. Rhinotrematids from
South America likewise appear to be oviparous, and free-living aquatic lar-
vae have been found for some species. Although egg-laying sites are unknown,
females presumably oviposit on land near water and the larvae make their way to
water. The developmental mode is not known for uraeotyphlids, from southern
India, but presumably they also lay eggs on land and have aquatic larvae. Some
species of the large, widespread family Caeciliidae also oviposit on land and have
free-living aquatic larvae.

1.3.1.3 Terrestrial and/or fossorial

Many caecilians burrow underground, although some forage at night on the
ground surface. Some terrestrial and fossorial caeciliids from South America,
Africa, India, and the Seychelles lay direct-developing eggs. In some of these,
females have been found coiled around their eggs. Some terrestrial and fossor-
ial caeciliids are viviparous. All members of the family Scolecomorphidae from
Africa are terrestrial. One is viviparous; in the other five species the mode is
unknown.

1.3.2 Salamanders
Salamander lifestyles include fully aquatic, combined aquatic and terrestrial,
terrestrial, arboreal, and fossorial. Body shapes of aquatic salamanders range
from the slender and eel-like sirenids and amphiumids to the flattened and
robust cryptobranchids. Many permanently aquatic species retain external gills
as adults. Most arboreal salamanders are small and have extensively webbed feet;
some have prehensile tails. Burrowing salamanders generally have long slender
bodies and tails, reduced limbs and feet, and small body size.
8 | Amphibian ecology and conservation

As a group salamanders exhibit diverse reproductive modes. Some salaman-

ders go through a complex life cycle with aquatic larvae and metamorphosis.
Others are paedomorphic: they become sexually mature and reproduce while
retaining juvenile characteristics. Still others have direct-developing eggs or give
birth to live young.

1.3.2.1 Aquatic
Some aquatic salamanders lay eggs in still or slowly flowing water. Siren and
Pseudobranchus from southeastern USA and northeastern Mexico live in swamps,
lakes, marshes, and sluggish streams, where they attach their eggs to vegetation.
The larvae develop into eel-like paedomorphic adults: they lack eyelids, have
external gills, and their skin resembles larval skin. When their habitat dries out,
sirenids secrete a mucous cocoon and burrow into the mud where they estivate
until conditions improve. The Mexican axolotl (Ambystoma mexicanum) is per-
manently aquatic and paedomorphic. Its larvae fail to metamorphose fully, and
the gonads mature in the larval body form.
Proteus anguinus, from southeastern Europe, lives in subterranean lakes and
streams of limestone caves where it lays aquatic eggs that hatch into aquatic lar-
vae. Females attend their eggs. In North America, Typhlomolge and Haideotriton
also live in cave waters and lay aquatic eggs that hatch into aquatic larvae. All
these species are paedomorphic.
Some aquatic salamanders oviposit in flowing water of cold streams. Male
hellbenders (Cryptobranchus alleganiensis) from eastern North America con-
struct nests under rocks. More than one female might lay her strings of eggs in
the nest. The male guards the eggs through their early stages. Likewise, male
Andrias japonicus from Japan and male Andrias davidianus from central China
guard their nests. In all three cryptobranchid species, aquatic larvae undergo
incomplete metamorphosis. The adults retain certain larval features such as lid-
less eyes and the absence of a tongue pad.

1.3.2.2 Combination of aquatic and terrestrial

In Eurasia, many salamandrids (e.g. Triturus) live on land but lay their eggs in
ponds, lakes, or streams. Likewise, most Asian hynobiids are terrestrial as adults
but migrate to aquatic sites to lay aquatic eggs that hatch into aquatic larvae. The
same is true for many North American terrestrial salamanders. Tiger salaman-
ders (Ambystoma tigrinum) and spotted salamanders (Ambystoma maculatum)
migrate to breeding ponds in the spring. Once the aquatic larvae metamorphose
and leave the water, they return to their aqueous beginnings only during breed-
ing season.
 1 Amphibian diversity and life history | 9

In contrast, female marbled salamanders (Ambystoma opacum) lay their eggs

under leaf litter or logs in depressions on dry ground. They stay until their eggs
hatch when the nests flood during winter rains. Following aquatic larval devel-
opment and metamorphosis, marbled salamanders are completely terrestrial.
Female Amphiuma from the southeastern USA lay large yolky eggs in dried-
out swamps or in cavities under logs near ponds: places that will flood during
spring and summer rains. Females stay with their eggs until then. Once they
hatch, the well-developed larvae metamorphose within a few weeks. Adults live
in swamps or slow-moving streams, but move overland during rains. They sur-
vive droughts by burrowing underground for 2 years or more.
Some plethodontid salamanders lay eggs on land, and after hatching the lar-
vae make their way to water. Female Hemidactylium scutatum oviposit just above
or at the water line in bogs and swamps. After hatching, the larvae wriggle or flip
into the water. Some Desmognathus lay their eggs under rocks, logs, or leaf litter
at water’s edge. The newly hatched larvae stay with their mother in the nest site
for several days, then wriggle to water.
The complex life cycle of red-spotted newts (Notophthalmus viridescens) from
eastern North America involves several stages. Aquatic females attach their eggs
to underwater vegetation. The eggs hatch into aquatic larvae that eventually
metamorphose into an immature terrestrial stage called an eft. The orange-red
efts stay on land for 1–14 years. Eventually they return to ponds where they turn
dull green with a row of small red dots along each side of their body and trans-
form into the aquatic adult body form. In some populations, however, paedo-
morphic adults reproduce in the larval body form.

1.3.2.3 Terrestrial
Many species of salamander live under leaf litter or logs, retreating into crevices
or holes during dry conditions. Terrestrial salamanders, including many in the
temperate zone, have various ways of reproducing independent of water bodies.
The seepage salamander (Desmognathus aeneus) oviposits in a nest near water. The
larvae hatch at an advanced stage and metamorphose within a few days in the
nest without feeding. Other plethodontid salamanders (e.g. Desmognathus wrighti,
Bolitoglossa, and Plethodon) lay large, direct-developing eggs in cavities or inside
hollow logs. In many species, the female remains with the eggs; in a few species
the male remains instead. Montane populations of European fire salamanders
(Salamandra salamandra) often retain eggs in their oviducts. The young absorb
nutrients from their yolk and are born live, although lowland populations usually
have aquatic eggs and larvae. Salamandra atra are viviparous; after the developing
young exhaust their yolk reserves, they obtain nutrients from the female.
10 | Amphibian ecology and conservation

1.3.2.4 Fossorial
Most Oedipina, fossorial or semi-fossorial plethodontid salamanders ranging
from Mexico to northern South America, lay direct-developing eggs under-
ground. Some fossorial plethodontids (e.g. Lineatriton lineola) attend their
direct-developing eggs.

1.3.2.5 Arboreal
Neotropical arboreal plethodontids (e.g. Bolitoglossa and Nototriton) lay their
eggs under mats of mosses and liverworts on tree branches and in bromeliads.
The eggs undergo direct development, and some female Bolitoglossa attend their
eggs. Aneides lugubris from western North America lays direct-developing eggs
as high as 10 m above the ground.

1.3.3 Anurans
Anuran lifestyles include purely aquatic, aquatic and terrestrial, terrestrial, arbor-
eal, and fossorial. A frog with relatively short hind legs is most likely a terrestrial
hopper or fossorial species. One with long hind legs is likely to be aquatic, arbor-
eal, or a jumping terrestrial species. Aquatic anurans tend to have their eyes on the
top of their heads rather than at the sides, and they often have fully webbed feet.
Some have flattened bodies. Arboreal frogs generally have expanded pads on the
ends of their toes. Many fossorial species have small heads with pointed snouts
and depressed bodies. Some have spadelike tubercles on their hind feet used for
burrowing.
Anurans have evolved remarkably diverse life histories, from aquatic eggs to
viviparity. In between those extremes, frogs from numerous families on many
continents lay their eggs out of water yet have aquatic larvae. In the section head-
ings below, the first designation refers to post-metamorphic stages, the second
to egg and/or larval stages.

1.3.3.1 Aquatic/aquatic
Many aquatic frogs lay eggs that hatch into tadpoles. African clawed frogs
(Xenopus) attach their eggs to submerged vegetation in standing water. The
South American paradox frog (Pseudis paradoxa) oviposits among vegetation
in shallow water of ponds and lakes. Aquatic larvae grow to 25 cm—the largest
of any frog—then they metamorphose into relatively small juveniles. Tailed
frogs (Ascaphus truei) from northwestern USA and adjacent Canada live in cold,
torrential streams where they lay eggs under rocks. In some areas larvae require
several years to metamorphose.
 1 Amphibian diversity and life history | 11

Some fully aquatic anurans brood their young. Female South American Pipa
carry eggs embedded in their backs. In some species of Pipa the eggs hatch into
tadpoles. In others they undergo direct development. Female Australian gastric-
brooding frogs (Rheobatrachus) swallowed their late-stage eggs or early-stage
larvae, and the tadpoles absorbed yolk reserves while developing in their mothers’
stomachs. No Rheobatrachus have been seen since the 1980s; both species are
assumed extinct.

1.3.3.2 Terrestrial/aquatic
Terrestrial anurans from many families lay aquatic eggs, and the aquatic larvae
metamorphose into terrestrial juveniles. Species that oviposit in standing water
produce clutches in compact masses (some ranids), floating rafts on the water
surface (some ranids), strings (most Bufo and some pelobatids), scattered indi-
vidually or in small packets on the bottom substrate (Bombina and Discoglossus),
attached individually or in small groups onto submerged plants (some species
of Pseudacris, Acris, Hyla, and Spea), or as a film on the water surface (some
microhylids).
Some anurans breed in moderately fast streams or mountain torrents. Female
Atelopus from Central and South America lay their eggs in strings attached to
rocks. The larvae have ventral sucker-like discs that allow them to adhere to
rocks while feeding. Tadpoles of bufonid stream-breeding Ansonia from Asia
and Werneria from Africa have similar suckers. Other stream-adapted frogs,
such as many ranids, lay large eggs in compact masses attached to rocks in areas
where the current is slow and the eggs are less likely to be swept away.
Some terrestrial anurans produce foam nests in which their eggs are sus-
pended. Male Leptodactylus, Physalaemus, and Pleurodema from Central and
South America kick their hind legs during amplexus, whipping the females’
eggs and mucus and their sperm and mucus into foamy masses (Figure 1.2a).
The outermost layer of foam dries quickly and provides some protection against
desiccation and predation. Some foam-nesters produce their nests on the water
surface, others in cavities or holes next to ponds. Leptodactylus bufonius con-
structs mud nests at the margin of temporary ponds and deposits its foam nests
inside (Figure 1.2b). The eggs hatch into tadpoles that remain in the nests until
rains dissolve the nests and flood the area. Some Australian myobatrachids also
construct foam nests on the water surface.
Some terrestrial frogs oviposit in very small bodies of water on land.
Brazilian Bufo castaneoticus lay their eggs in water-fi lled fruit capsules of the
Brazil nut tree, and the tadpoles feed on detritus. Eupsophus from Chile and
12 | Amphibian ecology and conservation

(a) (b)

(c) (d)

Fig. 1.2 Representatives of four anuran modes of reproduction. (a) Pleurodema borelli
pair constructing a foam nest on the surface of water, from Argentina, (b) Leptodactylus
bufonius mud nest by the edge of a depression, from Argentina, (c) Hyla bokermanni eggs
on a leaf above water, from Ecuador, (d) male Rhinoderma darwinii brooding tadpoles in
its vocal sac, from Chile. Photographs by Martha L. Crump.

Crinia georgiana from Australia oviposit in small water-fi lled depressions,

seeps, or crevices on the ground where non-feeding larvae absorb their large
yolks before metamorphosing.
Temperate and tropical frogs with terrestrial eggs and aquatic larvae accom-
plish this feat in diverse ways. Dendrobatids lay their eggs on land, but then a
parent transports the larvae to water. In a few species of Dendrobates, the female
also provides her young with unfertilized eggs as food. Rhinoderma rufum, from
Chile, lays its eggs on land. The male takes late-stage eggs into his vocal sac
 1 Amphibian diversity and life history | 13

where they hatch, then transports the larvae to water. In Europe, male midwife
toads (Alytes) carry their eggs wrapped around their hind legs. Eventually they
hop to ponds where the eggs hatch into aquatic larvae.

1.3.3.3 Arboreal/aquatic
Taxonomically diverse arboreal frogs lay their eggs on vegetation overhanging
water. After the eggs hatch, the larvae fall into the water below where they continue
to develop. Agalychnis, Phyllomedusa, and some Hyla from the New World trop-
ics lay their eggs over standing water (Figure 1.2c), and neotropical centrolenids
oviposit over flowing water. In some centrolenids, a parent protects the eggs from
predators and keeps them moist by resting on them. Female Afrixalus from sub-
Saharan Africa oviposit on leaves above water, then fold the leaf edges together and
glue them in place with oviductal secretions. Some arboreal Old World rhacoph-
orids and hyperoliids construct foam nests on vegetation overhanging temporary
pools or slow-moving streams.
Water-filled basins offer oviposition sites that presumably lessen the risk of
eggs getting swept away and reduce predation. Males of several neotropical
gladiator frogs (e.g. Hyla boans and Hyla rosenbergi) construct basins beside
streams or rivers. Water seeps in and fills the nests, and the frogs lay eggs as a
surface film. After developing in the basin, the tadpoles metamorphose into
froglets that take to the trees.
Some arboreal anurans oviposit in water-filled tree holes and axils of aer-
ial plants. The eggs of many of these frogs (e.g. Anodonthyla, Platypelis, and
Plethodonthyla from Madagascar) have large amounts of yolk. The tadpoles typ-
ically lack mouthparts, and they are non-feeding. In contrast, female Osteopilus
brunneus from Jamaica lay eggs in water-filled leaf axils of bromeliads and con-
tinue to deposit about 250 more eggs in the bromeliad every few days throughout
the tadpoles’ development. The tadpoles—up to about 170 in a clutch—feed on
the later-arriving eggs until they metamorphose into arboreal froglets.
Some arboreal frogs attach their eggs to the walls of water-filled cavities in
trees. After hatching, the tadpoles drop into the water. In Chirixalus eiffingeri,
an Asian rhacophorid, the female returns periodically and deposits fresh eggs
for her tadpoles to eat. In others of this reproductive mode, the aquatic larvae
feed on algae and debris.
In the New World tropics, female Flectonotus carry their eggs in dorsal
pouches. After the eggs hatch as advanced tadpoles, the females transport
them to water-filled bromeliads or bamboo where they complete development.
Females of some neotropical Gastrotheca also brood their eggs in dorsal pouches
and transport the tadpoles to aquatic sites.
14 | Amphibian ecology and conservation

1.3.3.4 Fossorial/aquatic
Scaphiopus and Spea, North American spadefoot toads, spend much of their
lives underground but emerge following heavy rains and lay their eggs in newly
formed ponds. The tadpoles develop quickly, which increases the probability
of metamorphosing before the ponds dry. Rhinophrynus dorsalis, from south-
ern Texas to Costa Rica, likewise lives underground and emerges after the first
heavy rains to breed in temporary ponds. Female Hemisis marmoratum from
Africa lay their eggs in subterranean chambers and stay with their eggs until
after they hatch. At that point, the female digs a tunnel into adjacent water for
the tadpoles.

1.3.3.5 Terrestrial/non-aquatic
Terrestrial anurans exhibit diverse life histories that free them from aquatic
breeding sites. Adenomera from South America deposits foam nests under logs
or in terrestrial cavities, and non-feeding tadpoles develop in the nests until they
metamorphose. Neotropical Eleutherodactylus, Oreophrynella from Guyana and
southern Venezuela, and New Guinean microhylids lay direct-developing eggs
under logs or leaf litter. Many attend their eggs.
Some completely terrestrial anurans brood their young. Female Assa dar-
lingtoni from Australia attend their terrestrial eggs. When the eggs are about
12 days old, the father climbs into the egg mass, rupturing the capsules. The
newly hatched tadpoles wriggle into brood pouches, one along each side of
the male’s body. He broods his non-feeding larvae until they metamorph-
ose into froglets. Female Darwin’s frogs (Rhinoderma darwinii) from Chile
and Argentina lay their eggs on moist ground. Just before the eggs hatch the
males gobble them into their mouths and into the vocal sacs (Figure 1.2d).
The young ingest secretions from the vocal-sac lining and emerge from their
fathers’ mouths as froglets.
Several Nectophrynoides, African bufonids, retain eggs in their oviducts and
give birth to live young. In Nectophrynoides occidentalis, after depleting their
yolk reserves the developing embryos feed on “uterine milk” secretion produced
by glands in the mother’s oviduct walls. These frogs live at high elevations,
exposed to long periods of cold and drought. The females have a 9-month gesta-
tion period during which they estivate underground.

1.3.3.6 Arboreal/non-aquatic
Some neotropical Eleutherodactylus lay their direct-developing eggs in tree
holes, bromeliads, moss, or on leaves. Some attend their eggs, others do not.
Eleutherodactylus jasperi from Puerto Rico lived in arboreal bromeliads and gave
 1 Amphibian diversity and life history | 15

birth to live young. The direct-developing eggs were retained in the oviducts,
and nutrition came entirely from the embryo’s yolk reserves. This species has not
been seen since 1981 and is assumed extinct.
Female Cryptobatrachus, Stefania, and Hemiphractus, neotropical hylids,
carry their direct-developing eggs exposed on their backs, secured by mucous
gland secretions. Females of some Gastrotheca brood direct-developing eggs in
dorsal pouches that protect the developing embryos from predators and des-
iccation and also function in gaseous exchange between the females and their
embryos.

1.3.3.7 Fossorial/non-aquatic
The burrowing microhylid Synapturanus salseri from Colombia lays its eggs
in burrows just below the root mat on the forest floor. Non-feeding tadpoles
hatch at an advanced stage and absorb their yolk reserves. The Brazilian bur-
rowing leptodactylid Cycloramphus stejnegeri likewise oviposits in underground
nests and has non-feeding tadpoles. Other fossorial anurans, such as Geocrinia
and Arenophryne (Australian myobatrachids), Callulops (New Guinean micro-
hylids), and Breviceps (African microhylids), lay direct-developing eggs in
underground burrows. Female Breviceps stay with their eggs and presumably
keep them moist.

1.4 Amphibian declines and why they matter

The world is experiencing a “biodiversity crisis”: rapid and accelerating loss of
species and habitat (Ehrlich and Ehrlich 1981; Myers 1990; Raven 1990; Wilson
1992). Amphibians are part of this overall loss. Populations of amphibians are
declining and disappearing worldwide at an increasing rate as compared to pre-
1980 decades, even from protected areas (Blaustein and Wake 1990; Phillips
1994; Stuart et al. 2004). During the late 1980s and early 1990s, many declines
seemed mysterious because there was no obvious cause. Skeptics argued that
declines might be simply natural population fluctuations. Since the late 1980s,
scientists worldwide have focused on determining the extent of declines and
identifying the causes. We now know these declines are real.
The International Union for the Conservation of Nature (IUCN) assesses
the status of species on a global scale and maintains and updates a catalog of
taxa that face a high risk of global extinction: the IUCN Red List of Threatened
Species. The 2008 update lists 30% of described amphibians as threatened with
extinction (IUCN 2008). Since 1500, at least 39 species of amphibians have
become extinct.
16 | Amphibian ecology and conservation

Scientists have hypothesized six major threats to amphibians: habitat modi-

fication and destruction, commercial over-exploitation, introduced species,
environmental contaminants, global climate change, and emerging infectious
diseases, especially the chytrid fungus Batrachochytrium dendrobatidis (Collins
and Storfer 2003). Most agree the primary threat is habitat modification and
destruction. For the past 100 years, human population growth has been expo-
nential and has occurred largely in areas with the highest amphibian species
richness: the tropics and subtropics (Gallant et al. 2007). As a result, these land-
scapes are being heavily modified to support agriculture and other human activ-
ities. The chytrid fungus also is exerting a major impact in many areas and on
many species (Smith et al. 2006). Thus far the chytrid has caused the decline
or extinction of about 200 species of frog (Skerratt et al. 2007). Many factors
make the chytrid a significant concern, including its wide distribution in both
the New and Old Worlds, its rapid spread and high virulence, and the fact that
it infects a broad diversity of host species (Daszak et al. 1999).
Why should we care if we lose amphibians? It is for the same basic reasons we
should care if other animals and plants disappear: economics, ecosystem func-
tion, esthetics, and ethics (Noss and Cooperrider 1994; Groom et al. 2006).

1.4.1 Economics
Selfishly, we should care if we lose amphibians because we use them for our own
benefit, including for food and as pets. We use literally tonnes of frogs each year
in medical research and teaching. We have isolated novel chemical compounds
from granular glands of anuran skin and have used these compounds to develop
new drugs.

1.4.2 Ecosystem function

Amphibians play a key role in energy flow and nutrient cycling because they
serve as both predator and prey. By eating huge quantities of algae, tadpoles
reduce the rate of natural eutrophication, the over-enrichment of water with
nutrients, which leads to excessive algal growth and oxygen depletion. Most
adult amphibians eat insects and other arthropods. As ectotherms, amphib-
ians are efficient at converting food into growth and reproduction. Unlike
endothermic birds and mammals that generate heat metabolically, amphibians
expend relatively little energy to maintain themselves. Birds and mammals
use up to 98% of their ingested energy to maintain their body temperatures,
leaving as little as 2% to be converted to new animal tissue: food for preda-
tors. In contrast, amphibians convert about 50% of their energy gained from
food into new tissue, which is transferred to the next level in the food chain
 1 Amphibian diversity and life history | 17

(Pough et al. 2004). If amphibians disappeared, would the world be overrun

with houseflies, mosquitoes, and crop-eating insect pests? Would their preda-
tors go extinct?

1.4.3 Esthetics
Imagine the silence of rainy spring evenings without the lively croaking of male
frogs. The monotonous roads without spring migrations of salamanders. People
worldwide consider frogs to be good luck because of their association with rain.
Amphibians provide inspiration for our artistic endeavors, from literature to
music and the visual arts.

1.4.4 Ethics
Every species is a unique product of evolution. In 1982 the United Nations
General Assembly adopted the World Charter for Nature, which states: “Every
form of life is unique, warranting respect regardless of its worth to man, and,
to accord other organisms such recognition, man must be guided by a moral
code of action” (Noss and Cooperrider 1994). More than 100 nations signed
the charter. Like all other living species, amphibians have intrinsic value and a
right to exist.
Amphibians, amazing descendants of terrestrial pioneers, are fighting for
their lives in a world greatly modified by humans.

1.5 References
Blaustein, A. R. and Wake, D. B. (1990). Declining amphibian populations: a global phe-
nomenon? Trends in Ecology and Evolution, 5, 203–4.
Collins, J. P. and Storfer, A. (2003). Global amphibian declines: sorting the hypotheses.
Diversity and Distributions, 9, 89–98.
Crump, M. L. (1995). Parental care. In H. Heatwole and B. K. Sullivan (eds), Amphibian
Biology, vol. 2: Social Behaviour, pp. 518–67. Surrey Beatty and Sons, Chipping
Norton, NSW.
Crump, M. L. (1996). Parental care among the Amphibia. In J. S. Rosenblatt and
C. T. Snowdon (eds), Advances in the Study of Behavior, vol. 25: Parental Care: Evolution,
Mechanisms, and Adaptive Significance, pp. 109–44. Academic Press, New York.
Daszak, P., Berger, L., Cunningham, A. A., Hyatt, A. D., Green, D. E., and Speare, R.
(1999). Emerging infectious diseases and amphibian population declines. Emerging
Infectious Diseases, 5, 735–48.
Duellman, W. E. (1970). The Hylid Frogs of Middle America, vol. 1. Monograph of the
Museum of Natural History, Number 1. University of Kansas, Lawrence, KA.
Duellman, W. E. (1999). Global distribution of amphibians: patterns, conservation, and
future challenges. In W. E. Duellman (ed.), Patterns of Distribution of Amphibians: a
Global Perspective, pp. 1–30. John Hopkins University Press, Baltimore, MD.
18 | Amphibian ecology and conservation

Duellman, W. E. (2007). Amphibian life histories: their utilization in phylogeny and clas-
sification. In H. Heatwole and M. J. Tyler (eds), Amphibian Biology, vol. 7. Systematics,
pp. 2843–92. Surrey Beatty and Sons, Chipping Norton, NSW.
Duellman, W. E. and Trueb, L. (1986). Biology of Amphibians. McGraw-Hill, NewYork.
Ehrlich, P. R. and Ehrlich, A. H. (1981). Extinction: The Causes and Consequences of the
Disappearance of Species. Random House, New York.
Gallant, A. L., Klaver, R. W., Casper, G. S., and Lannoo, M. J. (2007). Global rates of
habitat loss and implications for amphibian conservation. Copeia, 2007, 967–79.
Groom, M., Meffe, G. K., and Carroll, C. R. (2006). Principles of Conservation Biology,
3rd edn. Sinauer Associates, Sunderland, MA.
Haddad, C. F. B. and Prado, C. P. A. (2005). Reproductive modes in frogs and their unex-
pected diversity in the Atlantic Forest of Brazil. BioScience, 55, 207–17.
IUCN (International Union for the Conservation of Nature) (2008). Red List of
Threatened Species. IUCN, Gland. http://www.iucn.org/themes/ssc/redlist.htm.
Myers, N. (1990). Mass extinctions: what can the past tell us about the present and the
future? Global and Planetary Change, 82, 175–85.
Noss, R. F. and Cooperrider, A. Y. (1994). Saving Nature’s Legacy: Protecting and Restoring
Biodiversity. Island Press, Washington DC.
Phillips, K. (1994). Tracking the Vanishing Frogs: an Ecological Mystery. St. Martin’s Press,
New York.
Pough, F. H., Andrews, R. M., Cadle, J. E., Crump, M. L., Savitzky, A. H., and Wells, K. D.
(2004). Herpetology, 3rd edn. Prentice Hall, Upper Saddle River, NJ.
Raven, P. H. (1990). The politics of preserving biodiversity. BioScience, 40, 769–74.
Salthe, S. N. (1969). Reproductive modes and the number and sizes of ova in the urodeles.
American Midland Naturalist, 81. 467–90.
Salthe, S. N. and Duellman, W. E. (1973). Quantitative constraints associated with
reproductive mode in anurans. In J. L. Vial (ed.), Evolutionary Biology of the Anurans,
pp. 229–49. University of Missouri Press, Columbia, MO.
Skerratt, L. F., Berger, L., Speare, R., Cashins, S., McDonald, K. R., Phillott, A. D.,
Hines, H. B., and Kenyon, N. (2007). Spread of chytridiomycosis has caused the rapid
global decline and extinction of frogs. EcoHealth, 4, 125–34.
Smith, K. F., Sax, D. F., and Lafferty, K. D. (2006). Evidence for the role of infectious
disease in species extinction and endangerment. Conservation Biology, 20, 1349–57.
Stuart, S. N., Chanson, J. S., Cox, N. A., Young, B. E., Rodrigues, A. S.L., Fischman, D. L.,
and Waller, W. (2004). Status and trends of amphibian declines and extinctions world-
wide. Science, 302, 1783–6.
Wake, M. H. (1977). The reproductive biology of caecilians: an evolutionary perspec-
tive. In D. H. Taylor and S. I. Guttman (eds), The Reproductive Biology of Amphibians,
pp. 73–101. Plenum, New York.
Wake, M. H. (1982). Diversity within a framework of constraints. Amphibian repro-
ductive modes. In D. Mossakowski and G. Roth (eds), Environmental Adaptation and
Evolution, pp. 87–106. Gustav Fischer, New York.
Wake, M. H. (1992). Reproduction in caecilians. In W. C. Hamlett (ed.), Reproductive
Biology of South American Vertebrates, pp. 112–20. Springer-Verlag, New York.
 1 Amphibian diversity and life history | 19

Wake, M. H. (1993). Evolution of oviductal gestation in amphibians. Journal of

Experimental Zoology, 266, 394–413.
Wells, K. D. (2007). The Ecology and Behavior of Amphibians. University of Chicago
Press, Chicago, IL.
Wilkinson, M. and Nussbaum, R. A. (1998). Caecilian viviparity and amniote origins.
Journal of Natural History, 32, 1403–9.
Wilson, E. O. (1992). The Diversity of Life. The Belknap Press of Harvard University
Press, Cambridge, MA.
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 2
Setting objectives in field studies
Dan Cogălniceanu and Claude Miaud

2.1 Basic concepts for a good start

Considerable financial and human resources may be wasted due to poor research
design and implementation. In addition, poor planning can result in taking
non-repeatable or unreliable data that are of no or limited use, and leads to the
paradox of “data-rich, information-poor.” Rigorous study planning and the def-
inition of clear and realistic goals can help avoid wasted effort. Before undertak-
ing field studies, biologists first must define their objectives and then assess the
availability of resources necessary to accomplish them. This is a key stage, and
any uncertainty or vagueness at this point could prove detrimental to the success
and usefulness of the study.
There are two different approaches in scientific investigation: inductive and
deductive methods. The inductive or exploratory method relies on gathering data
without first identifying hypotheses to be tested; results may then be explained
based on the data gathered. However, science involves more than accumulating
data and then searching for patterns. A useful study must start with an obser-
vation that identifies a problem or question to be resolved. The observation
should have biological significance and be based on current evolutionary theory
(Wolff and Krebs 2008). For example, amphibian populations are declining:
what are the causes? Two related species hybridize on just a narrow contact zone:
why? Habitats are fragmented by human activity: what effects does this have on
population persistence, and why (e.g. dispersal abilities, food resources, popula-
tion demography, genetics, susceptibility to predators, and human activities)?
As science is an activity focused on understanding natural processes and, espe-
cially in modern times, in devising solutions to problems, the hypothesis-based
deductive method was developed (James and McCulloch 1985). An hypothesis
is a statement related to an observation which may be true, but for which proof
has not yet been found. The function of the hypothesis is to direct the search
22 | Amphibian ecology and conservation

for order among facts. Fieldwork then serves to test one’s hypothesis, thereby
scientifically demonstrating correlation, causation, or the absence thereof. The
hypothesis must of necessity regard some facts as more significant than others,
based on the researcher’s previous experience, familiarity with the literature,
and individual interpretation.
As scientific knowledge is rarely complete, any list of potential alternative
hypotheses is also unlikely to be complete. Therefore, alternative hypotheses for
the particular problem or question being considered must also be formulated.
Thus, it is necessary to conduct research with both the hypothesis and alter-
native hypothesis in mind, as more than one cause may be contributing to any
single effect (Wolff and Krebs 2008). For example, Jaeger (1972) used enclos-
ures to test the mechanism of interspecific competition between two species of
plethodontid salamanders, hypothesizing that it resulted from either differential
exploitation of food resources or through interference. Both primary and alter-
native hypotheses are formulated and tested so that researchers can determine
which explanations best fit the results obtained. Unsupported hypotheses can be
rejected, but even the most parsimonious hypothesis may not be fully accepted
because there still may be underlying explanations as yet untested (Senar 2004).
Repeated experimentation involving alternative hypotheses eventually allows
researchers to gain a measure of confidence in the validity of empirically sup-
ported hypotheses (Jaeger and Halliday 1998). Both hypotheses and data are
essential for credible science since, as Krebs (1999) concluded, hypotheses with-
out data are not very useful, and data without hypotheses are useful only for an
inductive approach.
Ecology is an empirical science that requires data from the “real” world,
and a field study is a tool which can ultimately lead to the acceptance or rejec-
tion of an hypothesis. After repeated experimentation and validation, data
and information from many field studies are synthesized and organized into
concepts of how nature functions. These concepts are the result of the integra-
tion between what scientists think they know (based on previous research and
observation) and newly acquired data (Ford 2000). Asking the right question
is important, because the type of question asked and the particular techniques
and methods used in hypothesis-driven research strongly influence what is
discovered and the direction of future research. During the course of this
feedback process, scientists come to a better understanding of the phenomena
they study (Figure 2.1).
Scientists present what they want to accomplish during a field study in terms
of goals and objectives. A goal is a statement that explains what the study is
designed to accomplish. It is usually a broad and general statement, inclusive of
 2 Setting objectives in field studies | 23

Analysis and hypothesis testing

New theories and Data and

hypotheses information

Synthesis and creativity

Analysis and hypothesis testing

New theories and Data and

hypotheses information

Synthesis and creativity

Analysis and hypothesis testing

Theories and Data and

hypotheses information

Synthesis and creativity

Fig. 2.1 The cycle of scientific investigation and the shift towards the spiral of knowledge.

a long-term direction. The goal is then split into specific, measurable objectives
that indicate how the goal can be achieved within a specified time frame, and
the expected results.
Defining a study’s objectives clearly is the first and probably the most
important single step in research planning, and is a key element in a success-
ful project. The goals of research can be non-applied (i.e. aimed at increasing
or changing existing knowledge) or applied (i.e. focused on solving practical
questions), or both. Rather than addressing an effect, a study should focus
on the cause of the phenomenon in question. Separating effects from causes
is a major challenge when setting objectives. For example, acid rain has well-
known causes, but much research today focuses on the effects or seeks solutions
to limit its impact. In another example, many recent studies have reported on
amphibian declines (Stuart et al. 2004), but many fewer have identified spe-
cific causes (e.g. Becker et al. 2007). It is important that amphibian biologists
do not limit their focus to specific taxa; otherwise, they lose sight of the fact
that similar effects may be occurring in other taxa. Amphibian biologists need
to establish closer links with researchers studying similar problems in other
taxa (Halliday 2005).
24 | Amphibian ecology and conservation

Ideally, the cycle of scientific investigation should proceed from data and infor-
mation gathering and analysis, to quantifying knowledge, and to conceptual
understanding. A logical, stepwise approach of scientific inquiry (Lehner 1996)
should be followed: (1) perceive that a problem/question exists; (2) formulate a
possible explanation (i.e. devise an hypothesis); (3) formulate alternative hypoth-
eses; (4) identify the best approach to test the hypothesis (i.e. theoretical models,
experiments, or field observations); (5) collect and analyze data; (6) support or
reject the hypothesis; and (7) understand the meaning and implications of the
results. The original hypothesis can then be modified, experiments repeated and,
with time, conceptual understanding attained.

2.2 Steps required for a successful study

Ecological systems are large and complex and, at times, unpredictable. The more
complex the system, the more uncertainties arise. Data are most often obtained
within the parameters of a limited number of samples, restricted time, or specific
area, and then applied to larger scales. By carefully framing the hypothesis and
deciding the most practical, meaningful, and objective method of study, degree
of error can be minimized (Hayek 1994). The following section presents some of
the most important issues that must be considered when planning a field study.

2.2.1 Temporal and spatial scales

The issue of scale has three components (Schneider 2001): (1) pressing problems
in ecology often exist within timescales of decades or centuries, and cover large
areas; (2) most data can be gathered directly for only short periods of time and
over small areas; (3) patterns measured at small scales do not necessarily hold
true at larger scales; nor do processes observed at smaller scales necessarily exist
at larger ones. Thus, an inappropriate scale limits the inference of the results.
Spatial and temporal scales can be classified as gradient, interval, discrete,
and continuous (Bernstein and Goldfarb 1995). Setting temporal and spatial
scales involves defining their boundaries and helps to answer three practical
questions: where, for how long, and how often? Selecting the appropriate scale
is difficult because of environmental heterogeneity in both time and space.
Amphibian populations and communities also vary widely throughout time
and space, since they are influenced by a multitude of environmental variables
(Meyer et al. 1998; Pechmann et al. 1991; Pellet et al. 2006).
Temporal scales are rarely discussed explicitly, but they are often assumed
to span years rather than generations. Many time-dependent phenomena such
as extinction, predator–prey interactions, competition, and succession reveal
 2 Setting objectives in field studies | 25

important insights if considered on a generational scale (Frankham and Brook

2004). The current distribution and abundance of animal species inhabiting an
area is the result, in part, of the impact of geological processes (e.g. plate tecton-
ics, orogenesis, sea-level changes, major catastrophes), ecological processes (e.g.
competition, predation, climate), life-history traits (e.g. dispersal), and recent
human impacts (e.g. changes in vegetation and habitat structure, overexploi-
tation, introduction of nonindigenous species). For example, in the Northern
Hemisphere the Pleistocene–Holocene post-glacial recolonizations of species
and human activities (e.g. habitat destruction and alteration) are the two major
factors shaping the current distribution of amphibians. Objectives of a time-
scale component of a study plan should include such factors as short-term (less
than one generation), intermediate (more than one to a few generations), and
long-term (many generations, and inclusive of a large spatial scale and monitor-
ing activities).
Spatial scales and objectives are strongly interconnected. Selection of a study
site includes such factors as the complexity of local macro- and microhabitats
and, at large spatial scales, biogeographical provinces and landscapes (Morrison
2002). Of great importance to the overall study plan are the criteria for describ-
ing vegetation, whether gross (e.g. foliage height, diversity), physiognomy (phys-
ical structure), or floristic (plant taxonomic description). The relative usefulness
of structural or floristic measures depends on the spatial scale of the analysis.
For example, whereas most studies do not require a detailed description of plant
taxa, species lists might be essential for characterizing microhabitat and trophic
(resource) availability.
The importance of choosing the correct scale can be exemplified by a study
assessing population fluctuations within a particular species: the first step would
be to understand the distribution of the species in order to thoroughly sample all
areas, rather than by biasing results by sampling in only one small portion of the
range. In another example, a study of the movement patterns of a species may
focus on individual distributional patterns, daily or weekly movements, sea-
sonal migrations related to reproduction or dispersal, or on region-wide range
shifts through years or decades.

2.2.2 Choosing the model species

Many amphibian species have restricted distributions and complex habitat
requirements. Some amphibian populations, especially among temperate pond-
breeding species, are maintained by episodic reproduction that occurs in a spor-
adic and unpredictable manner (Alford and Richards 1999). If a number of
different species is available for study, selecting an experimental species should
26 | Amphibian ecology and conservation

be based on an understanding of its life history (i.e. feeding, habitat use, disper-
sal abilities, reproduction, behavior, predators, phylogeography, population gen-
etics) and its suitability in resolving the particular question being asked (Wolff
and Krebs 2008). For example, a species with low detectability is not a good
choice for a mark–recapture study since it will require a considerable sampling
effort and entail potential capture bias (Weir et al. 2005). The role of keystone
species (e.g. Eleutherodactylus bransfordii in Costa Rican forests), umbrella spe-
cies (e.g. Rana sylvatica in the Milwaukee river basin), or flagship species (e.g.
Salamandra lanzai in the Western Alps) can also be considered. Rare or threat-
ened species are often selected because of conservation applications, but research
on them could incur potential risks due to ethical considerations. Researchers
should avoid choosing a rare or threatened species if common or less vulnerable
surrogate species can be selected.

2.2.3 Pilot/desk study

A preliminary pilot study usually helps to develop and test realistic and achiev-
able objectives, and avoids later shortcomings and failures. Usually a pilot study
is carried out on short temporal and small spatial scales, and allows the testing of
conceptual models and methods. What might seem simple during office plan-
ning might prove completely different or unworkable in the field. Even a simple
review of the literature can prove helpful in avoiding mistakes, however.

2.2.4 Elaborate a conceptual model

A conceptual model (e.g. a simple box diagram showing components and link-
ages) is a simplified model of the system to be studied. There are no ideal methods
to employ; instead, a multitude of models are available to choose from with
various degrees of complexity. Conceptual models are helpful in that they can
be used to select the variables to be measured that might be considered import-
ant to the study. Since professionals with diverse backgrounds have different
philosophies or approaches to using models, it is recommended that each team
member contribute knowledge of and/or expertise with various models, then
develop an integrated study model from which to work (Maher et al. 1994).
After model development, many of the design questions become more obvi-
ous. A model need not embrace all components of the system; it needs only to
be adequate for the scope of the investigation. There is always a possible risk that
the conceptual model is inappropriate or over-simplified; thus, a slightly more
complex model may need to be developed. Adopting a more complex model
focuses researchers on collecting additional data that might prove important,
despite the extra costs involved with sampling and measurements. In other
 2 Setting objectives in field studies | 27

words, it might be better to collect more data than appears necessary at first,
than to discover later that some important parameter was omitted. A pilot study
helps to avoid under- or over-collecting data. Although conceptual models help
researchers identify what to measure, the timing of studies is determined by the
natural history of the species of interest.

2.2.5 The SMART approach

Specific objectives allow for greater chances of conducting a successful research
project. SMART stands for specific, measurable, attainable, relevant, and time
(Piotrow et al. 1997), an approach used in project writing that also helps in for-
mulating specific and measurable objectives within study plans. SMART helps
devise objectives that are clear and concise, indicates what is to be achieved,
addresses only attainable results, indicates when each stage will be completed,
and is not encumbered by idealistic aspirations.

• S: The specific part of an objective defines what will be done and where it will
occur. When setting objectives, ask simple questions that can be answered,
and avoid ambiguities, the use of buzzwords or jargon (e.g. “cutting-edge”),
and pompous phrasing.
• M: Measurable is an attribute of an activity or its results. The source of
and mechanism for collecting measurable data are identified, and collec-
tion of these data is determined feasible. If the objective of the study is to
document trends (i.e. an increase or decrease of one or more measurable
variables), then a baseline is required to act as a reference point (e.g. habi-
tat availability, characteristics and use; amphibian community structure;
population size). If a baseline is not yet available then it will be useful to
first have it established.
• A: Attainable refers to the probability of conducting the proposed activ-
ities within the established time frame with the available resources and sup-
port. It also includes the external factors critical to success. Doing research
today, for example, can become difficult due, in part, to increasing admin-
istrative restrictions (Prathapan et al. 2008). Other external factors include
unforeseen costs, shifts in exchange rates, obtaining collecting and access
permits, changes in legislation, and political, security, and health (both
human and animal) issues. In coping with external factors, a risk analysis
is useful because it allows for planning alternative strategies.
• R: Some useful measures for the relevance of the study’s objectives are the
utility and value of the results for practical purpose (e.g. management of
protected areas, better conservation measures, and ecological restoration). It
28 | Amphibian ecology and conservation

may be difficult to determine how relevant the objective may be, especially
since the true relevance may not be apparent until the study is completed
(e.g. if a drought were to affect a long-term study of amphibian breeding).
Perhaps an easy way to evaluate the relevance is to answer the “so what”
question, thus avoiding undertaking studies of limited or no interest.
• T: Finally, the time required to complete the project will depend on the
parameters discussed above.

2.2.6 Applying the SMART approach to

plan an amphibian inventory
In the following example, the SMART approach is used as the basis for setting
up an inventory of amphibian species in a national park or reserve.
• Specific: determine whether to inventory all amphibian species, or only
those of special interest, such as rare, threatened, or endemic species. Set
the spatial limits of the study, such as the administrative boundary of the
park or reserve, or specific habitats of interest (e.g. temporary ponds; roads
in areas which bisect amphibian dispersal corridors; high elevations con-
taining unique habitats or species richness, such as cloud forests).
• Measurable: select parameters to be measured (e.g. presence/not detected,
relative abundance, density, sex or life stage, percentage of area occupied),
select methods appropriate for each species (especially taking detection
probabilities into account), and decide habitat parameters (e.g. tempera-
ture, humidity, vegetative cover, characteristics of aquatic habitats).
• Attainable: identify resources and available support (e.g. funding, work
force) and administrative considerations (e.g. collecting and access per-
mits, training staff for field data collection, animal care and use require-
ments, security issues).
• Relevance: provide concise statements as to the importance of the research,
and whether it has practical applications. The relevance of the proposed
research is particularly important in field studies, especially if it will aid
in the management of protected areas and species conservation. Utility
also assists in determining research approaches. For example, incorpor-
ating measures of abundance with detection probabilities, in addition to
occupancy models, is much more informative in determining status than
occupancy models alone.
• Time: the research schedule (planning, fieldwork, data analysis, report
and publication preparation) should be clearly stated based on the previ-
ously outlined parameters and limiting factors.
 2 Setting objectives in field studies | 29

2.2.7 Experimental versus field studies

Biological communities, apart from their high internal complexity, are subject
to random, naturally occurring fluctuations involving both physical and biotic
parameters (e.g. heat, cold, drought, effects of disease, and spread of invasive
species). Stochastic fluctuations such as these are a major source of statistical
variance in nature, resulting in a shift by some researchers towards less complex
and more controlled field and laboratory experimental designs (see Chapter 6).
Such a reductionist approach can be framed within a simpler conceptual model
where the number of variables of interest is reduced in return for minimizing
uncontrolled environmental fluctuation.
Still, there is an experimental continuum between laboratory and field studies
(Figure 2.2). Perhaps some of the most powerful experimental tools which can
provide the statistical rigor required for hypothesis testing are outdoor experi-
ments (Fauth 1998; Rowe and Dunson 1994; Chapter 6). Controlled experi-
ments maximize a researcher’s ability to detect a response to variables of interest
(e.g. food, density of individuals, hydroperiod). The best approach is to use
observations in nature coupled with experimental analysis. For example, Wilbur
(1997) used both natural and artificial ponds to investigate complex food webs in
temporary ponds, and their effects on the larval amphibian community.

2.2.8 Methods for sampling, data storage, and analysis

Selecting an appropriate research technique is not an easy task, especially since
“trendy” methods may not be the best ones to use. Methods selected must be
adequate for the proposed objectives, and it is best to test them first to determine

Least Most Least

Mathematical
model
Control and precision

Laboratory
Unpredictability

experiment Simple
Realism

Outdoor
Complexity

experiment

Unrestricted field
experiment

Complex Most Least Most

Fig. 2.2 Trade-offs between complex and simple experiments.

30 | Amphibian ecology and conservation

efficiency and effectiveness. Researchers should adopt complimentary models,

applicable and relevant methods, and an altogether SMART use of study plans,
resources, and results. The most important elements in any study plan are accurate
and precise methods that meet the objectives of the study. A cost-benefit analysis
can prove helpful in the final selection of the methods (Arntzen et al. 1995, 2004).
The sampling design should be both efficient (a better use of sampling effort
in obtaining results) and simple (easily comprehended and easily implemented)
(Scott and Köhl 1993). Statistical analyses often have biases or limitations in
ecological studies (Krebs 1999; Pollock et al. 2002). Statistical significance may
not be the equivalent of biological significance. Focusing too much on statistical
issues may prove deceptive or lead to inaccurate conclusions. One way to min-
imize the potential of error is to incorporate power analysis into study designs
(Yoccoz 1991). Statistical power refers to the ability of a test to correctly reject
a null hypothesis, and is frequently evaluated in the context of the sample size
required to detect an effect of a given magnitude (Michener 2000).
When using probability statistics, it is possible to make two kinds of error:
a researcher may claim there is a difference when one does not exist, or can fail
to detect a difference when one does exist (Underwood 1997). The first type of
error is estimated by the traditional probability value (0.05) associated with
statistical tests. If a researcher rejects the null hypothesis at this level, then there
is still a 5% chance that he/she is in error.
The second type of error is more difficult to estimate because it depends on
the sample size, the magnitude of effect, and sample variability. Researchers
are likely to correctly reject a null hypothesis with larger sample sizes, larger
effect sizes, or less variable samples. Alternatively, a higher critical value may be
selected (e.g. 0.1) as an indication of statistical significance.
Statistical power is a measure of the proportion of time that a researcher
would correctly reject the null hypothesis if an experiment could be repeated
an infinite number of times; statistical power is usually estimated by computer
simulation. The goal of a power analysis is to define a level of confidence in the
research results; it can also be used in determining trends and in setting optimal
sample size. A discussion of power analysis is available through StatSoft (www.
statsoft.com/textbook/stpowan.html).

2.3 Trade-offs and pitfalls

There are several pitfalls that can and should be avoided when establishing object-
ives in field studies (Bardwell 1991). The most common ones are: (1) addressing
the wrong problem; (2) stating the problem in a way that no solution is possible;
 2 Setting objectives in field studies | 31

(3) prematurely accepting a solution as the only possible answer; and (4) using
data and information that are either incorrect (e.g. inaccurate information in the
scientific literature) or irrelevant. There are several additional pitfalls that may be
avoided by careful planning and a thorough understanding of the questions to be
asked (Tucker et al. 2005). Four of the most frequent are listed below.
1) The statistical framework might be inadequate, since many techniques
developed in the context of controlled experimentation are sometimes
incorrectly applied to field data, resulting in an inappropriate use of the
null hypothesis (Johnson 1999).
2) Researchers and technicians might differ in their skills, use non-comparable
methods, or have different personal goals. Training prior to the start of field
collection of data and a comparison of each person’s abilities helps to min-
imize these problems.
3) Methods may be changed during a study. This could lead to an incompati-
bility of data sets and limit the interpretation of results.
4) The locations of permanent sample sites are not properly recorded so that
different areas are subsequently revisited or sampled.
When designing fieldwork, researchers need to be aware of potential options
and trade-offs, and try to balance them (Hairston 1989). Examples include:
(1) complexity versus simplicity (e.g. choices ranging from theoretical models to
field experiments), (2) confidence in results versus general application (e.g. high
confidence can be achieved at short temporal and small spatial scales and with
relatively simple goals and conceptual models, but the results will be of limited
value), and (3) replication versus sophistication of experimental design, recog-
nizing that it is impossible to simultaneously maximize precision, realism, and
generality (Levins 1968).

2.4 Ethical issues

While ecological field studies have generated a wealth of useful and important
data, the environmental impacts of these studies have rarely been quantified. A
basic assumption is that the relative benefits of research outweigh the potential
short-term costs to the study animals or habitats (Farnsworth and Rosovsky
1993). Even the simple act of observation, however, may affect the behavior
of the study organisms. Repeatedly visiting different sites during fieldwork
can have negative impacts, either by spreading or introducing nonindigenous
species (Whinam et al. 2005) or diseases, such as amphibian chytrid fungus
(Garner et al. 2005), or by microhabitat alteration (e.g. turning logs). Preventive
32 | Amphibian ecology and conservation

measures, such as equipment disinfection (Chapter 26) and routine checks for
unwanted “passengers” (e.g. seeds) are now widely used (ARG-UK 2008).
Controversies continue regarding the negative effects of some common tech-
niques. For example, toe-clipping, historically one of the most widely used mark-
ing techniques, has recently received much criticism (Chapter 8; May 2004;
McCarthy and Parris 2004). Critics, however, have not provided much evalu-
ation of the impacts of alternative procedures. Apart from dorsal or ventral pat-
tern mapping by photography or computer imaging, which are non-invasive, all
other marking techniques have disadvantages (Phillott et al. 2007). The effects
of toe-clipping vary among species, and therefore must be assessed accordingly.
Other marking methods for amphibians are available, although they are not as
economic or as easy to use, but which have fewer risks (Chapter 8; Ferner 2007).
Toe-clipping also may be prohibited by regulatory constraints; information on
regulations is best obtained and evaluated during the planning stage. Thus, there
may be a trade-off between the risks associated with methodology and the know-
ledge to be gained, even when the species may be benefited (Funk et al. 2005).
Field studies often involve years of hard work. In the end, the results may
provide few insights compared to the amount of effort to acquire them, unless
careful planning precedes the initiation of research activities. Careful planning
optimizes researcher effort and helps ensure that the data recorded will be stat-
istically accurate, with beneficial results in advancing knowledge of amphibian
ecology and conservation biology.

2.5 Acknowledgments
Cristina Vâlcu, Tibor Hartel, Marian Griffey, and Ken Dodd provided helpful
comments on previous versions of this chapter.

2.6 References
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ARG-UK (Amphibian and Reptile Groups of the UK) (2008). Amphibian Disease
Precautions: a Guide for UK Fieldworkers. ARG-UK Advice Note 4, pp. 1–5. www.
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Bardwell, L. V. (1991). Problem-framing: a perspective of environmental problem solv-

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Part 2
Larvae
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 3
Morphology of amphibian larvae
Roy W. McDiarmid and Ronald Altig

3.1 Background
The larvae of amphibians are non-reproductive and usually aquatic. Most
undergo metamorphosis prior to attaining an adult morphology and sexual
maturity. Species within each amphibian order that develop by other modes (e.g.
direct development; Altig and Johnston 1989; Thibaudeau and Altig 1999) have
non-feeding larvae or embryos, and we do not discuss them in this chapter.
Amphibian larvae have some generalized morphological features that are use-
ful for identification. In contrast to fish, they lack bony supports in the tail fins,
and the vent is a longitudinal slit (not obvious in tadpoles). Amphibian larvae
also lack eyelids, and most have external gills that are visible at some stage in
their ontogeny.
Most caecilians (Gymnophiona) whether terrestrial or aquatic as adults,
have aquatic larvae that look grossly like the legless, elongate adults. The larvae
of salamanders (Caudata) look much like the adults. Unlike the condition in
caecilians and frogs, salamanders may occur in larval (i.e. larval morphology,
non-reproductive, and metamorphic) or larviform (i.e. larval morphology,
reproductive, may or may not metamorphose) states. Larviform salamanders
may exist as pedotypes (i.e. larval relative to the normal developmental tra-
jectory of the taxon, reproductive, will metamorphose if the environmental
conditions change to the detriment of being in the larval environment; some
ambystomatids and salamandrids; terminology of Reilly et al. 1997) or pedo-
morphs (i.e. larval relative to the developmental pattern of their ancestors, do
not metamorphose; all amphiumids, cryptobranchids, proteids, sirenids, and
some plethodontids). Larval and larviform salamanders grossly resemble meta-
morphosed individuals in general body form but retain a number of larval fea-
tures. Pond-adapted forms have a more bulky body and larger gills and tail fins
than the more streamlined, stream-adapted forms.
40 | Amphibian ecology and conservation

The larvae of frogs and toads (Order Anura), called tadpoles, are grossly dif-
ferent from adults and have many developmental (Altig and Johnston 1989) and
morphological (e.g. Altig and McDiarmid 1999a; also various morphologies doc-
umented in staging tables, see Duellman and Trueb 1986, pp. 128–9) features not
seen in other amphibian larvae. Tadpoles live in many kinds of habitats; the most
common types of tadpoles are found in lentic or lotic water, spend most of their
time on the bottom, and feed by rasping material from submerged surfaces.
Because amphibians are ectotherms, their inherent developmental rates are
modified by temperature and other environmental variables; size is thus an
inaccurate estimator of chronological age. Consequently, biologists describe
tadpole ontogeny using a staging table that divides their development into rec-
ognizable stages based on the attainment of specific morphological landmarks.
With such a table the degree of development of morphological features of tad-
poles can be compared among populations and across taxa occurring in the
same or different habitats regardless of chronological age or attainable size.
Larval amphibians are exceptionally variable within and among species, although
the degree and patterns of that variation are poorly documented and their sources
rarely investigated. The many papers on induced morphological changes published
in recent years (e.g. Relyea and Auld 2005, among many others) have made it abun-
dantly clear that every tadpole of a given taxon collected at any site is a variant
within the broad phenotypic range of its taxon. Although results from mesocosm
experiments with controlled combinations and densities of species provide some
insight into understanding phenotypic variation, predicting morphological vari-
ation from random samples of ponds is highly unlikely. The presence of different
sets of predators in natural situations complicates the picture even more, and one
has to keep these factors in mind when evaluating the morphology of tadpoles.
We mention in passing that less is known about amphibian eggs (e.g. Altig
and McDiarmid 2007), hatchlings (Gosner stages 21–24; Altig 1972), and
metamorphs than is known about tadpoles (stages 25–41) and other amphibian
larvae. We urge workers to preserve and describe positively identified samples of
these stages. Here we summarize data on the morphology, ontogeny, and diver-
sity of larvae in each amphibian order.

3.2 Larval caecilians

3.2.1 Morphology and ontogeny
The vermiform body (Figure 3.1) has primary annuli homologous to the costal
folds of salamanders that form during early development; secondary and tertiary
annuli may form later. External gills with branched rami (Figures 3.1a and c) are
 3 Morphology of amphibian larvae | 41

(a) (b)

(c) (d)

Fig. 3.1 Morphology of a larval caecilian. (a) Embryo of Ichthyophis glutinosus,

modified from figure in Sarasin and Sarasin (1887–1890); (b) heads of embryos of
Ichthyophis kohtaoensis, stages 27 (left) and 33 (right), modified from drawings in
Dünker et al. (2000); (c) hatchling of I. kohtaoensis, modified from photograph by
A. Summers on front cover, Journal of Morphology 2000, 243(1); and (d) larva of
Ichthyophis banannicus showing the gill slit (upper arrow) and tail fin (lower arrow),
photograph by R. Nussbaum.

present during embryological development and lost at hatching (Figure 3.1b).

The single gill slit (Figure 3.1d) closes at metamorphosis, and a small fin is present
on a short tail (Figure 3.1d). The sensory tentacle, situated adjacent or anterior
to the eye of caecilians, develops at metamorphosis. Some caecilians have small
bony scales (i.e. osteoderms) embedded in the skin in the grooves between the
annuli. These scales that are quite small when they first appear, get progressively
larger as they develop, beginning in the posterior annuli and moving anteriorly.
The definitive pattern of occurrence varies among species. Neuromasts, or lat-
eral line organs, are present throughout larval life; labial folds, which modify the
shape and size of the mouth opening during suction feeding, are present as they
are in larval salamanders. The most complete information on early ontogeny
can be found in Dünker et al. (2000) (also see Sarasin and Sarasin 1887–1890;
Brauer 1897, 1899; Sammouri et al. 1990).
42 | Amphibian ecology and conservation

3.2.2 Coloration
Most caecilians are somewhat drab shades of gray, brown, black, or blue. A few
species are more brightly colored and slightly banded or striped (terminology of
Altig and Channing 1993).

3.2.3 Diversity
Larvae of species of caecilians in the families Rhinatrematidae, Ichthyophiidae,
and some Caeciilidae that are known are similar to and grossly resemble adults
in general morphology. The paucity of ontogenetic data, however, makes fur-
ther comparisons impossible.

3.3 Larval and larviform salamanders

3.3.1 Morphology and ontogeny
With the exceptions of pedomorphs in the families Amphiumidae (cylin-
drical, elongate body with four tiny limbs, each with one to three tiny digits)
and Sirenidae (cylindrical, elongate bodies with front limbs only, each with
three or four fingers), salamander larvae have a typical quadruped morphology
(Figure 3.2a) similar to that of the adults once all four limbs develop. Costal
grooves divide the myotomic muscle bundles of the trunk into costal folds. Gills
of various shapes are often prominent (Figures 3.2a, b, and d), one to four gill
slits open during larval life eventually close in metamorphic taxa, gill rakers are
prominent to absent, and a gular fold that typically is free from adjacent throat
tissue is usually present. Fleshy labial folds modify mouth size and shape during
suction feeding, and neuromasts are present, although sometimes difficult to
see without special techniques (Lannoo 1985). Major metamorphic modifica-
tions include the loss of tail fins, gills, gill slits, gular folds, and neuromasts,
the development of eyelids, and many other integumentary, osteological, and
physiological changes.
Pond-inhabiting larvae (e.g. most species of Ambystomatidae and Hynobiidae)
have robust bodies and heads, tall dorsal fins that can originate as far anterior
as the back of the head, and long gill rami with plumose fimbriae. Stream-
inhabiting larvae (e.g. certain species of Hynobiidae, Plethodontidae, and some
Salamandridae) are more streamlined; they have low fins that usually originate
near the tail/body junction, and shorter, less plumose gills. In species within the
Rhyacotritonidae and desmognathine Plethodontidae, the gill rami are excep-
tionally short with few fimbriae. Gill rami are branched in species in the fam-
ilies Amphiumidae (gills lost soon after hatching) and Sirenidae (gills persist
 3 Morphology of amphibian larvae | 43

(a) Gm Df Vf

TL

Gf Cf Cg
V

SVL (d)
Gm
(b) (c)
Lb N
Lf
Gf
Gm

B Gr
Ll
B Is

Fig. 3.2 Measurements and body parts of a larval salamander. (a) Lateral (upper)
and ventral (lower) views of a typical Ambystoma salamander larvae, drawing by
D. Karges; (b) dorsal view of the head and anterior body region of a hatchling
Ambystoma maculatum, photograph by A.M. Richmond; (c) ventral view of head
of A. maculatum; and (d) stylized drawing of gill structure of a larval salamander,
modified from drawing in Pfingsten and Downs (1989). B, balancer; Cf, costal fold;
Cg, costal groove; Df, dorsal fin; Gf, gular fold; Gm, gill ramus with fimbriae attached
to posterior surface; Gr, gill rakers; Is, interbranchial septum; Lb, limb bud; Lf, labial
fold; Ll, lateral line organs (neuromasts); N, naris; SVL, snout–vent length; TL, total
length; V, vent; Vf, ventral fin.

throughout life although they atrophy during aestivation); rami in larvae of

all other families are non-branched. Larvae of sirenid salamanders have low,
fleshy, pigmented fins restricted to the tail, as in adults, and hatchlings have a
transparent dorsal fin that originates well forward on the body. Amphiumid
larvae have a very short, low caudal fin that is lost soon after hatching. All other
salamander larvae have tail fins throughout their ontogeny. Regression of the
44 | Amphibian ecology and conservation

dorsal fin usually starts long before other metamorphic changes are noticeable.
Fleshy flaps occur on the trailing edges of the hind legs of Onychodactylus lar-
vae. Keratinized toe tips are found in a number of taxa but are most common in
stream inhabitants. Sirenids have keratinized jaw sheaths (upper and lower) as
do some ambystomatid larvae (lower).
Hatching occurs before the limbs are fully developed, and the front limbs
usually develop faster than the hind ones. Some hatchlings (e.g. in the families
Ambystomatidae and Salamandridae) have a balancer, a fleshy projection on
each side of the lower part of the head (Figures 3.2b and c), that is lost soon after
hatching. Staging tables (compilation in Duellman and Trueb 1986, p. 128)
are available for several species (e.g. Ambystoma maculatum, Harrison 1969;
Ambystoma mexicanum, Cano-Martinez et al. 1994; and Hynobius nigrescens,
Iwasawa and Yamashita 1991).

3.3.2 Coloration
In contrast to Stereochilus marginatus (Plethodontidae), Rhyacotriton spp.
(Rhyacotritonidae), and most pedomorphs, most of which retain something
similar to the larval coloration as adults, larval salamanders often have a color-
ation distinct from that of the metamorph or the adult. Larval sirenids are jet
black with contrasting stripes and bands of red or yellow, while adults have either
a gray or black ground color usually overlain by speckles and small blotches of
gold to greenish iridophores. Color and pattern (i.e. coloration) in larvae of most
species can vary considerably during ontogeny, throughout a day, and among
sites in response to substrate color, temperature, and water clarity. Colors are
typically muted grays, browns, and blacks, and patterns range from none (uni-
colored), blotched, and mottled through striped (longitudinal or diagonal con-
trasting markings) and banded (transverse contrasting markings). The dorsum
of the tail muscle of small Ambystoma is often banded, and the pattern may
be retained throughout ontogeny (e.g. Ambystoma talpoideum) or change to a
totally different pattern sometime after hatching and then again after metamor-
phosis. In some species and populations, larval Desmognathus (Plethodontidae)
have a distinct pattern that is kept throughout life. Although colors are usually
more muted, the adult patterns in other salamanders (e.g. Ambystoma tigrinum
group, many plethodontids) may appear at metamorphosis or a different pattern
may appear (e.g. most Ambystoma) after metamorphosis and slowly develop into
the adult pattern, which is achieved long before sexual maturity.

3.3.3 Diversity
Larval salamanders show much less ecomorphological diversity than tadpoles.
By definition, pedotypes and pedomorphs retain a larval morphology even
 3 Morphology of amphibian larvae | 45

though they become reproductive, and species that metamorphose and are
adapted for either pond or flowing water are the most easily recognized groups.
Cannibal morphotypes with enlarged heads and altered dentition occur in some
species (Ambystomatidae, Hynobiidae). Pond inhabitants often do not over-
winter, whereas some stream inhabitants may grow as larvae for several years
before undergoing metamorphosis. In some parts of their range Notophthalmus
viridescens (Salamandridae) larvae metamorphose into a brilliantly colored eft
that lives on the forest floor for several years before returning to the ponds for an
aquatic existence as a reproductive adult.

3.4 Anuran tadpoles

3.4.1 Morphology and ontogeny
The transition between the body and tail across all stages and taxa of tadpoles is
difficult to define. The demarcation between the two is most consistently and
accurately described as the juncture of the axis of the tail myotomes with the
posterior body surface (Figure 3.3a, bottom). The tails of most tadpoles lack
vertebrae and are composed of dorsal and ventral fins and a long series of pro-
gressively smaller myotomic muscle bundles surrounding the notochord. The
tadpoles of some species of Megophryidae do have tail vertebrae (e.g. Haas et al.
2006; Handrigan et al. 2007). The shapes and extents of the fins vary among
taxa and habitats (i.e. tallest in pond dwellers, lowest in fast-water and semi-
terrestrial forms). The eyes are either dorsal (i.e. lie totally within the dorsal sil-
houette; Figure 3.3b, left) or lateral (i.e. included as part of the dorsal silhouette;
Figure 3.3b, right). All free-living tadpoles have a spiracle(s) through which water
that has been pumped in through the mouth by the buccopharyngeal muscula-
ture and passed over the gills and food filtering system passes out of the body. In
the vast majority of species, the spiracle is single and situated somewhere on the
left side of the body (Figure 3.3a). Tadpoles of Ascaphus (Leiopelmatidae) have
a single, ventral spiracle on the chest, while those of Bombina (Bombinatoridae)
and Alytes and Discoglossus (Alytidae) have a single spiracle located almost
midventrally on the abdomen. In microhylid larvae, the midventral spiracle is
located at the posterior part of the abdomen or near the vent. The tadpoles of
pipids, rhinophrynids and the leptodactylid genus Lepidobatrachus have dual
lateral spiracles. The spiracles of Lepidobatrachus develop differently from the
other two taxa (Ruibal and Thomas 1988).
Scent-laden water passes through the nares, over the olfactory epithelium of
the nasal sacs, and into the buccal cavity via the internal nares. The shape of the
external apertures varies from round to elliptical and may have a variety of papillae
46 | Amphibian ecology and conservation

(a)

IND
TMW

IOD
BL TaL

TMH MTH

TL
OD SP LB VT TMA

(b) (c)

Fig. 3.3 Measurements and body parts of a tadpole. (a) Dorsal (upper) and lateral
(lower) views of a typical tadpole, drawing of Rana sp. by D. Karges; (b) dorsal (left)
and lateral (right) eye positions of tadpoles, stylized drawings by D. Karges; and
(c) examples of medial (left, Bufo boreas) and dextral (right, Rana catesbeiana) vent
tubes; white arrow, plane of ventral fin; black arrow, outflow of vent tube. BL, body
length; IND, internarial distance; IOD, interorbital distance; LB, hind limb bud; MTH,
maximum tail height; OD, oral disc; SP, spiracle; TMA, tail muscle axis; TMH, tail muscle
height; TMW, tail muscle width; TL, total length; TaL, tail length; VT, vent tube.

associated with the margins. A vent tube extends posteriorly from the midventral
abdomen. Two major types are recognized: dextral, where the aperture lies to the
right of the sagittal plane of the tail fin (e.g. hylids and ranids), and medial, where
the aperture lines parallel with the plane of the tail fin (e.g. bufonids and scaphi-
opodids; Figure 3.3c). As with the spiracular tube configurations, there are many
subtle variations in the shape and position of the vent tube.
The lateral line system (i.e. neuromasts; Hall et al. 2002; Lannoo 1985) is
composed of many depressions in the skin with sensory cells in the center that
 3 Morphology of amphibian larvae | 47

signal the patterns of water flow over various parts of the body and tail. The
distribution and arrangement of neuromasts may be useful in distinguish-
ing between closely related species. In darkly pigmented tadpoles these sites
are often pale and obvious, but evaluation of stitch patterns in most tadpoles
requires separating the epidermis from the underlying dermis, clearing in gly-
cerin, and viewing the skin with dark-field illumination (see Lannoo 1985).
The oral apparatus, the composite of upper and lower labia and all kerati-
nized mouthparts, is highly variable across taxa and ecological types. The most
common oral apparatus (Figure 3.4a and c) occurs in many taxa in lentic and
lotic sites. An assembly of the two infralabial and two Meckel’s cartilages with
three joints forms the lower jaw that is surmounted by a serrated, keratinized
jaw sheath. The supralabial cartilage of the upper jaw is surmounted by a simi-
lar keratinized jaw sheath, and during a bite, the lower jaw passes totally behind
the upper (Figure 3.4d). The interactions of the serrated margins of the sheaths
serve as cutting/gouging surfaces when a tadpole feeds. The highly variable
shapes of the jaw sheaths suggest different feeding abilities.
The face of the oral disc has fleshy transverse tooth ridges (Figure 3.4a,
c, and d) surmounted by a row(s) of keratinized labial teeth. In most cases,
several replacement teeth are interdigitated below a presently erupted tooth
(Figure 3.4b, lower right), and they successively move into position as the
erupted tooth wears out. The tooth rows are numbered from the anterior edge
of the disc toward the mouth on the upper labium and from the mouth to
the posterior edge of the disc on the lower labium. A fractional designation
indicates the number of tooth rows on each labium; some rows have naturally
occurring medial gaps denoted parenthetically. For example, a Labial Tooth
Row Formula (LTRF) of 2(2)/3(1) indicates two upper rows with a gap in the
second one and three lower rows with a gap in the first one (Figures 3.4a and
c). Some tadpoles lack tooth rows (i.e. 0/0), and the maximum LTRF known
is 17/21 in a tadpole of an undescribed hylid frog from the Guayana Highlands
of southern Venezuela.
The papillate margins of the oral disc may be complete and encircle the disc
(e.g. tadpoles of Scaphiopodidae, Pelobatidae, and many stream-inhabiting tad-
poles of several families), have a medial dorsal gap (most common; Figure 3.4a),
or have both dorsal and ventral gaps (e.g. Bufonidae and those of some Hylidae,
Mantellidae, Ranidae, and Rhacophoridae; Figure 3.4c). Although the number
of rows of papillae on different parts of the disc margin may vary, the lengths of
the papillae are typically somewhat uniform; several species of Phrynobatrachus
(Ranidae) have exceptionally elongate papillae along the posterior margin of the
disc. Submarginal papillae occur on the face of the disc away from the margin
(a) (b)
UJS LJS
A-1 A-2
G
SRC
MZJ
MP IRC

A-1
UJS
LJS P-1
P-2
MZT P-3

TS
C
H
TR S-1
B
P-1 P-2 P-3 TR SP EM S-2

(c) (d)

1
2

Fig. 3.4 Components of the oral disc of a tadpole. (a) Oral disc of a typical tadpole,
schematic drawing by D. Karges; (b) sagittal sections of the oral apparatus of a common
benthic tadpole (upper) and a tooth ridge (lower left), schematic drawings modified
from ones in Heron-Royer and Van Bambeke (1889); two labial teeth of Hyla chrysoscelis
(lower right) removed from a tooth series and in natural position; (c) scanning electron
photomicrograph of the oral apparatus of a tadpole of Bufo fowleri, actual oral disc
width, about 2.3 mm, photograph by M. Penuel-Matthews; and (d) lateral view of
the mouthparts of H. chrysoscelis (1, upper jaw sheath; 2, lower jaw sheath; 3, lower
tooth rows; 4, upper tooth row). A-1, A-2, anterior tooth rows 1 and 2; P-1, P-2,
P-3, posterior tooth rows 1 to 3; S-1, S-2, sheaths of presently erupted (1) and first
replacement (2) teeth; B, body of first replacement tooth; C, cusps on first replacement
tooth; EM, lateral emargination in oral disc; G, dorsal gap in marginal papillae; H, head of
presently erupted tooth; IRC, infrarostral cartilage; LJS, lower jaw sheath; M, mouth; MP,
marginal papillae; MZJ, mitotic zone for production of jaw sheath; MZT, mitotic zone for
production of labial teeth; SP, submarginal papillae; SRC, suprarostral cartilage; TR, tooth
ridge; TS, tooth series; UJS, upper jaw sheath.
 3 Morphology of amphibian larvae | 49

and form various patterns (Figure 3.4a). The margin of the disc may be emar-
ginate (i.e. indented; Figure 3.4a) or not.
Hatchlings (Gosner 1960; stages 21–24) usually have external gills but lack
eyes and limb buds. The forelimb buds develop beneath the operculum after it
closes, and the hind-limb buds grow from the posteroventral intersection of the
body and tail muscle (Figure 3.3a, bottom). Staging tables have been made for
a number of taxa (Duellman and Trueb 1986, pp. 128–129), but using a com-
mon table allows for meaningful comparisons among taxa. Gosner (1960; gen-
eral) and Nieuwkoop and Faber (1956; Xenopus) are the two most commonly
cited. Recent summaries of tadpole morphology and terminology are included
in Altig (2007b) and Altig and McDiarmid (1999a, 1999b).

3.4.2 Coloration
Except for notations in descriptions, surprisingly little has been written
about tadpole coloration. As in other larval amphibians, three basic popula-
tions of pigment-containing cells interact to produce both color and pattern.
Melanophores contain melanins that produce browns and blacks, iridophores
contain reflective guanine crystals that produce whites and silvers, and xan-
thophores contain carotenoids that produce yellows and reds. The pigments
are retained inside the cells and can be dispersed in various patterns under the
influence of temperature and light. Altig and Channing (1993) summarized the
diversity of colorations in tadpoles, and Caldwell (1982) tested the functions of
coloration in tadpoles experimentally.

3.4.3 Diversity
Most morphological characters of tadpoles reflect their ecology. Suctorial tad-
poles in a number of families have streamlined bodies and mouthparts modi-
fied to maintain position in fast-flowing water as they feed. A typical increase
in the number of tooth rows, to a maximum of 17/21, is usually accompanied
by a larger oral disc with complete marginal papillae. Other unusual morpho-
logical structures found in stream-inhabiting tadpoles include a belly modified
as a sucker (a few bufonid and ranid species), and lateral sacs (or lymphatic
sacs) on the ventrolateral parts of the body of other stream-dwelling tadpoles
(Arthroleptidae). Tadpoles of Mertensophryne (Bufonidae) have a hollow crown
on the head that encircles the eyes and nares, and tadpoles of Schismaderma
carens (Bufonidae) have a semicircular, transverse flap of skin behind the eyes.
Suspension-feeding tadpoles in the families Microhylidae, Pipidae, and
Rhinophrynidae have reduced, soft mouthparts that lack keratinized struc-
tures. They usually hang in midwater and capture suspended particles as water
is pumped in through the mouth and out the spiracles. Even so, not all tadpoles
50 | Amphibian ecology and conservation

that lack keratinized mouthparts are suspension feeders, and tadpoles with
keratinized mouthparts that are infected with the amphibian chytrid fungus
(Batrachochytrium dendrobatidis) often lose most or all such structures.
Carnivores and other macrophagous feeders have a diversity of mouth-
parts related to how they feed. For example, tadpoles of the leptodactylid frog
Lepidobatrachus have huge mouths but almost no soft or keratinized mouth-
parts; they engulf entire organisms, including other tadpoles. Tadpoles of
other leptodactylid frogs, Ceratophrys spp., have huge jaws and many tooth
rows and efficiently tear their victims to pieces. Carnivorous tadpoles of Spea
(Scaphiopodidae) feed similarly. A number of other tadpoles (e.g. Hylidae: Hyla
leucophyllata group; Ranidae: Occidozyga) lack all or most tooth rows but have
huge jaw sheaths. Tadpoles that occupy tree holes and bromeliad tanks (e.g.
some species of Dendrobatidae, Hylidae, and Rhacophoridae) are of several
morphological types (Lannoo et al. 1987; Lehtinen et al. 2004) and have differ-
ent diets; some are non-feeding, several are detritovores or macrophagous car-
nivores, many eat frog eggs (fertilized or not) of their own (cannibals) or other
species, and some eat only trophic eggs supplied by their mother. Surface-film
feeders have large oral discs, but keratinized structures are reduced or absent;
the disc is turned upward (i.e. umbelliform) and captures material carried in
the surface film. The oral discs of these surface-feeding tadpoles may be formed
primarily from the lower labium (e.g. Microhylidae) or from parts of both labia
(e.g. Megophryidae). This convergent morphology occurs in six families, and
most tadpoles of this sort occur only in the slow reaches of streams.
Attempts have been made to define ecomorphological guilds or groups of taxa
with suites of common morphological characters that are presumed to reflect
a common ecology (e.g. Altig and Johnston 1989). Because of the lack of eco-
logical data for many species and an incomplete understanding of how some of
their morphologies actually function, we advise caution in assigning species to
specific guilds without some knowledge of their natural history. For example,
the morphologies of Mantidactylus lugubris (Altig and McDiarmid 2006) and
of some taxa that occur in phytotelms suggest that one might find them in fast-
flowing water. In fact, tadpoles of M. lugubris live in leaf packs in slow-flowing
water.

3.5 Summary
Amphibian larvae show considerable morphological diversity from the relatively
conserved forms of caecilians and salamanders to the unusual and often novel struc-
tures found in tadpoles of frogs and toads. The extreme variability of tadpoles is
 3 Morphology of amphibian larvae | 51

almost certainly a product of ontogenetic challenges, the recently discovered influ-

ences of predators and competitors (e.g. Relyea and Auld 2005), and the selective
effects of different habitats. The morphological variability manifest under these
different conditions makes identifications and ecological evaluations especially dif-
ficult. This situation, combined with our lack of understanding of geographic vari-
ation in larval amphibians, especially tadpoles, emphasizes the relative poor state
of our knowledge of larval biology. Much remains to be learned. The discovery of
various anomalies (e.g. Drake et al. 2007), often with a weak understanding of their
causes (Altig 2007a; see also Lannoo 2008), adds yet another impediment to our
total understanding of larval morphology.

3.6 References
Altig, R. (1972). Notes on the larvae and premetamorphic tadpoles of four Hyla and
three Rana with notes on tadpole color patterns. Journal of the Elisha Mitchell Scientific
Society, 88, 113–19.
Altig, R. (2007a). Comments on the descriptions and evaluations of tadpole mouthpart
anomalies. Herpetological Conservation and Biology, 2, 1–4.
Altig, R. (2007b). A primer for the morphology of anuran tadpoles. Herpetological
Conservation and Biology, 2, 73–6.
Altig, R. and Channing, A. (1993). Hypothesis: functional significance of colour and
pattern of anuran tadpoles. Herpetological Journal, 3, 73–5.
Altig, R. and Johnston, G. F. (1989). Guilds of anuran larvae: relationships among devel-
opmental modes, morphologies, and habitats. Herpetological Monographs, 3, 81–109.
Altig, R. and McDiarmid, R. W. (1999a). Body plan: development and morphology. In
R. W. McDiarmid and R. Altig (eds), Tadpoles, the Biology of Anuran Larvae, pp. 24–51.
University of Chicago Press, Chicago, IL.
Altig, R. and McDiarmid, R. W. (1999b). Diversity: familial and generic characteriza-
tions. In R. W. McDiarmid and R. Altig (eds), Tadpoles, the Biology of Anuran Larvae,
pp. 295–337. University of Chicago Press, Chicago, IL.
Altig, R. and McDiarmid, R. W. (2006). Descriptions and biological notes on three
unusual mantellid tadpoles (Amphibia: Anura: Mantellidae) from southeastern
Madagascar. Proceedings of the Biological Society of Washington, 119, 418–25.
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 4
Larval sampling
David K. Skelly and Jonathan L. Richardson

4.1 Introduction
Most amphibian species are metamorphic, and among those, the majority have
larvae that are fully aquatic (Duellman and Trueb 1986). These larvae are found
in an enormous variety of contexts ranging from bromeliads and tree holes, to
brackish pools and the largest rivers and lakes (Duellman and Trueb 1986). Add
to this mix of environments the fact that amphibian larvae differ dramatically in
their microhabitat use, from the water surface to benthic mud, and researchers
sampling larval amphibians are confronted with a non-trivial challenge of match-
ing techniques with study goals and logistic limits. Fortunately, there is a wealth
of experience that can be tapped when making decisions about where, when, and
how to sample (Shaffer et al. 1994; Olson et al. 1997).

4.1.1 Why sample larvae?

The reasons to sample amphibian larvae are many, but most emphasize either
strategic or logistic considerations. From the strategic perspective, larvae represent
a critical life history stage. Many of the reasons that motivate scientists to study
amphibians are related to the dynamics and fate of their populations. Monitoring
of larval cohorts can provide critical information regarding the trajectory of a
population and the factors that may affect abundance and distribution. Larvae
also represent concrete evidence of breeding. While many studies of anurans use
male calling as an index of breeding distribution, males can call from wetlands
where no breeding takes place. Larval surveys are less likely to overestimate breed-
ing distribution.
For many species larvae are also convenient. Many adult amphibians are found
at low density spread across large areas of terrestrial habitat. In addition they are
often cryptic, arboreal, or fossorial, making it extremely difficult to sample their
56 | Amphibian ecology and conservation

populations systematically. For amphibians with fully metamorphic life histories

and aquatic larvae, breeding represents the point in the life cycle where a spe-
cies can be indexed in a relatively small, defined area. The techniques described
below offer a variety of systematic approaches to estimating population attributes
that would be extremely challenging to determine for the adult stages of many
amphibian species.
Because of their strategic and logistic advantages, amphibian larvae have been
used in a wide range of research aimed at addressing fundamental questions in
fields ranging from ecology and evolution to physiology and developmental biol-
ogy. Well before amphibian larvae were widely studied by scientists concerned
about declines and species extinctions, larval amphibians were held up as a model
system by biologists.

4.1.2 Target responses

Once the motivation for sampling larvae is known, the target response vari-
ables may be selected (Table 4.1). Information on species occupancy is frequently
sought in both basic and applied settings. Presence and absence data can be
important in determining species richness within a wetland as well as providing
evidence for changes in species range. Any of the techniques described in this
chapter can be used to estimate species presence and absence. However, if presence/
absence is the sole motivation for sampling, some techniques such as dip-netting
(described below) may be much more efficient than others.

Table 4.1 Larval amphibian sampling methods and resulting response variables
Response Metrics Sampling methods Inferences

Species Presence/absence All methods Species distribution,

occupancy richness
Relative Catch per unit Time- or Comparing
density effort (CPUE) area-constrained among populations
sampling, trapping, and species
litterbags
Density Individuals per Area- or Comparing among
unit area or volume-constrained populations and
volume sampling including species cohort
box and pipe survival
sampling
Population Total number of Extrapolation from area- Population dynamics
size individuals in or volume-constrained extinction risk
population samples, mark–recapture
 4 Larval sampling | 57

Beyond estimating presence and absence, sampling can be directed at estimat-

ing density. Any technique which can be scaled by effort (sometimes expressed as
catch per unit effort, or CPUE) can offer estimates of relative density; that is, the
density of a given species relative to another. As one example, the number of larvae
from one species recovered during 30 person minutes of dip-netting may be com-
pared with a second species as an index of their relative density (both expressed
as individuals recovered per person minute). A smaller number of techniques,
identified below, allow direct estimates of the number of individuals per area, or
per volume. In general, these techniques operate on the principle that a defined
area or volume is sampled effectively. As one example, each use of a standard box
sampler covers 0.5 m2. After completing a set of 10 box samples within a wet-
land, the number of larvae of some species recovered from 5 m2 can be expressed
as the number of individuals per square meter. Finally, using mark–recapture
techniques, estimates of the total number of individuals within a larval cohort
may be made.

4.1.3 Timing
Amphibian larvae range from practically immobile, yolk-laden hatchlings, to
30-cm-long salamander larvae capable of moving extremely rapidly. Deciding
when to sample during the larval period is best done in conjunction with deci-
sions about how to sample. Small larvae move slowly and are often found in shal-
low areas. These attributes can make sampling relatively straightforward using
a variety of techniques. This is something to consider if your study aims allow
flexibility in the developmental stage sampled. It also suggests that effective lar-
val sampling depends on a close knowledge of the life histories of the species to
be sampled and their developmental progress within a given year. Late snowpack
melt, droughts, and a particularly cold or warm spring can all shift the timing of
larval development substantially. In tropical climates the time of year can be far
less important than the onset of wet season rains in triggering breeding and the
timing of larval sampling.

4.1.4 Sampling effort

How much is enough? In any sampling context, researchers are confronted with
the task of deciding how to allocate effort. As expected, a satisfactory answer
depends on study goal and tolerance of uncertainty. Any of the techniques
described below can be scaled in effort. The effort adopted, however, will require
direct estimates of how species detection, larval density estimates, or whatever
the target variable is, changes with increased effort. In our own research, we have
intentionally varied per wetland sampling effort (samples per visit, number of
58 | Amphibian ecology and conservation

visits, timing of visits) to determine the sensitivity of target responses (e.g. Werner
et al. 2007). There is no shortcut here; rules of thumb can be misleading resulting
in wasted effort and more collection than necessary or, more likely, incomplete
and inaccurate information (Skelly et al. 2003).
If you are new to a system, plan to learn during your initial sampling.
Intentionally trying multiple techniques and different degrees of sampling effort
will provide information that will ultimately save you time and produce more
reliable information. Before you step in the water, be prepared to spend more time
in each habitat if it is needed and to consider using multiple techniques to capture
the range of species and larval stages you intend to study.

4.2 Sampling techniques

4.2.1 Box/pipe sampler
4.2.1.1 Description
These area-based samplers are dropped rapidly through the water column in
areas of less than 1 m in depth. The earliest versions were sheet metal or ply-
wood boxes, 0.5  1.0 m, used in conjunction with a metal frame net designed
to fit snugly within the width of the box (Harris et al. 1988). Repeated sweeps
of the trapped volume of water are used to clear and count the amphibian lar-
vae within. A second form, the pipe sampler (Skelly 1996), ranges in size but is
typically constructed of an approximately 1 m length of aluminum pipe, 30 cm
or so diameter. Polyvinyl-coated aluminum pipe manufactured for air handling
in commercial heating and cooling applications is particularly effective. Pipe
samplers are used with dip nets constructed to measure half of the diameter of
the pipe. Entrapped larvae are cleared using repeated circular sweeps of the water
volume (Figure 4.1a).
Researchers have developed algorithms to ensure complete, or at least consist-
ent, clearance of animals. As one example, Werner et al. (2007) report sweeping
the water column of a pipe sampler a minimum of 10 times, and for at least 10 null
sweeps after the last collected animal has been removed from the net. For both
types of sampler, multiple samples are collected in a single wetland. Researchers
can use grids to lay out sampling points which can be distributed among habitat
types within a wetland. Alternatively, a minimum distance between sampling
points can be specified and placement of individual samples set haphazardly
within that constraint. If desired, data can be kept individually for each box or
pipe providing information on spatial distribution, variation in local density, pat-
terns of species coincidence, or association with particular microenvironments
(Freidenburg 2003).
 (a)

(b)

(c)

(d)

Fig 4.1 Representative sampling techniques targeting larval amphibians in aquatic

habitats: (a) Pipe sampling in which a pipe is used to trap larvae that are then cleared
using a dip net (section 4.2.1); (b) leaf litterbags are used for sampling salamander
larvae in stream habitats (from Waldron et al. 2003; section 4.2.4); (c) a collapsible
funnel trap deployed along a pond edge (section 4.2.5); (d) a larval Ambystoma
talpoideum salamander marked with a lateral orange visible implant elastomer (VIE) tag
(photograph courtesy of Kristen Landolt of Murray State University; section 4.2.6).
60 | Amphibian ecology and conservation

4.2.1.2 Application
Box samplers are most effective within vernal ponds with an open water column and
simple bottom substrates such as decaying leaves. Pipe samplers were later developed
to capture the advantages of a box sampler while enabling the sampling of a wider
variety of environments including those where water is interspersed with emergent
vegetation (Skelly 1996). We are unaware of the use of area-based larval amphibian
samplers within stream environments, although the use of comparable samplers for
benthic macroinvertebrates suggests the potential for such an application.

4.2.1.3 Considerations
The major advantage of box and pipe samplers is that sampling of a known area
of wetland bottom provides direct estimates of larval density (individuals/m2). If
depth within each pipe is recorded, estimates of density per unit volume can be
determined. In either case, if wetland bottom area (or volume) is known, research-
ers have extrapolated pipe- and box-sample-based density estimates into estimates
of the size of entire larval cohorts (e.g. Werner et al. 2007).
A major disadvantage of these area-based samplers is their time-intensiveness.
Repeatedly placing and clearing a box or pipe is relatively slow and methodical
work even for experienced users. In addition, when sampling takes place in remote
areas that require hiking into and out of, box and pipe samplers can be inconveni-
ent because of their size.

4.2.2 Dip net

4.2.2.1 Description
Dip nets are probably the most common sampling tools used to collect amphibian
larvae. Dip-netting can be relatively unstructured if the goal is simply to capture
representatives of a particular species. Alternatively, dip-net surveys in which the
elapsed time (Werner et al. 2007) or number of sweeps (Gunzburger 2007) is
counted can be used to provide estimates of relative abundance of species. With
calibration from other methods such as pipe sampling, time-constrained dip-net
surveys have been used to provide per-unit-area density estimates (Werner et al.
2007). Effective dip-netting depends strongly on the species targeted and the
environment type sampled. However, most neophytes tend to collect a great deal
of substrate along with each sweep. With experience, users can learn how far into
the water column and substrate the net needs to pass to capture the larvae without
burying them in an overabundance of mud and vegetation.
Dip nets used in larval amphibian sampling range in size and shape from small
aquarium nets used in confined volumes of water (Thoms et al. 1997) to very large
 4 Larval sampling | 61

nets approaching the size of some small seines (S. Cortwright, personal communi-
cation). Regardless of net size, it is common for amphibian biologists to construct
their own nets or to customize store-bought nets. Dip nets used for larval amphib-
ian sampling can have much shallower net bags and tend to require finer mesh
than those used for fish and other larger organisms. A shallow net bag facilitates
rapid processing of each sweep and can speed sampling significantly. Researchers
can also construct net dimensions that fit the structure of the environments they
sample (e.g. narrower nets may be appropriate for dipping out of marshes with
emergent vegetation, small stream pools, or tire ruts).

4.2.2.2 Application
Dip nets are used in most of the places where amphibian larvae are found. Nets
can be of lighter construction in vernal ponds (e.g. using mosquito mesh for the
net bag) compared with those used in streams and other places with abrasive
substrates.

4.2.2.3 Considerations
Dip-netting is fast, requires a minimum of equipment, and can be performed in
a wide range of environments. Nets can be constructed inexpensively to perform
well in particular contexts. On the downside, as much as any technique, dip-
netting effectively relies on experience. An experienced user can vastly outper-
form a beginner working side by side. A second disadvantage relates to population
density estimates. While dip-netting can be used by itself to estimate relative
density in terms of catch per unit effort (where effort is often measured as time
spent sampling), estimates of area- or volume-based density must rely on other
techniques or through calibration from samples collected using other methods in
the same or comparable environments (Werner et al. 2007).

4.2.3 Seine
4.2.3.1 Description
Seines have long been used to collect amphibian larvae (Routman 1984). They are
particularly effective in sampling open, deeper areas that cannot easily be sampled
using box and pipe samplers or dip nets. Seining typically requires at least two
people. One person at each end sets the seine in a line and then begins moving in
an agreed direction until a position is reached where they move together and begin
gathering the ends of the seine up and out of the water, forcing the entrapped lar-
vae down into the middle. This is done most easily if the seine is being gathered
onto the shore. Sometimes this is not possible in which case, some sort of floating
platform such as a foam bucket float can be used. When the captured sample is
62 | Amphibian ecology and conservation

concentrated in the center of the seine, it is picked up and moved onto the shore
where the contents are sorted and the larvae are processed as desired. Most seines
used for amphibian larvae are relatively small, on the order of 3–5 m long and 1 m
or so deep (Shaffer et al. 1994). Larger seines often have a bag sewn in the middle.
The bag can greatly increase sampling effectiveness, but may also require a third
person to keep it from getting caught or rolled up in vegetation.

4.2.3.2 Application
Seines are typically used in open-water areas of ponds and lakes, although large
stream pools may also be sampled using seines in conditions where flow is not too
great.

4.2.3.3 Considerations
Often seines are the only means of sampling larger amphibian larvae that live
in open-water regions of ponds (e.g. large Ambystomatid salamander larvae). A
major disadvantage of seines is that they are hard to handle in vegetation-choked
ponds and lakes often frequented by larval amphibians. In such an environment,
it can take over half an hour to sort through the vegetation and muck gathered
in a 5-min seine haul. During this sorting process, smaller amphibian larvae can
be hard to detect (and seine mesh is often coarse enough to enable their escape)
meaning that seines are most often used for large larvae. As with dip-netting,
seines are typically used to estimate catch per unit effort, although it is possible to
estimate number captured per unit area in some conditions (Shaffer et al. 1994).

4.2.4 Leaf litterbags

4.2.4.1 Description
Leaf litterbag sampling is a relatively new method for sampling stream habitats for
amphibians, particularly salamander larvae and adults (Pauley and Little 1998).
Litterbags create an artificial habitat refugium reproducing leaf packs commonly
found within streams, yet enclosed within mesh bags that can be easily removed
and sampled for salamanders. Leaf litterbags are constructed using plastic mesh
netting. Mesh gauge varies, but the 1.9 cm mesh (commonly found in deer-exclusion
netting and similar products) has proven useful. The mesh is cut into squares, with
50  50, 70  70, and 90  90 cm mesh sections representing small, medium,
and large bags, respectively. Several rocks are then placed on the netting to anchor
the bag, followed by leaves, needles, and other material likely to be found within
a particular stream environment. Once filled with litter, the corners of the mesh
are pulled together and cinched at the top using a cable tie (Figure 4.1b). Colored
flagging may be added to facilitate retrieval; this can be critical in contexts where
 4 Larval sampling | 63

the bag may be displaced downstream by a high-flow event. Litterbags tend to

work best when deployed in locations where debris packs are likely to naturally
accumulate (e.g. pools, channel bends). In higher-flow environments, bags can be
secured by partially covering them with larger rocks or by tethering bags to roots
or pinning them using a stake driven into the substrate (Waldron et al. 2003;
Talley and Crisman 2007). Bags are sampled by placing a dip net underneath the
bag in the water column, quickly removing the bag from the water, and placing
it over a light-colored dishpan (Pauley and Little 1998; Jung et al. 2000). While
gently shaking the bag, salamander larvae and adults will often fall into the dish-
pan where species identification and enumeration can take place. The litter pile
should also be examined to ensure that all captured individuals are included. The
litterbag can then be reassembled and deployed again, although the leaf litter may
need to be replaced to compensate for decomposition over time.

4.2.4.2 Application
Litterbags have been used in stream environments where debris collects, and this
technique targets species known to utilize leaf packs. They are easy to deploy,
and quick and inexpensive to construct. They also manage to exploit an attribute
of species that makes them otherwise hard to sample. In the absence of litterbag
sampling, larvae of many stream-dwelling species are difficult to collect. They are
also able to capture more secretive or uncommon species that might be missed in a
dipnet survey. While litterbag sampling can produce species presence and relative
density data for a stream reach, it does not likely provide accurate estimates of abso-
lute population size or density, since it is unclear what exact area of the stream is
being sampled with each bag (Chalmers and Droege 2002; Waldron et al. 2003).

4.2.4.3 Considerations
Litterbags are a highly specialized sampling tool. They are useful only for species
that dwell in streams and use leaf packs. But if such a species is being targeted, the
advantages of litterbags are substantial. Litterbag size is an important consider-
ation, as medium and large bags can capture more individuals and species; how-
ever, the size of the focal stream may only accommodate a smaller bag (Waldron
et al. 2003; Talley and Crisman 2007). Additionally, samples can be biased if
potential competitors or predatory individuals colonize the bag. Researchers
can discourage predatory (usually larger) adults and species by using finer mesh
or submerging bags in deeper water, leading to a preferential capture of larvae
(Waldron et al. 2003). Finally, the utility of litterbags may vary seasonally, as the
abundance of natural leaf pack habitats can vary throughout the year, depending
on the surrounding habitat cover.
64 | Amphibian ecology and conservation

4.2.5 Trapping
4.2.5.1 Description
Traps can be an effective means to capture amphibian larvae, requiring the
researcher to simply deploy the traps and check them after a time period sufficient
to have captured resident larvae. Most traps used by amphibian researchers are
of a funnel design, which channels larvae into a large holding section that can be
accessed by the researcher to recover captured animals. Commercially available
wire minnow traps have been used in many amphibian studies (e.g. Fronzuto and
Verrell 2000; Ghioca and Smith 2007). Home-made funnel traps using plastic
bottles (e.g. 2-L plastic drinks bottles) have also been used successfully (Calef
1973; Richter 1995). Collapsible traps made of fine nylon mesh and available
commercially (Promar, Gardena, CA, USA; Figure 4.1c), have capture rates equal
to or better than traditional wire minnow traps (Adams et al. 1997; C. Pearl,
personal communication). The finer mesh of these collapsible traps allows for the
retention of much smaller larvae, and the compact size and weight make them
suitable for backcountry work. Pyramid-shaped crayfish traps (Johnson and
Barichivich 2004) are an alternative to minnow and collapsible mesh traps that
can be particularly effective when it is important for part of the trap to extend
above the water surface. In all cases, traps are deployed by dropping each in a
predetermined area of the pond with a line attached and tied to a tree or float to
make locating and retrieving it easier.

4.2.5.2 Application
Traps can be effective in capturing amphibian larvae present within many aquatic
habitats with, perhaps, the exception of fast-moving water. Trapping is particu-
larly suited to detection of species presence, and to estimate catch per unit effort
(a metric of relative abundance). Trapping is sometimes the only suitable method
in deep water, steep-sided pond basins, or frozen ponds where wading in to con-
duct sampling is not feasible. Additionally, it may be easier to sample habitats
with structurally complex bottoms or vegetation-choked areas using traps, where
seining and dip-netting may be difficult. Lastly, funnel trapping will often cap-
ture rare, secretive, or more nocturnal species not detected using other methods
conducted in a short time period and during the daytime.

4.2.5.3 Considerations
Whereas traps can be left overnight, this practice requires a sampling location to
be revisited within a short time period (usually 12–24 h) to avoid trap mortality,
especially of non-target species or life stages. It is especially important, when traps
 4 Larval sampling | 65

are to be left for extended periods, to keep part of the trap above water to allow
access to water surface for trapped animals. Secondly, wire mesh size in commer-
cial minnow traps (around 6  6 mm) may be too large to effectively trap smaller
larvae, in which case sealing the trap with window screening composed of finer
mesh or the use of nylon collapsible traps can address this issue. Also, any trapping
methods and resulting data are based on an assumption of equal capture prob-
ability among individuals and populations. This can be violated if the presence
of conspecifics, competitors, or predators in the cage alters capture probabilities.
There could also be community-level biases if populations being sampled differ
in species composition. For instance, predators present in one habitat but not the
other may alter the behaviour of a target species and subsequent capture probabil-
ities. There can be other specific sampling biases in trapping certain species and
in some habitat types (e.g. playa wetlands; Ghioca and Smith 2007). Weather,
larval size and developmental stage, and resource availability can all affect capture
rates using traps (Adams et al. 1997). Some researchers use baits when trapping
amphibian larvae. However, for at least some species, it appears that baiting traps
does not increase trap effectiveness (Adams et al. 1997).

4.2.6 Mark–recapture
4.2.6.1 Description
Mark–recapture techniques are commonly used in studies of amphibian adult
populations, but can also be useful for the larval stage as well. However, rapid
growth often accompanied by a dramatic shift in body design can render marking
methods (often developed for juvenile and adults; see Chapter 8) unsuitable for
larval amphibians. Successful marking techniques for larvae include temporary
injectible organic dyes (Seale and Borass 1974) and externally staining dyes, which
typically stain amphibian larvae for less than 24 h (Pfennig 1999; Jung et al. 2002;
Harris et al. 2003), but can also slow growth rates (Travis 1981). More permanent,
yet onerous, marking techniques using paint sprays, stains, dimethyl sulfoxide,
and Super Glue (Ireland 1989) have largely been replaced by more convenient and
robust visible implant elastomer (VIE) tagging (Figure 4.1d). Passive integrated
transponder (PIT) tags may have limited use in all but the largest larval species due
to tag size (down to about 8 mm) and surgery required to implant, although the
development of smaller “injectable” PIT tags, applied using a hypodermic needle,
may expand the potential for this technique (Biomark, Boise, ID, USA).
VIE tagging appears to hold the most promise for amphibians, balancing the
ease of marking and longevity of the actual mark. Elastomer marks consist of a
silicone-based polymer material that is injected subcutaneously and cures into a
66 | Amphibian ecology and conservation

pliable and biologically inert solid (Northwest Marine Technology, Shaw Island,
WA, USA). Whereas some elastomer colors are visible to the naked eye when pre-
sent under translucent skin, fluorescent colors are often used and easily detected
using an ultraviolet light source (portable lights are available for field purposes).
Lowe (2003) used VIE to mark larvae of the spring salamander (Gyrinophilus
porphyriticus) and has indicated that marks can still be seen 10 years after mark-
ing (W. Lowe, personal communication). Fading of the elastomer does not
appear to be a common problem, although marks can migrate from the point
of injection or be lost altogether. Grant (2008) found that wood frog (Rana syl-
vatica) tadpoles can retain marks through metamorphosis and that larger marks
( 2 mm) were more likely to migrate in two larval stream salamander species.
Additionally, it was indicated that stream salamanders could be marked without
anesthesia, while wood frog tadpoles required anesthesia and also had poorer
mark retention (E. Grant, personal communication).

4.2.6.2 Application
Mark–recapture studies can be conducted in just about any habitat type, assuming
that the same method of capture is used for each sampling period. Assuming that
a sufficient proportion of the population is originally marked, mark–recapture
techniques can provide robust estimates of absolute population size, especially
when combined with robust capture–recapture estimation models (Chapter 24).
Jung et al. (2002) found that mark–recapture methods provided the most accurate
estimates of population size for two species of tadpoles in desert pool habitats.

4.2.6.3 Considerations
Regardless of technique, small amphibian larvae are relatively difficult to mark.
The VIE technique will be more useful for species with larger larvae, or at least
with individuals farther along in development. Gyrinophilus porphyriticus larvae
as small as 2 cm in total length have been marked successfully, as well as R. syl-
vatica individuals down to 2.5 cm in total length. Additionally, any substantial
loss of tags within a marked cohort can seriously bias population size estimates.
Consider this when deciding which technique to use and for what exact purpose.

4.3 Other techniques

A number of additional techniques have been used in sampling amphibian lar-
vae. Because they have limited application or because they have been used rela-
tively infrequently, we mention them only briefly here and point readers to sources
where more detailed descriptions are available.
 4 Larval sampling | 67

4.3.1 Bottom net

A bottom net is placed on the bottom in a set position such that, when triggered,
floats on an upper frame carry the net to the surface, entrapping larvae in open-
water areas of small ponds (Shaffer et al. 1994). This area-based sampling tech-
nique has been used relatively little for amphibians, but could be effective in water
where other techniques are not feasible.

4.3.2 Electroshocking
Electroshocking, developed initially to sample fish, is used primarily within
streams and rivers. However, it can stun and facilitate capture of amphibian lar-
vae as well (Brown and May 2007). It appears to be most effective for lentic species
found in slow-moving parts of rivers (Shaffer et al. 1994). Many stream-dwelling
amphibian species use retreats or bury themselves in the substrate in ways that
prevent their detection and capture even if stunned during electroshocking.

4.3.3 Visual encounter survey

Visual encounter surveys, discussed more thoroughly in Chapter 15, may also be
used, with some considerations, to sample larvae. Visual encounter surveys alone
will likely be insufficient to detect a significant proportion of larvae present in dark
or turbid water (such as a tannin-rich vernal pool, an algae-/vegetation-choked
pond, or a stream with turbulent waters). Larval behavior and microhabitat pref-
erence (e.g. aggregation, crypsis, water-column basking, hiding in the substrate,
preference for deep water or thick vegetation) can lead to species-specific and
highly variable rates of detection. However, where water conditions allow, and the
potential for confusing species is low, visual encounter surveys can be an efficient
technique.

4.4 Conclusions
As in many aspects of field biology, the techniques used for sampling amphib-
ian larvae are often passed without criticism or comment from one generation
of researchers to the next. In many cases, there has been little effort to ask why
one technique should be used as opposed to its alternatives or how a given
technique may be most effectively applied. The many techniques outlined in
this chapter are connected through the references listed below to an enormous
cumulative effort to understand the most effective and efficient means to esti-
mate the presence and density of larvae. Most of the techniques require little
equipment, and that equipment is typically relatively inexpensive. Neither are
the techniques difficult to master. Collectively, this means that there is little
68 | Amphibian ecology and conservation

reason not to try multiple techniques and to calibrate and understand the con-
sequences of altering the timing and intensity of sampling. The modest effort
to do so will greatly increase the reliability of the information gathered and, in
all probability, lead to unforeseen insights into the biology of the species being
studied.

4.5 Acknowledgments
We thank S. Cortwright, M. McPeek, E. Werner, and H. Wilbur for teaching us
about larval amphibian sampling. M. Adams, E. Grant, B. Hossack, W. Lowe,
and C. Pearl provided helpful insight and details into the techniques they use.

4.6 References
Adams, M.J., Richter, K.O., and Leonard, W.P. (1997). Surveying and monitoring
amphibians using aquatic funnel traps. In D.H. Olson, W.P. Leonard, and R. Bury
(eds), Sampling Amphibians in Lentic Habitats: Methods and Approaches for the Pacific
Northwest: Northwest Fauna Number 4, pp. 47–54. Society for Northwestern Vertebrate
Biology, Olympia, WA.
Brown, L.R. and May, J.T. (2007). Aquatic vertebrate assemblages of the Upper Clear
Creek Watershed, California. Western North American Naturalist, 67, 439–51.
Calef, G.W. (1973). Natural mortality of tadpoles in a population of Rana aurora. Ecology,
54, 741–58.
Chalmers, R.J. and Droege. S. (2002). Leaf litter bags as an index to populations of north-
ern two-lined salamanders (Eurycea bislineata). Wildlife Society Bulletin, 30, 71–4.
Duellman, W.E. and Trueb, L. (1986). Biology of Amphibians. John Hopkins University
Press, Baltimore, MD.
Freidenburg, L.K. (2003). Spatial Ecology of the Wood Frog (Rana sylvatica). PhD
Dissertation, University of Connecticut, Storrs, CT.
Fronzuto, J. and Verrell, P. (2000) Sampling aquatic salamanders: tests of the efficiency
of two funnel traps. Journal of Herpetology, 34, 146–7.
Ghioca, D.M. and Smith, L.M. (2007). Biases in trapping larval amphibians in playa
wetlands. Journal of Wildlife Management, 71, 991–5.
Grant, E.H.C. (2008). Visual implant elastomer mark retention through metamorphosis
in amphibian larvae. Journal of Wildlife Management, 72, 1247–52.
Gunzburger, M.S. (2007). Evaluation of seven aquatic sampling methods for amphibians
and other aquatic fauna. Applied Herpetology, 4, 47–63.
Harris, R.N, Alford, R.A., and Wilbur, H.M. (1988). Density and phenology of
Notophthalmus viridescens dorsalis in a natural pond. Herpetologica, 44, 234–42.
Harris, R.N., Vess, T.J., Hammond, J.I., and Lindermuth, C.J. (2003). Context-
dependent kin discrimination in larval four-toed salamanders Hemidactylium scutatum
(Caudata: Plethodontidae). Herpetologica, 59, 164–77.
 4 Larval sampling | 69

Ireland, P.H. (1989). Larval survivorship in two populations of Ambystoma maculatum.

Journal of Herpetology, 23, 209–15.
Johnson, S.A. and Barichivich, W.J. (2004). A simple technique for trapping Siren lac-
ertina, Amphiuma means, and other aquatic vertebrates. Journal of Freshwater Ecology,
19, 263–9.
Jung, R.E., Droege, S., Sauer, J.R., and Landy R.B. (2000). Evaluation of terrestrial
and streamside salamander monitoring techniques at Shenandoah National Park.
Environmental Monitoring and Assessment, 63, 65–79.
Jung, R.E., Dayton, G.H., Williamson, S.J., Sauer, J.R., and Droege, S. (2002). An
evaluation of population index and estimation techniques for tadpoles in desert pools.
Journal of Herpetology, 36, 465–72.
Lowe, W.H. (2003). Linking dispersal to local population dynamics: a case study using a
headwater salamander system. Ecology, 84, 2145–54.
Olson, D.H., Leonard, W.P., and Bury, R.B. (eds) (1997). Sampling Amphibians in Lentic
Habitats: Methods and Approaches for the Pacific Northwest: Northwest Fauna Number 4.
Society for Northwestern Vertebrate Biology, Olympia, WA.
Pauley, T.K. and Little, M. (1998). A new technique to monitor larval and juvenile sala-
manders in stream habitats. Banisteria,12, 32–6.
Pfennig, D.W. (1999). Cannibalistic tadpoles that pose the greatest threat to kin are most
likely to discriminate kin. Proceedings of the Royal Society of London Series B Biological
Sciences 266, 57–61.
Richter, K.O. (1995). A simple aquatic funnel trap and its application to wetland amphib-
ian monitoring. Herpetological Review, 26, 90–1.
Routman, E.J. (1984). A modified seining technique for single person sampling of deep or
cold water. Herpetological Review, 15, 72–3.
Seale, D. and Borass, M. (1974). A permanent mark for amphibian larvae. Herpetologica,
30, 160–2.
Shaffer, H.B., Alford, R.A., Woodward, B.D., Richards, S.J., Altig, R.G., and Gascon, C.
(1994). Quantitative sampling of amphibian larvae. In W.R. Heyer, M.A. Donnelly,
R.W. McDiarmid, L.C. Hayek, and M.S. Foster (eds), Measuring and Monitoring
Biological Diversity: Standard Methods for Amphibians, pp. 130–41. Smithsonian
Institution Press, Washington DC.
Skelly, D.K. (1996). Pond drying, predators, and the distribution of Pseudacris tadpoles.
Copeia, 1996, 599–605.
Skelly, D.K., Yurewicz, K.L., Werner, E.E., and Relyea, R.A. (2003). Estimating decline
and distributional change in amphibians. Conservation Biology, 17, 744–51.
Talley, B.L. and Crisman, T.L. (2007). Leaf litterbag sampling for larval plethodontid
salamander populations in Georgia. Environmental Monitoring and Assessment, 132,
509–15.
Thoms, C., Corkran, C.C., and Olson, D.H. (1997). Basic amphibian survey for inven-
tory and monitoring in lentic habitats. In D.H. Olson, W.P. Leonard, and R. Bury
(eds), Sampling Amphibians in Lentic Habitats: Methods and Approaches for the Pacific
Northwest: Northwest Fauna Number 4, pp. 35–46. Society for Northwestern Vertebrate
Biology, Olympia, WA.
70 | Amphibian ecology and conservation

Travis, J. (1981). The effect of staining on the growth of Hyla gratiosa tadpoles. Copeia,
1981, 193–6.
Waldron, J.L., Dodd, Jr., C.K., and Corser, J.D. (2003). Leaf litterbags: factors affecting
capture of stream-dwelling salamanders. Applied Herpetology, 1, 23–36.
Werner, E.E., Skelly, D.K., Relyea, R.A., and Yurewicz, K.L. (2007). Amphibian species
richness across environmental gradients. Oikos, 116, 1697–1721.
 5
Dietary assessments of larval amphibians
Matt R. Whiles and Ronald Altig

5.1 Background
Diet analyses of larval amphibians provide information on foraging patterns,
nutritional requirements, and trophic interactions in aquatic food webs, infor-
mation that is critical for successful conservation and management. Numerous
amphibian species around the globe are declining or presumed extinct (Stuart
et al. 2004). Thus, there is an urgent need for detailed information on larval
diets so that we can understand whether food-related factors are limiting wild
populations and facilitate successful rearing of species in captivity.
Some species of all three orders of living amphibians have free-living, feeding,
larval forms. Larval caecilians and salamanders, both of which occur in a variety
of lentic and lotic habitats, are all carnivores that eat small aquatic organisms
by suction-feeding, and their diets and trophic status are thus relatively easily
assessed. Anuran larvae (tadpoles) occur in almost every conceivable type of
freshwater habitat and show great diversity in feeding modes. As such, morpho-
logical diversity of the oral structures, which glean materials from substrata,
and the buccopharyngeal apparatus, which selectively captures food particles,
of tadpoles is large.
Whereas a few tadpoles are macrocarnivores (e.g. Lepidobatrachus,
Leptodactylidae), most either rasp materials associated with substrata such as
biofilms, periphyton, and deposited organic particles (e.g. ranids and many
hylids), harvest naturally suspended particles in the water column (e.g. micro-
hylids, pipids, and rhinophrynids), capture particles in the surface film (i.e.
neustonic tadpoles with umbelliform oral structures), or consume conspecific
or heterospecific eggs. Most of these feeding modes need further study (Altig
et al. 2007). Although many tadpoles are classified as general herbivores or
detritivores, they likely obtain a fair amount of energy and nutrients from more
72 | Amphibian ecology and conservation

easily assimilated heterotrophic components of their diets (e.g. animal parts

and microbes; Altig et al. 2007).
Herein, we focus on descriptive and observational methods for assessing diet
and trophic status of larval amphibians. However, other approaches that are
beyond the scope of this review can also provide valuable insight into the trophic
ecology of larval amphibians. In particular, manipulative approaches such as
cafeteria experiments can be used to examine amphibian food preferences (e.g.
Kupferberg 1997) and field enclosure/exclosure experiments can illuminate
functional roles and trophic interactions (e.g. Solomon et al. 2004). Combined
approaches, such as experimental manipulations performed in conjunction with
gut content analyses (e.g. Ranvestel et al. 2004; Whiles et al. 2006) have been
used to examine amphibian ecology at multiple scales, from individual foraging
to roles of amphibians in ecosystem processes. Manipulative approaches for
studying various aspects of amphibian ecology are discussed in Chapters 6 and
12 in this volume.

5.2 Larval caecilians and salamanders

With some modifications (e.g. different foraging strategies; benthic versus nek-
tonic and day versus night feeding), general habitats (e.g. lentic versus lotic),
and morphologies (e.g. gape size and size and number of gill rakers), the method
of food capture is essentially the same in all larval caecilians and salamanders.
These groups form a negative pressure by rapidly depressing the throat as they
open the mouth so as to suck food items into the buccal cavity. Plant materials
found in the guts of these groups are generally considered as incidental. As such,
gut content analyses can be effective for assessing diet in these groups, although
robust sampling strategies that account for spatiotemporal foraging patterns
and feeding behaviors should be used.

5.3 Anuran tadpoles

Most tadpoles appear to be omnivorous, although this contention is debated
and the true trophic status of many tadpoles has not been accurately assessed
(Altig et al. 2007). When tadpoles consume a range of food types that vary
in digestibility and nutritional quality, simply quantifying the constituents of
their diet may provide limited information on the relative importance of spe-
cific foods to growth and development. Accurate assessments of the trophic sta-
tus of omnivorous tadpoles require information on assimilation which can be
obtained through isotopic analyses, fatty acid analyses, or by applying ecological
 5 Dietary assessments of larvae | 73

efficiencies (e.g. assimilation and/or production efficiencies) to gut content data.

Consummatory diet information can be useful for studying feeding behaviors
and the types of materials that the oral apparatus and buccopharyngeal struc-
tures can harvest and capture.

5.4 Assessing food sources and diets

5.4.1 Category I: preparatory studies
Along with analyses of larval amphibians and/or their gut contents, many stud-
ies also include some assessment of food availability. In most freshwater habitats,
food resources are dynamic. For example, algal blooms can occur over short
time periods, and thus frequent sampling of potential food items is necessary.
Coupled with studies of foraging behavior, information on the seasonal phen-
ology of resources can guide the timing of sampling and aid in data interpret-
ation. On an even shorter diurnal time scale, samples of larvae and their prey
collected during daylight may not accurately reflect trophic interactions of sala-
manders active in the dark.
Sampling protocols, including number of samples collected at a given time,
apportioning samples among available micro- or mesohabitats (e.g. pools versus
riffles in a stream, macrophyte beds versus non-vegetated areas of a pond), will
vary with the specific system examined and associated questions. Preliminary
sampling can be used to obtain estimates of variability for a priori power ana-
lyses to assist with decisions regarding sampling intensities (e.g. Steidl et al.
1997).
Diets of many larvae change through development, and thus size and stage
should be accounted for when sampling. Larval size is important, but does not
always reflect age. For this reason, a staging table based on the sequence of acqui-
sition of discrete morphological markers should be used. Dünker and Wake
(2000) provide a staging table for caecilians, and those for salamander larvae
and tadpoles are summarized in Duellman and Trueb (1986).
Collection and preservation techniques for amphibians are often taxonomic-
ally specific (see Heyer et al. 1994). For collection of potential food items, Hauer
and Lamberti (2006) provide a wealth of information on proper collection tech-
niques of materials ranging from detritus to algae. Smith (2001), Thorpe and
Covich (2001), and Merritt et al. (2008) provide methods and taxonomic keys
for invertebrate prey. Prey items should be identified to the lowest taxonomic
level possible. For both amphibian larvae and prey, specimens should be placed
in a readily accessible, cataloged collection. This allows others to verify identifi-
cations and use materials for future studies.
74 | Amphibian ecology and conservation

5.4.2 Category II: gut contents

Most studies of amphibian diets have relied on examinations of gut contents.
However, gut-content data should be interpreted cautiously because the types
and amounts of materials present are a function of the time and place of collec-
tion (time of day, season, age of the individual, etc.) and euthanization and pres-
ervation methods. While gut contents acquired with robust sampling designs
that account for spatial and temporal variations can be reliable indicators of
foraging patterns and functional roles of larval amphibians, they may not be
good indicators of true trophic status because of differential assimilation of
materials (Altig et al. 2007). As noted above, we assert that larval caecilians and
salamanders are the only groups for which true trophic position can be assessed
via examination of gut contents alone. All methods have limitations, so the best
approach is to assess diets and trophic relations using multiple approaches (e.g.
examination of stomach contents combined with stable-isotopic analyses of tis-
sues). Further, approaches should be iterative to account for time lags between
ingestion of available foods and actual changes in isotopic signatures or fatty
acid profiles, which are a function of tissue turnover rates.
Gut contents can be removed from larger larval salamanders and caecilians
without killing the individual. Although gut contents may be removed with for-
ceps from large individuals that are anesthetized, the safest and most effective
techniques involve forced regurgitation. This is generally accomplished with
stomach flushing or gastric lavage, a technique that involves displacing materials
from the stomach and back out the mouth with a low-pressure stream of water
(Solé et al. 2005). Approaches vary and should be adjusted for an individual’s
size and mouth and gut morphologies, but generally involve gently forcing water
from a syringe through a lubricated (water or a water-based lubricant) catheter
tube that is inserted into the esophagus. During this procedure, the animal is
held with the head facing down and over a vessel where displaced materials and
fluid are collected. Contents can then be rinsed through an appropriately sized
sieve and preserved in ethanol or buffered formalin. Excess water should be
removed before preservation. If performed correctly, this technique is harmless
and very effective because fresh materials are collected from the stomach.

5.4.3 Procedures: anurans and small predators

For other amphibians (e.g. tadpoles, small salamanders), specimens are generally
sacrificed, in which case individuals should be collected as gently as possible, anes-
thetized with MS-222®, Orajel®, or similar materials, and quickly euthanized in
a humane manner to avoid vomiting and/or continued digestion of food. The
gut and its contents can be removed in the field or later from properly preserved
 5 Dietary assessments of larvae | 75

specimens. In cases where fatty acid or isotope analyses are also planned, speci-
mens may need to be frozen rather than placed in fluid preservatives. Freezing
leads to distortion of some materials, and removal of guts and processing or pres-
ervation should take place immediately after specimens are thawed.

5.4.4 Processing and analysis of gut contents

Processing methods for gut contents must account for differences in gut tracts
among taxa. Most tadpoles have long, coiled guts that are typically several times
their body length, whereas salamanders and caecilians have shorter guts with a
well-developed stomach that is typical of carnivores. These differences warrant
different approaches.
For salamanders and caecilians, prey items are removed from the gut, identi-
fied to the desired taxonomic level, and counted and measured. In cases where
guts contain many small prey items any number of subsampling techniques can
be used, so long as they are unbiased toward different types of prey. A simple
technique involves spreading the gut contents evenly in a Petri dish with num-
bered grids and then using a table of random numbers to select grids in which
contents are identified and measured until a representative subsample has been
examined. Protocols for degree of subsampling range from fixed counts (e.g.
100 individuals) to proportion of total examined (e.g. one-eighth of the total
grids or area examined). Methods should be calibrated by progressively exam-
ining larger portions of a subset of samples to assess the accuracy of various
subsample sizes. For most purposes, expressing prey in terms of mass or volume
is best, although simple numerical analyses are sufficient for some assessments
of foraging behaviors and prey availability. Mass analyses are more appropriate
for assessments of trophic status and nutrition because they can be converted
to organic content or caloric equivalents. Mass is generally estimated by actual
weighing (generally as dry mass (DM) or ash-free dry mass (AFDM), which
is a proxy for organic content; see methods below) or use of length/mass rela-
tionships, which are generally in the form: M  aLb where M is the mass of the
organism, L is a linear dimension such as length, and a and b are regression con-
stants (reviewed by Benke et al. 1999). Length/mass relationships for a variety
of potential prey items can also be found in Rogers et al. (1976), Bottrell et al.
(1976), Dumont et al. (1975), and Culver et al. (1985). In many cases, prey items
are fragmented, and thus care must be taken to reassemble them or estimate
total lengths. Body length and head width, which are the most common meas-
ures used, are generally performed with a dissecting microscope equipped with
an ocular micrometer, graduated stage, or image-analysis capabilities. Mass is
usually expressed as DM or AFDM. For more in-depth analyses, caloric content
76 | Amphibian ecology and conservation

is very informative. Estimates of caloric contents for many types of prey are
available (e.g. Cummins and Wuychek 1971; Rodgers and Qadri 1977), but
these estimates tend to be quite variable. Depending on the level of precision
desired, analyses such as bomb calorimetry should be performed on actual prey
items or other materials from the guts or specimens collected from the same
habitats at the same time. As with DM and AFDM, size/caloric-content rela-
tionships can be developed.
While collection and identification of gut contents from salamanders and
caecilians is relatively straightforward, tadpoles present a challenge because
their long guts contain mixtures of materials of different digestibility that are at
different stages of digestion. In this case, the most recently ingested materials
from the foregut should be the foci of analyses. This is generally accomplished
by removing a section of the foregut that is a consistent proportion of the total
gut length (e.g. the anterior quarter of the gut) and preparing it for examination
under a microscope (for identification of individual food particles) or for ana-
lyses of caloric content, fatty acid profiles, or isotopic composition (see methods
for fatty acids and isotopes below). Obviously, method of collection and preser-
vation can limit the types of analyses that can be performed on gut contents.
For identification of foregut contents, materials are generally rinsed from
the gut, slide-mounted, and examined under a microscope. One rather simple
and effective method is to place materials in glycerin and gently homogenize
them. If needed, water can be added and the materials sonicated to further
break up contents. Depending on the abundance of particles, either the entire
sample or a subsample is filtered with low vacuum on to a 0.45 μm gridded
membrane filter to obtain a reasonable dispersion of particles across the filter
(e.g. 10–20 particles/grid). Filters should be allowed to dry at ambient tem-
perature for 2–3 h to remove excess water, and then two ot three drops of Type
A immersion oil, which clears the filter, are added. After approximately 24 h
the filters will clear and can then be placed on a slide and covered with a cover-
slip, which is sealed with enamel nail polish. Slide-mounted materials are gen-
erally subsampled (e.g. some fi xed number of fields of view examined, a fi xed
number of grids examined, or all particles along transects are identified and
measured). Measurements of individual particles can be made using methods
ranging from an ocular micrometer to sophisticated image analysis systems.
Data on identified food types are generally expressed as area or converted to
mass, or, in the case of algae, biovolume (Lowe and LaLiberte 2006; Steinman
et al. 2006). There are many variations for mounting and analyzing gut con-
tents (for recent examples using various taxonomic groups, see Evans-White et al.
2003; Ranvestel et al. 2004; Rosi-Marshall and Wallace 2002). Regardless of
 5 Dietary assessments of larvae | 77

modifications, the key points are to avoid damaging materials beyond recogni-
tion and to obtain an evenly dispersed, representative sample.
Masses of materials in guts can be estimated with methods ranging from
simply weighing materials to using size/mass relationships (e.g. body length/
mass relationships for prey; see above section on salamander and caecilian guts)
either developed by the investigator or gleaned from the literature. For direct
weighing, DM or AFDM estimates are best. Dry mass estimates are generally
developed by drying materials at 55–60°C to constant mass. For AFDM, dried
materials are weighed, then ashed in a furnace (≈500°C) for 1–2 h, and weighed
again; mass of remaining ash is subtracted from the original dry mass to obtain
AFDM. In either case, samples should be allowed to cool in a dessicator before
weighing so that moisture is not absorbed in the cooling process. Actual drying
and ashing times will vary with sample size. Small amounts of materials can
be processed in this manner on small pieces of heavy duty aluminum foil or
glass fiber filters. Development of wet mass/DM or AFDM relationships can
streamline procedures and decrease the number of samples that are destroyed.
Likewise, if caloric equivalents are derived through calorimetry, similar rela-
tionships can be developed.
Gut content data expressed as mass or area (or in some cases biovolume for
algae (see Lowe and LaLiberte 2006) can be combined with estimates of assimi-
lation efficiencies for more accurate assessments of trophic status. Procedures
vary, but a simple approach is to express food types as percent of total gut con-
tents and multiply each by its respective assimilation efficiency. Resultant values
for the food types are then summed and the percentage contribution of each to
assimilated materials can be calculated. If caloric content is estimated, indices of
prey or food-type importance can be calculated using basically the same basic
procedure; masses of each food type in the gut are multiplied by their corre-
sponding caloric density and percentage caloric contribution of each food type is
the total number of calories attributable to a given food type divided by the total
caloric content in the gut. Regester et al. (2008) provide examples of these types
of analyses and related procedures applied to pond-breeding ambystomatid com-
munities. Examples using various freshwater omnivores can be found in Bowen
et al. (1995) and Evans-White et al. (2003). Regester et al. (2008) and Skelly and
Golon (2003) provide examples of methods for estimating assimilation efficien-
cies of amphibian larvae.
In cases where algae are obviously consumed, analyses of photosynthetic pig-
ments may be performed on gut contents to estimate algal biomass. For this pro-
cedure, specimens and/or their gut contents must be frozen immediately upon
collection and kept dark and frozen prior to analyses. Chlorophyll a, which can
78 | Amphibian ecology and conservation

be used to estimate algal biomass, can be extracted from samples of gut contents
with an organic solvent and concentration is then estimated with spectropho-
tometry, fluorometry, or high-performance liquid chromatography. Steinman
et al. (2006) provide a review of methods to estimate algal biomass via photo-
synthetic pigment analyses. Although informative to a degree, pigment analyses
alone cannot account for the digestibility of algal materials, and some tadpoles
will re-ingest feces containing undigested algal materials. The ability of tad-
poles to digest algal materials can be assessed through microscopic examination
of cells in feces (e.g. whether full chloroplasts are present) or culturing algae
from feces can identify the quantity and types of materials that are still viable
after passing through guts (Peterson and Boulton 1999). Simple counts of prey
items or food types in guts may not provide reliable information on trophic sta-
tus, but can be valuable for assessing feeding preferences and interactions with
other consumers. A variety of indices, including Horn’s index and Morisita’s
index, can be applied to gut-content data to quantify diet overlap among species
(see Brower et al. 1998; Krebs 2001; Chapter 18). Likewise, if data on the abun-
dance of prey and/or food type in the environment are also available, feeding
selectivity can be assessed and quantified using a variety of available indices.
Relatively simple and commonly used indices include Ivlev’s index of electivity
(Ivlev 1961) and the selectivity index (Chesson 1983).

5.5 Category III: assimilatory diet

Analyses of isotopic composition and fatty acid profiles can provide reliable
information on diets and trophic status because they reflect assimilation over
long periods of time. However, these approaches can be more costly and compli-
cated than simple gut analyses because they require expensive analytical equip-
ment, and they also have their own limitations, some of which are discussed
below. In most cases, samples are simply collected and preserved in a manner
that does not alter isotopic or fatty acid composition, depending on the analyses
to be performed, and then sent to an analytical laboratory for analyses.

5.5.1 Stable-isotope analysis

Isotopes of carbon (13C) and nitrogen (15N) are commonly used in studies of
freshwater food webs because they allow for tracking transfers of organic car-
bon and nitrogen from living plant and detrital sources to primary consumers
and predators (Peterson and Fry 1987; Fry 2006). Other isotopes, including
deuterium (2H or D) and 18O, are gaining increasing attention from ecologists
for tracing movements of organisms. We focus on carbon and nitrogen, as they
 5 Dietary assessments of larvae | 79

can provide valuable information on assimilatory diets and methods for their
analyses are standardized. Stable-isotope analyses focus on ratios of heavier
(e.g. 15N and 14C) and lighter isotopes (14N and 13C). Ratios are expressed as ,
which is parts per thousand (‰) deviation from a standard, calculated as 13C
or 15N  [(Rsample  Rstandard)/Rstandard]  1000 where R  13C/12C or 15N/14N.
Common standards are Pee Dee belemnite limestone for C and atmospheric N
(‰ value set to 0). A positive (N) or less negative (C) value indicates the sample
is enriched with the heavy isotope.
Carbon isotope signatures of consumers are generally similar to those of their
food source or sources (i.e. you are what you eat), which can have 13C val-
ues ranging from slightly higher than atmospheric CO2 (−8‰) to −40‰ for a
variety of reasons that influence degree of fractionation (see Peterson and Fry
1987; Fry 2006; Hershey et al. 2006). In particular, in streams, water velocity
can influence carbon isotopic compositions of foods such as algae (Finlay et al.
1999). Differences in C3 and C4 photosynthetic pathways result in differences
among some plants and their detritus. Likewise, there are often measurable dif-
ferences in carbon isotope ratios between terrestrial and aquatic autotrophs and
their detritus.
Nitrogen is more often used to assess trophic position, as 15N shows a fairly
consistent change with each trophic step (≈3.4‰ increase in 15N with each
trophic step up from primary consumer to predator; Vander Zanden and
Rasmussen 1999; Post 2002). This fairly predictable fractionation results
from the tendency for organisms to excrete more of the lighter nitrogen isotope
than the heavy one. However, fractionation can vary and there is evidence that
the increase in 15N with each trophic step is considerably less than 3.4‰ in
some tropical stream food webs (Kilham and Pringle 2000). In general, basal
resources such as periphyton and detritus in freshwater habitats will have 15N
values ranging from near 0 to 3‰, primary consumers range from 3 to 6‰,
and predators from 6 to 12‰, but values can vary considerably for a variety
of reasons (Fry 2006). Whiles et al. (2006) and Verburg et al. (2007) provide
examples of isotopic analyses of stream food webs with abundant and diverse
tadpole assemblages.
Most studies examine naturally occurring isotopic ratios (natural abundance
studies) in consumer tissues and food resources, but additions of enriched
materials (e.g. tracer studies using 15N enriched ammonium or nitrate or 13C
enriched acetate) are also used, particularly when natural isotopic ratios among
food types are similar. Tracer studies can be informative for assessing diets and
trophic interactions, as well as examining roles of consumers in biogeochem-
ical cycling, because materials (e.g. N or C) are followed from basal resources
80 | Amphibian ecology and conservation

through top consumers. Stable-isotope tracer additions have been used to exam-
ine carbon cycling through consumers in streams (Hall 1995), nitrogen cycling
in stream food webs (Hall et al. 1998), and movement of nutrients from streams
to riparian habitats via consumers (Sanzone et al. 2003). Tracer studies could
be particularly informative for assessing roles of amphibians in freshwater eco-
system processes, as well as material and energy exchanges between aquatic and
terrestrial systems.

5.5.2 Procedures
Stable-isotope analyses can be performed on entire organisms or, in the case of
larger individuals, muscle tissue (generally 1–2 g wet weight). While muscle tissue
is the usual focus for larger animals, blood and other tissues such as liver or skin
are sometimes examined because they have different incorporation and turn-
over rates, and can thus be used to assess temporal patterns of food availability
and assimilation (Dalerum and Angerbjorn 2005). For example, while isotopic
analyses of muscle tissues may reflect assimilation over a period of months, liver
or blood plasma, which turnover much more rapidly, can reflect a period of days
or weeks. This isotopic memory is an important consideration, as diets of some
amphibians can change considerably through development and with changing
resource availability.
Muscle tissues from large individuals can be sampled non-lethally using ster-
ile biopsy punches, which are available from many surgical supply companies. If
biopsies are collected, wounds should be treated with an appropriate antibiotic
before releasing the specimen. Live amphibians and prey items that are to be proc-
essed in their entirety should be allowed to clear their guts as much as possible
by placing them in filtered water from the same habitat for approximately 12 h.
Alternatively, gut tracts can be removed before processing, but this can be a tedi-
ous process for small specimens. The best methods for preservation of samples for
isotopic analyses are freezing or drying at low temperature (55–60°C to constant
mass). In the field, samples should be placed on ice or in liquid nitrogen and then
transferred to a freezer (−80°C) or drying oven when available. Dried samples
should be stored in a desiccant such as drierite or silica. Cross-contamination
must be avoided throughout sampling and processing of all materials.
Comprehensive analyses of food webs require sampling of all potential food
items that are available for larval amphibians or their prey. Sampling of prey,
algae, biofilms, detritus, and other potential resources simply requires collection
of representative samples. These samples can be collected using standard proced-
ures (e.g. Hauer and Lamberti 2006), but care must be taken to avoid cross-
contamination. Sampling of fine particles from substrates or the water column
 5 Dietary assessments of larvae | 81

can be collected and processed on glass-fiber filters. As with any other sampling,
replicates should be collected to account for natural variability. In flowing waters,
areas of different velocities should be sampled, as current velocity can alter the
carbon isotope compositions of periphyton (Finlay et al. 1999).
Prior to isotopic analyses, all samples are dried to constant weight at 55–60°C,
ground into a fine powder, weighed, and packed into tin capsules. Samples of
particulates on glass-fiber filter can either be carefully scraped from the filters,
or a portion of the filter, with sampled material on it, can be packed into the
tin capsule. While muscle tissue is the usual focus for larger animals, for most
prey items (e.g. aquatic invertebrates) the entire organism is processed, or, in
the case of very small prey, individuals are combined. Samples for N isotope
analysis should contain at least approximately 100 μg of DM of N; samples
for C isotope analysis should contain approximately 2 mg of DM of C. Sample
sizes for analysis of both C and N isotopes vary with C:N ratio of the sample,
with larger sample sizes needed (e.g. 10–60 mg) for materials with high C:N
ratio (e.g. sediments with low %N). Samples of basal resources and sediments
from calcareous regions should be processed to remove carbonates before ana-
lysis. Carbonates can easily be removed by acidification via exposure to HCl
vapors (Yamamuro and Kayanne 1995). Isotopic analyses are performed using
a continuous-flow isotope-ratio mass spectrometer.
Analyses and interpretation of stable-isotope data range from simply com-
paring values of resources and consumers to assess relative contributions, to
quantifying trophic status (Vander Zanden et al. 2000; Post 2002) or niche
breadth (Layman et al. 2007). Depending on the nature of the system, stable-
isotope analyses will sometimes reveal clear relationships among consumers
and resources. In other cases, environmental complexities and high degrees
of omnivory can confound results. In cases where multiple food sources are
likely contributing to the isotopic composition of a consumer, as would be the
case with most tadpoles, mixing models can be used to assess relative contribu-
tions of different foods. Simple two-source mixing models can be used to esti-
mate relative contributions of two resources (e.g. periphyton and detritus; see
Hershey et al. 2006), whereas more complex models are necessary when more
than two food sources are likely.

5.5.3 Fatty acid analyses

Fatty acids are the essential foundations of energy storage lipids in consumers,
but they are not readily synthesized by animals. As such, they must be obtained
through their food. Differences in fatty acid content among food types can
contribute to many of the qualitative differences among larval food resources in
82 | Amphibian ecology and conservation

terms of their importance for growth and development. Given their nutritional
value, that they are conserved, and that various resources and consumers have
unique combinations of fatty acids, fatty acid profiles can be good indicators
of diet and trophic status. Analyses of fatty acid profiles have been successfully
used to assess food web structure in marine, lake, and stream systems (Muller-
Navarra et al. 2000; Stübing et al. 2003; Sushchik et al. 2003; as well as aquatic–
terrestrial food web linkages, Koussoroplis et al. 2008). In some cases, fatty acid
analyses can provide detailed taxonomic information, including specific types
of algae assimilated (e.g. Napolitano et al. 1997).
Samples of muscle tissues (large amphibians), whole organisms (small
amphibians, prey items), and basal resources (algae, detritus) can be collected
in the same manner as described above for isotopic analyses, except that all
materials should be frozen at −80°C as quickly as possible. Moisture content
of materials is quantified with freeze drying so that lipids are not altered or
volatilized. Lipids are extracted from homogenized materials using chloroform/
methanol solvent extraction (Bligh and Dyer 1957). Analyses involve separation
of polar and non-polar lipids with a solid-phase extraction system, conversion
into fatty acid methyl esters, and separation using a gas chromatograph with a
flame ionization or mass spectrometer detector.
Discriminant function analysis and similar approaches can be used to assess
trophic interactions among sampled resources and consumers (Budge et al.
2002). As with isotopic analyses, resolution will vary among systems, and in
situations where trophic interactions are complex multiple approaches (e.g. fatty
acid analyses combined with isotopic analyses and/or gut content analyses) will
obviously produce more robust results.

5.6 Summary
Understanding the food habits of larval amphibians is actually the first step in
understanding the interactions of amphibians and the communities in which
they live and ultimately their ecological significance (Altig et al. 2007). Given the
current status of many amphibian species and populations, and the likelihood
that past and ongoing declines have ecosystem-level consequences (Ranvestel
et al. 2004; Whiles et al. 2006), there is urgent need for detailed, quantitative
information on the myriad ecological roles of larvae. The methods reviewed
herein provide the basic tools for assessing diet, nutritional ecology, and trophic
interactions. Choosing the proper technique ultimately depends on the research
question and resource limitations. Because each approach has inherent limita-
tions, we recommend combined approaches.
 5 Dietary assessments of larvae | 83

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 6
Aquatic mesocosms
Raymond D. Semlitsch and Michelle D. Boone

6.1 Introduction
We have often told our students that any experiment can be easily criticized for
lack of realism because all experiments, whether in the laboratory or field, by
design are contrived by humans and are not natural. However, in spite of such
criticism the more challenging issue is coming up with new venues or innova-
tive designs to answer important ecological or conservation questions and to
balance trade-offs. As amphibian ecology has become more experimental, we
have been confronted with numerous trade-offs concerning the choice of venue,
design, realism, and replication. Each choice is often coupled with benefits as
well as limitations. As in many rapidly developing fields, criticism is an expected
and healthy process that pushes investigators to think about their choices and
develop solutions. The use of mesocosms in studies of aquatic ecology across
an array of taxonomic groups has been central in that debate (e.g. Kimball and
Levin, 1985; Carpenter 1996; Schindler 1998). Within the field of amphibian
biology, a number of reviews also have stimulated a healthy discussion on the use
of experiments (Hairston 1989a, 1989b), and in particular, use of mesocosms
(e.g. Jaeger and Walls 1989; Rowe and Dunson 1994; Skelly and Kiesecker
2001; Skelly 2002).
The purpose of our chapter is to provide background on the use of aquatic
mesocosms in ecological and conservation studies of amphibians. We describe
the history, various types, set-ups, and designs and present selected case studies
to emphasize their versatility. We also provide examples and comments on limi-
tations of the technique and strengths of inference that come with the results.
Our goal is to provide readers with an effective technique to answer a range of
ecological and conservation questions.
88 | Amphibian ecology and conservation

6.2 Historical background

The use of aquatic mesocosms in amphibian ecology was born out of the need
to move away from purely descriptive field studies and correlative analyses
and towards more manipulative studies that could test hypotheses to establish
cause and effect (see Wilbur 1987). Studies using aquatic mesocosms are often
viewed as the best compromise between the variability of natural wetlands, con-
founding factors, and the lack of control that is common in field studies and
the ability to manipulate single variables, replicate treatments, and randomly
assign treatments to experimental units in laboratory experiments. They have
sometimes been referred to as “hybrid” or “semi-field” experiments (Rowe and
Dunson 1994). Aquatic mesocosms differ from “microcosms” in that they are
self-sustaining once established and they do not need input of additional nutri-
ents or resources after initial set-up, thus allowing study organisms to complete
critical phases of their life cycle. To be effective, mesocosms must be capable of
rearing populations of amphibian larvae, simulate important components of the
aquatic ecosystem, and be reproducible and affordable. For purposes of this dis-
cussion, we refer primarily to two common types of aquatic mesocosm: cages or
pens (Figure 6.1) that are placed in a natural pond or stream to capture location
effects such as habitat variability and containers including plastic tubs, wading
pools, or cattle watering tanks (Figure 6.2) that are filled with water and have
components of aquatic ecosystems added to simulate natural variation. Artificial
streams fit in the category of container mesocosms but have a flow-through or
recirculating water source or system. Any of these types can be large or small and
contain few or many components of a natural system; in this way they form a
continuum between laboratory microcosms and natural field studies.
To the best of our knowledge, Warren Brockelman was the first to use
pens placed in natural ponds for experimental amphibian studies in 1966–7
in Michigan, USA (Brockelman 1969). He used wooden framed pens (0.61 m
wide  2.44 m long  0.6 m high) covered with fiberglass window screening
and with lids, half of which had screen bottoms and half had bottoms open to
sediments. His study addressed the effects of intraspecific competition on Bufo
americanus tadpole growth, development, and survival.
The first use of containers for experimental amphibian studies was by Henry
Wilbur and Joe Travis in 1977–9 in North Carolina (Wilbur and Travis 1984).
They used cattle watering tanks (galvanized steel, round, 3.58 m2 surface area,
0.61 cm deep) buried in the ground flush to the surface and filled with natural
pond water. Replicate tanks were positioned in different habitat types to test dif-
ferences in the colonization of aquatic habitats by invertebrates and amphibians,
 6 Aquatic mesocosms | 89

Fig. 6.1 Examples of different cages used to rear amphibian larvae.

Full-Sun Treatment
High-Shade Treatment

Low-Shade Treatment

Fig. 6.2 Examples of different cattle tanks and pools used to rear amphibian larvae.
90 | Amphibian ecology and conservation

and the formation of community structure. In 1980, one of Henry Wilbur’s

graduate students, Peter Morin, subsequently developed a more efficient and
novel venue for experiments using cattle tanks to manipulate factors important
to community regulation (Morin 1981). He placed cattle tanks (epoxy-coated
galvanized steel, round, 1.52 m diameter, 1000 L) in a field, above ground, so
that they could be filled, manipulated, and easily drained to initiate new experi-
ments each year. Each tank was filled with tap water and then each received an
equal amount of dried litter collected from the edge of a natural pond (550 g)
and Purina Trout Chow (50 g) for substrate and a source of nutrients. Equal
amounts of inoculum of plankton derived from pooled plankton collections
from eight natural ponds were added to each tank to initiate a diverse food web
and prey base for larvae. This latter approach of using cattle tanks as aquatic
mesocosms that could be renewed for each question and study has been critical
in the advancement experimental amphibian ecology.

6.3 Why use mesocosms?

Studies in nature and in the laboratory allow researchers to answer important
questions, but both have limitations (Diamond 1986). While laboratory stud-
ies allow us to maximize control and manipulate factors so that cause–effect
relationships can be established, the laboratory lacks realism and complexity,
reducing its application in the real world. In contrast, field studies offer realism
and can encompass the complexity of natural food webs, but it can be difficult
to distinguish cause–effect relationships and there can be high levels of vari-
ation among replicates. Mesocosms are the middle ground. Proponents argue
that mesocosms offer the best of both worlds: control, the ability to determine
cause–effect relationships, natural food webs, and realism (Wilbur 1987; Rowe
and Dunson 1994; Drake et al. 1996; Boone and James 2005). Critics, however,
contend that mesocosms offer little gain over laboratory studies in that they are
still artificial and may have limited application to the real world, yet they require
more work and time, and they have greater variation among replicates than
laboratory studies (Carpenter 1996; Skelly 2002). Certainly, mesocosms would
not be mistaken for natural ponds, but they incorporate many natural elements
that allow them to mimic some but not all of the processes occurring in larger
natural ponds or lakes (Schindler 1998).
There are a number of reasons why mesocosms can be a useful technique in
your research program. First, mesocosms allow you to incorporate some compo-
nents of natural systems into your experiments—the changes in season, photo-
period, temperature, and habitat that animals experience in nature—something
 6 Aquatic mesocosms | 91

that you typically cannot alter in the laboratory. Secondly, if you have evaluated
a mechanism within or between organism(s), or developed a theoretical model,
then a logical next step is to see if your predictions hold in a more complex and nat-
ural environment; mesocosm studies are a logical next step. Thirdly, if you want to
evaluate single factors in isolation, a mesocosm may be ideal. Fourthly, manipula-
tions in mesocosms can help you eliminate less important factors and find system
drivers, so that you can design a better, more efficient large-scale field study.
If you need a short-term answer to a basic experimental question, then a labora-
tory experiment can offer an expedient answer: can trematodes infect limb buds?
What concentration of an insecticide is lethal to tadpoles or zooplankton? Do
overwintered bullfrog tadpoles prey upon recently hatched tadpoles? If your ques-
tion is site- or habitat-specific or requires a vast area to encompass the entire ecosys-
tem, then field experiments may be the best way to go: can ponds in city parks and
golf courses support native fauna? What effects do forest-management practices
have on amphibian communities? Studies in mesocosms provide a useful surro-
gate for the field and allow us to evaluate single and multiple factors that may be
important at a larger scale, which allows us to better design larger field studies and
provides insight into the system: do changes in food webs from eutrophication
cause increases in number of trematodes and in the number of infected amphib-
ians? Does a sublethal pesticide used on golf courses cause changes in aquatic
community dynamics? Although mesocosms are often referred to as a “black box,”
suggesting that the mechanisms underlying the outcome are not clear, additional
studies can be done to discern mechanisms by behavioral observations, periodic
sampling of covariates (e.g. temperature, primary production, dissolved carbon),
or with a paired laboratory study (e.g. prey palatability).
Mesocosms are useful in addressing a variety of questions in ecology, evolu-
tion, behavior, and conservation (see case studies below). We surveyed the litera-
ture from 2004 to 2007 through the Ecological Society of America (including
the journals Ecology, Ecological Applications, Frontiers in Ecology and Evolution,
and Ecological Monographs), Oecologia, Evolution, Animal Behavior, and Behavior
by searching the terms “amphibian” or “frog” or “salamander” and then select-
ing all studies that used mesocosms. A total of 30 articles using mesocosms or
enclosures were found only in the journals Oecologia or of the Ecological Society
of America. The studies addressed a range of questions in ecology (e.g. species
interactions, population biology, community structure), evolution (e.g. pheno-
typic plasticity), behavior (e.g. oviposition selection), and conservation (e.g.
effects of pathogens, habitat fragmentation, pesticides). Whereas mesocosms
have been historically used for basic questions of ecology and evolution, they are
becoming more frequently used in the field of conservation and have enormous
92 | Amphibian ecology and conservation

potential for use in regulatory fields such as ecotoxicology (reviewed by Boone

and James 2005).
The appropriateness of a mesocosm as a representative of a natural system may
depend on many things, including the size of the mesocosm and the species or
community of interest. For studies with amphibian, invertebrate, and plankton
communities, aquatic mesocosms ranging from 200 to more than 1500 L (see
Figure 6.3a) were self-sustaining and maintained a similar temperature regime

(a)
14

Mesocosms
12 Enclosures

10
Number of studies

0
<120 200–450 750–1300 >1500
Volume (L)
(b)
12

10
Number of studies

0
0 0.1–0.3 0.875–1.8
Leaf litter (kg)

Fig. 6.3 Abundance of mesocosm studies conducted: (a) using different volumes and
(b) with different amounts of leaf litter (see text for methods and results).
 6 Aquatic mesocosms | 93

to natural communities. This is an important limitation because mesocosms

of these sizes may not be appropriate for studies with larger species like wide-
ranging predatory fish or larger-scale ecosystem processes like nutrient cycling.
What is important for effective use is that the amphibians of interest, inverte-
brate prey, and plankton resources can complete major portions of their life cycle
and be self-sustaining in the mesocosm. Isolated, temporary pond communities
range in size from tire ruts to large shallow wetlands, and have characteristics
that differ from larger lake systems that may be deeper and experience turnover
and temperature inversions. Therefore, effective use of mesocosms is likely lim-
ited to species and processes that occur in shallow, temporary wetlands.
Additionally, there is evidence indicating that results generated from meso-
cosm experiments have high predictability to the field. Mesocosm studies evalu-
ating the role of competition, predation, and pond drying (e.g. Wilbur 1972,
1987; Morin 1981, 1983; Semlitsch 1987) have proven useful in understanding
the structure and regulation of amphibian communities. Long-term monitoring
at a natural wetland (Rainbow Bay) in South Carolina, USA, has shown that
community structure is determined by competitive and predatory interactions
occurring along a gradient of pond drying (Semlitsch et al. 1996), which was
predicted by mesocosm research. Manipulations with sublethal pesticide expos-
ure in mesocosms show zooplankton populations are reduced and periphyton
abundance increases for short periods of time, which can result in positive effects
on tadpoles despite the pesticide having sublethal but negative effects on tadpoles
directly (e.g. Boone and James 2003; Boone and Bridges-Britton 2006). These
studies suggest that changes in the food web may be more important than direct
negative effects of the contaminant alone (Mills and Semlitsch 2004). When
these studies were replicated under more natural field conditions in ponds, simi-
lar outcomes were found despite the fact that predators were not excluded and
that zooplankton populations could have recovered more quickly due to ephippia
present in the soil sediments (Boone et al. 2004). Studies of dragonfly preda-
tion on tadpoles have documented qualitatively similar results between natural
pools and mesocosms (Gascon 1992) and between different sizes of mesocosms
(Gascon and Travis 1992). Such examples lend strong support to the usefulness
of mesocosms, particularly given that mesocosm manipulations require less time,
money, and effort to conduct, relative to the large field studies.

6.4 Types of mesocosm

There are a number of types of mesocosm, including cattle watering tanks or wad-
ing pools (Figure 6.2) and field enclosures (Figure 6.1). Cattle tank ponds can be
94 | Amphibian ecology and conservation

purchased in galvanized steel (which must be sealed with an epoxy paint or lined)
and polyethylene plastic (Behlen or Rubbermaid produce tanks in blue or black).
The polyethylene ponds are most commonly used today because they are long-
lasting (10 years), are not prone to rusting, and do not require sealing as metal tanks
do. Wading pools are useful and cheap but short-lived (1–2 years) and usually made
of PVC/vinyl, which can leach phthalates, chemicals that are associated with endo-
crine disruption. Water tests found that Behlen polyethylene tanks did not leach
estrogenic compounds (S.I. Storrs, personal communication). However, wading
pools can be especially useful to address short-term behavioral questions related to
factors that may influence oviposition (Vonesh and Buck 2007) or plasticity in tad-
pole morphological development associated with predators and competition which
may occur in a matter of weeks (Relyea 2004; Relyea and Auld 2005).
Field caging studies enable us to study a discrete location or habitat while
moving toward greater realism. Field cages can range in size from small to large.
In our survey, most of the cages were relatively small (2.4–120 L; Garcia et al.
2004; Griffis-Kyle and Ritchie 2007; Ireland et al. 2007) or large (pond size or
sections; Scott 1990; Loman 2004; Boone et al. 2004). Aquatic field cages are
often made of fine screen mesh, such as fiberglass window screen, mosquito net-
ting, or plastic meshes, which are exposed to the larger matrix allowing for water
exchange and influx of aquatic life smaller than the mesh size. Alternatively,
field cages can also be made of polyethylene plastics that will prevent the flow
of materials into the enclosure after initial set-up and also prevent things like
pesticides or predators from moving into or out of the enclosure. These field
cages whether made of permeable or impermeable material can be attached to
untreated lumber into a box or rectangle to be placed in a pond (Figure 6.2). If
the screen, mesh, or fabric has rigidity, it is also possible to use it to construct
circular cages to hold animals (Figure 6.2). Alternatively, “bags” can be sewn or
fabricated that can be supported with an external structure like multiple stakes,
drinking water pipe, and/or floatation devices (Figure 6.2).
The advantage of field cages is that you can study sites of particular interest
and ask questions in the relevant environment giving your study greater realism
and the ability to manipulate factors of interest. In contrast, field cages often do
not represent independent observations (unlike wading pools or tanks) given
that there is the potential for exchange among enclosures. For example, if a
researcher was examining how a fish predator may influence growth and devel-
opment of tadpoles, the presence of fish in the pond or the presence/absence of
fish as a treatment could influence all the enclosures if there was water exchange.
However, these potential problems can be eliminated or minimized with choice
of mesh and some design considerations for the cages.
 6 Aquatic mesocosms | 95

6.5 Setting up mesocosms

There are a number of ways to set-up mesocosms for larval amphibian studies,
with cattle tank ponds and wading pools generally established with a standard-
ized volume of water (tap or well water), a nutrient base (leaves or grass and/
or rabbit chow), and plankton from natural ponds. Larger volumes of water
(900–1500 L) have the advantage of being more stable environments, especially
in terms of temperature extremes in summer, and less prone to predator inva-
sion. Smaller volumes of water can be shaded and raised above the ground to
reduce likelihood of predators moving through. In our survey mentioned above,
most mesocosm studies were conducted in tanks holding 750–1300 L of water
(Figure 6.1). Water is typically allowed to sit between 2 days and 2 weeks so that
chlorine from the tap water will dissipate and so that algal and zooplankton
inoculum can establish.
The addition of leaf litter, grass, rabbit chow, and/or some other nutrient source
is a standard practice in mesocosm experiments. Natural litter substrates serve as
a slow-release nutrient source. Rabbit chow serves as a fast-release nutrient source.
Both of these will support the aquatic food web throughout the field season and
can influence other relevant factors like the amount of ultraviolet radiation pene-
tration of the water (by influencing abundance of dissolved organic compounds)
and dissolved oxygen. Researchers have used different types and amounts of
nutrients, which can impact the aquatic system. In our survey, we found that
roughly an equal number of studies used relatively low or high amounts of leaf lit-
ter (Figure 6.3). Most enclosure studies did not add nutrients unless it was being
explicitly tested, presumably because there were adequate flow of nutrients and
food into the enclosures. However, leaf litter or vegetation can be added to serve
as refugia for amphibian prey within the enclosures.
The amount and type of nutrient addition can have an effect on the aquatic
community because it sets the energy base for the system. A recent study by
Williams et al. (2008) using dead grass or deciduous leaf litter found higher
productivity in systems with grass, which resulted in greater tadpole growth. In
studies manipulating the amount of leaf litter, Rubbo et al. (2008) and Boone
(unpublished data) showed that increasing the amount of leaf litter increased
the productivity of the system, and hence the survival and growth of tadpoles.
Additionally, Rubbo and Kiesecker (2004) found the species from which the
leaf litter originated had an effect on amphibian biomass and survival, with oak
leaves having positive effects on amphibians compared to maple leaves. The type
of leaf litter or grass can influence the nutrient base of the community and influ-
ence the algal and bacterial communities that flourish there. Consideration of
96 | Amphibian ecology and conservation

the type of natural system you are attempting to represent (e.g. forested pond,
grassy pond, or marsh) is the best guide to selecting the appropriate nutrient
base to add.
It is important to establish the primary components of an aquatic food web in
mesocosm tanks in order for them to be self-sustaining. A complex community
of both phyto- and zooplankton is critical to help balance nutrient exchange,
bacterial growth, and nitrogenous waste production as well as serve as a food
source for amphibian larvae. Temporary wetlands are often an ideal source of
inoculum for mesocosms because there will be fewer insect predators and no
fish. All inoculum added to mesocosms should be strained to eliminate detri-
mental insect or fish larvae, and eggs or larvae of non-target amphibian species.
Several additions over time and from varying natural ponds will help ensure
adequate diversity and complexity.

6.6 Common experimental designs

Establishing cause–effect relationships is the hallmark of mesocosm approaches
and allows complex multifactorial designs. Designs using pools or tanks allow
clear tests of each factor independently. Mesocosm studies allow researchers to
address how incremental changes in a treatment level impact the study organ-
ism and how different treatments may interact with one another. This level of
manipulation would be difficult to achieve under most field conditions, but is
a critical tool for understanding how amphibian populations and communities
function.
Replicate tanks or pools are physically separate pond communities that can
be established with standardized procedures but diverge if treatment differences
occur. Each tank represents an independent replicate and experimental unit.
Cages located within ponds are typically not independent from one another;
therefore, the question of interest will determine how these field experiments
are replicated. If you are interested in how a particular type of habitat influences
an amphibian community, then you should replicate at the level of pond-habitat
type. For instance, to examine whether amphibians can survive larval develop-
ment in golf course ponds, Boone et al. (2008) used multiple golf course and
reference ponds with replicate cages both within and between ponds. Using
a single golf course and reference pond with replicate enclosures within each
pond would not have clarified whether golf-course management affected larval
amphibians; instead, it would only confirm that the two ponds are different
for some reason, perhaps due to golf course management, historically differ-
ent abiotic and biotic factors present in the pond, or chance. Replicate cages in
 6 Aquatic mesocosms | 97

the latter case would constitute “pseudoreplicates” (Hurlbert 1984) and would
increase the probability of making a type I error or determining there is a sig-
nificant difference related to treatment when there is not. However, if you are
interested in how overwintered bullfrog tadpoles may influence the amphibian
community, then manipulating bullfrog abundance in replicate cages in a single
pond would be acceptable. Replication in field cage studies requires replicating
at the level of your question and factor of influence (e.g. golf course), which
will likely be at separate locations. Replication is also necessary within ponds to
determine variation among specific cage locations (e.g. aspect, slope, or shading
along a shoreline). Split-plot or nested designs are especially useful in studies
using cages in ponds and may allow the testing of hypotheses not testable with
less sophisticated designs (Gunzburger 2007).
The number of replicates you should use is also an important consideration.
Typically there is a trade-off between the number of replicates and the total
size of the study related to logistical and financial constraints. However, the
more divergent your replicates, the greater the variation within your treatments,
and the less your ability to detect significant differences between treatments.
Therefore, consideration of the level of variation inherent in your replicates will
influence the number of replicates that you need. While laboratory studies fre-
quently have five or more replicates because of the ease of replication, mesocosm
tank studies will often be limited to three to five replicates (Boone and James
2005), which is typically sufficient to detect significant differences in behavior,
morphology, development, and survival. You can estimate a priori how many
replicates you should use by determining your desired statistical power (1−;
a power of 0.8 or greater is desirable) based on your  criteria and the effect
size (size of the difference). Using a statistical program like SAS (Proc Power)
or using online calculators (e.g. www.math.yorku.ca/SCS/Online/power/) you
can determine the ideal number of replicates to use based on the system you are
studying.
The random assignment of treatments is one of the most critical steps of all
true experiments and should be applied to mesocosm studies. Randomizing
your treatments is extremely important to avoid confounding variation
among replicate tanks or cages with variation among treatments. You can use
a random number chart or a random number generator in SAS (Proc Plan).
Treatments may be randomly assigned across all tanks or cages (completely
random design), or they may be blocked so that experimental units are grouped
along a known gradient that may cause variation (e.g. shading) not relevant to
the experimental question and which can be removed statistically (randomized
complete block).
98 | Amphibian ecology and conservation

6.7 Case studies

6.7.1 Community ecology
The study of community ecology has had a long history of experimental studies
to help disentangle the complex interactions among species. Morin (1981) used
three species of anuran tadpoles (Scaphiopus holbrookii, Pseudacris crucifer, and
Bufo terrestris) as competitors and adult newts (Notophthalmus viridescens) as a
predator to address the role of interspecific competition. Tadpoles were reared
together from hatching to metamorphosis in 16 cattle tanks (epoxy-coated gal-
vanized steel, 1.52 m diameter, 1000 L) at constant initial densities in all tanks.
Newts were added in four densities (none, two, four, and eight per tank) to vary
the intensity of predation and replicated four times each. The results showed
that in the absence of newt predators Scaphiopus (62%) and Bufo (33%) meta-
morphs were relatively abundant, followed by Pseudacris (5%). However, in
the presence of a high density of newts, Pseudacris (68%) metamorphs were
most abundant followed by Scaphiopus (20%) and Bufo (12%). The rarity of
Pseudacris in tanks without newts was best explained by intense interspecific
competition with Scaphiopus and Bufo. The increased survival and larger body
mass of Pseudacris in tanks with newts was best explained by release from inter-
specific competition due to newt predation on the preferred prey of Scaphiopus
and Bufo tadpoles. Morin suggested that newts acted as a ‘keystone’ predator
to prevent exclusion of competitively inferior species by reducing the survival
and density of competitively superior species. These results demonstrate that
predation and competition could interact to produce deterministic patterns of
community structure in amphibians.
In a subsequent experiment, Wilbur (1987) tested the interaction effects of
competition, predation, and disturbance on the structure of a similar aquatic
amphibian community. He used four species of anuran tadpoles (Rana sphe-
nocephala, S. holbrookii, P. crucifer, and B. terrestris) as competitors and adult
newts (Notophthalmus viridescens) as a predator. Tadpoles of the four species
were reared together in 36 cattle tanks (epoxy-coated galvanized steel, 1.52 m
diameter, 1000 L) at a constant high or low density. Four newts were added to
half the tanks and ponds were drained at three rates; either 50 days, 100 days,
or control tanks kept at 50 cm deep. All 12 treatment combinations were repli-
cated in three spatial blocks to account for physical differences across a field were
tanks were positioned. The results showed that Scaphiopus was least sensitive to
intraspecific density, metamorphosed quickly in fast-drying tanks, and was the
competitive dominant in tanks without newt predators, but suffered highest
predation from newts. Rana was most successful at low density but could not
 6 Aquatic mesocosms | 99

grow fast enough to metamorphose in fast-drying tanks and suffered high pre-
dation by newts. Bufo were very sensitive to high density and produced few met-
amorphs without newt predators. This effect was reversed by newts selectively
preying on Scaphiopus and Rana tadpoles, especially in rapidly drying tanks.
Pseudacris performed poorly in all slow-drying tanks but showed moderate suc-
cess in high-density tanks where newts had removed most competitors. The
overall conclusion of this study was that all three factors interacted with each
other and with species’ life history to determine community structure. Further,
it suggested that competition dominates simple habitats of short duration and
predation becomes increasingly important in ponds with longer hydroperiods
that permit the establishment of diverse predators. Both of the studies described
above were influential in focusing attention on the complex interactions of pre-
dation, competition, and pond drying in aquatic communities rather than his-
torical single-factor arguments.

6.7.2 Evolutionary ecology

The “common-garden” experimental approach to evolutionary studies has been
important for clarifying genetic and environmental interactions by measuring
genotype performance in a simplified controlled environment (Clausen et al.
1947). Parris et al. (1999) examined the larval performance of hybrid and par-
ental genotypes of the southern and plains leopard frog (Rana sphenocephala
and Rana blairi) in a common-garden approach using cattle tanks. They estab-
lished 56 experimental populations in cattle tanks (polyethylene, round, 1.83 m
diameter) by adding 960 L of tap water, 1.0 kg of deciduous leaf litter, and mul-
tiple additions of plankton from natural ponds. Six genotypes of hatchling tad-
poles were added to tanks at a low (20 per tank) or high (60 per tank) density to
simulate a competitive gradient. The 12 treatment combinations were replicated
either three or five times in the 56 tanks (Parris et al. 1999). Survival was the
same among all genotypes, but decreased with increasing density. Body mass
at metamorphosis was the same among genotypes, but decreased with increas-
ing density. Tadpoles of R. sphenocephala had the shortest larval period while
the other parental species R. blairi had the longest larval period. Ponds with
hybrid genotypes produced a greater proportion of metamorphs than those with
R. blairi tadpoles but a smaller proportion than ponds with R. sphenocephala
tadpoles. Their results demonstrate equal or increased larval performance of
hybrids relative to parental genotypes in cattle tank environments. Studies such
as the one described above are critical for determining whether or not natural
hybridization could potentially lead to the production of recombinant geno-
types possessing novel adaptations.
100 | Amphibian ecology and conservation

6.7.3 Ecotoxicology
The field of toxicology historically has taken a reductionist approach by using
single species and chemical testing in the laboratory. However, there is growing
effort to develop multispecies, community, and ecosystem-level experimental
approaches that better mimic real-world problems (Kimball and Levin 1985;
Rowe and Dunson 1994; Boone and James 2005; Semlitsch and Bridges 2005).
A model study by Boone et al. (2007) examined the role that chemical con-
tamination, competition, and predation play singly and in combination in an
aquatic amphibian community. They established replicate aquatic communities
in 64 polyethylene cattle tanks (round, 1.85 m diameter, 0.6 cm height, 1480 L
volume) by adding 1000 L of tap water, 1.0 kg of leaf litter, plankton from nat-
ural ponds, 60 Bufo americanus tadpoles, 30 R. sphenocephala tadpoles, and 10
Ambystoma maculatum salamander larvae to each. Four factors were manipulated
and replicated four times in a fully factorial design: no or two bluegill sunfish,
no or six overwintered bullfrog tadpoles, 0 or 2.5 mg/L carbaryl (an insecticide),
and 0 or 10 mg/L ammonium nitrate fertilizer. The results showed that bluegills
had the largest impact on the community by eliminating B. americanus and A.
maculatum and reducing the abundance of R. sphenocephala. Chemical contam-
inants had the second strongest effect on the community with the insecticide
reducing A. maculatum abundance by 50% and increasing the mass of anurans
(R. sphenocephala and B. americanus) at metamorphosis; the fertilizer positively
influenced time and mass at metamorphosis for both anuran and salamander
larvae. Presence of overwintered bullfrog tadpoles reduced mass and increased
time to metamorphosis of the anurans. Although both bluegill and overwin-
tered bullfrog tadpoles had negative effects on the amphibian community, they
performed better in the presence of one another and in contaminated ponds.
These results indicate that predicting complex interactions from single-factor
effects may not be straightforward. Further, the research supports the hypoth-
esis that combinations of factors can contribute to population declines.

6.7.4 Land use and management

Only recently have ecologists begun to use mesocosms to address questions con-
cerning human land use and habitat fragmentation. Hocking and Semlitsch
(2007) tested the effects of timber harvest on oviposition-site selection by gray
treefrogs (Hyla versicolor) in four experimental forest treatments. Five plastic
wading pools (1.5 m diameter, 30 cm deep) were placed above ground in each of
the four forest treatments: (1) a clear-cut with high coarse woody debris (CWD),
(2) a clear-cut with low CWD, (3) a partial cut with 50% canopy removal,
 6 Aquatic mesocosms | 101

and (4) a control. The five pools were placed at three distances from the nat-
ural breeding pond in each treatment (see Figure 1 in Hocking and Semlitsch
2007): one pool at 10 m, two pools at 64 m, and two pools at 115 m from
a natural breeding pond. The four forest treatments were positioned around
three replicate natural breeding ponds for a total of 60 wading pools. Pools
filled naturally with rainwater by April. Following the first oviposition event,
all pools were checked every 24–48 h for treefrog eggs. Eggs were removed
and counted. The results indicated that treefrogs laid significantly more eggs in
the clear-cuts (low-CWD 77 185 eggs; high-CWD 51 990 eggs) than in either
the partial (13 553 eggs) or the control (14 068 eggs) treatments. Further, the
interaction of forest  isolation treatment showed that oviposition in both clear-
cut treatments decreased with increasing isolation whereas oviposition was the
same at all levels of isolation in the partial and control treatments. Hocking and
Semlitsch (2007) suggested that the strong preference for open-canopy pools
likely benefits the larval stage because of higher-quality food and warmer water
temperatures but that isolation of breeding pools beyond 50–100 m can inhibit
oviposition by female treefrogs. They also add that the possible benefits to one
stage (tadpoles) might be diminished by the risk of desiccation to the terrestrial
juvenile stage after metamorphosis. Thus, mesocosms can be used to address
land-use questions beyond the boundary of aquatic ecosystems.

6.8 Conclusion
Although we are both advocates for the use of mesocosms in amphibian ecology
and conservation, we also agree that a pluralistic approach to any question is
likely necessary for complete understanding. The use of laboratory experiments
to understand mechanisms as well as field studies to anchor mesocosm results
back to the real world are critical. The use of cattle watering tanks, wading
pools, and cages is a powerful technique in any researcher’s toolbox. The choice
of mesocosm should reflect knowledge of the natural history of the species and
system being studied. If used properly, at the correct scale for the questions one
is asking, many questions can be clearly answered and results can have strong
inferences back to natural systems. However, we caution readers that neither
type of mesocosm described here can address all ecological and conservation
questions alone. One has only to read Schindler (1998) to realize that most
large-scale ecosystem processes cannot be replicated in cattle tanks or cages.
But, for many population- and community-level studies of amphibians, meso-
cosms offer an excellent approach.
102 | Amphibian ecology and conservation

6.9 References
Boone, M. D. and Bridges-Britton, C. M. (2006). Examining multiple sublethal con-
taminants on the gray treefrog, Hyla versicolor: effects of an insecticide, herbicide, and
fertilizer. Environmental Contamination and Chemistry, 25, 3261–5.
Boone, M. D. and James, S. M. (2003). Interactions of an insecticide, herbicide, and
natural stressors in amphibian community mesocosms. Ecological Applications, 13,
829–41.
Boone, M. D. and James, S. M. (2005). Use of aquatic and terrestrial mesocosms in eco-
toxicology. Applied Herpetology, 2, 231–57.
Boone, M. D., Semlitsch, R. D., Fairchild, J. F., and Rothermel, B. B. (2004). Effects of
an insecticide on amphibians in large-scale experimental ponds. Ecological Applications,
14, 685–91.
Boone, M. D., Semlitsch, R. D., Little, E. E., and Doyle, M. C. (2007). Multiple stressors
in amphibian communities: interactive effects of chemical contamination, bullfrog
tadpoles, and bluegill sunfish. Ecological Applications, 17, 291–301.
Boone, M. D., Semlitsch, R. D., and Mosby, C. (2008). Suitability of golf course ponds
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 7
Water-quality criteria for amphibians
Donald W. Sparling

7.1 Introduction
Most amphibian species spend portions or even their entire life cycle in water.
Whether their habitats are streams, wetlands, vernal ponds, farm ponds, or
larger bodies of water, amphibians are directly affected by several natural and
anthropogenic chemical and physical characteristics. Dissolved oxygen, tem-
perature, pH, salinity and water conductivity, organic carbons, and pollutants
are important factors of their habitats that can affect survival, growth, mat-
uration, and physical development. In addition to direct effects, these char-
acteristics interact with other factors such as predators, prey, parasites, and
competitors to affect populations. Just as one example, the incidence of infest-
ation of tadpoles by Ribeioria ondatrae, a trematode parasite that causes limb
malformations, has been linked to the nutrient status of ponds (Johnson and
Chase 2004). Field studies of amphibians should at least include a description
of naturally occurring environmental conditions. In some cases, this descrip-
tion should also include analysis of environmental contaminants. This chapter
discusses chemical and physical factors that are most important to aquatic life
stages of amphibians and directs the reader to reviews for more complicated
issues dealing with water quality.
Because of space limitations, the descriptions of these factors and the methods
used in analyzing them are abbreviated. More complete descriptions of physio-
logical requirements can be found in Feder and Bruggren (1992) and McDiarmid
and Altig (1999). I recommend US Geological Survey (USGS) (2008a) and
Eaton et al. (2005) for standardized protocols of water-quality analysis.

7.2 Dissolved oxygen

Whereas free oxygen in the atmosphere occurs as O2 and is a relatively abun-
dant gas, it must be dissolved in water to be useful to aquatic aerobic organisms.
106 | Amphibian ecology and conservation

These organisms take in oxygen through moist membranes such as gill epithe-
lium, dermis, or other structures. Aside from for rare exceptions, only aquatic
life stages of amphibians potentially face oxygen problems; atmospheric oxy-
gen is sufficient for terrestrial forms. Some species of amphibian, such as tailed
frogs (Ascaphus spp.), live in swift-moving lotic environments where water is
constantly mixed with air and oxygen concentrations are usually near satur-
ation. Others, however, inhabit quiescent, warm water, lentic environments
where oxygen concentrations can fluctuate or persist at low concentrations.
Under laboratory conditions, oxygen concentrations below 4 mg/L are deemed
stressful to amphibian larvae and other aquatic organisms (ASTM 1988) and
prolonged exposure to such hypoxic conditions in the field can be considered
adverse. However, there are differences among species and animals can accli-
mate over time to low oxygen concentrations.
Many amphibians have anatomical features, behaviors, and physiological
processes that allow survival under temporary hypoxic conditions. For example,
members of the families Ranidae and Ambystomatidae assimilate oxygen
through dermal uptake, gills, and lungs. Under normoxic conditions (non-
stressful oxygen concentrations), approximately 70% of oxygen uptake by these
larvae is through the dermis, 20% via the gills, and 10% through the lungs
(Gatten et al. 1984). In contrast, Bufo spp. and Ascaphus spp. do not inflate their
lungs until just before metamorphosis (Feder 1984) and Plethondontids lack
lungs entirely.
In hypoxic conditions, larvae with lungs often swim to the surface and gulp
air into the lungs and pulmonary uptake of oxygen becomes more important.
Surfacing, however, incurs metabolic costs and exposes tadpoles to predators such
as turtles (Feder 1983). However, in an experiment using aquariums, McIntyre
and McCollum (2000) found that the alternative behavior of not coming to the
surface could also incur risks. Young, pre-lunged bullfrog (Rana catesbeiana)
tadpoles experienced a higher degree of predation in high-oxygen tanks than in
low-oxygen ones because predacious tiger salamanders (Ambystoma tigrinum)
spent a greater proportion of time searching for tadpoles at the bottom of the
tanks under high-oxygen conditions than when the dissolved oxygen was low.
Hypoxia can induce similar physiological responses in amphibians as in other
vertebrates including changes in blood pH, build-up of lactate in muscles, leth-
argy, and, under severe conditions, mortality (Ultsch et al. 1999). Chronic and
intermittent hypoxia can reduce growth and developmental rates and delay
hatching of salamander embryos (Valls and Mills 2007).
Several factors affect the concentration and measurement of dissolved oxygen.
Oxygen concentrations can vary widely through the course of a day, especially
 7 Water-quality criteria for amphibians | 107

in warm, eutrophic bodies of water. In ponds, a major source of oxygen comes

from photosynthesis by algae and other green plants. Because photosynthesis
is driven by water temperature and sunlight, dissolved oxygen concentrations
are frequently lowest at dawn, increase several-fold during the course of the day,
reach maximum concentrations in mid afternoon, and decrease sharply during
the night due to biological oxygen demand (Figure 7.1). Cloudy or cool weather
may reduce the extremes in concentrations. These daily variations demonstrate
the need to record measurements under similar times of day and sunlight when
comparing oxygen concentrations among ponds or through time. Measurements
taken in early morning may be most meaningful with regard to environmental
stress.
In lotic conditions, especially in shallow headwater streams inhabited by
many species of salamander, dissolved oxygen concentrations are relatively uni-
form throughout the water column. In lentic bodies, however, there is often a
declining gradient from the surface, where mixing with air is maximal, to the
bottom, where anaerobic decay is greatest. Because larvae of different species
may occupy different water depths, oxygen concentrations should be taken at
least several centimeters below the surface or at the depth commonly used by
the species of concern.
The measurement of dissolved oxygen is relatively straightforward with one
of the many commercially available electronic dissolved-oxygen meters. For

15

12
Dissolved oxygen (mg/L)

0
12:00 4 8 12:00 4 8 12:00
am am am
Time of day

Fig. 7.1 Hypothetical variation in dissolved oxygen concentration in a warm, semi-eutrophic,

lentic body of water.
108 | Amphibian ecology and conservation

example, YSI Instruments, Orion, Accumet, and Oakton are a few of the com-
panies that manufacture portable field meters. These almost always come with
temperature sensors and may be bundled with conductivity and pH sensors.
Colorimeter kits (e.g. LaMotte, Orion, Chemetrics) are available and tend to be
less expensive than electronic meters but require reagents for every test and may
be less sensitive. When collecting water for oxygen analysis it is important to fill
plastic or glass bottles to the very top without adding air and to keep the bottle
cool until analysis. Ideally, oxygen should be analyzed on site to obtain the most
accurate values.

7.3 Temperature
Because amphibians are poikilotherms they have limited ability to regulate
their body temperature and are greatly affected by the temperature of their sur-
rounding environment. Water temperature, therefore, is extremely important in
affecting metabolic rates, other physiological processes, and behavior.
As a class, amphibians demonstrate a wide tolerance to temperature regi-
mens: some species are able to withstand “temperature changes of 30°C on a
daily basis and up to about 35°C on a seasonal basis” (Rome et al. 1992, p. 183).
Wood frogs (Rana sylvatica) have the most northern distribution of any North
American species (Conant and Collins 1998) and live above the Arctic Circle.
Couch’s spadefoot (Scaphiophus couchii) inhabits very arid, hot desert areas in
the west where temperatures may exceed 45°C. Some species are eurythermic;
for example, American bullfrogs range from central Mexico to northern Maine
and Nova Scotia and tiger salamanders can be found from southern Texas into
Alberta and Saskatchewan. Given time to acclimate, adult and larval amphib-
ians in temperate and northern climes can survive near and even subzero tem-
peratures by becoming dormant and greatly reducing their metabolic rates
(Voituron et al. 2003). Those that inhabit dry, hot regions often burrow into the
ground, enmesh themselves in cocoons, and estivate (Pinder et al. 1992). Some
species, such as northern leopard frogs (Rana pipiens), can shunt water from vital
organs to prevent tissue damage from ice crystal formation and concentrate glu-
cose in organs to serve as a type of anti-freeze (Tattersall and Ultsch 2008).
In general, between 10 and 40°C, each 10° increase in ambient tempera-
ture increases metabolism by 1.4–2.4 times (Rome et al. 1992). Higher meta-
bolic rates require greater oxygen; however, oxygen concentrations decrease
as water temperatures increase (see below). At temperatures near 0°C most
amphibians are very sluggish. At high temperatures, say above 30–35°C for
some species but as low as 25–30°C for less tolerant animals, thermal stress can
 7 Water-quality criteria for amphibians | 109

result in reduced mobility, abnormally high heart rates, and eventually death.
Subtle interactions between temperature and other stressors can also occur. For
example, Anderson et al. (2001) demonstrated complex relationships between
predators and temperature in Pacific treefrogs (Pseudacris regilla). At 25.7°C
tadpoles grew faster and exceeded the size limitation of their insect predators
more quickly than tadpoles raised at 9.9°C. However, tadpoles of similar size
encountered greater predation rates at the warmer temperature. Temperature
can have a strong influence on life history progress (Camp and Marshall 2000)
and microhabitat use (Crawford and Semlitsch 2008) in both aquatic and ter-
restrial amphibians.
There is a direct relationship between water temperature and dissolved oxygen
concentrations. At sea level, where the partial pressure of oxygen, PO, is highest,
the concentration of dissolved oxygen is around 14 ppm at 0°C and declines as
water temperatures increase (Figure 7.2). The same general relationship exists
at higher elevations except that PO saturation concentrations decline. Because
oxygen demand increases as water increases in temperature, physiological stress
can be very high in warm, low oxygenated waters.
Measuring water temperature is straightforward with any digital or analog
thermometer. Many electronic meters used for other water chemistry parameters
such as pH, conductivity, or dissolved oxygen come equipped with thermom-
eters. Some thermometers automatically record minimum and maximum tem-
peratures between readings so that a range can be ascertained. More sophisticated
systems include data loggers that keep a continuous record of temperatures.

15
Dissolved oxygen (mg/L)

13

11

5
0 5 10 15 20 25 30
Temperature (°C)

Fig. 7.2 Relationship between water temperature and dissolved oxygen concentration
at sea level.
110 | Amphibian ecology and conservation

7.4 pH
pH, the negative log of the hydrogen ion concentration in water, is another key
characteristic in describing amphibian habitats. The pH scale ranges from 0 to 14
which corresponds to a solution of 1 M H (100) to 1 M OH with all H bound
with oxygen as a hydroxide. A pH of 7 defines neutral solutions, those with lower
pH values are acidic, and those with higher values are alkaline. For ecological
purposes, pH ranges of 6.0–7.5 are generally considered to be circumneutral or
within a range that should present no harm to most aquatic organisms. A corre-
sponding term, alkalinity, is the ability of water to buffer changes in pH.
A key factor in determining pH is the type of bedrock found within the
watershed. Sedimentary rock such as sandstone, limestone, and dolomite are
high in carbonates and bicarbonates that bind with hydrogen ions and form
natural buffers. Alkalinity is typically high in these waters. Watersheds with a
bedrock of igneous rock such as granite or basalt typically have low alkalinity
due to the absence of carbonates.
Another natural factor affecting pH is the concentration of organic acids
including tannic, fulvic, and humic acids. These organic compounds are weak
acids in that they have low dissociation constants compared to mineral acids.
Organic acids can be important in maintaining a low pH in isolated water bod-
ies with high organic matter such as fens and bogs, but they also serve to some
degree as buffers against anthropogenic sources of acidity.
Two major sources of anthropogenic acidity are acid deposition and acid
mine drainage. Acid deposition comes from the production of nitrous oxides
(NOx) and sulfates (SO42) during the combustion of fossil fuels. These mol-
ecules combine with hydrogen in the air and are deposited on to land and water
either with rain or attached to dust and organic particles that precipitate in
dry form. Once in water the hydrogen and anions dissociate and free hydrogen
ions increase acidity. Acid deposition can be of major concern in wetlands and
streams that lie downwind of major industrial centers or cities and in water-
sheds with low alkalinity. The US Environmental Protection Agency (USEPA)
(2008) is a good reference for current aspects of acid rain and deposition. Acid
mine drainage is most prevalent in unreclaimed mined areas. Inversion of soil
during mining places layers high in sulfides towards the surface and oxidation
of these soils results in acidic seeps and leaching. The USGS (2008b) provides a
good review of acid mine drainage.
Many review articles have been written on the adverse effects of acid depos-
ition and anthropogenic acidity on amphibians (e.g. Sparling 1995; Rowe
and Freda 2000). Variation in sensitivity to low pH occurs at the life stages,
 7 Water-quality criteria for amphibians | 111

population, and species levels. At pH values equal to or below 4.5, embryonic

development may cease entirely. At somewhat higher pH values (≈4.5–5.0)
development continues but hatching is curtailed (Dunson and Connell 1982).
Interspecific differences to low pH have been found in several studies (e.g.
Gosner and Black 1957; Sadinski and Dunson 1992). The critical pH or that
which can cause significant increases in mortality for amphibian embryos
ranges from 5.0 to 3.5 (Freda 1990). Tolerant species include carpenter’s frogs
(Rana virgatipes), wood frogs, and pine barren’s treefrog (Hyla andersoni).
Tolerance to low pH may be acquired in that animals living in acidic envi-
ronments for many generations may have higher tolerance to reduced pH
than those inhabiting circumneutral waters (Andren et al. 1989). The pri-
mary mechanism of toxicity due to low pH is interference with ion transport.
Other effects include compromised immune systems (Brodkin et al. 2003), an
inability of embryos to hatch, reduced growth, and delayed metamorphosis.
Acidic waters can be treated to increase pH. In watersheds with low alkalinity
the addition of limestone to streams can substantially increase pH. Hard water with
high calcium and magnesium concentrations can affect the toxic effects of acidity
on embryos and larvae. Calcium can increase amphibian tolerance to low pH by
reducing the loss of plasma ions. For example, adding 10 mg/L dissolved calcium
to water increased hatching of Jefferson salamander (Ambystoma jeffersonianum)
embryos at pH 4.5 compared with 1 mg/L (Freda and Dunson 1985). Conversely,
calcium reduced hatching in wood frogs at pH 4.5. Freda (1990) explained that the
difference in response to calcium was related to a curling effect seen in embryos
that is elicited by H and by calcium. Embryos that can hatch even though curled
are benefited by calcium whereas those that cannot hatch when curled suffer.
Metals are more soluble with reduced pH and may interact with acidity to
influence toxicity. Aluminum is particularly of concern because it is the most
common metal in the Earth’s crust, is soluble with low pH, and changes its spe-
ciation form as pH varies (reviewed by Sparling and Lowe 1996). Aluminum
interacts with acidity in complex ways (e.g. Skei and Dolmen 2006) and a com-
plete review of this interaction is beyond the scope of this chapter.
A common way of measuring pH is through one of the many commercially
available pH meters. A typical meter will accurately measure pH to 0.01 pH
units. If budgets are limited, however, pH papers with different ranges can be
purchased and they are accurate to about 0.2 pH units. As with other water-
quality measurements, it is usually preferable to assess water several centimeters
below the surface. Sealed probes can be submerged to the desired depth or water
can be collected in narrow-mouthed bottles for open or pH papers. The meas-
urement of pH is temperature sensitive and most meters are set to 25°C for
112 | Amphibian ecology and conservation

optimal accuracy. For field measurements, therefore, I recommend meters with

automatic temperature compensation (ATC); these simultaneously record tem-
perature and adjust the pH values accordingly.

7.5 Conductivity, hardness, and salinity

Conductivity is the ability of water to carry an electrical current and is due to
the total concentration of anions and cations in water, collectively called total
dissolved solids (TDS) in water. It is measured in units of micro-Siemens per
centimeter or μS/cm. For comparison, ultrapure water has a conductivity of
5.5  103 μS/cm, drinking water around 5–50 μS/cm and sea water around
5000 μS/cm. Waters that have conductivity ranging between 150 and 500 μS/cm
are adequate for aquatic organisms (USEPA; www.epa.gov/volunteer/stream/
vms59.html).
Hardness can be measured as either the concentration (mg/L) of calcium ions
or the sum of calcium and magnesium ions in water. These cations help buffer
the effects of acidity and alter the toxic effects of dissolved metals (Horne and
Dunson 1995b). Hard water is traditionally defined as a calcium-equivalent
concentration of 80–120 mg/L and soft water as 0–20 mg/L. Moderate to hard
water tends to be better for amphibians than soft water because calcium and
magnesium can ameliorate the toxic effects of metals and pH, improve osmo-
regulation, and are required for proper formation of bones and other physio-
logical processes (Ultsch et al. 1999).
Salinity is the concentration of chloride salts in water and is usually measured
in parts per thousand (g/L or ‰), molarity (mM), or osmoles (mOsm). Most
amphibians have low tolerance to salinity because they are not good osmoregula-
tors (Gomez-Mestre et al. 2004). Balinksy (1981) listed 64 species of anurans and
13 species of urodeles that are tolerant to brackish water, although none are truly
marine. Among the most tolerant species are cane toads (Bufo marinus), crab-
eating frog (Rana cancrivora), African clawed frog (Xenopus laevis), European
green toad (Bufo viridis), and some Batrachoseps spp. salamanders (Shoemaker et al.
1992). Euryhaline species can live in water that is 600–800 mOsm (70–93 ppm
NaCl) but adults of most others do best at 200–300 mOsm (23–34 ppm NaCl;
Shoemaker et al. 1992). Relatively little data are available on tadpoles but, in gen-
eral, the lower the salinity the better. Road salts may pose problems for amphib-
ians inhabiting ponds located near roads (Karraker et al. 2008).
As with other water-quality factors mentioned above, there are electronic
meters and colorimeter kits available to measure these parameters.
 7 Water-quality criteria for amphibians | 113

7.6 Total and dissolved organic carbon

Organic carbon comes from organic molecules as contrasted to inorganic car-
bon that is derived from carbon dioxide and carbonates. Total organic car-
bon includes all sizes of organic molecules, both dissolved and non-dissolved.
Dissolved organic carbon consists of organic molecules smaller than 0.45 μm
in diameter. This carbon comes from many sources including surface run-off,
aerial deposition, and decomposition of aquatic organisms. Elevated concen-
trations of organic carbon, along with suspended soil particles, are principal
constituents of total suspended solids (TSS) and indicate potential problems
with run-off into streams and lakes. Organic carbons are used as nutrients by
bacteria and can increase biological oxygen demand. However, moderate levels
of organic carbon are important because these molecules are a source of food
for microorganisms. They also bind with certain pollutants such as metals and
reduce their availability to aquatic organisms.
Dissolved organic carbon (DOC) is ecologically important to amphibians.
Banks et al. (2007) showed a positive relationship between DOC concentration
in wetlands of Acadia National Park (range 2–18 mg/L) and the concentration of
methylated mercury (MeHg) in sediments and northern green frog (Rana clami-
tans) and bullfrog tadpoles. Because MeHg is more bioavailable and toxic than
inorganic mercury, DOC in this situation can be detrimental to amphibians. In
contrast, some forms of DOC, such as tannins, cause a brownish coloration to
the water. These DOCs reduce light penetration into water which may curtail
primary productivity (Carpenter et al. 2001). They also significantly suppress
penetration of ultraviolet-B radiation which has been linked to malformations.
Diamond et al. (2005) determined that DOC reduced ultraviolet-B penetration
in the top 1 cm of water up to 87% and Calfee et al. (2006) determined that DOC
protected salamander embryos from harmful ultraviolet-B exposure. Freda (1991)
demonstrated that DOC can mitigate the toxicity of aluminum and other metals
in acidified waters. However, humic and fulvic acids in excess of 10–20 mg/L can
be toxic to amphibians (Freda et al. 1989; Horne and Dunson 1995a).
The analysis of TOC and DOC is a bit more complex than the other ana-
lytes so far described. Briefly, the first step is to separate inorganic and organic
carbons in the water. This can be done by acidifying the water, thus converting
carbonates to carbon dioxide which is then sparged from the water. The remain-
ing carbon is considered organic. An aliquot of this is filtered through a 0.45 μm
membrane and the carbon in the filtrate is DOC. Both TOC and DOC can be
measured on a carbon analyzer or other instruments.
114 | Amphibian ecology and conservation

Colored DOCs (tannic, humic, and fulvic acids) can be measured indir-
ectly by comparing the color of water to a set of standards available in kit form.
Turbidity, which is affected by the total amount of inorganic and organic sus-
pended particles, can be measured with a turibidimeter that measures light
penetration through a standardized vial in units called nephelometric turbidity
units (NTUs).

7.7 Pollutants
The number of types of pollutants or contaminants that can affect amphibian
populations is enormous and beyond the scope of this brief chapter. Sparling
(2003) surveyed the potential effects of contaminants on amphibians and
Sparling et al. (2000) provided an extensive review of contaminant effects on
amphibians and reptiles. Here I list a brief summary of contaminant classes and
their significance.

7.7.1 Fertilizers and nitrogenous compounds

There are several ways of measuring ammonia and nitrates in water, includ-
ing: (1) ion chromatography (for nitrates); (2) ion-specific probes coupled with
a pH meter that registers millivolt output; (3) colorimetry; (4) test papers;
and (5) other methods for sewage treatment operations and large volumes of
material. Of the first four methods, ion-specific probes are less expensive than
purchasing an ion chromatograph and are more sensitive and accurate than col-
orimetry or test papers. Test papers, although least expensive, are more limited
in their resolution and detection limits than the other methods.

7.7.2 Pesticides
Pesticides are chemicals that are used to control plant and animal species con-
sidered to be noxious or unwanted by humans. The types of pesticides can be
distinguished by intended target and by chemical class, which usually corres-
ponds to primary mode of action. By target the primary classes are insecticides,
fungicides, herbicides, and rodenticides.
Very roughly and with exceptions, insecticides are most acutely toxic to
amphibians and other aquatic organisms of these classes. However, insecti-
cides, fungicides, and herbicides exert a wide variety of sublethal or chronic
effects that affect individual and population health. Rodenticides are not of
much concern to amphibians, simply because of how they are used. There is a
large and growing number of chemical families used as pesticides (see Cowman
and Mazanti 2000 for a more complete review). Of greatest concern due to
 7 Water-quality criteria for amphibians | 115

their widespread usage are organophosphates, carbamates, pyrethrins, and a few

organochlorine insecticides. These are neurotoxins and either function at the
synapse between neurons (organophospates and carbamates) or by affecting the
transmission of action potentials down axons. In addition to direct mortality,
pesticides can reduce growth, inhibit development, affect escape and predatory
behavior, cause genotoxicity, affect gonad development, and alter other physio-
logical processes (see Cowman and Mazanti 2000; Sparling 2003) and inter-
act with other stressors (e.g. McIntyre and McCollum 2000; Relyea and Mills
2001). Aerial spraying of pesticides results in airborne particles that can travel
many kilometers from the point of application to affect remote populations of
amphibians (Davidson et al. 2001; Sparling et al. 2001).
There is no field method to determine the presence or concentration of pesti-
cides in that their analyses are complex and often expensive. Matrix samples
(sediment, water, or organisms) need to be collected and brought to a laboratory
where the pesticides are extracted and analyzed using high-performance liquid
chromatography (HPLC), gas chromatography, or other methods, all following
recognized protocols.

7.7.3 Metals
Metals and metalloids such as zinc, copper, lead, chromium, arsenic, cadmium,
mercury, selenium, and others are naturally occurring elements but are also
released through many different industrial processes at concentrations that can
be toxic to amphibians. Linder and Grillitsch (2000) reviewed the ecotoxicol-
ogy of metals on amphibians and reptiles and showed that effects can range
from direct mortality to a host of sublethal and potentially debilitating effects.
Toxicity is affected by pH in that metal solubility increases with acidity and sol-
uble forms of metals are more bioavailable than non-soluble forms.
Ion-specific probes are available for lead, silver, and copper, but the method
for measuring is more complicated than inserting the probe directly into a water
sample and should be done under laboratory conditions. Fortunately, metals are
very stable, and once samples have been acidified to pH
2 they can be stored
for several months before analysis. Colorimetric methods exist for iron, man-
ganese, aluminum, molybdenum, and copper, but most metals are analyzed
through atomic absorption spectrophotometry or ion-coupled spectrophotom-
etry (ICP) in a laboratory.

7.7.4 Organic pollutants and halogenated hydrocarbons

For this chapter, I include polycyclic aromatic hydrocarbons (PAHs), poly-
chlorinated biphenyls (PCBs), dioxins, furans, and chlorinated pesticides
116 | Amphibian ecology and conservation

(e.g. DDT, toxaphene, dieldrin) in this category. All of these chemicals are
organically based and some have chlorine or other halogens incorporated into
the molecular structure. PAHs include benzene, toluene, benzo[a]pyrene, and
hundreds of other molecules that form through natural processes of decompos-
ition, but also come from combustion of fossil fuels, processing of petroleum,
and other sources. PCBs were used as lubricating fluids and in electrical trans-
formers until the 1970s when their use in North America was banned. Dioxins
and furans are formed during forest fires but also through the combustion
of fossil fuels and are strong carcinogens. Similarly, many of the chlorinated
hydrocarbon pesticides have been banned because they are extremely persist-
ent in the environment and cause cancer, genotoxicity, endocrine disruption,
and other harmful effects to humans and wildlife. Nevertheless, because they
are extremely persistent, they can still be found throughout the world. At most
environmentally realistic concentrations these chemicals produce a variety of
sublethal effects including genotoxicity, inhibition of growth and develop-
ment, endocrine disruption, skeletal defects and malformations, and reduced
hatching success (reviewed by Sparling 2000). Sophisticated laboratory methods
including HPLC or gas chromatography are required for the analysis of these
chemicals.

7.7.5 Pharmaceuticals
Ecotoxicologists are just becoming aware of a myriad of chemicals that pass
through our waste-treatment plants and enter natural bodies of waters essen-
tially intact. These include prescription and non-prescription medicines, anti-
biotics, caffeine, and other products that pass through human bodies or are
disposed of into toilets and down drains. These chemicals can exert numerous
effects such as endocrine disruption and many physiological and behavioral
changes that have yet to be defined. Because our awareness of these substances is
so new, methods for determining their concentrations in field-collected matrices
are often not available. Others can be determined through gas chromatography/
mass spectrophotometry.

7.8 Summary and conclusions

In summary, students of amphibian ecology and behavior must be aware of
various chemical and physical factors. These, in addition to vegetation, sub-
strate composition, structural elements in the environment, water depth, stream
flow, and the presence of other vertebrate and invertebrate species constitute the
basic elements of habitat assessment for amphibians. There are many different
 7 Water-quality criteria for amphibians | 117

methods of measuring these factors and the choice of method should be based
on desired accuracy and precision as well as economics.

7.9 References
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(Ambystoma) species to ultraviolet radiation. Journal of Herpetology, 40, 35–42.
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Amphibians and Reptiles, pp. 233–68. SETAC Press, Pensacola, FL.
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for the Examination of Water and Waste Water, 21st edn. American Public Health
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Feder, M. E. and Burggren, W. W. (1992). Environmental Physiology of the Amphibians.
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amphibians Bufo bufo and Triturus vulgaris. Canadian Journal of Zoology, 84, 1668–77.
Sparling, D. W. (1995). Acidic deposition: a review of biological effects. In D. Hoffman,
B. A. Rattner, G. A. Burton, and J. Cairns Jr (eds), Handbook of Ecotoxicology,
pp. 301–29. Lewis Publishers, Boca Raton, FL.
Sparling, D. W. (2000). Ecotoxicology of organic contaminants to amphibians. In
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Reptiles, pp. 461–94. SETAC Press, Pensacola, FL.
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118–26.
 Part 3
Juveniles and adults
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 8
Measuring and marking
post-metamorphic amphibians
John W. Ferner

8.1 Introduction
In this chapter, I review marking and measuring techniques for post-metamorphic
amphibians; larvae are covered in Chapter 4 in this volume. Measuring the size
of individuals is important for determining age, categorizing life history stages
(juvenile or adult), and calculating growth rates (larval measurements are covered
in Chapter 3). More field and behavioral studies requiring marking are now being
conducted than in the past, and often investigators need very specific marking
techniques and non-invasive ways of identifying individuals. Additionally, ethical
issues relative to certain techniques, such as radioactive tags and mutilation pro-
cedures, are now more commonly considered as, for example, when considering
toe-clipping. Some older techniques are used much less often than they used to
be. Major advancements in biotelemetry (see Chapter 11) and passive integrated
transponder (PIT) tags have added needed flexibility to the choice of marking
methods.
Recent reviews of marking and identification techniques include Baker and
Gent (1998) and Ferner (2007) for amphibians and reptiles, and Donnelly et al.
(1994) for amphibians. Additional references are found in a number of papers
published in a special edition of Mertensiella (Henle and Veith 1997).
Some criteria for ideal marks or tags are as follows (after Ferner 2007):
• they should not affect the survivorship or behavior of the organism;
• they should allow the animal to be as free from stress or pain as possible;
• they should identify the animal as a particular individual, if desirable;
• they should last indefinitely, or at least the duration of the study;
• they should be easily read and/or observable;
124 | Amphibian ecology and conservation

• they should be adaptable to organisms of different sizes;

• they should be easy to use in both laboratory and field, and use easily
obtained material at minimal cost.
There are no techniques that satisfy all of these criteria. Selecting a technique
requires deciding which are the most feasible and scientifically accurate in meet-
ing the objectives of the particular study. Two or more marks often are used to
meet multiple study objectives or to serve as a back-up to the primary mark (e.g.
simultaneous toe-clipping and photographing of dorsal patterns). Techniques

Table 8.1 Sources of materials for marking amphibian species (as reviewed in Ferner
2007 unless otherwise indicated).
Technique item Source of materials

Adhesive (small wounds) Vetbond®

®
Bandage (New Skin ) Medtech (www.newskinproducts.com)
Beads, balls, and elastic Ball Chain Manufacturing Co. (www.ballchain.com)
for waistbands Bead Industries (www.beadindustries.com)
B. Toucan (www.btoucan.com)
Fluorescent pigments for Scientific Marking Materials, PO Box 23122,
branding Seattle, WA 98124, USA
Radiant Color Company, 2800 Radiant Ave.,
Richmond, CA 94804, USA (+1 800 Radiant;
fax +1 510 233 9138)
Fluorescent elastomer dyes; Northwest Marine Technology, PO Box 427, 976
visual implant elastomers (VIEs) Ben Nevis Road, Shaw Island, WA 98286, USA
(www.nmt.us)
PIT tags Destron Fearing Corporation/Digital Angel,
St. Paul, MN, USA (www.destronfearing.com,
www.digitalangel.com)
Also see www.biomark.com
PIT pack Battery powered Destron Fearing transceiver
(model FS 2001 A-ISO) and custom-built
antenna (Blomquist et al. 2008)
Scanner (for PIT tags) AVID Marketing, Norco, CA USA
Fluorescent tags (VIAlpha Northwest Marine Technology
Numeric Tags) (see address above)
Ultraviolet lamp for UVP, San Gabriel, CA, USA (www.uvp.com)
detecting fluorescents
PIT, passive integrated transponder.
 8 Working with post-metamorphic amphibians | 125

described for use with one group of amphibians might be adapted for a dif-
ferent group. Therefore, investigators might benefit by considering all of the
techniques available, regardless of how originally used. Further, there may be a
need for standardizing marking techniques in comparative studies, or those that
might be carried on by other researchers in the future.
In the following text, I emphasize the use of marking techniques for adult
amphibians which have proved most successful. When available, information
on the advantages and disadvantages of each technique has been given. This
chapter provides information adequate for considering techniques without
consulting the original source. However, once a technique has been tentatively
selected, it is advisable to consult the primary literature if time and facilities
permit. The use of complex techniques, such as telemetry or PIT tags, requires
careful review of the original sources. Sources for materials used with various
techniques are given in Table 8.1, although availability and contact information
are likely to change constantly.

8.2 Toe-clipping
8.2.1 Anurans
Although several toe-clipping methods are proposed in the literature, that of
Martof (1953) is most widely used. He cut toes of Rana clamitans with scissors,
trying to avoid damage to webbing between them. Little blood loss or loss of
swimming power was observed and no regeneration of the digits was found.
This system assigns serial numbers to the digits of the feet. No more than two
toes were removed from any one foot. The left hind foot indicated units, and
for those larger than five, combinations of two digits (the fifth and one other)
were excised. For example, clipping the fifth and third toes designated tens and
the front feet denoted hundreds. With this arrangement, 6399 individuals can
be marked in series; the greatest number can be indicated by removing the two
outer digits on each foot. The only concern Martof expressed about this pro-
cedure was that confusion might arise where only a single toe on a single hand
foot was removed with recapture of a frog with an injured foot. He suggested
alleviating this problem with a special designation, such as a zero marking by
removal of the second and fourth toes on the other hind foot. Waichman (1992)
also proposed a numbering system for toe-clipping that letters the four limbs
(A through D) and numbers the toes. This scheme makes all 959 combinations
available using two and three toes, but not removing more than two toes for each
limb (see Table 8.2).
Table 8.2 Alphanumeric code for toe-clipping amphibians and reptiles from Waichman (1992)
One toe Two toes A2A5 A3D2 A5B5 B1D4 B4C1
A2B1 A3D3 A5C1 B1D5 B4C2
A2B2 A3D4 A5C2 B2B3 B4C3
A1 A1A2 A2B3 A3D5 A5C3 B2B3 B4C4
A2 A1A3 A2B4 A4A5 A5C4 B2B4 B4C5
A3 A1A4 A2B5 A4B1 A5C5 B2B5 B4D1
A4 A1A5 A2C1 A4B2 A5D1 B2D1 B4D2
A5 A1B1 A2C2 A4B3 A5D2 B2D2 B4D3
B1 A1B2 A2C3 A4B4 A5D3 B2D3 B4D4
B2 A1B3 A2C4 A4B5 A5D3 B2D4 B4D5
B3 A1B4 A2C5 A4C1 A5D4 B2D5 B5C1
B4 A1B5 A2D1 A4C2 A5D5 B3B4 B5C2
B5 A1C1 A2D2 A4C3 B1B2 B3B5 B5C2
C1 A1C2 A2D3 A4C4 B1B3 B3C1 B5C3
C2 A1C3 A2D4 A4C5 B1B4 B3C2 B5C4
C3 A1C4 A2D5 A4D1 B1B5 B3C3 B5C5
C4 A1C5 A3A4 A4D2 B1C1 B3C4 B5D1
C5 A1D1 A3A5 A4D3 B1C2 B3C5 B5D2
D1 A1D2 A3C1 A4D4 B1C3 B3D1 B5D3
D2 A1D3 A3C2 A4D5 B1C4 B3D2 B5D4
D3 A1D4 A3C3 A5B1 B1C5 B3D3 B5D5
D4 A1D5 A3C4 A5B2 B1D1 B3D4 C1C2
D5 A1A3 A3C5 A5B3 B1D2 B3D5 C1C3
A1A4 A3D1 A5B4 B1D3 B4B5 C1C4
C1C5 C5D3 A1A2C3 A1A4B2 A1A5D1 A2A4B5 A2A5D4
C1D1 C5D4 A1A2C4 A1A4B3 A1A5D2 A2A4C1 A2A5D5
C1D2 C5D5 A1A2C5 A1A4B4 A1A5D3 A2A4C2 A3A4B1
C1D3 D1D2 A1A2D1 A1A4B5 A1A5D4 A2A4C3 A3A4B2
C1D4 D1D3 A1A2D2 A1A4C1 A1A5D5 A2A4C4 A3A4B3
C1D5 D1D4 A1A2D3 A1A4C2 A2A3B1 A2A4C5 A3A4B4
C2C3 D1D5 A1A2D4 A1A4C3 A2A3B2 A2A4D1 A3A4B5
C2C4 D2D3 A1A2D5 A1A4C4 A2A3B3 A2A4D2 A3A4C1
C2C5 D2D4 A1A3B1 A1A4C5 A2A3B4 A2A4D3 A3A4C2
C3C4 D2D5 A1A3B2 A1A4D1 A2A3B5 A2A4D4 A3A4C3
C3C5 D3D4 A1A3B3 A1A4D2 A2A3C1 A2A4D5 A3A4C4
C3D1 D3D5 A1A3B4 A1A4D3 A2A3C2 A2A5B1 A3A4C5
C3D2 D4D5 A1A3B5 A1A4D4 A2A3C3 A2A5B2 A3A4D1
C3D3 A1A3C1 A1A4D5 A2A3C4 A2A5B3 A3A4D2
C3D4 Three toes A1A3C2 A1A5B1 A2A3C5 A2A5B4 A3A4D3
C3D5 A1A3C3 A1A5B2 A2A3D1 A2A5B5 A3A4D4
C4C5 A1A3C4 A1A5B3 A2A3D2 A2A5C1 A3A4D5
C4D1 A1A2B1 A1A3C5 A1A5B4 A2A3D3 A2A5C2 A3A5B1
C4D2 A1A2B2 A1A3D1 A1A5B5 A2A3D4 A2A5C3 A3A5B2
C4D3 A1A2B3 A1A3D2 A1A5C1 A2A3D5 A2A5C4 A3A5B3
C4D4 A1A2B4 A1A3D3 A1A5C2 A2A4B1 A2A5C5 A3A5B4
C4D5 A1A2B5 A1A3D4 A1A5C3 A2A4B2 A2A5D1 A3A5B5
C5D1 A1A2C1 A1A3D5 A1A5C4 A2A4B3 A2A5D2 A3A5C1
C5D2 A1A2C2 A1A4B1 A1A5C5 A2A4B4 A2A5D3 and so on
Codes A5 and B5 are available only for animals with five foretoes.
128 | Amphibian ecology and conservation

Regardless of the numbering system used, the potential for toe regeneration
must be considered (see Table 8.3). In addition, the thumbs of the forefeet are
required by males during amplexus, and they should not be clipped (for example,
Briggs and Storm 1970). Another potential problem is understanding how toe-
clipping may stress an animal and affect resource acquisition. Daugherty (1976)
indicated a problem with weight loss in toe-clipped Rana pipiens. The most ser-
ious criticism of toe-clipping anurans was raised by Clarke (1972), who noted
that the probability of recapturing Bufo fowleri decreased as the number of digits
excised increased; researchers today do not clip as many toes as Clarke, however.
Halliday (1995) reported that studies of Atelopus elegans, Atelopus carbonerensis,
Bufo marinus, Bufo granulosus, and Bufo bufo provided little or no evidence for
any impact on survival after toe-clipping. An assessment and concern about use
of toe-clipping of Hyla labialis was provided by Lüddecke and Amezquita (1999).
Fewer than 1% of toe-clipped Rana pretiosa showed any sign of infection, and no
mortality was found (Reaser and Dexter 1996). They stressed that hygienic, ster-
ile techniques must be used, and that impacts should be carefully monitored in

Table 8.3 Digit-regeneration occurrence and times for selected toe-clipped amphibian
species
Species Time interval for regeneration

Anurans
Bufo hemiophrys Several months but recognizable
Hyla regilla Only slight after 1 year
Hyperolius viridiflavus ferniquei Complete in newly metamorphosed
Rana catesbeiana None in 3-year study
Rana erythraea Slow or nonexistent over 4 years
Caudates
Ambystoma opacum Some in short period
Taricha spp. Several years
Taricha granulosa Indefinitely, if kept below or at 10C
Triturus cristatus Slow compared to tail clips
Triturus vulgaris Up to 10 months; little winter regeneration
(Griffiths 1984)
Plethodon cinereus 7 months in laboratory
Plethodon glutinosus At least 2 years
Plethodon wehrlei 50% after 100 days; regenerated digits
identifiable by lack of pigmentation
Cryptobranchus alleganiensis 1 year
Batrochoseps spp. Slow; regenerated toes recognizable
After review by Ferner (2007) unless otherwise indicated.
 8 Working with post-metamorphic amphibians | 129

studies using toe-clipping. Despite these potential problems, toe-clipping is still

the most common marking technique used for anurans.

8.2.2 Salamanders
Toe-clipping has been the common method of marking salamanders in recent
years, and in most cases something similar to Martof ’s (1953) system of mark-
ing anurans is used. Another scheme (as above) was provided by Waichman
(1992) (Table 8.2). The potential of toe regeneration needs to be considered in
salamanders, but whether or not it is a deterrent to using toe-clipping depends
upon the species and study objectives. Table 8.3 summarizes reports in the lit-
erature concerning the regeneration of digits in salamanders.
Some salamanders, such as juvenile (
2.5 cm snout–vent length, or SVL)
dusky salamanders (Desmognathus fuscus), have toes too small for precise clip-
ping. In such cases, animals have been marked successfully by clipping small
pieces of the tail at either right angles to the longitudinal axis of the body or in
the transverse plane at different angles (Orser and Shine 1972). This technique
allowed groups of marked juveniles to be distinguished for at least 1 month
before regeneration became a problem.
Mutilation techniques, such as toe-clipping, may use some form of local or
general anesthesia. Peterman and Semlitsch (2006) stressed the importance of
properly buffering solutions of the anesthetic MS-222 in plethodontid salaman-
ders, and the effect of various concentrations on the time to become anesthe-
tized and recover. They found induction time usually decreased, and the time
for recovery increased, with increasing concentrations of MS-222. This study
should be consulted before using this anesthetic.
Davis and Ovaska (2001) found that toe-clipping in Plethodon vehiculum
resulted in less weight gain than animals marked with fluorescent tags or used
as controls. These authors used unique combinations of three clipped toes per
salamander, removing no more than one toe per foot. The number of ambigu-
ous marks in toe-clipped individuals increased dramatically in frequency com-
pared with the fluorescent tagged animals after 50 days, but the majority of both
retained useful marks throughout the 87-week field study.

8.2.3 Ethical issues related to toe-clipping of amphibians

In recent years there has been increasing concern over the possible impact of vari-
ous marking techniques on animals. Mutilation techniques such as toe-clipping
have been reviewed in several studies to determine their impact on survival. In
addition, ethical issues relative to the possibility of inflicting pain on organisms
are a concern. University Institutional Animal Care Committees (IACUC) in
130 | Amphibian ecology and conservation

the USA may request documentation on marking techniques and consideration

of several alternatives. For guidelines see www.ssarherps.org/pages/ethics.php.
In Europe legislation is often even more strict and a license is required before any
invasive surgical procedure including scientific studies using PIT tagging.
Parris and McCarthy (2001) reported evidence that toe-clipping anurans
decreases the rate of return of animals in mark–recapture studies. They encour-
aged clipping as few toes as possible, and stated that researchers must control for
the impact of clipping on the rate of recapture. They further pointed out that
these results have “important implications for the ethical treatment of animals,
the continued use of toe-clipping to mark species of conservation concern, and the
removal of multiple toes from an individual frog or toad” (McCarthy and Parris
2004). The debate on toe-clipping anurans continues (summarized by Ferner
2007 and Phillott et al. 2008).

8.3 Branding
8.3.1 Anurans
Kaplan (1958) described a branding technique for frogs involving the incorp-
oration of India ink into scarified skin of the venter. Numbers were etched in
the skin with a hypodermic needle and then filled with India ink. Erythema
disappeared quickly, no infection was observed, and the numerals remained
distinguishable for more than 3 months. Kaplan also described an electric tat-
tooing technique as an improvement upon the scarification method. Higgins
India Ink™ was found most effective if mixed with a drop of glycerin to aid its
spreading into the skin. Surplus ink was wiped away, leaving a clear, permanent
mark. Some initial inflammation was observed in the frogs, but was only rarely
prolonged. Additional branding techniques are provided in Table 8.4.

Table 8.4 Branding techniques used for marking amphibian species (as reviewed in
Ferner 2007)
Species Description of branding technique

Anurans Scarring with needle or electric tattoo marker then

filled with India ink
Heated chromium nickel wire
Silver nitrate (75%), potassium nitrate (25%)
Ascaphus truei Freeze branding with copper wire cooled in dry ice
Bufo bufo Alcian Blue dye solution using a “Panjet®” innoculator
Eleutherodactylus podiciferus Pressurized fluorescent granular powder
 8 Working with post-metamorphic amphibians | 131

8.3.2 Salamanders
Salamander branding techniques involve heat or freeze brands, or the application
of pigments or dyes into scarified skin. Taber et al. (1975) branded Cryptobranchus
alleganiensis with heated 1.5-cm-high numerical brands of thin nickel-chrome
wire. These remained clear through a 2-year study, but some faded and required
rebranding. Bull et al. (1983) tested freeze-branding application times while mark-
ing Ambystoma macrodactylum. Letter brands of 5 mm high made with silver-tipped
copper rods were immersed for 5 min in liquid nitrogen. A 0.75-s application pro-
duced the best results, with maximum clarity occurring at 3 months. Woolley
(1962) used a black Carter’s felt ink pencil to mark salamanders. Dilute acetic acid
or ammonium hydroxide was used to remove mucus on the salamander’s tail before
application. These marks lasted at least a month.
Taylor and Deegan (1982) injected a fluorescent pigment into red eft
(Notophthalmus viridescens) dermis using a compressed air spray gun with minimal
mortality. After 39 days, individuals began succumbing to water mold (Saprolegnia)
infections, but these could not be linked to the use of fluorescent branding.
Nishikawa and Service (1988) described a technique using dry fluorescent
dust applied with pressurized air on Plethodon jordoni and Plethodon glutino-
sus. The technique required four to 10 colours of inert fluorescent pigments,
canisters, a spray gun with 6.35 mm nozzle, a hose, a single-stage regulator,
and pressurized air. Animals were placed on a dry enamel pan, and the spray
gun was held 1 cm away from the skin surface. Marks were applied using lower
pressures (25 psi) for smaller animals and greater pressures (40 psi) for larger
animals, depending on the size of the fluorescent particles used. Three to four
marks could be applied to each animal on either side of the body (cranial and
caudal to the forelimb, mid-body, and cranial and caudal to the hind limb) for
a maximum of 10 possible locations. Nishikawa and Service (1988) reported
higher recapture rates than for any salamander toe-clipping studies performed
to that time.

8.4 Tagging and banding

The use of any external tag or band must be considered carefully, as the device may
impede the movement of the animal or snag on the substrate and vegetation.

8.4.1 Anurans
Kaplan (1958) used aluminum toe bands to tag frogs (“butt-end” bird band,
#1242, size 2½). These numbered, cylindrical bands were placed around a toe, and
the two ends were pressed together with pliers. The bands were tightened so as not
132 | Amphibian ecology and conservation

to restrict circulation, but they pierced the webbing of the foot. These remained
fixed indefinitely and caused no apparent hindrance in the movement of the frog.
A variety of tags and bands used for anurans is summarized in Table 8.5.
McAllister et al. (2004) recommended against the knee tags used by Watson
et al. (2003) in R. pretiosa due to skin and muscle lacerations in 33% of the
recaptured animals. Emlen (1968) reported no differences in behavior, mor-
tality, emigration rates, or weight loss between tagged and untagged American
bullfrogs (Rana catesbeiana). Due to soiling and staining, seasonal replacement
of waistbands was necessary. However, Robertson (1984) reported a negative
impact using Emlen’s (1968) waistband design on 10 species of Australian hylids
and leptodactylids.
Windmiller (1996) suspended yarn tags in a plastic bag filled with fluorescent
pigment which was pressed into the yarn, and attached to the dorsal integument
of juvenile ranid frogs. The pigment trails were followed at night by illuminat-
ing them with a longwave (366 nm) ultraviolet lamp (Blak-Ray ® model ML-49)
while wearing safety/ultraviolet-enhancing glasses. Buchan et al. (2005) used
soft VIAlpha Numeric Tags (US $1.00 per tag) and an injector (US$120) to
insert the tag. Buchan et al. (2005) found high survivorship, retention, and
readability of these tags in both the laboratory and field.

Table 8.5 Tagging and banding techniques used for marking amphibian species (as
reviewed in Ferner 2007 unless otherwise specified)
Species Description of tagging/banding technique

Anurans Eppendorf microcentrifuge tubes filled with Cyalume™ luminescent

fluid glued to dorsum
Soft VIAlpha Numeric Tags® (fluorescent) with imprinted letters and
numbers inserted into sartorius muscle
Bufo bufo Knee-tagging (Elmberg 1989; Kuhn 1994)
Rana catesbeiana Painted nylon waistband around pelvic girdle
Rana catesbeiana and Fluorescent acrylic yarn held between squares of self-adhering
Rana clamitans verterinary bandage and glued to dorsum
Rana clamitans Waistband using surgical thread sewn through piece of surveyor’s
flagging
Rana pretiosa Numeric-coded fingerling tags attached over knee with elastic
thread
Uperoleia rugosa 1-mm squares Scotchlite® reflective sheeting attached with
cyanoacrylate tissue cement
Xenopus laevis Glass beads attached to forelimb with surgical wire
 8 Working with post-metamorphic amphibians | 133

Visual implant elastomers (VIEs), as described for salamanders below, are

a very useful mark for anuran larvae. Their potential for use in marking adult
anurans may not be fully appreciated.

8.4.2 Salamanders
Woolley (1973) tagged Eurycea lucifuga and Eurycea longicauda with a sub-
cutaneous injection of two parts Liquitex acrylic polymer to one part distilled
water. This mixture was injected into the lateral proximal caudal region with
a 22-gauge hypodermic needle, leaving a mark 7–10 mm in diameter. Woolley
observed no adverse effects with this procedure and found slight fading in very
few individuals. The author suggested that a series of acrylic colours be used to
differentiate individuals.
Subcutaneous injections of fluorescent, elastomer dyes were used in Plethodon
vehiculum by Davis and Ovaska (2001). Red, orange, or yellow dyes (VIEs)
were injected into six locations in various combinations to create 816 unique
marks. The elastomer was mixed with a hardener following the manufacturer’s
instructions, and 0.1 mL was placed in a 0.3 mL syringe. The authors recom-
mend fluorescent marking or pattern mapping over toe-clipping in studies of
plethodontid salamanders.
Bailey (2004) evaluated VIE marking in Eurycea bislineata by documenting
mark retention, salamander survivorship, and growth rates. No adverse effects
and no loss of marks were found over the 11-month study. Misreading marks
in the field by observers was found to be a factor in some cases, so a training
period for investigators is recommended to minimize observer bias. Rittenhouse
et al. (2006) used the same technique with powdered fluorescent pigments with
newly metamorphosed Ambystoma maculatum, as described for Rana sylvatica
(see Table 8.4). No temporal effects of powder-dusting were detected.

8.4.3 Caecilians
Gower et al. (2006) reviewed the use of alphanumeric fluorescent tags (VIAlpha
tags) on the Indian caecilian Gegeneophis ramaswamii. They found that making
an incision with a scalpel blade before using the injector increased the efficiency
of tagging, and that anesthesia was needed to quiet specimens. Equipment ster-
ilization between uses minimizes the risk of spreading pathogens.

8.5 Trailing devices

As with bands and tags, the use of trailing devises must be carefully considered
because of the potential to impair the movement of animals. They may be very
134 | Amphibian ecology and conservation

Table 8.6 Trailing devices used for marking amphibian species (as reviewed in Ferner
2007 unless otherwise indicated)
Species Description of trailing technique

Ambystoma tigrinum Aquatic forms tagged with trailing plastic float on

monofilament line sutured in tail
Bufo bufo Sewing thread on bobbin fastened around waist with
elastic band (Sinsch 1988)
Bufo valliceps Cotton thread bobbin on rigid plastic tubing holder on
elastic band
Leptodactylus labyrinthicus Cotton thread spool-and-line quilting cocoon on 1 cm
inguinal elastic band
Rana pipiens Nylon sewing thread on rigid plastic tubing holder on
elastic band
Rana sylvatica Metamorphs dipped in fluorescent powder pigments
leaving trail

difficult to attach, and they may snag on the substrate and vegetation. Still, they
are useful in certain applications. Table 8.6 reviews these techniques.

8.6 Pattern mapping

The patterns and markings of many species are distinctive and yet have a high
degree of individual variation. Photographing or sketching these patterns may be
useful for individual recognition on subsequent recaptures (Meyer and Grosse
1997; Streich et al. 1997; Winkler and Heunisch 1997).

8.6.1 Anurans
While more commonly used in salamanders, the patterns of anurans may be
unique enough to make individual recognition possible. Wengert and Gabrial
(2006) used photographs of chin-spot patterns in Rana mucosa and found a high
success rate in reidentification over a 3-month study; changes in spot pattern
over time were observed so regular recaptures might minimize misidentifica-
tions using this technique. The effectiveness of using photographic identifica-
tion on long-term studies of adult anurans has yet to be determined.
Pointing out concerns in using artificial or invasive marks with the protected
Leiopelma archeyi (Archey’s frog) in New Zealand, Bradfield (2004) developed a
highly accurate photographic identification technique using natural markings.
This study is a model on how to develop and test a photographic pattern mapping
 8 Working with post-metamorphic amphibians | 135

technique. A series of six digital photographs were taken from dorsal, ventral,
cranial (facial), caudal, right lateral, and left lateral views. Photographs using a
flash were most successful in discerning usable characteristics of the various black
markings. A filing system for the photos used subgroupings which substantially
reduced the amount of time needed to identify recaptures. Intra- and interobserver
consistency of identification was tested and found to be high. Overall, 99.2% of
identifications were successful once recaptures were assigned to their subgroups.

8.6.2 Salamanders
Naturally occurring variation in morphology was used for Triturus cristatus and
Triturus vulgaris by photographing belly patterns (Hagstrom 1973). With this
technique, one must be sure that the patterns are both recognizably different
and constant through time. Ontogenetic belly pattern variation of T. cristatus
was documented by Arntzen and Teunis (1993). Healy (1974) found the vari-
ation in dorsal spot patterns of Notophthalmus viridescens useful in identifying
individuals. This technique requires recapture and handling which may not be
consistent with the animals’ behavior.
Digital photographs of dorsal spot patterns were tested for their effectiveness
in the identification of Eurycea bislineata by Bailey (2004). Observers were given
color printouts of the dorsal view of each animal with the SVL written below
the image for size reference. The observers compared these photographs to test
animals and were timed as to how long it took them to identify each animal.
Photo-identification rates were very high (about 94% correct), but were poten-
tially lower if observers were also given un-marked (photographed) individuals
in the attempted comparisons. Use of Adobe Photoshop® was effective in com-
paring qualities of integument patterns of vertebrates in digital photographs
and may be useful in matching images (L. Rifai, personal communication).
Sweeney et al. (1994) describe the use of computer analysis for the belly patterns
of T. cristatus. Computer applications for animal pattern analysis can now be
found on websites such as www.conservationresearch.co.uk/.
Loafman (1991) photographed the natural variation of spot patterns in adult
Ambystoma maculatum to successfully identify about 97% of the recaptures.
Adult A. opacum were photographed in a box and successfully reidentified
using their distinctive bar pattern over a 1-year period; using head patterns
alone, 80% of the adults could be distinguished from one another (Doody
1995).
The use of spot patterns for identification of A. maculatum is reviewed by
Grant and Nanjappa (2006) with special attention to possible errors in this
technique. They quantified error rates in specific mapping techniques, modified
136 | Amphibian ecology and conservation

techniques to address sources of error, compared methods relative to observer

bias in both the field and laboratory, and determined the effort needed to search
databases for specific individuals. They provided very specific suggestions for
researchers dealing with large samples of specimens identified by patterns.

8.7 Passive integrated transponder (PIT) tags

An extensive review and evaluation of a microchip marking system was given by
Camper and Dixon (1988). The 10 mm  2.1 mm PIT tags are encased in glass
and encoded with an alpha-numeric code which is read by a portable reader with
a hand wand. The authors tested tags on 95 individuals from a wide range of
species, and implanted tags with a metal syringe having a 12-gauge needle. This
baseline study experienced only one failed PIT due to a cracked glass cover, a 6%
error rate in the readings, a reading success on the first pass of the wand of 92%
and a migration rate of a tag within the animal of 36% (with possible reduction
of first-pass reading-rate success) (Camper and Dixon 1988). Examples of spe-
cies of amphibians studied with PIT tags are given in Table 8.7.

Table 8.7 Use of PIT tags for marking amphibian species (as reviewed in Ferner 2007
unless otherwise indicated)
Species Description of technique

Anurans
Bufo bufo Needle injection into pinched dorsum, massaged
caudally to base of spine
Limnodynastes peronii and Inserted with needle behind front limb along side of
Litoria aurea body
Rana catesbeiana, Rana clamitans, Needle injected into pinched ventral side
and Rana pipiens
Rana pretiosa Inserted through 2 mm incision on dorsum 1–2 cm
caudal to eyes, then massaged back along spine
Rana sylvatica Inserted through 2 m incision above the scapula;
“PIT pack” used for detection (Blomquist et al. 2008)
Rana temporaria Needle injection into pinched dorsum, massaged
caudally to base of spine

Caudates
Ambystoma opacum Inserted into body cavity at 3 mm incision 5 mm
cranial to hind limb
Taricha torosa Needle insertion into abdomen
 8 Working with post-metamorphic amphibians | 137

8.7.1 Anurans
All anurans in the Camper and Dixon (1988) project had the PIT tags implanted
intra-abdominally using the implanter syringe with the needle dipped in 70%
ethanol before implantation. Wounds were cleaned after injection with 70% etha-
nol and sealed with Krazy Glue®. Synthetic liquid bandages can also be used.
Ireland et al. (2003) found that the tag migrated on its own to the caudal
lymph space between the hind legs in all but 5% of the frogs they studied. A
field scanner was used for identification and the authors believed the technique
to be the best available for marking medium-to-large-sized anurans. The pri-
mary drawback is the cost of the AVID microchips (≈US$8.00) and scanner
(≈$1000). McAllister et al. (2004) inserted 12-mm tags (125 kHz model) on
frogs 42 mm and massaged them back along the body to the base of the spine
to minimize the chance of their being lost through the incision prior to healing
(about 2 weeks).
Pyke (2005) provides a good review on the use of PIT tags and reports on the
marking of 3000 individuals of nine species. The tags were supplied by Trovan
and are “individually-packaged needles inside hermetically sealed packages.”
The resulting small wound was sealed with Vetbond® (n-butyl cyanoacrylate
adhesive) and fewer than 1% of the animals exhibited any sort of distress call
during the procedure. As with previous studies, Pyke (2005) found little imme-
diate impact or effect on reproduction and long-term survival.

8.7.2 Salamanders
Ott and Scott (1999) anesthetized salamanders (see Table 8.6) for about 10
min with 2-phenoxy ethanol (30 drops/500 ml distilled H2O) and sealed the
incision with New Skin® liquid bandage. Recovery from anesthesia was in con-
tainers of distilled H2O with the head above water for a 3–4-h period until the
animals appeared active when prodded gently. Where no adverse impacts of PIT
tags were reported, Ott and Scott (1999) pointed out the disadvantage of cost
and marking time.

8.7.3 Caecilians
PIT tags probably hold the most promise for marking caecilians, but it is only
appropriate for larger specimens and requires perfecting the injection technique.
Inasmuch as the long salamanders Siren and Amphiuma have been marked suc-
cessfully over a period of more than 5 years (C. K. Dodd Jr, personal commu-
nication), PIT tagging holds promise for studies of caecilians. Since caecilians
may be less likely to be recaptured than some other amphibians, the cost of lost
tags should be considered.
138 | Amphibian ecology and conservation

8.8 Taking measurements

It is important to age specimens, place them in a class size (i.e. juvenile or
adult), and document growth rates that precise SVL and tail length measure-
ments be taken. Salamanders and caecilians may be placed on flat transparent
surfaces or restrained in clear plastic tubes with a millimeter scale or metric
stick used for measuring. Snout–vent distances are taken from the tip of the
snout to the caudal end of the cloacal aperture. Tail lengths are taken from the
caudal end of the cloacal aperture to the tip of the tail. Any regenerated por-
tion of the tail should be noted and measured separately. Anurans are usually
measured from the tip of the snout to the caudal projection of the urostyle as
the vent is often not easily found. Calipers are useful in measuring the lengths
of anurans.
Body mass should also be measured in grams after the specimen is patted dry
of any surface moisture. A specimen bag or customized bin on a scale may be
used to contain the animal. Use of an electronic or spring scale (such as Pesola®)
should be selected to obtain a weight to the nearest 0.10 g if possible. As many
amphibians evacuate the bladder or cloaca upon capture, investigators should
allow this to happen before weighing the specimen.

8.9 Recommendations
After extensive review of the literature and considering the criteria for selecting
a marking technique listed in the Introduction to this chapter, the following are
recommended.

• Toe-clipping remains the method of choice for most studies of anurans

with necessary consideration of the impact on the animals and long-term
retention of the clip.
• The emerging technique of choice with caudates is the use of VIE which
may also be used with anurans in the future. Non-invasive techniques,
particularly pattern recognition, are to be considered as a possibility for all
amphibians.
• When financial considerations and practicality dictate, PIT tagging is an
increasingly popular method for long-term studies.

In summary, it is essential that all techniques be considered and understood

thoroughly before use (the advantages, disadvantages, sources of bias), be prac-
ticed with specimens in captivity using good hygiene, and be monitored for the
impact of the mark on the survival and behavior of the amphibian.
 8 Working with post-metamorphic amphibians | 139

8.10 References
Arntzen, J. W. and Teunis, S. F. M. (1993). A six year study on the population dynamics of
the crested newt (Triturus cristatus) following the colonization of a newly created pond.
Herpetological Journal, 3, 99–110.
Bailey, L. L. (2004). Evaluating elastomer marking and photo identification methods for
terrestrial salamanders: marking effects and observer bias. Herpetological Review, 35,
38–41.
Baker, J. and Gent, T. (1998). Marking and recognition of animals. In T. Gent and
S. Gibson (eds), Herpetofauna Worker’s Manual, pp. 45–54. Joint Nature Conservation
Committee, Peterborough.
Blomquist, S. M., Zydlewski, J. D., and Hunter, M. L. (2008). Efficacy of PIT tags for
tracking the terrestrial anurans Rana pipiens and Rana sylvatica. Herpetological Review,
39, 174–9.
Bradfield, K. S. (2004). Photographic Identification of Individual Archey’s Frogs, Leiopelma
archeyi, from Natural Markings. DOC Science Internal Series 191. New Zealand
Department of Conservation, Wellington.
Briggs, J. L. and Storm, R. L. (1970). Growth and population structure of the cascade
frog, Rana cascadae Slater. Herpetologica, 26, 283–300.
Buchan, A., Sun, L., and Wagner, R. S. (2005). Using alpha numeric fluorescent tags for
individual identification of amphibians. Herpetological Review, 36, 43–44.
Bull, E. L., Wallace, R., and Bennett, D. H. (1983). Freeze-branding: a long term mark-
ing technique on long-toed salamanders. Herpetological Review, 14, 81–2.
Camper, J. D. and Dixon, J. R. (1988). Evaluation of a microchip marking system for
amphibians and reptiles. Texas Parks and Wildlife Department, Research Publication,
7100–59, 1–22.
Clarke, R. D. (1972). The effect of toe clipping on survival in Fowler’s toad (Bufo wood-
housei fowleri). Copeia, 1972, 182–5.
Daugherty, C. H. (1976). Freeze branding as a technique for marking anurans. Copeia,
1976, 836–8.
Davis, T. M. and Ovaska, K. (2001). Individual recognition of amphibians: effects of
toe clipping and fluorescent tagging on the salamander Plethodon vehiculum. Journal of
Herpetology, 35, 217–25.
Donnelly, M. A., Guyer, C., Juterbock, J. E., and Alford, R. A. (1994). Techniques for mark-
ing amphibians. In W. R. Heyer, M. A. Donnelly, R. W. McDiarmid, L. C. Hayek, and
M. S. Foster (eds), Measuring and Monitoring Biological Diversity, Standard Methods for
Amphibians, appendix 2, pp. 277–284. Smithsonian Institution Press, Washington DC.
Doody, J. S. (1995). A photographic mark-recapture method for patterned amphibians.
Herpetological Review, 26, 19–21.
Elmberg, J. (1989). Knee-tagging: a new marking technique for anurans. Amphibia-
Reptilia, 10, 101–4.
Emlen, S. T. (1968). A technique for marking anuran amphibians for behavioral studies.
Herpetologica, 24, 172–3.
Ferner, J. W. (2007). A Review of Marking and Individual Recognition Techniques for
Amphibians and Reptiles. Herpetological Circular No. 35. Society for the Study of
Amphibians and Reptiles, Salt Lake City, UT.
140 | Amphibian ecology and conservation

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 9
Egg mass and nest counts
Peter W. C. Paton and Reid N. Harris

9.1 Background: using egg mass and

nest counts to monitor populations
There is a critical need to develop statistically sensitive monitoring programs to
assess changes in the distribution and abundance of amphibians and determine
underlying mechanisms affecting populations (Alford and Richards 1999;
Marsh 2001; Storfer 2003; Petranka et al. 2004, 2006). However, assessing
trends is challenging because amphibian populations may experience dramatic
fluctuations (Pechmann et al. 1989) and all species have imperfect detection
probabilities (Bailey et al. 2004). Biologists interested in monitoring long-term
amphibian population trends have a number of methods available (Heyer et al.
1994), but selecting the most appropriate survey and analysis techniques can be
difficult (Williams et al. 2002).
Techniques used to assess amphibian populations during the breeding season
include surveys of calling anurans (Weir et al. 2005), dip-net sweeps to cap-
ture larvae in aquatic systems (Werner et al. 2007), drift fences with pit falls
(Pechmann et al. 1989), egg-mass counts (Adams et al., 1998; Lips et al. 2001;
Dodd 2003; Rödel and Ernst 2004), and nest counts (Corser and Dodd 2004;
Harris 2005). Advantages of calling surveys include the ability to survey many
breeding wetlands rapidly and most anurans can be detected, but urodeles are
not sampled because they do not vocalize. Dip-net surveys assess the occurrence
and density of larval amphibians, but are labor-intensive, require knowledge of
local breeding phenology, and require expertise in larval identification. Drift-
fence arrays can accurately assess changes in adult population size and prod-
uctivity for individual ponds; however, they are expensive because fences are
difficult to install and traps need to be checked often.
Many amphibians oviposit spawn in clumped aggregations, rather than
individual eggs. These spawn clumps are usually called “egg masses” in the
144 | Amphibian ecology and conservation

North America literature (e.g. Paton and Crouch 2002) and in the European
literature as “spawn clumps” (Griffith and Raper 1994), “batches” (Barton and
Rafinski 2006), “clutches” (Ficetola et al. 2006), or “egg masses” (Sofianidou
and Kyriakopoulou-Sklavounou 1983; Waringer-Löschenkohl 1991; Hartel
2008).
Nest counts can monitor populations in a similar manner to egg-mass counts.
Both types of count estimate annual breeding effort of populations and provide
an index of population size. In addition, both counts can test for effects of cli-
mate on population-level breeding effort (Corser and Dodd 2004), habitat rela-
tionships (Chalmers and Loftin 2006), population presence, and life-history
information (e.g. relationships between egg size and clutch size, and between
egg size and embryonic survival). Comparisons of egg size and clutch size among
populations can investigate maternal investment patterns as adaptations to local
environmental conditions. For nest counts, behavioural information can often
be gained, such as frequency of solitary and communal nesting and patterns of
nest attendance or brooding.
In the following sections we describe characteristics that make a species suit-
able for egg mass or nest counts. We also discuss factors that need to be consid-
ered when designing studies to collect and analyze these types of survey data.
Both egg mass and nest counts can be used to study habitat use over a variety of
spatial scales, thus are good candidates for landscape and metapopulation stud-
ies designed to manage and conserve at-risk species.

9.2 Oviposition strategies

Amphibians exhibit up to 39 different reproductive modes based on factors
such as oviposition site (e.g. aquatic, terrestrial, live-bearing), egg type, and
mode of development (Wells 2007). Worldwide, at least 10 families of anurans
and some urodeles have terrestrial oviposition (Wells 2007; see Appendix 9.1).
Although terrestrial oviposition strategies for many species have been described,
using egg counts to monitor their populations typically are not considered due
to low detection probabilities and potential negative impacts on populations by
disturbing eggs in nests. However, we review several case studies below where
nest-count surveys were used to monitor populations because detection prob-
abilities were high and negative impacts were low, which suggests that a similar
approach might be used for other species.
About 80% of anuran families deposit eggs in standing water (Wells 2007).
Species that oviposit in smaller wetlands, such as vernal pools (e.g. Ranidae,
Microhylidae), are probably best suited for egg-mass counts because biologists
 9 Egg mass and nest counts | 145

can search sites completely. For example, Egan and Paton (2004) found that
most pond-breeding amphibian sites were less than 0.05 ha in southern New
England, USA. Oviposition sites selected by some tropical species are extremely
small bodies of water, such as treeholes, aerial plants, or water-filled seedpods
(Wells 2007). However, although their oviposition habitat has been described,
to our knowledge no one has used egg-mass counts to monitor tropical amphib-
ian populations. Searching for egg masses of species that breed in large lakes is
probably not feasible due to low detection probabilities, and thus other monitor-
ing strategies should be employed. For stream-breeding species, egg masses can
be detected (e.g. Ascaphidae), although stream monitoring programs typically
search for juveniles and adults (Adams and Bury 2002).
Some anurans lay eggs in foamy nests on the water’s surface or in water-filled
basins (e.g. members of the subfamily Leptodactylinae; Wells 2007), which can
be highly visible on the surface of shallow, temporary pools. Also, rhacophorid
and hyperoliid frogs build foam nests in trees and other substrates. Thus, one
possible technique to monitor their populations might be to attempt to count
foam nests, but given the difficulties of finding the nests of most foam-nesting
species, this is probably not a cost-effective technique for most species.
There are many arboreal tropical species that deposit eggs on leaves, branches,
or tree trunks above standing water (see summary in Wells 2007). Neotropical
hylids ov