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Biostatistics with R
An Introductory Guide for Field Biologists

Biostatistics with R provides a straightforward introduction on how to analyse

data from the wide field of biological research, including nature protection and
global change monitoring. The book is centred around traditional statistical
approaches, focusing on those prevailing in research publications. The authors
cover t tests, ANOVA and regression models, but also the advanced methods of
generalised linear models and classification and regression trees. Chapters usually
start with several useful case examples, describing the structure of typical datasets
and proposing research-related questions. All chapters are supplemented by
example datasets and thoroughly explained, step-by-step R code demonstrating
the analytical procedures and interpretation of results. The authors also provide
examples of how to appropriately describe statistical procedures and results of
analyses in research papers. This accessible textbook will serve a broad audience
of interested readers, from students, researchers or professionals looking to
improve their everyday statistical practice, to lecturers of introductory under-
graduate courses. Additional resources are provided on www.cambridge.org/
biostatistics.

Jan Lepš is Professor of Ecology in the Department of Botany, Faculty of Science,

University of South Bohemia, České Budějovice, Czech Republic and senior
researcher in the Biology Centre of the Czech Academy of Sciences in České
Budějovice. His main research interests include plant functional ecology, particu-
larly the mechanisms of species coexistence and stability, and ecological data
analysis. He has taught many ecological and statistical courses and supervised
more than 80 student theses, from undergraduate to PhD.

Petr Šmilauer is Associate Professor of Ecology in the Department of Ecosystem

Biology, Faculty of Science, University of South Bohemia, České Budějovice,
Czech Republic. His main research interests are multivariate statistical analysis,
modern regression methods and the role of arbuscular mycorrhizal symbiosis in
the functioning of plant communities. He is co-author of multivariate analysis
software Canoco 5, CANOCO for Windows 4.5 and TWINSPAN for Windows.
‘We will never have a textbook of statistics for biologists that satisfies everybody. However,
this book may come closest. It is based on many years of field research and the teaching of
statistical methods by both authors. All useful classic and advanced statistical concepts and
methods are explained and illustrated with data examples and R programming procedures.
Besides traditional topics that are covered in the premier textbooks of biometry/biostatistics
(e.g. R. R. Sokal & F. J. Rohlf, J. H. Zar), two extensive chapters on multivariate methods in
classification and ordination add to the strength of this book. The text was originally published
in Czech in 2016. The English edition has been substantially updated and two new chapters
‘Survival Analysis’ and ‘Classification and Regression Trees’ have been added. The book will
be essential reading for undergraduate and graduate students, professional researchers, and
informed managers of natural resources.’
Marcel Rejmánek,
Department of Evolution and Ecology, University of California, Davis, CA, USA
Biostatistics with R
An Introductory Guide for Field Biologists

JAN LEPŠ
University of South Bohemia, Czech Republic

PETR ŠMILAUER
University of South Bohemia, Czech Republic
University Printing House, Cambridge CB2 8BS, United Kingdom
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Cambridge University Press is part of the University of Cambridge.

It furthers the University’s mission by disseminating knowledge in the pursuit of
education, learning, and research at the highest international levels of excellence.

www.cambridge.org
Information on this title: www.cambridge.org/9781108480383
DOI: 10.1017/9781108616041
© Jan Lepš and Petr Šmilauer 2020
This publication is in copyright. Subject to statutory exception
and to the provisions of relevant collective licensing agreements,
no reproduction of any part may take place without the written
permission of Cambridge University Press.
First published 2020
Printed in the United Kingdom by TJ International Ltd, Padstow Cornwall
A catalogue record for this publication is available from the British Library.
ISBN 978-1-108-48038-3 Hardback
ISBN 978-1-108-72734-1 Paperback
Additional resources for this publication at www.cambridge.org/biostatistics
Cambridge University Press has no responsibility for the persistence or accuracy of
URLs for external or third-party internet websites referred to in this publication
and does not guarantee that any content on such websites is, or will remain,
accurate or appropriate.
 Contents

Preface page xiii

Acknowledgements xvii

1 BasicStatistical Terms, Sample Statistics 1

1.1 Cases, Variables and Data Types 1
1.2 Population and Random Sample 3
1.3 Sample Statistics 4
1.4 Precision of Mean Estimate, Standard Error of Mean 9
1.5 Graphical Summary of Individual Variables 10
1.6 Random Variables, Distribution, Distribution Function,
Density Distribution 10
1.7 Example Data 13
1.8 How to Proceed in R 13
1.9 Reporting Analyses 17
1.10 Recommended Reading 18

2 Testing Hypotheses, Goodness-of-Fit Test 19

2.1 Principles of Hypothesis Testing 19
2.2 Possible Errors in Statistical Tests of Hypotheses 21
2.3 Null Models with Parameters Estimated from the Data:
Testing Hardy–Weinberg Equilibrium 26
2.4 Sample Size 26
2.5 Critical Values and Significance Level 27
2.6 Too Good to Be True 29
2.7 Bayesian Statistics: What is It? 30
2.8 The Dark Side of Significance Testing 32
2.9 Example Data 35
2.10 How to Proceed in R 35
2.11 Reporting Analyses 37
2.12 Recommended Reading 37

3 Contingency Tables 39
3.1 Two-Way Contingency Tables 39
3.2 Measures of Association Strength 44
3.3 Multidimensional Contingency Tables 46
3.4 Statistical and Causal Relationship 47
3.5 Visualising Contingency Tables 49
3.6 Example Data 50
3.7 How to Proceed in R 50
vi Table of Contents

3.8 Reporting Analyses 54

3.9 Recommended Reading 54

4 Normal Distribution 55
4.1 Main Properties of a Normal Distribution 55
4.2 Skewness and Kurtosis 56
4.3 Standardised Normal Distribution 57
4.4 Verifying the Normality of a Data Distribution 58
4.5 Example Data 60
4.6 How to Proceed in R 60
4.7 Reporting Analyses 63
4.8 Recommended Reading 64

5 Student’s t Distribution 65
5.1 Use Case Examples 65
5.2 t Distribution and its Relation to the Normal Distribution 66
5.3 Single Sample Test and Paired t Test 67
5.4 One-Sided Tests 70
5.5 Confidence Interval of the Mean 72
5.6 Test Assumptions 73
5.7 Reporting Data Variability and Mean Estimate Precision 74
5.8 How Large Should a Sample Size Be? 77
5.9 Example Data 79
5.10 How to Proceed in R 79
5.11 Reporting Analyses 82
5.12 Recommended Reading 83

6 Comparing Two Samples 84

6.1 Use Case Examples 84
6.2 Testing for Differences in Variance 85
6.3 Comparing Means 87
6.4 Example Data 88
6.5 How to Proceed in R 88
6.6 Reporting Analyses 91
6.7 Recommended Reading 91

7 Non-parametric Methods for Two Samples 92

7.1 Mann–Whitney Test 93
7.2 Wilcoxon Test for Paired Observations 95
7.3 Using Rank-Based Tests 97
7.4 Permutation Tests 97
7.5 Example Data 99
7.6 How to Proceed in R 99
7.7 Reporting Analyses 102
7.8 Recommended Reading 103
 Table of Contents vii

8 One-Way Analysis of Variance (ANOVA) and Kruskal–Wallis Test 104

8.1 Use Case Examples 104
8.2 ANOVA: A Method for Comparing More Than Two Means 104
8.3 Test Assumptions 105
8.4 Sum of Squares Decomposition and the F Statistic 106
8.5 ANOVA for Two Groups and the Two-Sample t Test 108
8.6 Fixed and Random Effects 108
8.7 F Test Power 109
8.8 Violating ANOVA Assumptions 110
8.9 Multiple Comparisons 111
8.10 Non-parametric ANOVA: Kruskal–Wallis Test 115
8.11 Example Data 116
8.12 How to Proceed in R 117
8.13 Reporting Analyses 127
8.14 Recommended Reading 128

9 Two-Way Analysis of Variance 129

9.1 Use Case Examples 129
9.2 Factorial Design 130
9.3 Sum of Squares Decomposition and Test Statistics 132
9.4 Two-Way ANOVA with and without Interactions 134
9.5 Two-Way ANOVA with No Replicates 135
9.6 Experimental Design 135
9.7 Multiple Comparisons 137
9.8 Non-parametric Methods 138
9.9 Example Data 139
9.10 How to Proceed in R 139
9.11 Reporting Analyses 149
9.12 Recommended Reading 150

10 Data Transformations for Analysis of Variance 151

10.1 Assumptions of ANOVA and their Possible Violations 151
10.2 Log-transformation 153
10.3 Arcsine Transformation 156
10.4 Square-Root and Box–Cox Transformation 156
10.5 Concluding Remarks 157
10.6 Example Data 158
10.7 How to Proceed in R 158
10.8 Reporting Analyses 163
10.9 Recommended Reading 163

11 Hierarchical ANOVA, Split-Plot ANOVA, Repeated Measurements 164

11.1 Hierarchical ANOVA 164
11.2 Split-Plot ANOVA 167
11.3 ANOVA for Repeated Measurements 169
viii Table of Contents

11.4 Example Data 171

11.5 How to Proceed in R 171
11.6 Reporting Analyses 181
11.7 Recommended Reading 182

12 Simple Linear Regression: Dependency Between Two Quantitative

Variables 183
12.1 Use Case Examples 183
12.2 Regression and Correlation 184
12.3 Simple Linear Regression 184
12.4 Testing Hypotheses 187
12.5 Confidence and Prediction Intervals 190
12.6 Regression Diagnostics and Transforming Data in Regression 190
12.7 Regression Through the Origin 195
12.8 Predictor with Random Variation 197
12.9 Linear Calibration 197
12.10 Example Data 198
12.11 How to Proceed in R 198
12.12 Reporting Analyses 204
12.13 Recommended Reading 205

13 Correlation: Relationship Between Two Quantitative Variables 206

13.1 Use Case Examples 206
13.2 Correlation as a Dependency Statistic for Two Variables on an Equal
Footing 206
13.3 Test Power 209
13.4 Non-parametric Methods 212
13.5 Interpreting Correlations 212
13.6 Statistical Dependency and Causality 213
13.7 Example Data 216
13.8 How to Proceed in R 216
13.9 Reporting Analyses 218
13.10 Recommended Reading 218

14 Multiple Regression and General Linear Models 219

14.1 Use Case Examples 219
14.2 Dependency of a Response Variable on Multiple Predictors 219
14.3 Partial Correlation 223
14.4 General Linear Models and Analysis of Covariance 224
14.5 Example Data 225
14.6 How to Proceed in R 226
14.7 Reporting Analyses 237
14.8 Recommended Reading 238
 Table of Contents ix

15 Generalised Linear Models 239

15.1 Use Case Examples 239
15.2 Properties of Generalised Linear Models 240
15.3 Analysis of Deviance 242
15.4 Overdispersion 243
15.5 Log-linear Models 243
15.6 Predictor Selection 244
15.7 Example Data 245
15.8 How to Proceed in R 246
15.9 Reporting Analyses 250
15.10 Recommended Reading 251

16 Regression Models for Non-linear Relationships 252

16.1 Use Case Examples 252
16.2 Introduction 253
16.3 Polynomial Regression 253
16.4 Non-linear Regression 255
16.5 Example Data 256
16.6 How to Proceed in R 256
16.7 Reporting Analyses 259
16.8 Recommended Reading 260

17 Structural Equation Models 261

17.1 Use Case Examples 261
17.2 SEMs and Path Analysis 261
17.3 Example Data 265
17.4 How to Proceed in R 265
17.5 Reporting Analyses 272
17.6 Recommended Reading 272

18 Discrete Distributions and Spatial Point Patterns 274

18.1 Use Case Examples 274
18.2 Poisson Distribution 274
18.3 Comparing the Variance with the Mean to Measure Spatial
Distribution 276
18.4 Spatial Pattern Analyses Based on the K-function 279
18.5 Binomial Distribution 280
18.6 Example Data 283
18.7 How to Proceed in R 283
18.8 Reporting Analyses 289
18.9 Recommended Reading 289

19 Survival Analysis 290

19.1 Use Case Examples 290
19.2 Survival Function and Hazard Rate 291
x Table of Contents

19.3 Differences in Survival Among Groups 293

19.4 Cox Proportional Hazard Model 293
19.5 Example Data 295
19.6 How to Proceed in R 295
19.7 Reporting Analyses 302
19.8 Recommended Reading 302

20 Classification and Regression Trees 303

20.1 Use Case Examples 303
20.2 Introducing CART 304
20.3 Pruning the Tree and Crossvalidation 306
20.4 Competing and Surrogate Predictors 307
20.5 Example Data 308
20.6 How to Proceed in R 309
20.7 Reporting Analyses 316
20.8 Recommended Reading 316

21 Classification 317
21.1 Use Case Examples 317
21.2 Aims and Properties of Classification 317
21.3 Input Data 319
21.4 Similarity and Distance 319
21.5 Clustering Algorithms 320
21.6 Displaying Results 320
21.7 Divisive Methods 321
21.8 Example Data 322
21.9 How to Proceed in R 322
21.10 Other Software 324
21.11 Reporting Analyses 325
21.12 Recommended Reading 325

22 Ordination 326
22.1 Use Case Examples 327
22.2 Unconstrained Ordination Methods 327
22.3 Constrained Ordination Methods 330
22.4 Discriminant Analysis 331
22.5 Example Data 333
22.6 How to Proceed in R 333
22.7 Alternative Software 340
22.8 Reporting Analyses 341
22.9 Recommended Reading 341

Appendix A: First Steps with R Software 343

A.1 Starting and Ending R, Command Line, Organising Data 343
A.2 Managing Your Data 349
 Table of Contents xi

A.3 Data Types in R 351

A.4 Importing Data into R 357
A.5 Simple Graphics 359
A.6 Frameworks for R 360
A.7 Other Introductions to Work with R 362

Index 363
Preface

Modern biology is a quantitative science. A biologist weighs, measures and

counts, whether she works with aphid or fish individuals, with plant communities
or with nuclear DNA. Every number obtained in this way, however, is affected by
random variation. Aphid counts repeatedly obtained from the same plant individ-
ual will differ. The counts of aphids obtained from different plants will differ
more, even if those plants belong to the same species, and samples coming from
plants of different species are likely to differ even more. Similar differences will
be found in the nuclear DNA content of plants from the same population, in
nitrogen content of soil samples taken from the same or different sites, or in the
population densities of copepods across repeated samplings from the same lake.
We say that our data contain a random component: the values we obtain are
random quantities, with a part of their variation resulting from randomness.
But what actually is this randomness? In posing such a question, we
move into the realm of philosophy or to axioms of probability theory. But what is
probability? A biologist is usually happy with a pragmatic concept: we consider
an event to be random if we do not have a causal explanation for it. Statistics is a
research field which provides recipes for how to work with data containing
random components, and how to distinguish deterministic patterns from random
variation. Popular wisdom says that statistics is a branch of science where precise
work is carried out with imprecise numbers. But the term statistics has multiple
meanings. The layman sees it as an assorted collection of values (football league
statistics of goals and points, statistics of MP voting, statistics of cars passing
along a highway, etc.). Statistics is also a research field (often called mathematical
statistics) providing tools for obtaining useful information from such datasets. It is
xiv Preface

a separate branch of science, to a certain extent representing an application of probability

theory. The term statistic (often in singular form) is also used in another sense: a numerical
characteristic computed from data. For example, the well-known arithmetic average is a
statistic characterising a given data sample.
In scientific thinking, we can distinguish deductive and inductive approaches. The
deductive approach leads us from known facts to their consequences. Sherlock Holmes may
use the facts that a room is locked, has no windows and is empty to deduce that the room must
have been locked from the outside. Mathematics is a typical example of a deductive system:
based on axioms, we can use a purely logical (deductive) path to derive further statements,
which are always correct if the initial axioms are also correct (unless we made a mistake in the
derivation). Using the deductive approach, we proceed in a purely logical manner and do not
need any comparison with the situation in real terms.
The inductive approach is different: we try to find general rules based on many
observations. If we tread upon 1-cm-thick ice one hundred times and the ice breaks each time,
we can conclude that ice of this thickness is unable to carry the weight of a grown person. We
conclude this using inductive thinking. We could, however, also employ the deductive
approach by using known physical laws, strength measurements of ice and the known weight
of a grown person. But usually, when treading on thin ice, we do not know its exact thickness
and sometimes the ice breaks and sometimes it does not. Usually we find, only after breaking
through it, that the ice was quite thin. Sometimes even thicker ice breaks, but such an event is
affected by many circumstances we are not able to quantify (ice structure, care in treading, etc.)
and we therefore consider them as random. Using many observations, however, we can estimate
the probability of breaking through ice based on its thickness by using the methods of
mathematical statistics. Statistics is therefore a tool of inductive thinking in such cases, where
the outcome of an experiment (or observation) is affected by random variability.
Thanks to advances in computer technology, statistics is now available to all
biologists. Statistical analysis of data is a necessary prerequisite of manuscript acceptance
in most biological journals. These days, it is impossible to fully understand most of the
research papers in biological journals without understanding the basic principles of statistics.
All biologists must plan their observations and experiments, as only correctly collected data
can be useful when answering their questions with the aid of statistical methods. To collect
your data correctly, you need to have a basic understanding of statistics.
A knowledge of statistics has therefore become essential for successful enquiry in
almost all fields of biology. But statistics are also often misused. Some even say that there are
three kinds of lies: a non-intentional lie, an intentional lie and statistics. We can ‘adorn’ bad
data by employing a complex statistical method so that the result looks like a substantial
contribution to our knowledge (even finding its way into prestigious journals). Another
common case of statistical misuse is interpreting statistical (‘correlational’) dependency as
causal. In this way, one can ‘prove’ almost anything. A knowledge of statistics also allows
biologists to differentiate statements which provide new and useful information from those
where statistics are used to simply mask a lack of information, or are misused to support
incorrect statements.
The way statistics are used in the everyday practice of biology changed substantially
with the increased availability of statistical software. Today, everyone can evaluate her/his
data on a personal computer; the results are just a few mouse clicks away. While your
 Preface xv

computer will (almost) always offer some results, often in the form of a nice-looking graph,
this rather convenient process is not without its dangers. There are users who present the
results provided to them by statistical programs without ever understanding what was
computed. Our book therefore tries not only to teach you how to analyse your data, but also
how to understand what the results of statistical processing mean.
What is biostatistics? We do not think that this is a separate research field. In using
this term, we simply imply a focus on the application of statistics to biological problems.
Alternatively, the term biometry is sometimes used in a similar sense. In our book, we place
an emphasis on understanding the principles of the methods presented and the rules of their
use, not on the mathematical derivation of the methods. We present individual methods in a
way that we believe is convenient for biologists: we first show a few examples of biological
problems that can be solved by a given method, and only then do we present its principles and
assumptions. In our explanations we assume that the reader has attended an introductory
undergraduate mathematical course, including the basics of the theory of probability. Even so,
we try to avoid complex mathematical explanations whenever possible.
This book provides only basic information. We recommend that all readers continue
a more detailed exploration of those methods of interest to them. The three most recom-
mended textbooks for this are Quinn & Keough (2002), Sokal & Rohlf (2012) and Zar (2010).
The first and last of these more closely reflect the mind of the biologist, as their authors have
themselves participated in ecological research. In this book, we adopt some ideas from Zar’s
textbook about the sequence in which to present selected topics. After every chapter, we give
page ranges for the three referred textbooks, each containing additional information
about the particular methods. Our book is only a slight extension of a one-term course
(2 hours lectures + 2 hours practicals per week) in Biostatistics, and therefore sufficient detail
is lacking on some of the statistical methods useful for biologists. This primarily concerns the
use of multivariate statistical analysis, traditionally addressed in separate textbooks and
courses.
We assume that our readers will evaluate their data using a personal computer and we
illustrate the required steps and the format of results using two different types of software. The
program R lacks some of the user-friendliness provided by alternative statistical packages, but
offers practically all known statistical methods, including the most modern ones, for free
(more details at cran.r‑project.org), and so it became de facto a standard tool, prevailing in
published biological research papers. We assume that the reader will have a basic working
knowledge of R, including working with its user interface, importing data or exporting results.
The knowledge required is, however, summarised in Appendix A of this book, which can be
found after the last chapter. The program Statistica represents software for the less demanding
user, with a convenient range of menu choices and extensive dialogue boxes, as well as an
easily accessible and modifiable graphical presentation of results. Instructions for its use are
available to the reader at the textbook’s website: www.cambridge.org/biostatistics.
Example data used throughout this book are available at the same website, but also
from our own university’s web address: www.prf.jcu.cz/biostat-data-eng.xlsx.
Note that in most of our ‘use case examples’ (and often also in the example data), the
actual (or suggested) number of replicates is very low, perhaps too low to provide reasonable
support for a real-world study. This is just to make the data easily tractable while we
demonstrate the computation of test statistics. For real-world studies, we recommend the
xvi Preface

reader strives to attain more extensive datasets. If there is no citation for our example dataset,
such data are not real.
In each chapter, we also show how the results derived from statistical software can be
presented in research papers and also how to describe the particular statistical methods there.
In this book, we will most frequently refer to the following three statistical textbooks
providing more details about the methods:

• J. H. Zar (2010) Biostatistical Analysis, 5th edn. Pearson, San Francisco, CA.
• G. P. Quinn & M. J. Keough (2002) Experimental Design and Data Analysis for Biologists.
Cambridge University Press, Cambridge.
• R. R. Sokal & E. J. Rohlf (2012) Biometry, 4th edn. W. H. Freeman, San Francisco, CA.
Other useful textbooks include:

• R. H. Green (1979) Sampling Design and Statistical Methods for Environmental Biologists.
Wiley, New York.
• R. H. G. Jongmann, C. J. F. ter Braak & O. F. R. van Tongeren (1995) Data Analysis in
Community and Landscape Ecology. Cambridge University Press, Cambridge.
• P. Šmilauer & J. Lepš (2014) Multivariate Analysis of Ecological Data Using Canoco 5,
2nd edn. Cambridge University Press, Cambridge.
More advanced readers will find the following textbook useful:

• R. Mead (1990) The Design of Experiments. Statistical Principles for Practical Application.
Cambridge University Press, Cambridge.
Where appropriate, we cite additional books and papers at the end of the correspond-
ing chapter.