PREVENTING PREDATION: EVOLUTION AND ADAPTIVE PLASTICITY
IN MORPHOLOGICAL DEFENSE OF AN INVASIVE SPECIES
By
Andrea Lynn-Jaeger Miehls

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
DOCTOR OF PHILOSOPHY
Fisheries and Wildlife
Ecology, Evolutionary Biology, and Behavior
2012

ABSTRACT
PREVENTING PREDATION: EVOLUTION AND ADAPTIVE PLASTICITY
IN MORPHOLOGICAL DEFENSE OF AN INVASIVE SPECIES
By
Andrea Lynn-Jaeger Miehls

Invasive species are one of the leading threats to global biodiversity, but until recently,
little attention has been paid to how evolution contributes to invasive species effects despite
important applications to conservation and our general understanding of evolution in the wild.
Invasive species often experience strong selection upon introduction, which may result in
adaptive evolution that could facilitate their successful integration into food webs and their
effects on native species. Recent species invasions in North America offer a strong opportunity
to address fundamental questions in evolutionary ecology as well as advance our understanding
of invasive species effects. Bythotrephes longimanus (the spiny water flea) is a predatory
zooplankton with a conspicuous tail spine that invaded the Great Lakes region during the 1980s
and may be having large negative effects on fisheries. Previous field studies show the
morphology and life history of Bythotrephes strongly vary spatially and temporally, but the cause
is not known. Evolution by natural selection and phenotypic plasticity are potential sources of
Bythotrephes trait variation, but heretofore, these sources of variation have not been investigated.
My dissertation research investigated ecological and evolutionary factors that influence
morphological and life history variation in Bythotrephes. Using Bythotrephes collected from
Lake Michigan, I found moderate-to-high genetic variation in distal spine and body length and
maternal effects in both traits. Further, experiments revealed that spine length, body size, and
clutch size respond plastically to temperature but not to fish predator cues, with higher

temperature inducing mothers to have smaller clutches of larger offspring (longer absolute distal
spine and body length) that were better defended against predation. Although Bythotrephes use
temperature as the proximate cue of plasticity, it is likely that the trait changes represent
adaptations to varying fish predation risk which correlates with water temperature. I also found
temporally and spatially variable selection on distal spine length consistent with seasonal
changes in gape-limitation of fish predators and spatial heterogeneity of fish, respectively. Yet,
despite net selection for increased distal spine length, I observed little evidence of an
evolutionary response to selection based on comparisons of historic and contemporary wildcaptured individuals and retrieved spines from sediment cores. In a companion study of
Canadian Shield lakes, I identified gape-limited fish predators as agents of selection on
Bythotrephes distal spine length. Specifically, I found selection for increased distal spine length
in lakes dominated by a gape-limited fish predator and no significant selection in lakes
dominated by a non-gape-limited fish predator. A large difference (20%) in average distal spine
length between lakes of each predator type was consistent with the direction of selection,
suggesting potential local adaptation of distal spine length to gape-limited fish predation.
The results of my dissertation indicate Bythotrephes respond to fish predation through
multiple mechanisms, including phenotypic plasticity and evolutionary responses to selection.
These responses to predation likely promote Bythotrephes success as an invasive species, and
may also underlie negative effects on important Great Lakes fisheries through food web
interactions. More generally, the results of my dissertation suggest the effects of invasive species
may occur not just through their ecological interactions, but also through evolutionary and
phenotypically plastic trait modifications. As invasive species continue to affect biodiversity
worldwide, understanding the mechanisms behind invasive species effects is critical.

To my research assistants, for your curiosity, enthusiasm, and spirit
~ and ~
To my husband, for your strength, dedication, and unwavering support

iv

ACKNOWLEDGEMENTS

No graduate project is accomplished without the dedication, enthusiasm, and untiring
efforts of people. I express my deep gratitude to the many individuals and organizations that
have contributed to my dissertation research, graduate program, and other professional endeavors
during my time at Michigan State University.
I am grateful to the numerous organizations that have provided financial support for my
research, including the EPA STAR Fellowship Program, Great Lakes Fishery Commission,
National Science Foundation, NOAA Great Lakes Environmental Research Laboratory, and
Sigma Xi Grants-in-Aid of Research Program. Additionally, the Michigan State University
College of Agriculture and Natural Resources, Department of Fisheries and Wildlife (FW
Department), Ecology, Evolutionary Biology, and Behavior Program (EEBB Program), and
Graduate School have provided financial support. The contributions from these organizations
have allowed me to investigate a variety of research questions spanning the continuum from
basic to applied science, in many areas of ecology, evolution, aquatic science, and invasion
biology. I am grateful for the resources these organizations have invested toward the exploration
of our natural world.
I would also like to thank the FW Department and EEBB Program at MSU for the rich
learning environments they provided to me at MSU. The FW Department has been my academic
home for nine years and two graduate degrees. Being a member of the FW Department for that
time has been an incredibly rewarding experience. I feel privileged to have interacted with the
many outstanding researchers, educators, and other professionals comprising the faculty, staff,
and students. I thank the FW Department for fostering such a strong community in which

v

students can grow both professionally and personally. The EEBB Program provided an important
opportunity for me to interact with well-respected scientists and to develop my knowledge of
broader topics in ecology, evolutionary biology, and behavior. The required courses and weekly
seminars provided vital exposure to research that was an abundant source of ideas for my own
dissertation. I have enjoyed interacting with the EEBB community and thank the program for the
many ways it has enriched my experience at MSU.
My co-advisors, Dr. Andrew McAdam and Dr. Scott Peacor, have my warmest respect
and gratitude for all they have done in support of my research and professional development. I
thank both Andrew and Scott for their interest and enthusiasm while developing this project, for
the countless hours of guidance they have given, and the daily inspiration they have provided. I
thank Andrew specifically for his eagerness to embark on a project involving a creepy little
invertebrate far outside his taxonomic expertise, for his curiosity while pursuing this research,
for all that he has taught me about problem solving and creativity in science, and for his intuition
in knowing when to provide encouragement. I thank Scott specifically for the mentorship he has
provided during both of my graduate degrees, for his dedication to my professional development,
for teaching me to remember the big picture of any scientific endeavor, and for serving as a
model for integrity in research and personal interactions. I have learned so much from both of
my advisors and feel fortunate to have been a part of the labs of such outstanding researchers,
mentors, and people.
Many other individuals have played critical roles in my research. My committee
members, Dr. Doran Mason and Dr. Doug Schemske, have provided valuable guidance on my
research. I thank Doran for his continued commitment to my graduate work after serving as my
advisor during my Master’s degree, for the knowledge he has brought to my dissertation research

vi

regarding the Great Lakes, and for his contributions to my field work on Lake Michigan. I also
credit Doran for motivating one of the most significant (and best) decisions I have made
professionally – my decision to complete graduate work at MSU. I thank Doug for sharing his
expertise on evolutionary ecology and invasive species, for providing perceptive feedback on my
research, and for delivering one of the most thought-provoking class lectures I have attended – a
lecture that provided inspiration at a time when I needed it most. I also thank my co-authors, Dr.
Paul Bourdeau and Lindsey Valliant, for their contributions to the conceptual foundation,
analysis, and writing of my dissertation chapters. Paul’s contributions, in particular, have been
extensive and more deeply valued than co-authorship and chapter acknowledgements can
convey; Paul has been a sounding board for nearly all of my work and an essential source of
creativity and insight. I also thank the many additional collaborators on my research, including:
Dr. Kimberly Schulz, Dr. Natalie Kim, and Dr. Kevin Pangle for their assistance developing
rearing protocols for Bythotrephes – a task much easier in theory than practice; Dr. Christian
Laforsch and Quirin Herzog for additional experiments investigating the adaptive value of
phenotypic plasticity in Bythotrephes traits; Dr. Carla Cáceres for multiple rigorous discussions
of our experimental design and research results; Dr. Donna Kashian for her guidance involving
resurrection ecology methods and sediment core collection; Dr. Norman Yan and Dr. Joelle
Young for their assistance completing my research in the Canadian Shield lakes; and Dr. Steve
Pothoven and Dennis Donahue for coordinating Lake Michigan field work.
This research would not have been possible or anywhere nearly as enjoyable without the
hard work and dedication of my research assistants and volunteers (a.k.a. the Bytho Babes and
Bytho Dudes). Their enthusiasm was motivation to me daily and served as a constant reminder of
what I love about science. My incredible assistants and volunteers were: Matt Bach, Teresa

vii

Campton, Keali Chambers, Brittany Damschroder, Jillian Fejszes, Jason Fischer, Skye Fissette,
Brittany Gunther, Nicole Hedquist, Tyler Joseph, Stefanie Jubb, Lydia Kramer, Chris Long,
Monica Lucas, Ian McCririe, Claire Michael, Jennifer Pellegrini, Ariel Porty, Veronica Quesnell,
Andria Salas, Ben Staton, Marie Stevenson, Mariam Teimorzadeh, Elizabeth Throckmorton,
Aditi Upadhye, Brandon Vieder, and Miriam Weiss. I also thank the other research assistants and
volunteers in the Peacor Lab who provided assistance with my research: Natalie Davenport,
Anne Kellerman, Rachel Komosinski, Emily Reed, and Alex Sookhai.
I am fortunate to have been associated with multiple lab groups at MSU and the
University of Guelph that were a source of lively discussion, rigorous feedback, and uninhibited
fun during my dissertation research. I thank the McAdam Lab at MSU and the University of
Guelph for their conceptual contributions to my research and the knowledge they have shared
with me about small mammal research (I will always have an affection for “smammalers”
because of them). I thank the Holekamp Lab at MSU for their involvement in the early
conceptualization of my research project and for introducing me to the fascinating world of
hyena evolutionary ecology. Interacting with the McAdam and Holekamp Labs has clearly
shown the value of engaging with people addressing similar conceptual questions, but in wildly
different (at least superficially) study systems, for providing fresh perspectives and novel insight.
I thank the Peacor Lab and the Limnology Lab at MSU for the many chats (scientific and
otherwise) and never-ending fun. Whether during picnic table lunches, karaoke, or weekend
hijinks, this group (faculty, students, post-docs, and lab adoptees) has been the root of so many
wonderful memories. Lastly of the MSU groups, I thank my room 336 cube-mates for the
soundtrack their voices have provided – drifting above the cement block walls of our cubes – to
my daily work.

viii

One of the most enriching aspects of my time at MSU has been my involvement in
teaching. For the last year of my dissertation research, I have kept a Wordle of my teaching
philosophy posted above my office desk; the prominence of “knowledge”, “discovery”, and
“learning” are constant reminders of why the work that we do, and the sacrifices we make, are
worthwhile. I thank my students in FW 364, ISP 217L, ISP 221, and PLB 802 for continually
renewing in me the feeling of awe and wonder that the process of scientific discovery entails. I
also thank my many teaching mentors, including Drs. Bill Taylor, Jen Lau, Kelly Millenbah,
Richard Brandenburg, and my advisors, Drs. Andrew McAdam and Scott Peacor, for sharing the
joy of teaching with me and for supporting my teaching development. Dr. Bill Taylor, in
particular, has been instrumental in providing an abundance of teaching and leadership
opportunities; his influence on my professional development has been substantial and I am
deeply grateful for the tireless energy he has put toward my growth. I also thank my network of
mentors for professional development, including Drs. Mary Bremigan, Kendra Cheruvelil, John
Dettmers, Chuck Krueger, Mike Jones, Ace Sarnelle, and Pat Soranno.
One of my favorite quotations is from an essay written by David Quammen, who writes:
“People come into our lives and then they go out again. The entropy law, as applied to human
relations. Sometimes in their passing, though, they register an unimagined and far-reaching
influence.” Many of the people I encountered during previous work and educational experiences
have created just such an enduring influence on my life. My interactions at the Illinois Natural
History Survey’s Lake Michigan Biological Station formed the foundation for much of my field
and laboratory research during my PhD; I thank my close friends and colleagues there for their
continued encouragement of my research. The wise words of my mentors during my
undergraduate degree at Augustana College, Drs. Stephen Hager, Kevin Geedey, Cecilia Vogel,

ix

and Dara Wegmen-Geedey, and my mentor during high school, Jason Powell, still resonate
within me today. I owe unquantifiable thanks to these teachers for providing an enriching
educational environment that allowed me to grow my passion for science.
Lastly, I am indebted to my friends and family who have unselfishly and tirelessly
supported me throughout my graduate research. I thank my friends for the creativity and
diversity they bring into my life; it is an honor to have friends so dear that I consider them
family. I thank my parents, Barb and Doug Jaeger, for raising me in a household that cherished
teaching and learning, encouraged curiosity, and embraced my early scientific pursuits (even my
refrigerated green slime concoctions). I thank my brother, Jeff Jaeger, for giving me courage to
tackle challenges, confidence to pursue my goals, and for the boundless energy he brings to all
his interactions. Finally, I thank my husband, Scott Miehls, for always standing by my side, for
reminding me of all that is good in people, and for providing profound love and support. You
bring the color and light into my life.

x

TABLE OF CONTENTS

LIST OF TABLES ....................................................................................................................... xiii 
LIST OF FIGURES ...................................................................................................................... xv 
CHAPTER 1 ................................................................................................................................... 1 
Introduction ................................................................................................................................. 1 
Focus of Dissertation .................................................................................................................. 6 
Study Organism .......................................................................................................................... 7 
Chapter Outline ......................................................................................................................... 10 
Literature Cited ......................................................................................................................... 16
CHAPTER 2 ................................................................................................................................. 24 
Introduction ............................................................................................................................... 25 
Methods .................................................................................................................................... 27 
Results ....................................................................................................................................... 33 
Discussion ................................................................................................................................. 35 
Appendix ................................................................................................................................... 52 
Literature Cited ......................................................................................................................... 56
CHAPTER 3 ................................................................................................................................. 63 
Introduction ............................................................................................................................... 65 
Methods .................................................................................................................................... 67 
Results ....................................................................................................................................... 72 
Discussion ................................................................................................................................. 74 
Appendices................................................................................................................................ 89 
Literature Cited ......................................................................................................................... 98
CHAPTER 4 ............................................................................................................................... 102 
Introduction ............................................................................................................................. 104 
Methods .................................................................................................................................. 108 
xi

Results ..................................................................................................................................... 113 
Discussion ............................................................................................................................... 115 
Appendices.............................................................................................................................. 131 
Literature Cited ....................................................................................................................... 140
CHAPTER 5 ............................................................................................................................... 146 
Introduction ............................................................................................................................. 147 
Methods .................................................................................................................................. 150 
Results ..................................................................................................................................... 155 
Discussion ............................................................................................................................... 156 
Literature Cited ....................................................................................................................... 171
CHAPTER 6 ............................................................................................................................... 175 
Summary of Findings.............................................................................................................. 175 
Future Directions .................................................................................................................... 181 
Concluding Thoughts .............................................................................................................. 185 
Literature Cited ....................................................................................................................... 188 

xii

LIST OF TABLES
Table 2.1

Quantitative genetic variation in invasive species traits, including genetic
and additive genetic variation (Vg and Va, respectively), coefficients of
additive genetic variation (CVa), and broad- and narrow-sense
2

2

heritabilities (H and h , respectively) ................................................................42
Table 2.2

Genetic (Vg), maternal (Vm), and environmental (Ve) variance
components, coefficient of genetic variation (CVg), broad-sense
2

2

heritability (H ), and maternal effects (m ) for distal spine and body
length of Bythotrephes longimanus .....................................................................46
Table 3.1

Effect of fish kairomones (presence / absence) on the morphology and life
history of Bythotrephes longimanus raised at 9°C and 15°C. The
generation and instar of Bythotrephes used in each analysis is noted.
Significance of random effects for aquaria (all generations), clonal line (F1
and F2 generations), and clonal sub-line (F2 generation only) was assessed
using likelihood ratio tests. Gen: generation; Aq: aquaria; CL: clonal line;
SL: sub-line; N/A: not applicable ........................................................................92

Table 3.2

Effect of temperature (9°C versus 15°C) on the morphology and life
history of Bythotrephes longimanus. The generation and instar of
Bythotrephes used in each analysis is noted. Significance of random
effects for aquaria (all generations), clonal line (F1 and F2 generations),
and clonal sub-line (F2 generation only) was assessed using likelihood
ratio tests. Gen: generation; Aq: aquaria; CL: clonal line; SL: sub-line;
N/A: not applicable ..............................................................................................94

Table 4.1

Sample dates for paired monthly collections used to estimate natural
selection on Bythotrephes distal spine length during 2007-2009 and
average water temperature 1m below the surface. Note that the October 2,
2007 and November 3, 2008 collections are referred to in the text as
September and October collections, respectively, given the approximate
one-month interval between these collections and the previous collections.
Only one sampling trip was completed for these two time periods .....................124

Table 4.2

Natural selection on distal spine length of Bythotrephes longimanus
between the first two developmental (instar) stages in Lake Michigan for
multiple sites in an 8.0km transect from 2007-2009. Ave: average distal
spine length (mm); SD: standard deviation; N: sample size; SE: standard
error; S: selection differential (mm); SDp: propagated standard deviation
(calculated as the square root of the sum of the squared standard deviation
xiii

of distal spine length for each instar); i: selection intensity (calculated as
the selection differential divided by the propagated standard deviation); p:
p-value (*p < 0.05, **p < 0.01; ***p < 0.001) ....................................................131
Table 5.1

Bythotrephes invasion year, categorical fish abundance, and field
information for study lakes. Smelt are considered a gape-limited predator,
whereas cisco are considered a non-gape-limited predator of Bythotrephes.
Temperatures were taken 1m below the water surface at the time of
Bythotrephes collection. A: absent; L: low; M: medium; H: high .......................163

Table 5.2

Statistical results for the between and within cohort analyses of natural
selection on Bythotrephes distal spine length. The instar-by-fish
interaction assessed whether natural selection (effect of instar) differed by
fish predator. Note that the within cohort analysis did not include trip as a
fixed effect (see text). Lake (i.e., the six replicate study lakes) was
modeled as a random effect; the significance of lake was determined
through a likelihood ratio test. F: F-statistic; df: degrees of freedom; p: pvalue; Var: among-lake variance; Ratio: likelihood ratio ....................................164

Table 5.3

Natural selection on Bythotrephes distal spine length estimated within six
replicate study lakes; we found significant selection (i.e., significant effect
of instar) in all GLP lakes (Fairy, Mary, Peninsula) and no significant
selection in NGLP lakes (Boshkung, Harp, and Kashagawigamog). t and p
values are for the effect of instar on distal spine length. df: degrees of
freedom; S: selection differential (mm); SD: pooled standard deviation
within a lake; i: standardized selection differential (i.e., selection
intensity). Note that a second sample collection was not completed in
Kashagawigamog, precluding the estimation of selection within a cohort .........165

xiv

LIST OF FIGURES
Figure 2.1

Bythotrephes longimanus, the spiny water flea. The Bythotrephes tail
spine is comprised of the distal spine (measured from the tip of the spine
to the anterior end of the first set of instar barbs; solid line) and up to two
intercalary segments (for asexually produced Bythotrephes). Body length
was measured from the anterior edge of the eye to the base of the tail spine
along the midline of the body with segments spanning the head and eye
region, thorax, and abdomen (dashed line). The Bythotrephes pictured is a
third instar ............................................................................................................47

Figure 2.2

Clonal analysis design, with clonal sub-lines (F1 generation) nested within
clonal lines (wild-caught Bythotrephes); all analyses were completed using
morphological data from the F2 generation. Vg is the genetic variance,
represented by the variance in morphology of F2 individuals among clonal
lines, Vm is maternal variance, represented by the variance among sublines within clones, and Ve is the residual environmental variance within
sub-lines. Though two F1 young per clonal line and two F2 young per subline are pictured, all young born to clonal and sub-lines were reared.
Figure modified from Lynch and Walsh (1998) ..................................................48

Figure 2.3

Genetic (Vg), maternal (Vm), and environmental (Ve) variance components
for Bythotrephes distal spine length. Variance components did not differ
by month within 2008, but differed among years. Bars are the 95%
confidence intervals around the variance components ........................................49

Figure 2.4

Genetic (Vg), maternal (Vm), and environmental (Ve) variance components
for Bythotrephes body length. Variance components differed by month in
2008 and between years. Bars are the 95% confidence intervals around the
variance components ............................................................................................50

Figure 3.1

Bythotrephes longimanus, the spiny water flea. a) Bythotrephes
morphological traits (the individual pictured is a third instar); b) F2
offspring representing the average morphology of individuals born to
clonal lines raised at 9°C and 15°C .....................................................................84

Figure 3.2

Predator and temperature effects on Bythotrephes: a) distal spine length; b)
distal spine length versus body length of F2 offspring; c) body length; and
d) the ratio of distal spine to body length. Error bars are standard errors. C:
control treatment; F: fish treatment......................................................................85

xv

Figure 3.3

Predator and temperature effects on Bythotrephes: a) clutch size; and b)
days to first reproduction. Error bars are standard errors. C: control
treatment; F: fish treatment ..................................................................................87

Figure 3.4

Predator and temperature effects on Bythotrephes: a) S2 length; and b) S3
length. Error bars are standard errors. C: control treatment; F: fish
treatment ..............................................................................................................96

Figure 4.1

We estimated natural selection on distal spine length of Bythotrephes
longimanus, the spiny water flea (the individual pictured is a third instar) .........125

Figure 4.2

Net selection on Bythotrephes distal spine length as illustrated by the
change in average distal spine length pre-selection (first instar individuals)
and post-selection (second instar individuals) across all years and sites (net
selection differential: S = 0.08mm). Error bars are standard errors .....................126

Figure 4.3

Selection differentials (S) on Bythotrephes distal spine length for multiple
sites along an 8.0km transect in Lake Michigan during: a) 2007 (six sites:
July-September); b) 2008 (four sites: July-September; 1 site: October);
and c) 2009 (four sites: July-October). Note the different y-axis in panel
(a). Black bars: statistically significant (p < 0.05) selection differentials for
increased distal spine length; white bars: statistically significant (p < 0.05)
selection differentials for decreased distal spine length; gray bars: nonstatistically significant selection differentials ......................................................127

Figure 4.4

Distal spine length of: a) first instar wild-captured Bythotrephes from this
study (2007-2009) and previously published studies (1989: Burkhardt
1994; 1992-1993, 1996: Sullivan and Lehman 1998); and b) retrieved
spines collected from sediment cores (2007-2009); note that the 2007 and
2009 sediment core data are shown offset from the depth interval for
clarity. The solid line in panel (b) is the linear regression across all three
2
years (adjusted R = 0.04) ....................................................................................129

Figure 4.5

Morphological measurements used to test for a correction between distal
spine and body length of Bythotrephes longimanus. The individual
pictured is a third instar........................................................................................135

Figure 4.6

Spine morphology of Bythotrephes longimanus. The distal spine is present
at birth, whereas the second (S2) and third (S3) spine segments are added
with the first and second instar molts, respectively. We tested for a tradeoff between distal spine and segment two length using the morphological
measurements indicated below. The individual pictured is a third instar ............138

Figure 5.1

We estimated natural selection on: a) distal spine length of Bythotrephes
longimanus, the spiny water flea (the individual pictured is a third instar);
through b) a comparison between first and second instar Bythotrephes
xvi

between cohorts (solid arrows) and within a cohort (dashed arrow) using
paired sampling trips. The collection trips were separated by 3d, which
was equivalent to the duration of a single instar at the water temperature
sampled (~21°C). c) Epilimnion water temperature (°C) in Harp Lake
during the 2008 growing season. The open diamond represents the date of
first sample collection in Harp Lake ....................................................................166
Figure 5.2

a) Average distal spine length of first instar Bythotrephes within each lake.
b) and c) Natural selection on distal spine length assessed through a
comparison of first instar individuals (pre-selection phenotype) to second
instar individuals (post-selection phenotype) between cohorts (b) and
within a cohort (c). The difference between the pre- and post-selection
phenotype for each lake in panels b) and c) is equivalent to the selection
differential. There are no post-selection data for Kashagawigamog for the
within cohort analysis (c) because a second collection trip was not
completed. Error bars are standard errors. Kash: Kashagawigamog; ***p ≤
0.001; N.S.: non-significant .................................................................................168

xvii

CHAPTER 1

A new beginning: How species invasions provide insight into evolutionary ecology

Andrea Lynn-Jaeger Miehls

Introduction
As an evolutionary ecologist, I view the natural world through a lens of questions: What
are the determinants of trait phenotype? How do species traits affect interspecific interactions?
How fast do traits evolve in the wild? What conditions are conducive to adaptive evolution?
What conditions limit adaptive evolution? These questions have been the focus of the field of
evolutionary ecology for decades. They are critical questions, and they are also difficult
questions to address. For instance, consider predicting or measuring the rate of adaptive
evolution. For many species, we often lack information regarding the duration of selective
events, trait phenotype prior to selection, the identity of selective agents, and the amount of
genetic variation in traits before selection (but note that there are exceptions, including long-term
datasets for, e.g., collared flycatchers, Ficedula albicollis: Kruuk et al. 2001; Merilä et al. 2001;
great tits, Parus major: Boyce and Perrins 1987; red deer, Cervus elaphus: Kruuk et al. 2000;
2002; red squirrels, Tamiasciurus hudsonicus: McAdam and Boutin 2003; 2004; Soay sheep,
Ovis aries: Milner et al. 1999; 2000), all of which may hinder the prediction or measurement of
evolutionary rates. Moreover, many studies lack temporal or spatial replication (Kingsolver et al.
2001), which may reduce confidence in measurements or hinder the ability to generalize results.

1

With the recognition of these challenges to the study of evolutionary ecology has also
come the awareness that massive contemporary movements of species across the globe provide
incredible opportunities for the study of evolution. The introduction of species to new areas (i.e.,
species invasions) is occurring at an unprecedented rate due to anthropogenic movement of
species as well as anthropogenic alteration of the environment in ways that allow species to
expand their historic distributions (Mack et al. 2000; Kolar and Lodge 2001). Although the
ecology of species invasions has been well studied for decades (reviewed in, e.g., Elton 1958;
Mack et al. 2000; Pimentel et al. 2000; Ehrenfeld 2010), it was not until recently that the value of
species invasions for providing insight into evolutionary processes was widely appreciated
(reviewed in, e.g., Levin 2003; Cox 2004; Sax et al. 2005; Callaway and Maron 2006; Strauss et
al. 2006; Sax et al. 2007). However, it is interesting to note that, despite the recent merge of
evolutionary ecology and invasion biology, parallels between species introductions and historic
evolutionary events were recognized by Darwin (1859).
The primary value of the study of species invasions for our understanding of evolutionary
ecology is the ability to observe evolution in “real time”, free from many of the challenges that
often plague evolutionary investigations. For example, species invasions are often replicated
temporally and spatially, providing unplanned experiments (Grinnel 1919) for the study of
evolutionary processes over scales that would otherwise be nearly impossible (Sax et al. 2005;
2007). Moreover, the characteristics of species invasions are often well known, e.g., information
on the date of introduction and number of invading individuals is frequently available (Sax et al.
2005). Additionally, because of the interest in the ecology of species invasions, there are
frequently data available on population growth rates post-invasion and possibly even specimens
of invading individuals, often with their strong potential to answer evolutionary questions going

2

unrecognized (A. Miehls, pers. obs.). Finally, if information on the native range is available, a
wealth of evolutionary hypotheses can be generated based on differences in the ecology, and
thereby differences in selective pressures, between the native and invaded range. Thus, the study
of invasive species offers incredible opportunities to answer questions in evolutionary ecology
that otherwise are extremely challenging to address.
Moreover, the study of invasive species also entails an important application due to the
effects of invasive species on biodiversity and their economic cost. Species invasions are now
considered one of the world’s leading threats to biodiversity and ecosystem function (Mills et al.
1994; Vitousek et al. 1997; Pimentel et al. 2000; Kolar and Lodge 2001). Cost estimates for
control and remediation from the early 2000s, which are certainly lower than current-day costs,
indicate annual expenditures of over $141 billion in the United States and $248 billion
worldwide (Pimentel et al. 2000; Cox 2004). These costs are in addition to other, more difficult
to estimate, indirect costs to fisheries, navigation, and industry, and non-monetary costs to
spiritual and aesthetic value of ecosystems. Given the severe effects of species invasions,
invasive species are now considered part of a four-pronged ecological and evolutionary
“revolution” (Cox 2004), in combination with habitat destruction and degradation, overexploitation of plants and animals, and global climate change.
Evolutionary Ecology of Species Invasions
Invasive species experience many selective pressures in the invaded range, which they
may respond to through phenotypic plasticity (i.e., the ability of a genotype to produce different
phenotypes in response to differing environmental conditions; Baker 1965; Daehler 2003;
Richards et al. 2006; Pichancourt and van Klinken 2012) and evolution (Mooney and Cleland
2001; Lee 2002; Cox 2004; Lambrinos 2004; Strayer et al. 2006). Phenotypic plasticity is

3

considered advantageous for species invasions as plasticity enhances ecological niche breadth by
allowing organisms to express advantageous phenotypes in a broad range of environments
(Bradshaw 1965; Richards et al. 2006). When environmental conditions are similar between the
native and invaded range, phenotypic plasticity may pre-adapt (Schlaepfer et al. 2009) invasive
species, granting high fitness in the invaded range. However, invasive species will also
experience numerous new selective pressures, wrought by novel predators, prey, competitors,
and abiotic conditions in the invaded range. New selective pressures may result in evolution of
invasive species traits (Lee 2002; Cox 2004; Lambrinos 2004; Bossdorf et al. 2005). There is
growing evidence that evolution of invasive species traits may occur frequently, and that
evolution is critically important to the establishment success of many invasive species (Cox
2004; Lambrinos 2004). Importantly, evolution of invasive species traits is not restricted to
canalized traits (i.e., traits whose expression is unaffected by the developmental environment;
Stearns 1989). Evolution of phenotypic plasticity (either through de novo evolution of plasticity
or evolution of existing reaction norms) may also occur during species invasions (Sexton et al.
2002; Parker et al. 2003; Richards et al. 2006).
There is mounting evidence that phenotypic plasticity and evolution play a key role in the
pronounced effects of invasive species on ecosystems. Regarding phenotypic plasticity, there are
multiple examples that indicate phenotypic plasticity contributes to the competitive effects of
invasive species on native species. Many plants are more vigorous in the invaded range due to
phenotypic plasticity in resource acquisition traits (Chun et al. 2007; Zhao et al. 2010), which is
thought to aid in the competitive displacement of native species (Blossey and Notzold 1995;
Zhao et al. 2010). Behavioral plasticity in fish (e.g., American shad, Alosa sapidissima; Quinn
and Adams 1996) and morphological plasticity in the European wild rabbit (Oryctolagus

4

cuniculus; Williams and Moore 1989) may also increase their competitive ability and effects on
native species.
There is also growing evidence that evolution of invasive species traits contributes to
their ecological effects. For example, release of purple loosestrife (Lythrum salicaria) from
specialized herbivores in the native European range, and subsequent evolution of phenotypic
plasticity in North America, likely contributed to their competitive displacement of native
species (Blossey and Notzold 1995; Chun et al. 2007). Further, evolution of longer legs by cane
toads (Bufo marinus) has increased their range expansion rate in Australia from 10km yr-1 to
over 50km yr-1 (Phillips et al. 2006), thus magnifying their already-severe ecological effects on
native predators, prey, and competitors. Finally, Argentine ant (Linepithema humile) invasion in
North America involved a reduction in genetic diversity that was adaptive in the invaded range
due to decreased territoriality and intraspecific competition, which contributed to their
competitive displacement of native ant species (Tsutsui et al. 2000; Holway and Suarez 2004).
Whether invasive species respond to selective pressures in the invaded range through
phenotypic plasticity or evolution (including the evolution of plasticity) depends on the
frequency and direction of environmental change. When environmental change is persistent and
directional across multiple generations, evolutionary responses typically yield the highest fitness
(Lee and Gelembiuk 2008; Svanbäck et al. 2009). Conversely, when environmental change
involves short-term fluctuations (e.g., fluctuations within a generation, or between only a few
generations), phenotypic plasticity typically yields the highest fitness (Lee and Gelembiuk 2008;
Svanbäck et al. 2009). An important aspect of the fluctuating environments that select for
phenotypic plasticity is that the cues for plasticity (i.e., the environmental conditions that trigger
altered phenotype) must be consistently associated with selective environments (i.e., cues must

5

reliably predict selective environments; Hairston 1987), and plasticity must yield higher fitness
than constitutive trait expression across environments (Doughty and Reznick 2004). Thus,
whether invasive species respond to novel selection pressures in the invaded range through
phenotypic plasticity or evolution depends on temporal variation in selective pressures.

Focus of Dissertation
My dissertation focuses on evolutionary and phenotypically plastic morphological and
life history responses of an invasive species to predation. I used an invasive species in this work
because of the numerous advantages discussed above that species invasions provide toward
addressing fundamental questions in evolutionary ecology, as well as the important application
to conservation that the study of invasive species entails. My dissertation focuses on the
following questions:
General:

How do species respond to selection pressures in new environments? When are
phenotypically plastic vs. evolved responses expected?

Chapter 2: How do genetic variation and maternal effects in traits under selection vary
temporally?
Chapter 3: What environmental conditions are used as cues for phenotypic plasticity, and do
cues need to be directly related to selective agents, or can proxy cues be used?
Chapter 4: How does natural selection vary temporally and spatially, and what factors constrain
evolutionary responses to selection in the wild?
Chapter 5: Who are the agents of selection and how quickly do species traits evolve in the wild?

6

Study Organism
I used the recent invasion by Bythotrephes longimanus (the spiny water flea) in the
Laurentian Great Lakes as a model system for addressing these questions. Bythotrephes are
epilimnetic, planktivorous cladoceran zooplankton that were first found in Lake Ontario in 1982
(Barbiero and Tuchman 2004). Soon after their initial detection, Bythotrephes spread to Lakes
Huron (1984), Erie (1985), Michigan (1986), and Superior (1987; Mills et al. 1993), as well as
over 90 surrounding lakes (Strecker et al. 2006). Bythotrephes are an efficient predator of both
large and small-bodied zooplankton (Schulz and Yurista 1999; Bunnell et al. 2011), and are also
a relatively large and conspicuous prey item for many young and adult fish (Schneeberger 1991;
Pothoven et al. 2007; 2012).
Effects of Bythotrephes Invasion
The initial effects of the Bythotrephes invasion were well documented in the Great Lakes,
especially Lake Michigan (e.g., Lehman 1991; Lehman and Cáceres 1993; Schulz and Yurista
1999). The primary direct effect of Bythotrephes invasion was a shift in the size structure and
abundance of zooplankton communities (Wahlström and Westman 1999; Bunnell et al. 2011),
including the decline of four zooplankton species (Leptodora kindtii, Daphnia mendotae, D.
pulicaria, and D. retrocurva; Barbiero and Tuchman 2004). Additionally, there is evidence (e.g.,
Mills et al. 1992; Lehman and Cáceres 1993; Dumitru et al. 2001; Yan et al. 2001) that
Bythotrephes may be having indirect deleterious effects on juvenile and adult fish. Effects on
juvenile fish may occur through competition, as suggested by declines associated with failed
recruitment in yellow perch (Perca flavescens) in a small lake (Long Lake; Hoffman et al. 2001)
and Lake Michigan (Vanderploeg et al. 2002; Dettmers et al. 2003) closely following
Bythotrephes invasion. Bythotrephes can also affect fish by interfering with foraging ability,

7

leading to slower fish growth and higher mortality (Barnhisel and Kerfoot 2004; Compton and
Kerfoot 2004), and through direct physical injury following consumption, including stomach and
digestive tract punctures in up to 30-40% of cases for small fish (Compton and Kerfoot 2004).
Finally, recent precipitous declines in Lake Huron alewife (Johnson et al. 2005), and decreased
condition of Pacific salmon (Oncorhynchus spp.), have been linked to Bythotrephes via food web
interactions (Johnson et al. 2005; Eshenroder, R.L. pers. comm.).
Life History and Morphology of Bythotrephes
Bythotrephes reproduce by cyclic parthenogenesis and have a short generation time (1015 day, depending on water temperature; Branstrator 2005) that may yield up to ten or more
asexual generations during the growing season. The asexual generations are followed by sexual
reproduction at the end of the season (Yurista 1992; Pothoven et al. 2001; Branstrator 2005). The
Bythotrephes morphology features a large body size and long tail spine. The longest component
of the tail spine is the distal spine, which is present at birth and does not change with instar
development (i.e., the length of the distal segment does not grow; Burkhardt 1994; Sullivan and
Lehman 1998). Instead, during the first two molts, total spine length increases through the
addition of intercalary segments that each end with a paired barb (Branstrator 2005). The long
spine can comprise over 10% of adult body mass (Sullivan and Lehman 1998; Branstrator 2005)
and 80% of body length (Barnhisel and Harvey 1995). The length of the spine is considered a
key trait influencing Bythotrephes food web dynamics, serving as an effective deterrent against
predation by gape-limited fish (typically juvenile fish <100mm in length; Barnhisel 1991a;
Barnhisel 1991b; Schneeberger 1991; Baker et al. 1992; Barnhisel and Harvey 1995).
Potential for Evolution and Phenotypic Plasticity in Bythotrephes Traits

8

The morphology and life history of Bythotrephes in the Great Lakes strongly vary
temporally (Burkhardt 1994; Pothoven et al. 2001; 2003), but the cause is not known. In Lake
Michigan, for example, Bythotrephes tail spine and body length are shortest at the start of the
growing season (June-July), longest mid-growing season (August-September), and then decrease
at the end of the growing season (October onward; Pothoven et al. 2001). Observed seasonal
changes in Bythotrephes traits correlate with and have been theorized to be a defense against
gape-limited fish predation, which increases throughout the growing season and typically
comprises the dominant source of predation on Bythotrephes mid-to-late in the growing season
(Branstrator 2005).
Seasonal elongation of the tail spine and increased body size in Bythotrephes are
consistent with two explanations: 1) adaptive phenotypic plasticity to reduce the risk of gapelimited fish predation; and 2) natural selection by gape-limited and non-gape-limited fish
predators. Numerous studies of cladoceran zooplankton and other taxa (e.g., Dodson 1974;
Agrawal 2001; Laforsch and Tollrian 2004; Kishida et al. 2006) have shown plasticity in prey
morphology in response to gape-limited predators. These studies are suggestive that seasonal
variation in Bythotrephes traits may represent plastic responses to gape-limited predation.
Alternatively, seasonal variation in Bythotrephes traits may be due to temporally fluctuating
selection by gape-limited and non-gape-limited fish predators. Many studies indicate gapelimited predation selects for increased morphological size (e.g., Paine 1976; Case 1978; Day et
al. 2002; Zimmerman 2007; Urban 2008), whereas non-gape-limited predation selects for
decreased morphological size if larger individuals are preferentially consumed (e.g., Hambright
1991; Urban 2007; 2008). Thus, natural selection by temporally varying predator gape-limitation

9

may also explain seasonal variation in Bythotrephes traits. My dissertation explores these
mechanisms as drivers of phenotypic change in Bythotrephes traits.

Chapter Outline
Chapter 2: Genetic and maternal effects on tail spine and body length in the invasive spiny water
flea (Bythotrephes longimanus)
Chapter 2 provides a foundation for my entire dissertation by investigating genetic,
maternal, and environmental sources of phenotypic variation in Bythotrephes distal spine and
body length. I quantified these variance components during five time periods across three years
using a laboratory clonal analysis of wild-captured Bythotrephes from Lake Michigan. I found
that variance components for distal spine length were variable among but not within years. Spine
length was always heritable but was not always influenced by maternal effects. In contrast,
variance components for body length varied both within and among years, but likewise body
length was always heritable and not always influenced by maternal effects. Results indicate
Bythotrephes traits have heritable variation comparable to native species and other invasive
species that would enable an evolutionary response to natural selection. This chapter was
published in Evolutionary Applications:
Miehls, A.L.J., S.D. Peacor, and A.G. McAdam. 2012. Genetic and maternal effects on tail spine
and body length in the invasive spiny water flea (Bythotrephes longimanus). Evolutionary
Applications 5:306-316.
Chapter 3: Plastic response to a proxy cue of predation risk when direct cues are unreliable
Chapter 3 explores phenotypic plasticity in Bythotrephes morphological and life history
traits in response to temperature and fish cues. Investigating phenotypically plastic response of

10

Bythotrephes traits to their environment is critical for understanding: 1) the basis of previously
observed temporal trait variation; 2) possible adaptive plasticity in Bythotrephes traits that might
facilitate their invasion in novel ecosystems; and 3) whether plasticity may serve as a constraint
on an evolutionary response to selection. Using a laboratory experiment with Bythotrephes
collected from Lake Michigan, I found no effect of fish (Perca flavescens) cues on Bythotrephes
morphology or life history. In contrast, higher water temperature led to longer absolute spine and
body length, increased investment in morphological defense of offspring (measured as the ratio
of spine-to-body length), and decreased clutch size and age at reproduction. I argue that these
plastic responses are unlikely to be adaptive to temperature per se, but rather my findings
indicate that temperature serves as a proxy cue of predation risk.
Chapter 4: Evolutionary stasis despite selection on a heritable trait in an invasive zooplankton
Chapter 4 is the capstone chapter for the research on Bythotrephes evolutionary ecology
in Lake Michigan. Chapter 4 presents an analysis of temporal and spatial variation in natural
selection on Bythotrephes distal spine length in Lake Michigan and corresponding evolutionary
response to selection. I found that selection varied temporally, with selection for decreased distal
spine length early in the growing season, and strengthening selection for increased distal spine
length later in the growing season, a trend consistent with seasonal changes in the relative
strength of non-gape-limited and gape-limited fish predation. Selection also varied spatially,
which is consistent with well-known heterogeneity of fish predators in large lakes. Yet, despite
net selection for increased distal spine length (and known genetic basis of distal spine length;
Chapter 2), I observed little evidence of an evolutionary response to selection.
Chapter 5: Gape-limited predators as agents of selection on the defensive morphology of an
invasive species

11

Chapter 5 investigates the agents of selection on Bythotrephes distal spine length using a
set of Canadian Shield lakes dominated by either a gape-limited or non-gape-limited fish
predator. Chapter 5 is both a supporting chapter for Chapter 4 (by providing a test of selection by
gape-limited and non-gape-limited fish predators) and also a stand-alone exploration of possible
local adaptation of Bythotrephes distal spine length in response to different selective
environments. I found that natural selection varied by gape-limitation, with strong selection for
increased distal spine length in lakes dominated by gape-limited predation, and no significant
selection in lakes dominated by non-gape-limited predation. Distal spine length was 20% longer
in lakes dominated by gape-limited predation, suggesting local adaptation in response to
selection for increased distal spine length. As all study lakes were invaded less than twenty years
prior to my collections, my findings suggest rapid divergence in defensive phenotype in response
to selection from predators.
Implications of Dissertation Research
My dissertation research provides multiple contributions to the study of invasive species
and evolutionary ecology. Chapter 2 advances our current understanding of evolutionary ecology
by investigating genetic variation in quantitative traits of an invasive species, for which the vast
majority of previous work has focused on neutral genetic variation. Further, Chapter 2 quantifies
maternal variation, an under-appreciated source of phenotypic variation, and tests the stability of
genetic and maternal variation through time, a rarely completed analysis. Chapter 3 presents
what I believe to be the first evidence for a plastic response of a prey to a correlate of predation
risk, but no response to cues released from the predator. This work raises the possibility that
organismal use of proxy cues in nature may be more common than previously realized, if proxy
cues, due to their indirect and potentially surprising relationships to the adaptive responses they

12

elicit, are less often investigated than more obvious potential direct cues of plastic responses.
Chapter 5 demonstrates evolutionary stasis in an important trait governing interspecific
interactions that is under significant, directional selection and has a strong genetic basis. This
finding is based on one of the largest temporally and spatially replicated investigations of natural
selection (47 total estimates) ever completed, and also uses multiple lines of evidence (wild
captured individuals, paleoecology of retrieved spines) to show evolutionary stasis. This research
indicates evolutionary stasis can occur, even when an evolutionary response is expected due to
selection on genetically-based traits. Finally, Chapter 5 identifies gape-limited predators as
agents of selection; prior evidence for natural selection in the wild by predators on traits of prey,
especially invasive prey, is surprisingly limited. Moreover, the results of this chapter are
suggestive that substantial differences in prey phenotype can evolve rapidly (less than twenty
years) in response to predation.
Why a New Beginning?
I chose to precede the title of this chapter with “a new beginning”, a phrase not (entirely)
intended as substanceless, emotive language (although, undoubtedly the title reflects some
sentimentality as I conclude nine years of graduate study). Instead, this title is intended to echo
an exciting moment in invasion biology and evolutionary ecology, as the current convergence of
these fields brings about new discoveries in evolutionary ecology and new perspectives for
invasion biology. I began this chapter with a discussion of the value of invasive species as model
organisms for addressing major questions in evolutionary ecology. Ongoing work, including my
own dissertation research, indicates the study of invasive species can provide numerous insights
into evolutionary ecology that are otherwise incredibly challenging to attain. Thus, the study of
invasive species is catalyzing many new discoveries in the field of evolutionary ecology. Further,

13

what I mean by “new perspectives” is a re-conceptualization of species invasions as tremendous
learning opportunities. Of course, invasive species have caused incredible damage to ecosystems,
and I have no intention of glorifying or promoting these events. However, what I desire to
emphasize is the importance of learning from invasions and using that knowledge toward a better
end, whether it’s preventing future invasions or providing insight into processes governing the
natural world. This is an optimist’s perspective, making the best out of a bad situation. Finally,
on a more personal note, this dissertation defines a new beginning in my own life, as I conclude
one professional endeavor and eagerly begin the next.

14

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15

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22

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CHAPTER 2

Genetic and maternal effects on tail spine and body length in the invasive spiny water flea
(Bythotrephes longimanus)

Andrea Lynn-Jaeger Miehls, Scott D. Peacor, and Andrew G. McAdam

Abstract
Interest in the evolution of invasive species has grown in recent years, yet few studies
have investigated sources of variation in invasive species traits experiencing natural selection.
The spiny water flea, Bythotrephes longimanus, is an invasive zooplankton in the Great Lakes
that exhibits seasonal changes in tail spine and body length consistent with natural selection.
Evolution of Bythotrephes traits, however, depends on the presence and magnitude of
quantitative genetic variation, which could change within or across years. Clonal analysis of
wild-captured Bythotrephes indicated that variance components for distal spine length were
variable among but not within years. Spine length was always heritable but was not always
influenced by maternal effects. In contrast, variance components for body length varied both
within and among years, but likewise body length was always heritable and not always
influenced by maternal effects. Results indicate that important Bythotrephes traits have heritable
variation comparable to native species and other invasive species that would enable an
evolutionary response to natural selection. This evolutionary capacity could contribute to the
widespread success and dramatic effects of Bythotrephes invasion in systems with diverse biotic
and abiotic conditions.

24

Introduction
Invasive species are considered one of the leading threats to biodiversity and ecosystem
function. Whereas the myriad and often severely negative ecological effects of species invasions
have garnered considerable research attention for decades (reviewed in, e.g., Elton 1958; Mack
et al. 2000; Pimentel et al. 2000; Ehrenfeld 2010), research on the evolution of invasive species
has intensified only recently (reviewed in, e.g., Mooney and Cleland 2001; Lee 2002; Cox 2004;
Lambrinos 2004; Strayer et al. 2006). This growing body of research suggests invasive species
often evolve upon introduction into new environments due to novel selection pressures,
population bottlenecks, founder effects, and hybridization with related species in the invaded
range, as well as freedom from selection pressures in the native range (Lambrinos 2004;
Bossdorf et al. 2005). Further, there is growing evidence that evolution of invasive species may
play a key role in their pronounced ecological effects (Mooney and Cleland 2001; Lambrinos
2004; Strayer et al. 2006).
Although we are beginning to appreciate the importance of evolution to the ecological
effects of invasive species, we still have limited knowledge of the rate of and constraints on
invasive species evolution (but see Lavergne and Molofsky 2007; Dlugosch and Parker 2008;
Colautti et al. 2010). In particular, we know little about genetic variation in invasive species
traits despite its potential to constrain evolutionary rates (Lynch and Walsh 1998; Conner and
Hartl 2004; but see Blows and Hoffman 2005). A wealth of studies of neutral (molecular) genetic
variation in invasive species have been published (Dlugosch and Parker 2008), but there have
been few studies of genetic variation in quantitative traits (reviewed in Table 2.1; see also
Dlugosch and Parker 2008). Furthermore, the few studies that have addressed quantitative
genetic variation suggest no consistent pattern in the amount of genetic variation in invasive

25

species traits; quantitative genetic variation ranged from very low to exceptionally high (Table
2.1).
The amount of genetic variation for a given trait can also vary in space and time due to
genotype-by-environment interactions (Pigliucci 2001) or the erosion of genetic variation by
natural selection. For example, previous research with clonally reproducing zooplankton
suggests that genetic variation, as well as the ratio of genetic variation to total phenotypic
variation (i.e., heritability), can decline throughout the growing season due to clonal selection
(Lynch 1984; Pfrender and Lynch 2000). Thus, to understand how genetic variation affects the
evolution and persistence of invasive species, it is important that genetic variation be quantified
across a range of environmental conditions across the growing season.
Recent species introductions into the North American Great Lakes offer a strong
opportunity to investigate sources of variation in invasive species traits and their ramification for
ecological interactions. One invasive species considered to have potent ecological effects in the
Great Lakes is Bythotrephes longimanus, the spiny water flea (hereafter Bythotrephes; Figure
2.1), a predatory zooplankton native to Eurasia that was introduced to North America in the
1980s (Mills et al. 1993). The invasion of Bythotrephes has resulted in dramatic ecosystem
changes due to their central position in the food web as a dominant predator of zooplankton
(Bunnell et al. 2011) and common prey for fish (Pothoven et al. 2007). The primary ecological
effects of Bythotrephes involve alteration of zooplankton community abundance and
composition (Lehman 1991; Yan et al. 2002; Barbiero and Tuchman 2004), which may affect
phytoplankton communities (Strecker and Arnott 2008) and larval fish (Hoffman et al. 2001)
through trophic cascades.

26

In this study we used clonal analysis of wild-captured Bythotrephes raised across three
generations in captivity to measure quantitative genetic variation in two Bythotrephes traits,
namely their distal spine and body length, which affect their susceptibility to fish predators (see
Study Species, below) and thus govern their food web interactions. We assessed whether genetic
variation in Bythotrephes traits differed among five time periods across three years. Although the
initial Bythotrephes invasion may have involved a genetic bottleneck (based on analysis of
neutral genetic variation; Colautti et al. 2005), their rapid spread soon after invasion (Mills et al.
1993) as well as evidence for multiple introductions and gene flow among introduced
populations (Colautti et al. 2005) all suggest genetic variation in the Bythotrephes population
may be high. We, therefore, predicted Bythotrephes traits would contain significant genetic
variation. Following previous studies of clonal selection in other cladoceran zooplankton (e.g.,
Daphnia; Lynch 1984; Pfrender and Lynch 2000), we further predicted that genetic variation
would decline throughout the growing season.

Methods
Study Species
Bythotrephes reproduce by cyclic parthenogenesis and have a short generation time (1015 day, depending on water temperature; Branstrator 2005). They produce multiple asexual
generations during the growing season, which culminate in sexual reproduction at the end of the
season (Yurista 1992; Pothoven et al. 2001; Branstrator 2005). A conspicuous trait of
Bythotrephes is their long tail spine (Figure 2.1). The longest component of the tail spine is the
distal spine (i.e., spine section from the tip of the spine to the first set of instar barbs), which is
present at birth and does not change with instar development (i.e., the length of the distal

27

segment does not grow). Instead, during the first two molts, total spine length increases through
the addition of intercalary segments that each end with a paired barb (Branstrator 2005).
A large volume of research documenting fish predation on Bythotrephes indicates that
fish are size-specific predators and suggests that natural selection on Bythotrephes traits may be
occurring, although no study has explicitly measured natural selection. Bythotrephes are
consumed by both gape-limited fish (typically juvenile fish less than 100mm length) and nongape-limited fish (typically adult fish; Schneeberger 1991; Mills et al. 1992; Branstrator 2005;
Pothoven et al. 2007), with gape-limited predation dominating later in the growing season
(Branstrator 2005). Because gape-limited fish selectively choose prey based on prey size, lateseason gape-limited fish predation may be an important source of selection on the size of
Bythotrephes morphological traits. Field surveys showing a correlation between Bythotrephes
spine and body size and the abundance of gape-limited fish (Straile and Hälbich 2000), diet
analysis of wild-caught fish (Schneeberger 1991; Mills et al. 1992; Barnhisel and Harvey 1995),
variable spine and body size of Bythotrephes among lakes with different fish predation regimes
(Bilkovic and Lehman 1997; Sullivan and Lehman 1998), and evidence for selective feeding of
fish on Bythotrephes based on size of their morphological traits (Barnhisel 1991a; 1991b) all
suggest that fish may act as agents of natural selection on Bythotrephes morphological traits.
Field Collections
We collected Bythotrephes from Lake Michigan during 2007, 2008, and 2010 at either
the 60m (2007, 2008; sampling coordinates: 43º 13.80' N 86º 29.58' W) or 45m (2010; sampling
coordinates: 43º 12.40' N 86º 27.06' W) depth contours, approximately 12.5km and 9.5km,
respectively, west of Muskegon, MI. There was a single collection of 60 Bythotrephes midgrowing season (September 27) in 2007, three collections of approximately 300 Bythotrephes

28

each spanning the growing season (July 28, September 22, and November 3) during 2008, and a
single collection of 25 Bythotrephes early in the growing season (July 28) during 2010. Because
Bythotrephes resting eggs hatch only at the start of the growing season (Yurista 1992) and Lake
Michigan is well mixed at our sampling locations (Beletsky et al. 2007), we assumed that our
samples at different time periods represented repeated samples of the same population. We used
a conical zooplankton net with a 1m diameter opening and 363μm mesh size towed vertically
through the top 25m of the water column. On shipboard, Bythotrephes were placed into 60mL
clear glass jars (one per jar) within 10min of collection. Jars contained approximately 50mL of
Lake Michigan water previously passed through a 63µm sieve.
Laboratory Culturing
We reared Bythotrephes in biological incubators using methods derived from Kim and
Yan (2010) and personal communications (K. Schulz, State University of New York – College of
Environmental Science and Forestry, Syracuse, New York, USA). We maintained Bythotrephes
at 15ºC with a 13:11 light:dark cycle in Lake Michigan water passed through GF/F Whatman
filters. Water changes occurred every-other-day in 2007 and daily in 2008 and 2010. Each day
we fed Bythotrephes ad libitum with approximately 150 Artemia nauplii that were less than 48hr
old; in 2010 Bythotrephes were also given three Daphnia pulicaria daily.
Clonal Lines
We used clonal lines (Lynch and Walsh 1998) to raise Bythotrephes through the F2
generation (Figure 2.2). We initiated each clonal line with a wild-captured Bythotrephes; the
young (F1 generation) of the wild Bythotrephes were used to establish clonal sub-lines. We
assumed all wild-captured Bythotrephes were genetically distinct, whereas we assumed all young
born to a clonal line were genetically identical (Lynch and Walsh 1998). All young born in the

29

lab were transferred to individual culturing jars within 24hr of birth. In 2007 and 2008, F2
Bythotrephes were reared until they reproduced or died and preserved in 95% ethanol, whereas
in 2010, F2 Bythotrephes were preserved at birth (first instar). Consequently, in 2007 and 2008,
F2 Bythotrephes were preserved at various instar stages (1, 2, and 3). Whereas distal spine length
does not change through instar development, body size increases with each instar (Burkhardt
1994), so we accounted for instar stage in the body length analysis. Two of our sampling periods,
September 2007 and November 2008, lacked sufficient replication of instars among clonal and
sub-clonal lines to estimate sources of phenotypic variation. Thus, only body length data in July
and September 2008 and July 2010 were analyzed. However, clonal analyses of distal spine
length were performed for all time periods (i.e., September 2007, July through November 2008,
and July 2010).
Morphological Measurement
We photographed F2 Bythotrephes using a digital camera attached to a dissecting
microscope. Bythotrephes were oriented on their side such that the length of the body and tail
spine was along a single plane of focus and the eye, thorax, and abdomen regions of the body
were visible (Figure 2.1). Using ImageJ (Abramoff et al. 2004), we measured distal spine and
body length to the nearest 0.001mm of F2 Bythotrephes from the digital photographs. Whereas
our measurement of distal spine length (measured from the tip of the spine to the anterior end of
the first set of instar barbs; Figure 2.1) was standard for Bythotrephes research (e.g., Sullivan and
Lehman 1998), our measurement of body length was not. We noted that some Bythotrephes had
a bend at the junction of the thorax and abdomen (as in Figure 2.1) whereas others did not.
Differences in body position could, therefore, inflate measurement variation of body length

30

based on previously used measurement techniques (linear distance from the eye to the base of the
tail spine, e.g., Sullivan and Lehman 1998). As a result, we measured body length from the
anterior edge of the eye to the base of the tail spine along the midline of the body with segments
spanning the head and eye region, thorax, and abdomen (Figure 2.1). Although some organisms
are known to decrease in length after preservation in ethanol (e.g., larval fish; Moku et al. 2004),
our test of Bythotrephes morphological shrinkage in 95% ethanol revealed no significant
shrinkage of Bythotrephes distal spine or body length (Supplementary Material).
Heritability, Maternal Effects, and Statistical Analyses
Using our clonal breeding design, we quantified genetic, maternal, and environmental
variance components for distal spine and body length from statistical models of among clonalline, among sub-line, and within sub-line variation, respectively (Lynch 1985; Lynch and Walsh
1998; Figure 2.2). We fitted linear mixed effects models (LME) for each trait separately (i.e.,
distal spine or body length) using the nlme package (Pinheiro et al. 2009) in R version 2.10.1 (R
Development Core Team 2009). In each case, the trait measured in F2 offspring was modeled by
random effects for clonal line and sub-line nested within clonal line. We assessed the
significance of the random effects in two ways: 1) by obtaining 95% confidence intervals around
the random effects through bootstrapping (Potvin and Roff 1993); and 2) through model
comparisons using likelihood ratio tests (LRT). 95% confidence intervals were obtained by
randomly resampling our dataset of distal spine and body lengths (1000 iterations, accounting for
clonal and sub-line structuring), which created a distribution of variances around our random
effects. For the LRT, we fitted two additional models for each trait, with each model containing
successively fewer random effects. The first additional model for each trait contained the random
effect for clonal line but not the random effect for sub-line. The second additional model for each

31

trait was a linear model without either random effect. As Bythotrephes body size increases
during instar development, we included instar as a fixed effect (categorical for instars 1, 2, and 3)
in models of body length. The only fixed effect in models of distal spine length was the intercept.
We also tested for temporal differences in genetic and maternal variance components by
assessing differences by month within 2008 (July, September, and November) and assessing
differences across years. We compared a model containing time period (either month or year) as
a fixed effect and all random effects described above to a model containing the same fixed and
random effects, but which allowed clonal and sub-line variation to differ by time period using the
varIdent function in the nlme package (Pinheiro and Bates 2000). We used a LRT to assess
whether separate estimates of clonal and sub-line variation for each time period significantly
improved the fit of the model. Note that this approach differs from simply assessing the
significance of time period as a fixed effect, which would assess whether mean phenotypes (i.e.,
distal spine or body length) differ among time periods. In our analysis, we were testing whether
time period affected the clonal line and sub-line random effects in the model.
Descendants of clonal organisms are effectively linkage groups for their entire genotype
2

and, therefore, broad-sense heritability (H ) is the appropriate measure of inheritance (Lynch and
Walsh 1998; Conner and Hartl 2004). We estimated genetic (Vg), maternal (Vm), and
environmental (Ve) variation from the variance among clonal lines, variance among clonal sublines, and variance within clonal sub-lines, respectively, from our mixed-effect model analysis of
our clonal breeding design. Based on Houle (1992), we estimated the coefficient of genetic
variation (CVg) as:

32

CVg 

100 * V g
mean

,

accounting for clonal and sub-line structure in the trait mean. We used the variance components
2

to calculate H as:

H2 

Vg
.
Vg  Vm  Ve

2

2

Similar to the calculation of H , we calculated maternal effects (m ) as the ratio of
maternal variance (Vm) to total phenotypic variance (Vg+ Vm + Ve).

Results
We obtained 13 clonal lines that produced F2 Bythotrephes in September 2007, 18, 31,
and 4 clonal lines for July, September, and November 2008, respectively, and 17 clonal lines for
July 2010 (percent survival of clonal lines to the F2 generation during the five time periods was
22, 6, 9, 2, and 68%, respectively).
F2 Bythotrephes mean (± 1 SD; controlled for clonal and sub- line) distal spine length
was 4.72 ± 0.73mm in September 2007, 5.11 ± 0.50mm in 2008 (all months combined), and 5.70
± 0.34mm in July 2010. Distal spine length in 2010 was significantly greater than in 2007 (p <
0.001) and 2008 (p < 0.001); distal spine length in 2008 was also significantly greater than in
2007 (p < 0.05). Variance components for Bythotrephes distal spine length did not differ among
2

months within 2008 (likelihood ratio test: χ 2 = 3.3, p > 0.05), but differed across years
2

(likelihood ratio test: χ 2 = 74.5, p < 0.001). We found significant (based on 95% confidence
33

intervals and the LRT) genetic variation in Bythotrephes distal spine length in 2007, 2008, and
2010 (Table 2.2; Figure 2.3), corresponding to CVg estimates of 15.0, 8.1, and 4.1, respectively,
2

and H estimates of 0.76, 0.48, and 0.27, respectively (Table 2.2). We found significant maternal
2

variation in distal spine length in 2008 and 2010, corresponding to m estimates of 0.13 and 0.59,
respectively, but not in 2007 (Table 2.2; Figure 2.3).
F2 Bythotrephes mean body length for instars one, two, and three, was 1.53 ± 0.18mm,
1.79 ± 0.24mm, and 2.34 ± 0.15mm in July 2008 and 1.61 ± 0.29mm, 1.86 ± 0.24mm, and 2.21 ±
0.34mm in September 2008, respectively, and 1.58 ± 0.10mm for instar one in July 2010. Body
length for instars one, two, and three did not differ by time period (p > 0.05 for all comparisons).
In contrast to distal spine length, variance components for body length differed between months
2

within 2008 (likelihood ratio test: χ 1 = 13.4, p < 0.001) and also across years (likelihood ratio
2

test: χ 2 = 72.5, p < 0.001). We found significant genetic variation in Bythotrephes body length
in July 2008, September 2008, and July 2010 (Table 2.2; Figure 2.4), corresponding to CVg
2

estimates of 7.8, 13.4, and 3.5, respectively, and H estimates of 0.30, 0.48, and 0.13,
respectively (Table 2.2). We found significant maternal variation in body length in July 2008 and
2

July 2010, corresponding to m estimates of 0.23 and 0.44, respectively, but not in September
2008 (Table 2.2; Figure 2.4).

34

Discussion
A growing body of research suggests that evolution in traits important to interspecific
interactions of invasive species may play a key role in their effects on ecosystems (e.g., Mooney
and Cleland 2001; Lambrinos 2004; Strayer et al. 2006). Our research quantified genetic
variation in traits of the invasive zooplankton, Bythotrephes longimanus, a species that has
caused severe and widespread ecological effects in the Great Lakes. We found that the two traits
considered to have the greatest effect on food web interactions with fish, namely Bythotrephes
distal spine and body length, both had significant genetic and maternal variation and exhibited
moderate-to-high coefficients of genetic variation, broad-sense heritabilities, and maternal
effects (Table 2.2). Moreover, genetic variation for Bythotrephes traits did not decline over the
growing season, as is typical for cladoceran zooplankton, but instead remained similar for distal
spine length and increased for body length (Table 2.2).
Coefficients of genetic variation and heritability estimates for Bythotrephes distal spine
and body length are similar to other invasive species (Table 2.1) and heritability estimates fall
within the range of previously published broad-sense heritabilities for native cladoceran
zooplankton, suggesting that evolution of these important Bythotrephes traits may not be limited
by low genetic variation. Our range in heritability estimates (Table 2.2) for Bythotrephes distal
spine and body length is similar to the range of heritabilities (0.28 – 0.62; Pfrender and Lynch
2000) for body size of Daphnia pulex, a widespread native Daphnia species. The concordance of
heritability estimates between the invasive Bythotrephes and native Daphnia is significant given
that previous studies have suggested that low genetic variation relative to other sources of
variation may prohibit evolution of invasive species. Multiple source populations have already
been suggested for the Bythotrephes invasion based on evidence from neutral loci (Colautti et al.

35

2005) and may explain the presence of substantial genetic variation relative to other sources of
variation in the traits we investigated.
Two potential sources of bias to our study must be considered. First, our heritability
estimates were derived from a clonal analysis in the lab, which is typical for heritability
estimates for cladocerans (e.g., Lynch 1984; Lynch and Walsh 1998; Pfrender and Lynch 2000),
but which could introduce a bias due to experimentally controlled environmental conditions
(Conner et al. 2003; Wolff 2003; Calisi and Bentley 2009; but see, Weigensberg & Roff 1996).
Our estimates of environmental variation (Table 2.2) were consistently lower than genetic
variation for Bythotrephes distal spine length, but similar to genetic variation for Bythotrephes
body length and maternal variation for both traits. It is possible that low environmental variation
in the laboratory inflated our estimates of heritability and maternal effects as compared to wild
populations. Second, our analyses involved low sample sizes that resulted from the, at times,
poor survival of clonal lines and logistical constraints of the labor-intensive culturing of
Bythotrephes (Kim and Yan 2010), which necessitated lower sample sizes than studies of other
organisms (e.g., Daphnia). It is possible that poor survival could have reduced our estimates of
genetic and maternal variation if significant lab-based clonal selection occurred. However, there
was no relationship between the magnitude of genetic or maternal variation and clonal line
mortality; our two time periods with the lowest clonal line mortality, September 2007 and July
2010, represented the highest and lowest estimates of genetic variation in distal spine length. The
lack of relationship between mortality and sources of variation indicates that mortality was
random with respect to our clonal lines. Further, the absence of a significant difference in genetic
variation by time period within 2008 for distal spine length was unlikely to be due to low
statistical power, as these data were sufficient for detecting significant changes in genetic

36

variation in distal spine length across years and changes in genetic variation for body length both
within and across years. Thus, despite variable clonal line survival among time periods, we did
not have any evidence for clonal selection associated with our culturing approaches and the
remaining samples were sufficient to test our predictions.
Our findings did not support the prediction that genetic variation in Bythotrephes traits
would decrease throughout the growing season, despite the similarity in life history between
Bythotrephes and the other cladoceran zooplankton on which we based this prediction. Like most
cladoceran species, Bythotrephes reproduce by cyclic parthenogenesis (i.e., multiple rounds of
asexual reproduction culminating in sexual reproduction that produces resting eggs at the end of
the growing season; Yurista 1992; Pothoven et al. 2001; Branstrator 2005). After sexual
reproduction, genetic variation and broad-sense heritabilities for cladoceran traits are often high
(with heritabilities falling within the range of 0.4 – 0.6) as each resting egg hatchling is
genetically unique (Lynch 1984). During subsequent asexual reproduction, genetic variation
typically is reduced and broad-sense heritabilities decline due to clonal selection, approaching
zero at the end of the season (Lynch 1984). Contrary to the scenario of clonal selection eroding
genetic variation over the growing season, genetic variation for Bythotrephes distal spine length
did not differ during three sampling periods within one growing season (July – November 2008).
Furthermore, genetic variation in Bythotrephes body length increased between July and
September 2008. The increase of genetic variation in Bythotrephes body length was associated
with a decrease in maternal variation that together resulted in increased heritability, despite a
concomitant increase in environmental variation.
Multiple scenarios could explain why seasonal patterns in Bythotrephes genetic variation
are dissimilar to other cladoceran zooplankton. Previous research documenting declines in

37

genetic variation of Daphnia clones during the growing season (Lynch 1984) was associated
with strong and consistent natural selection. Conversely, temporal fluctuation in the direction of
selection across the growing season (Sasaki and Ellner 1997), such as from opposing selection
pressures by gape-limited and non-gape-limited fish predators whose predation rate varies
seasonally (Branstrator 2005), might maintain high levels of genetic variation in Bythotrephes
traits both within and across seasons. Alternatively, phenotypically plastic responses to changing
environmental conditions can prevent selection from eroding genetic variation (Ghalambor et al.
2007), and thus could explain the persistence of genetic variation in Bythotrephes traits. If clones
differ in their plastic responses to the environment (i.e., if clones differ in their reaction norms),
then certain clones may be favored in one set of environments and selected against in another set,
thus maintaining genetic variation (Pigliucci 2001). Indeed, Burkhardt (1994) and Pothoven et al.
(2003) suggested that Bythotrephes body size may be phenotypically plastic, potentially varying
in response to their abiotic (e.g., temperature) and biotic (e.g., predator or prey presence)
environment. Although our results for genetic variation in Bythotrephes distal spine and body
length suggest a more complex scenario than unidirectional clonal selection through the growing
season, to date, no study has estimated natural selection or quantified phenotypic plasticity
experimentally in Bythotrephes traits. Thus, knowledge of selection and plasticity would be
valuable for understanding why Bythotrephes distal spine and body length exhibit seasonal
patterns in genetic variation that are atypical for a cyclic parthenogen.
Genetic variation in Bythotrephes distal spine and body length has implications for how
Bythotrephes can respond to their food web interactions in the Great Lakes and could explain
their invasive success in ecosystems with varying biotic conditions. Genetic variation allows for
an evolutionary response to persistent environmental change across multiple generations (Lee

38

and Gelembiuk 2008; Svanbäck et al. 2009). Our finding of moderate-to-high heritabilities
throughout the growing season and across years suggests that Bythotrephes morphology could
evolve in response to gape-limited fish predation, which increases throughout the growing
season. Evolution of increased body size in response to gape-limited predation has previously
been shown in Ambystoma maculatum (spotted salamander) larvae (Urban 2008) and Daphnia
species (Spitze 1991). Moreover, Bythotrephes are abundant in a variety of lake environments
with differing fish predator species and relative abundance of gape- versus non-gape limited
predators (Bilkovic and Lehman 1997; Sullivan and Lehman 1998; Young and Yan 2008).
Concordantly, previous research has shown that distal spine and body length are variable among
lakes (Sullivan and Lehman 1998). Potentially, the variability in Bythotrephes spine and body
length represents local adaptation to differing fish predation regimes, if Bythotrephes in other
systems also maintain sufficient genetic variation to respond to selection from fish.
In addition to significant genetic variation, we also found significant maternal variation in
Bythotrephes distal spine and body length that can affect phenotypic change between
generations. The type of maternal effects we estimated are sometimes referred to as maternal
identity effects (Sakwińska 2004) or intergenerational environmental effects (Schwaegerle et al.
2000). In a clonal analysis, maternal identity effects occur when sisters within a clonal line
produce offspring that consistently differ despite being genetically identical and raised in a
common environment. Maternal identity effects can be caused by maternal responses to smallscale environmental variation, which all experiments (even highly controlled laboratory
experiments) inevitably involve (Schwaegerle et al. 2000), or by individual genetic differences
caused by mutation. Because maternal identity effects have been shown to affect traits related to

39

fitness (Sakwińska 2004), maternal identity effects may represent an important source of
phenotypic variation in fitness-related traits of Bythotrephes.
In conclusion, our research has quantified genetic variation in two important ecological
traits of an invasive species and has tested the stability of these sources of variation through time.
Genetic variation in Bythotrephes distal spine and body length could contribute to the success of
Bythotrephes at invading and dramatically altering ecosystems by permitting an evolutionary
response to selection. This capacity for phenotypic change could explain, in part, the ability of
Bythotrephes to invade lake ecosystems with variable biotic and abiotic conditions. As invasive
species continue to threaten biodiversity worldwide, elucidating both the ecological and
evolutionary foundations for their success will be critical.

40

Acknowledgements
We thank the McAdam Lab, Doug Schemske, and three anonymous reviewers for helpful
comments. Doran Mason, Dennis Donahue, Steven Pothoven, and the NOAA Great Lakes
Environmental Research Laboratory and NOAA Lake Michigan Field Station offered research
vessels and field support staff. Keali Chambers, Brittany Damschroder, Jason Fischer, Brittany
Gunther, Nicole Hedquist, Lydia Kramer, Ian McCririe, Scott Miehls, Jennifer Pellegrini,
Veronica Quesnell, Andria Salas, Ben Staton, Marie Stevenson, and Brandon Vieder helped to
collect data. Natalie Kim, Kevin Pangle, Kim Schulz, and Peder Yurista provided valuable help
with Bythotrephes culturing protocols. This work was supported by the Great Lakes Fishery
Commission, the National Science Foundation (DEB-0089809), and an EPA Science to Achieve
Results (STAR) fellowship (STAR Research Assistance Agreement No. FP91698801-0 awarded
by the U.S. Environmental Protection Agency). This work has not been formally reviewed by the
EPA and the views expressed in this document are solely those of the authors. SDP
acknowledges support from Michigan State University AgBioResearch. This is contribution
number 1603 of the NOAA Great Lakes Environmental Research Laboratory.

41

Tables
Table 2.1. Quantitative genetic variation in invasive species traits, including genetic and additive genetic variation (Vg and Va,
2

2

respectively), coefficients of additive genetic variation (CVa), and broad- and narrow-sense heritabilities (H and h , respectively).
Species

Trait

Anolis cristatellus

2

Number of circum-trunk scales

Vg

1

Va

CVa

H

1

0.48 Eales et al. 2010

Number of ventral scales
Eurytemora affinis

2

0.79

Lee et al. 2003

2,3

1.40

Lee et al. 2007

2,3

0.54

Lee et al. 2007

0.46

Lee et al. 2007
Boman et al. 2008

Survival to metamorphosis: 15 PSU
Survival to metamorphosis: 0 PSU
Survival to metamorphosis: 5 PSU

Survival to metamorphosis: 25 PSU
Leptinotarsa
decemlineata

2

Reference
h
0.27 Eales et al. 2010

2,3

4

Development time: 17°C

5.50

0.55

4

Development time: 23°C
Female adult weight: 17°C
Male adult weight: 17°C
Female adult weight: 23°C
Male adult weight: 23°C
5
Emergence body mass

7.11
5.60
8.65
14.64
4.23

0.52
0.23
0.62
0.55
0.07

0.01x10

Boman et al. 2008
Boman et al. 2008
Boman et al. 2008
Boman et al. 2008
Boman et al. 2008
0.05 Piiroinen et al. 2011

Overwintering body mass

0.05x10

-3

0.12 Piiroinen et al. 2011

-3

0.05x10

0.06 Piiroinen et al. 2011

Mass independent metabolic rate

-3

0.05x10

0.08 Piiroinen et al. 2011

Diapause behaviour

-3

0.08x10

0.02 Piiroinen et al. 2011

-3

0.17x10

0.05 Piiroinen et al. 2011

-3

1.69x10

0.16 Piiroinen et al. 2011

-3

5

5

Metabolic rate

5

5

Overwintering survival

5
5

Days until entered diapause

42

Table 2.1 (con’t)
Species
Mahonia genus
Phalaris

2

2

6

0.47

Lavergne and Molofsky 2007

6

arundinacea

0.27

Reference
Ross et al. 2009
Lavergne and Molofsky 2007

0.27

Lavergne and Molofsky 2007

0.49

Lavergne and Molofsky 2007

Trait
Seedling growth
6
Relative growth rate
Tillering rate

Vg
0.01

Va

CVa

Leaf number
Stem height

6
6

Root/shoot ratio

Below ground biomass

0.27

Lavergne and Molofsky 2007

0.26

Lavergne and Molofsky 2007

6

0.30

Lavergne and Molofsky 2007

0.08

Lavergne and Molofsky 2007
Brooks and Endler 2001a
Brooks and Endler 2001a
Brooks and Endler 2001a
Brooks and Endler 2001a
Brooks and Endler 2001a
Brooks and Endler 2001a
Brooks and Endler 2001a
Brooks and Endler 2001a
Brooks and Endler 2001a
Brooks and Endler 2001a
Brooks and Endler 2001a
Brooks and Endler 2001a
Brooks and Endler 2001a
Brooks and Endler 2001b
Brooks and Endler 2001b
Brooks and Endler 2001b

6

Potamopyrgus
antipodarum

h

6

Above ground biomass
Poecilia reticulata

H

Emergence time
Male body area
Male tail area
Male black area
Male fuzzy black area
Male iridescent area
Male orange area
Male orange chroma
Male orange brightness
Male total spot number
Male mean brightness
Male brightness contrast
Male mean chroma
Male color contrast
Female responsiveness to males
Female discrimination of males
7
Female preference for male traits

11.79
13.89
35.58
55.29
36.76
67.33
17.83
20.58
23.67
4.53
15.46
1.95
9.53

0.87
0.41
0.43
0.79
0.45
0.96
0.15
0.47
0.79
0.58
0.54
0.55
0.38
0.27
0.11
0.02

Size at first reproduction

8

0.74

0.09

Dybdahl and Kane 2005

8

Age at first reproduction

58.59

0.07

Dybdahl and Kane 2005

43

Table 2.1 (con’t)
Species

Trait

Vg
47.76

8

Total offspring

8

Reproduction rate

Va

0.22

CVa

2

H
0.20
0.12

9

2

h

Reference
Dybdahl and Kane 2005
Dybdahl and Kane 2005

Length at termination

0.07 Koskinen et al. 2002

Yolk-sac volume

Thymallus thymallus

0.04 Koskinen et al. 2002

9

9

0.21 Koskinen et al. 2002

Growth rate
9

Survival

0.12 Koskinen et al. 2002
9

0.37 Koskinen et al. 2002

9

Swim-up length

0.25 Koskinen et al. 2002

9

Hatching length

0.10 Koskinen et al. 2002

Incubation time

Note: Statistically significant values are presented in bold; values reported without reference to statistical significance are presented in
italics. We obtained values from a literature search using the ISI Web of Science® with the key words “invasive species” and “genetic
variation”. Only studies reporting univariate estimates of quantitative genetic trait variation of invasive species in their invaded range
were included.
1
2

2

Eales et al. (2010) derived h from mother-offspring regression, and thus values may be inflated due to maternal effects.
Only traits for invasive populations of E. affinis from Lee et al. (2003; 2007) were included.

3 2

H obtained by doubling intra-class correlations reported in Lee et al. (2007).

4

2

CVa and H averaged across invasive populations in Boman et al. (2008).
44

5

2

Va and h were calculated from Vsire, Vdam, Vpotato, and Vresid reported in Piiroinen et al. (2011) following Falconer and Mackay

(1996); only values for both males and females were included.
6 2

H determined from Figure 5 of Lavergne and Molofsky (2007) for invasive range.

7 2

h averaged across female preferences for the following male traits reported in Brooks and Endler (2001b): attractiveness, predicted

attractiveness, body size, tail area, black area, fuzzy black area, orange area, orange chroma, orange brightness, iridescent area, spot
number, mean brightness, brightness contrast, mean chroma, and color contrast.
8

2

Vg and H averaged across 12 and 15°C temperature treatments in Dybdahl and Kane (2005).

9 2

h averaged across populations in Koskinen et al. (2002).

45

Table 2.2. Genetic (Vg), maternal (Vm), and environmental (Ve) variance components, coefficient of genetic variation (CVg), broad2

2

sense heritability (H ), and maternal effects (m ) for distal spine and body length of Bythotrephes longimanus.
Time
Period

Vg

Distal spine length
2007 September 0.498
2008 Combined 0.172
2010 July
0.055
Body length
2008 July
0.016
2008 September 0.056
2010 July
0.003

95% C.I.
Lower Upper CVg

Vm

95% C.I.
Lower Upper

Ve

95% C.I.
Lower Upper

H

2

m

2

0.150
0.085
0.041

0.928
0.260
0.174

15.0
8.1
4.1

0.057
0.046
0.118

0.005
0.011
0.076

0.080 0.097
0.122 0.138
0.166 0.028

0.059
0.109
0.022

0.158
0.176
0.036

0.76
0.48
0.27

0.09
0.13
0.59

0.004
0.028
0.001

0.063
0.109
0.013

7.8
13.4
3.5

0.012
<0.001
0.012

0.003
0
0.005

0.042 0.025
0.013 0.060
0.022 0.012

0.017
0.044
0.009

0.036
0.080
0.015

0.30
0.48
0.13

0.23
0
0.44

Note: Variance components for distal spine length did not differ by month within 2008, but differed among years. Variance
components for body length differed by month in 2008 and between years. Statistically significant (p < 0.05) genetic and maternal
variance components based on likelihood ratio tests are presented in bold; 95% confidence intervals around the variance components
are given.

46

Figures

Figure 2.1. Bythotrephes longimanus, the spiny water flea. The Bythotrephes tail spine is
comprised of the distal spine (measured from the tip of the spine to the anterior end of the first
set of instar barbs; solid line) and up to two intercalary segments (for asexually produced
Bythotrephes). Body length was measured from the anterior edge of the eye to the base of the tail
spine along the midline of the body with segments spanning the head and eye region, thorax, and
abdomen (dashed line). The Bythotrephes pictured is a third instar.

47

Bythotrephes Clonal Analysis
Bythotrephes
Clone A

Bythotrephes
Clone B

Wild-captured
Bythotrephes
Sub-line (F1)
Grandoffspring (F2)
Vg

Vm

Ve

Figure 2.2. Clonal analysis design, with clonal sub-lines (F1 generation) nested within clonal
lines (wild-caught Bythotrephes); all analyses were completed using morphological data from the
F2 generation. Vg is the genetic variance, represented by the variance in morphology of F2
individuals among clonal lines, Vm is maternal variance, represented by the variance among sublines within clones, and Ve is the residual environmental variance within sub-lines. Though two
F1 young per clonal line and two F2 young per sub-line are pictured, all young born to clonal and
sub-lines were reared. Figure modified from Lynch and Walsh (1998).

48

Figure 2. Genetic (Vg), materna (Vm), and environmen (Ve) vari
.3.
al
ntal
iance compo
onents for
Bythotrep
phes distal spine length. Variance co
omponents d not differ by month w
did
r
within 2008, but
differed among years Bars are th 95% conf
a
s.
he
fidence interv around the variance component
vals
e
ts.

49

Figure 2. Genetic (Vg), materna (Vm), and environmen (Ve) vari
.4.
al
ntal
iance compo
onents for
Bythotrep
phes body le
ength. Varian components differed by month i 2008 and between yea
nce
d
in
ars.
Bars are the 95% con
nfidence inte
ervals around the varianc componen
d
ce
nts.

50

APPENDIX

51

Appendix
Appendix A. Shrinkage of Bythotrephes longimanus distal spine length and body length in 95%
ethanol.
Morphological shrinkage of organisms can occur in chemicals used for preservation (e.g.,
shrinkage of larval fish in 90% ethanol; Moku et al. 2004). Yan and Pawson (1998) found that
the length of Bythotrephes morphological traits changed in various concentrations of sugarformalin, a preservative derived from formaldehyde. However, no estimates of Bythotrephes
morphological shrinkage in 95% ethanol have been published. We, therefore, completed an
experiment to estimate shrinkage of Bythotrephes distal spine and body length in 95% ethanol.
We tested shrinkage of Bythotrephes distal spine and body length using Bythotrephes
collected from Lake Michigan on July 31, 2008 and transported live to Michigan State
University. Bythotrephes were photographed within five seconds of placement in ethanol using a
camera mounted to a dissecting microscope. After photography, Bythotrephes were stored
individually in ethanol for 126 days. Bythotrephes morphological traits were measured using
ImageJ image analysis software (Abramoff et al. 2004). Distal spine length was measured from
the tip of the spine to the anterior end of the first set of instar barbs and body length was
measured from the anterior edge of the eye to the base of the tail spine along the midline of the
body with segments spanning the head and eye region, thorax, and abdomen.
Although a percentage correction (final length / initial length * 100%) has previously
been used to estimate morphological shrinkage in chemical preservatives, using a percentage
correction assumes an isometric relationship between the numerator and the denominator. In the
absence of isometry, analysis of the ratio (where variation in the numerator is of primary interest)
can be confounded by variation in the denominator (Atchley et al. 1976; Albrecht et al. 1993;

52

Sokal and Rohlf 1995). To prevent the confounding of variation, we used ANCOVA-like models
(Packard and Boardman 1988; Jackson et al. 1990; García-Berthou 2001) that accounted for
allometric relationships, which we linearized through log-transformation to investigate shrinkage
of Bythotrephes distal spine and body length.
We defined linear models of final length (day 126; log-transformed, continuous variable)
predicted by initial length (day 1; log-transformed, continuous variable), instar (categorical for
instars 1, 2, and 3), and an interaction between initial length and instar using R (version 2.10.1; R
Development Core Team 2009). The interactions between initial length and instar were not
significant for distal spine (general linear model: F2, 42 = 0.01; p > 0.05) or body length (general
linear model: F2, 54 = 0.67; p > 0.05) and were, therefore, removed from both models. Nonsignificant interactions between traits and instar indicate that potential shrinkage of traits does
not vary by instar. Upon removal of the interactions, instar was not significant for distal spine
length (general linear model: F2, 44 = 1.4, p > 0.05), but was significant for body length (general
linear model: F2, 56 = 3.2, p = 0.05). Therefore, instar was removed from the final model of
distal spine length, but was retained in the final model of body length. The non-significant effect
of instar on final distal spine length suggests that distal spine length of first, second, and third
instars did not differ, whereas the significant effect of instar on final body length indicated
significant differences of body length among instars, as would be expected given that
Bythotrephes increase body length with each molt. Final length (log transformed) was
significantly positively correlated with initial length for distal spine (log transformed; linear
regression: t46 = 21.2; p < 0.001; intercept = 0.08 ± 0.08 mm; slope = 0.92 ± 0.04; adjusted rsquared = 0.91; values are reported as ± 1 SE) and body length (log transformed; linear

53

regression: t56 = 8.1; p < 0.001; intercept = -0.09 ± 0.09 mm; slope = 0.81 ± 0.10; adjusted rsquared = 0.84).
In order to avoid bias in slope and intercept values associated with error in our predictor
variable (i.e., log initial length), we used reduced major axis regression (RMA; Mitchell-Olds
and Shaw 1987; Sokal and Rohlf 1995; McArdle 2003) to estimate slopes and intercepts for
distal spine length and body length. The RMA slope for distal spine length was 0.97 (95% C.I.
0.94 to 1.12), whereas the slope for body length was 1.12 (95% C.I. 0.99 to 1.24); neither of the
slopes differed from 1. Additionally, the RMA intercepts for distal spine length (intercept = 0.08
± 0.08 mm) and body length (intercept = -0.09 ± 0.09 mm) were not significantly different from
0 (p > 0.05 for distal spine; p > 0.05 for body length). The slope equal to one and intercept equal
to zero indicates that final and initial length scale isometrically for both distal spine and body
length. As a result, there was no statistically significant shrinkage for Bythotrephes distal spine
and body length in 95% ethanol.

54

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CHAPTER 3

Plastic response to a proxy cue of predation risk when direct cues are unreliable

Andrea Lynn-Jaeger Miehls, Andrew G. McAdam, Paul E. Bourdeau, and Scott D. Peacor

Abstract
Phenotypic plasticity in response to proximate cues that directly affect fitness or cues
directly released by selective agents has been well documented. For example, prey respond to
light level when light level affects predation risk, and prey respond to cues released by predators
to reduce predation risk. Less well understood is the potential for organisms to use proximate
cues that are indirectly related to the selective agent (e.g., prey response to a cue that correlates
with, but does not directly affect, predation risk). Previous field studies indicate that body size
and spine length of an invasive cladoceran in Lake Michigan, Bythotrephes longimanus (the
spiny water flea), increase throughout the growing season, coincident with a decrease in clutch
size. Although the cause of the seasonal trait changes is not known, the changes are associated
with warmer water temperature and an increased risk of gape-limited fish predation. Using a
laboratory experiment, we found no effect of fish (Perca flavescens) chemical cues (kairomones)
on Bythotrephes morphology or life history. In contrast, higher water temperature led to longer
absolute spine and body length, increased investment in morphological defense of offspring
(measured as the ratio of spine-to-body length), and decreased clutch size and age at
reproduction. These plastic responses are unlikely to be adaptive to temperature per se, but rather
our findings indicate that temperature serves as a proxy cue of predation risk. Temperature
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correlates with risk of gape-limited juvenile fish predation, which is the dominant source of
predation on Bythotrephes later in the growing season. We argue that for Bythotrephes,
temperature is a more reliable cue of gape-limited predation risk than fish kairomones, as
kairomones from non-gape-limited adult fish (to which a plastic response would have little
effect) are present throughout the season. Organisms may, therefore, not only respond to changes
in abiotic factors because those factors directly affect risk, but also when the abiotic cues serve
as proxies signaling changes in predation risk.

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Introduction
Organisms respond to many biotic and abiotic environmental conditions through
phenotypic plasticity (i.e., the ability of a genotype to produce different phenotypes in response
to differing environmental conditions; Pigliucci 2001), but the environmental conditions to
which organisms respond, as well as the trait modifications that result, are highly variable among
species and even among populations within a species. Consequently, investigating which aspects
of the environment are used as cues for phenotypic plasticity and how plastic responses affect
fitness have been the focus of a large body of research. Biotic and abiotic environmental
conditions used as proximate cues for phenotypic plasticity typically either directly affect fitness
or are directly released by selective agents. For example, prey may use light level as a proximate
cue for anti-predator responses when light level directly affects predation risk (e.g., Orrock et al.
2004). Similarly, prey may use chemicals released by predators (termed kairomones) as
proximate cues for anti-predator responses (Tollrian and Dodson 1999; Laforsch and Tollrian
2004).
However, proximate cues do not necessarily have to directly affect fitness or be directly
released by selective agents, but may instead be factors that are correlated in time or space
(Hairston 1987). Theory (Hairston 1987) suggests prey may evolve to use correlated
environmental cues as proxies for predation risk when the correlated cues are reliable predictors
of predation risk. For example, an abiotic variable such as temperature may correlate with
predation risk, without directly affecting predation risk, and thus serve as a “proxy cue”. There is
general support for the use of proxy cues as reliable indicators of changing environmental
conditions (e.g., beginning or end of growing season), such as the use of photoperiod for

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breaking or inducing diapause (Stross 1966), but experimental support for proxy cues eliciting a
response in an organism to other organisms, such as predators, is lacking.
Bythotrephes longimanus (the spiny water flea; hereafter Bythotrephes; Figure 3.1a) are
cladoceran zooplankton native to Eurasia that were introduced to the Laurentian Great Lakes in
the 1980s (Mills et al. 1993). Bythotrephes are a dominant predator of zooplankton (Bunnell et
al. 2011) and common prey for fish (Pothoven et al. 2007). The morphology and life history of
Bythotrephes in the Great Lakes strongly vary temporally (Burkhardt 1994; Pothoven et al. 2001;
2003), but the cause is not known. In Lake Michigan, for example, Bythotrephes tail spine and
body length are shortest at the start of the growing season (June-July), longest mid-growing
season (August-September), and then decrease at the end of the growing season (October
onward; Pothoven et al. 2001).
Seasonal changes in Bythotrephes traits may be due to phenotypically plastic responses to
their biotic and/or abiotic environment, but heretofore cues for plasticity in Bythotrephes traits
have not been investigated. Observed seasonal changes in Bythotrephes traits correlate with and
have been theorized to be a defense against gape-limited fish predation (typically juvenile fish
<100mm length), which increases throughout the growing season and typically comprises the
dominant source of predation on Bythotrephes mid-to-late in the growing season (Branstrator
2005). Seasonal elongation of the tail spine and increased body size in Bythotrephes may thus
represent plastic responses to reduce the risk of gape-limited fish predation. Consequently, a
likely proximate cue for predation risk is kairomones released by gape-limited fish predators.
However, Bythotrephes are also consumed by non-gape-limited (adult) fish (Pothoven et al.
2007) throughout the growing season, which also release kairomones, and to which
morphological plastic responses would likely not be adaptive because predation by non-gape-

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limited predators is not affected by the morphology of Bythotrephes. The effectiveness of
induced defenses by Bythotrephes is thus contingent on the particular fish present, and therefore
fish kairomones may not be expected to be a reliable signal of predation risk. Alternatively,
water temperature correlates with both observed seasonal changes in Bythotrephes traits
(Burkhardt 1994) as well as risk of gape-limited fish predation, and thus, water temperature
could serve as a reliable proxy signal of predation risk from gape-limited fish. We, therefore,
conducted a study that tested fish predator kairomones and temperature as proximate cues of
Bythotrephes morphological and life history plasticity.

Methods
Methodological Overview
Using a laboratory experiment, we investigated the effect of fish predator kairomones and
temperature on Bythotrephes morphological and life history traits. Our experimental design
entailed raising Bythotrephes clonal lines in the presence or absence of fish kairomones (by using
water obtained from five fish and five control aquaria) at 9°C and 15°C (one incubator per
temperature). We raised eight Bythotrephes clonal lines per replicate aquarium per temperature
treatment, for a total of 40 clonal lines in each of the four temperature-fish kairomone treatments
(total of 160 clonal lines).
Bythotrephes Morphology and Life History
The longest section of Bythotrephes’ prominent tail spine is the distal spine, which
extends from the tip of the spine to the first pair of instar barbs (Figure 3.1a). The distal spine is
present at birth and does not change with instar development, although other traits, such as body
size, increase with each instar molt (Burkhardt 1994). Instead, during the first two molts, total

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spine length increases through the addition of intercalary segments separated by paired barbs
(Branstrator 2005). Bythotrephes reproduce by cyclic parthenogenesis, with multiple asexual
generations during the growing season culminating in sexual reproduction at the end of the
season (Yurista 1992; Pothoven et al. 2001).
Fish Kairomone Collection
We obtained predator fish kairomones from yellow perch (Perca flavescens; 50-120mm
total length), an important predator of Bythotrephes in the Great Lakes (Schneeberger 1991;
Baker et al. 1992). Perch were held in five replicate aquaria and fed Bythotrephes (see Appendix
A for detailed methods). Water was collected from the five aquaria containing fish and from five
identically treated control aquaria (that lacked only the perch) and frozen in 100mL aliquots
(frozen kairomone concentration of 1 fish: 2L water) for later experiments with Bythotrephes.
The effectiveness of our kairomones was examined by measuring the behavioral response of
Daphnia mendotae to preserved fish kairomones (see Appendix B for detailed methods).
Field Collection of Experimental Bythotrephes
We collected 160 Bythotrephes from Lake Michigan on July 28, 2010, a time period
representing the middle of the Bythotrephes growing season. The lake was stratified, with
average water temperatures in the epilimnion (0-12m deep) and metalimnion (13-25m deep) of
24ºC and 14ºC, respectively. Bythotrephes collection occurred at the 45m depth contour
(sampling coordinates: 43º 12.40' N 86º 27.06' W), approximately 9.5km west of Muskegon, MI.
We used a conical zooplankton net with a 1m diameter opening and 363μm mesh size towed
vertically through the epilimnion and metalimnion. On board the ship, Bythotrephes were placed
into 60mL glass jars (one per jar) within 10min of collection. We retained only first and second

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instar Bythotrephes with very small brood pouches (used to assess reproductive state). Jars
contained approximately 50mL of Lake Michigan water passed through a 63µm sieve.
Experimental Design
Wild-captured Bythotrephes were split equally between 9°C and 15°C temperature
treatments maintained in biological incubators (one incubator per temperature). The colder
temperature represented Lake Michigan water temperature at the beginning and end of the
Bythotrephes growing season, whereas the warmer temperature represented Lake Michigan
water temperature mid-growing season (Pothoven et al. 2001). Although water temperatures are
often greater than 15°C during the growing season (Pothoven et al. 2001), our previous
experience as well as others (Garton et al. 1990) indicate raising Bythotrephes at higher
temperature (20°C and greater) increases mortality dramatically, and thus we used a
conservatively low upper temperature limit.
Within each temperature treatment (i.e., incubator), half of the Bythotrephes were raised
in water containing fish kairomones and half in control water, for a total of eight wild-captured
Bythotrephes per replicate aquaria per temperature treatment. We completed daily water changes
to counter breakdown of kairomones. The frozen control and kairomone water was thawed each
day of the experiment; each 100mL aliquot was diluted with an additional 200mL of Lake
Michigan water passed through GF/F Whatman filters to a final kairomone concentration of 1
fish: 6L water. Bythotrephes were maintained with a 13:11 light:dark cycle and fed ad libitum
daily with approximately 150 Artemia nauplii less than 24hr old and three juvenile Daphnia
pulicaria (Kim and Yan 2010; pers. comm. K. Schulz, State University of New York – College
of Environmental Science and Forestry, Syracuse, New York, USA).

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Clonal Lines
We raised Bythotrephes in clonal lines (Lynch and Walsh 1998) through the F2
generation. Previous research has shown that cladoceran zooplankton exhibit plastic responses
within and among generations, often with the strongest responses occurring through maternal
effects (Tollrian 1995; Yurista 2000). Each clonal line was initiated with one wild-captured
Bythotrephes whose daughters (F1 generation) were used to establish sub-lines. All Bythotrephes
from a common clonal line were exposed to the same temperature and fish kairomone
treatments. Wild-captured and F1 Bythotrephes were preserved in 95% ethanol the day they
reproduced or died; all F2 Bythotrephes were preserved the day they were born. Approximately
one-third of wild-captured Bythotrephes in each temperature treatment, as well as one-quarter of
F1 Bythotrephes in the 15°C treatment and one-third in the 9°C treatment were culled randomly
due to a limited supply of kairomone water (a greater proportion of F1 Bythotrephes raised at
9°C were culled to reduce kairomone consumption due to their longer generation time; see
results).
Morphological and Life History Traits
We measured the following Bythotrephes morphological and life history traits: distal
spine length, S2 length (present for second and third instar Bythotrephes), S3 length (present for
third instar Bythotrephes), body length (Figure 3.1a), the ratio of distal spine-to-body length,
clutch size (i.e., number of offspring), days to first reproduction, and instar at first reproduction
(Bythotrephes can reproduce in either the second or third instar stage; Pothoven et al. 2001;
2003). To ensure that we analyzed individuals of differing life history strategies separately, we

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only considered second instar measurements for those individuals that first reproduced at their
second instar and only measured third instar individuals that first reproduced at their third instar.
Digital photographs of Bythotrephes (oriented as in Figure 3.1a) taken with a dissecting
microscope were analyzed with ImageJ (Abramoff et al. 2004) to measure morphological traits
to the nearest 0.001mm. Distal spine length was measured from the tip of the spine to the
anterior end of the first pair of instar barbs; S2 (and, similarly, S3) length was measured from the
anterior end of the first (second) pair of instar barbs to the anterior end of the second (third) pair
of instar barbs (Figure 3.1a; Sullivan and Lehman 1998). We measured body length from the
anterior edge of the eye to the base of the tail spine along the midline of the body with segments
spanning the head and eye region, thorax, and abdomen (Figure 3.1a; Miehls et al. 2012).
Statistical Analyses
The effect of temperature and fish kairomones on Bythotrephes morphology and life
history was tested statistically using linear mixed effects models (LME) fitted for each trait
separately using the nlme package (Pinheiro et al. 2009) in R version 2.12.2 (R Development
Core Team 2011). We first analyzed the effect of fish kairomone treatment within each
temperature treatment. When no significant effect of fish kairomone treatment was found, we
pooled the fish kairomone treatments and tested for a significant effect of temperature.
Models for traits of wild-captured Bythotrephes included temperature or fish kairomone
treatment as fixed effects and aquaria (i.e., aquaria of fish kairomone or control water origin) as a
random effect. Models for traits of F1 Bythotrephes included temperature or fish kairomone
treatment as fixed effects, and clonal line nested within aquaria as random effects. For F2
Bythotrephes, sub-line nested within clonal line was included as an additional random effect. We
assessed the significance of each random effect through model comparisons using likelihood

71

ratio tests (LRT). For the LRT, we fitted up to three additional models for each trait, with each
model containing successively fewer random effects to compare the fit of more complicated
models to the data versus the fit of a simpler model that excluded the random effect of interest.
Because Bythotrephes’ distal spine is present at birth (and does not change length with
instar development), distal spine length of wild-captured Bythotrephes should not have been
affected by our experimental treatments and any difference in distal spine length by experimental
treatment for F1 and F2 Bythotrephes represents maternal effects. Differences in first instar body
length by experimental treatment for F1 and F2 Bythotrephes also represent maternal effects.
Because no F1 Bythotrephes reproduced at their second instar in the colder temperature treatment
(see results), we do not present data for body length or the ratio of distal spine-to-body length for
second instar, F1 Bythotrephes in the colder temperature.

Results
Survival of Clonal Lines to F2 Generation
Lab-based mortality in our clonal rearing resulted in 21 and 17 clonal lines in our fish
kairomone and control treatments, respectively, that survived to produce F2 Bythotrephes at
15°C (75% and 68% survival, respectively), and 4 and 7 clonal lines in our fish kairomone and
control treatments, respectively, at 9°C (15% and 26% survival, respectively). Although fewer
clonal lines survived to the F2 generation in the colder temperature, the mortality rate (per day)
was identical between the temperature treatments, with approximately 1% and 2% of wildcaptured and F1 Bythotrephes, respectively, dying per day in both temperature treatments.
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Response to Predator Kairomones
Fish kairomones did not affect any of the Bythotrephes traits we measured (Figs. 2-3;
Appendix C: Table 3.1; Appendix D: Figure 3.1). The lack of response was consistent across
temperature treatments (15°C and 9°C), generations (wild-captured, F1, and F2 generations), and
instars (one, two, and three).
Response to Temperature
In contrast to fish kairomones, temperature had a strong effect on Bythotrephes
morphology and life history (Figs. 1-3; Appendix C: Table 3.2). Below, we report trait responses
to temperature for each generation (wild-captured, F1, and F2), as well as instar within each
generation for traits known to vary with instar development (i.e., body length and the ratio of
distal spine-to-body length).
Body length of second instar, wild-captured Bythotrephes (measured after producing
offspring; see methods) was greater in the higher temperature (Figure 3.2c); no other
morphological traits of reproductively mature (i.e., second or third instar), wild-captured
Bythotrephes showed a response to temperature (Figure 3.2; Appendix D: Figure 3.1).
Temperature also did not affect clutch size of wild-captured Bythotrephes (Figure 3.3a). Wildcaptured Bythotrephes produced offspring with longer distal spines (F1: Figure 3.2a) that also
exhibited greater investment in morphological defense (ratio of distal spine-to-body length; F1,
Instar 1: Figure 3.2d) in the higher temperature.
Body length of third instar, F1 Bythotrephes was greater at the higher temperature (Figure
3.2c), but no other morphological traits of reproductively mature F1 Bythotrephes showed a
response to temperature (Figure 3.2; Appendix D: Figure 3.1). Clutch size of F1 Bythotrephes
73

was smaller at the higher temperature (Figure 3.3a); the F2 offspring produced at the higher
temperature had longer distal spine (F2: Figure 3.2a) and body length (F2, Instar 1: Figure 3.2c),
and also exhibited greater investment in morphological defense (F2, Instar 1: Figure 3.2d). F1
Bythotrephes reproduced twice as quickly at the higher temperature (Figure 3.3b) and
reproduction occurred at both the second and third instar stages, whereas all F1 Bythotrephes
reproduced in their third instar at the colder temperature. Of note, greater distal spine-to-body
length of F2 offspring at the higher temperature was not a product of changes resulting from
growth. At both temperatures, body length increased proportionally more than distal spine length
with increased size (Figure 3.2b). Yet, F2 offspring at the higher temperature (which were larger
in absolute size) still showed greater ratios of distal spine-to-body length and also had markedly
longer distal spines than offspring of the same body size at the lower temperature (Figure 3.2b).

Discussion
Our experiment clearly showed a strong plastic response of a prey to a correlate of
predation risk, but no response to cues emanating directly from the predator. Response to a
correlate of predation risk (that does not directly affect predation risk), without a concomitant
response to cues directly released from the predator, has rarely, if ever been documented.
Support for the use of proxy cues of predation risk is provided by theory (Hairston 1987), which
suggests prey may evolve to use a correlated environmental cue as a proxy for predation risk
when the correlated cue is a reliable predictor of predation risk. For Bythotrephes in Lake
Michigan, temperature positively correlates with predation risk by gape-limited juvenile fish,

74

which are the dominant predators of Bythotrephes later in the growing season; thus, temperature
serves as a reliable predictor of size-dependent predation risk. Conversely, the presence of fish
kairomones might be an indicator of overall predation risk, but fish kairomone presence is not a
reliable predictor of size-dependent predation risk, to which plastically induced defenses would
be effective. Plastic induction of defenses in response to kairomones from non-gape-limited adult
fish, which are present throughout the growing season, would have little benefit. Here we found
striking response of Bythotrephes morphology and life history to temperature (Figure 3.1b), but
no response to kairomones from an important fish predator.
Field studies have documented temporal changes in Bythotrephes traits that correspond to
the trait plasticity we observed in response to temperature, thus suggesting plastic responses to
temperature might underlie observed seasonal variation in the wild. For example, field surveys in
multiple lakes have shown an increase in body size and distal spine length that positively
correlates with water temperature (Lake Constance: Straile and Hälbich 2000; Lake Michigan:
Burkhardt 1994; Pothoven et al. 2001; 2003). Further, multiple studies also documented
decreased clutch size and/or reproduction at younger instar stages that was associated with
warmer water temperature (Straile and Hälbich 2000; Pothoven et al. 2001; 2003), strongly
paralleling the trait changes we observed between our temperature treatments.
The lack of Bythotrephes response to fish kairomones may be surprising given the
numerous examples of cladoceran zooplankton (Tollrian and Dodson 1999) that plastically
respond to predator fish kairomones as well as the known importance of Bythotrephes traits (e.g.,
distal spine length) for deterring gape-limited fish predation (Barnhisel 1991a; 1991b). However,
we can rule out multiple potential methodological concerns. We are confident that our
representative fish predator, yellow perch (Perca flavescens), was an appropriate species to test

75

as yellow perch are an important predator of Bythotrephes in the Great Lakes (Schneeberger
1991; Baker et al. 1992) and also have a closely related species (European perch, Perca
fluviatilis) that preys upon Bythotrephes in their native range (Thorpe 1977; Straile and Hälbich
2000). We also confirmed that our freezing methodology effectively preserved fish kairomones
by demonstrating a behaviorally plastic response of D. mendotae (Appendix B) and observing
that the water containing fish kairomones (but not the control water) noticeably smelled of fish
(personal observation). Further, we used a fish kairomone concentration (1 fish: 6L water) that
previous studies (Reede 1995; Rose et al. 2001) using similarly sized fish (40-100mm length)
have shown to elicit strong responses in cladoceran zooplankton. The fish also were fed (and ate
as confirmed through diet analysis) Bythotrephes in order to increase the probability that fish
scent would represent a threat (Stabell et al. 2003; Bourdeau 2010). Finally, the trait magnitudes
of our laboratory-raised Bythotrephes fell within natural field variation of Bythotrephes traits,
suggesting that our Bythotrephes were not constrained by a laboratory condition that prevented
natural growth. Thus, we are confident that the lack of a Bythotrephes plastic response to fish
kairomones is a real, biologically meaningful result.
We argue that temperature is a proxy cue for predation risk that elicits adaptive
phenotypic responses based on three lines of reasoning: 1) the morphological and life history
responses we observed, which involved an altered reproductive strategy with temperature, are
not consistent with adaptive response to temperature per se; 2) the responses we observed are,
instead, consistent with adaptive response to risk of gape-limited fish predation, which positively
correlates with temperature; and 3) the use of the most common cue of fish predation risk,
kairomones, as a proximate cue for predation risk would not be adaptive. We discuss each of
these reasons below.

76

The observed plastic responses of Bythotrephes to temperature are unlikely to be adaptive
to temperature per se given four reasons. First, one of the most well-supported rules of plasticity
in response to temperature for ectotherms is the temperature-size rule, which states that
individuals (within a species) raised at higher temperatures grow quickly, but reach a smaller
final size than individuals raised at colder temperatures (Atkinson 1994; 1995); the temperaturesize rule has been supported for over 80% of ectotherms investigated (Atkinson 1994). Smaller
body size at higher temperature is physiologically adaptive for many ectotherms, as higher
temperature causes increased metabolic rate, and consequently results in a higher energetic
demand, which creates a cost to larger body size (Kozłowski et al. 2004). Indeed, many
cladoceran zooplankton (Gillooly and Dodson 2000; Hart and Bychek 2011) follow the
temperature-size rule. However, in contrast to the temperature-size rule, Bythotrephes had larger
body size and longer distal spine length at the higher temperature, suggesting plasticity in
Bythotrephes traits may not be physiologically adaptive to temperature per se. Second,
Bythotrephes also produced offspring with longer distal spines relative to body length (i.e., larger
ratio of distal spine-to-body length) at the higher temperature. Greater investment in distal spine
length (relative to body size) likely has no adaptive value to higher temperature per se, and may
even carry both a physiological cost (due to increased structural investment in a trait that has
little energetic return, i.e., a trait that does not contribute to resource acquisition) and physical
cost (due to increased weight which may promote sinking in warmer water, which is less viscous
than colder water; Atkinson 1994). Third, Bythotrephes clutch size decreased with temperature
(despite a concomitant increase in maternal body size), a finding that would have little adaptive
value to temperature per se and which is also opposite to another general rule, the size-fecundity
relationship (which states that larger individuals have greater fecundity; Stearns 1992; Roff

77

2002). Fourth, some F1 Bythotrephes at the higher temperature reproduced at their second instar
stage, whereas all F1 Bythotrephes at the colder temperature delayed reproduction to their third
instar stage. Although we might expect temperature to affect growth rate (such as the number of
days between instar molts; Branstrator 2005), it is unlikely that temperature should alter
development such that reproduction occurs at different stages.
The differences in Bythotrephes phenotype at different temperatures are consistent with
the use of temperature as a proxy for adaptive response to predation risk. Given the increase in
Bythotrephes distal spine length and the ratio of distal spine-to-body length with temperature,
Bythotrephes exhibit their greatest investment in morphological defense later in the growing
season, when water temperature is warmest. Predation by gape-limited juvenile fish is also
greatest later in the growing season (due to growth of juvenile fish from completely gape-limited
size classes early in the growing season into partially gape-limited classes mid-to-late in the
season), and can even be the dominant source of predation on Bythotrephes (Branstrator 2005).
Thus, temperature correlates with gape-limited predation, which allows temperature to serve as a
proxy for predation risk. Previous research has demonstrated that longer spines of Bythotrephes
(Barnhisel 1991a; 1991b) as well as other cladoceran zooplankton (Daphnia lumholtzi; Swaffar
and O’Brien 1996) provide protection against gape-limited fish predation. Correspondingly, our
observed plastic response of Bythotrephes traits to temperature would likely have adaptive value
for reducing the risk from gape-limited fish predation.
Critical to our argument, temperature can serve as a reliable cue of predation risk in this
predator-prey system, but fish kairomones are not reliable cues. Because adult fish are present
throughout the growing season (and, therefore, fish kairomones are always present), but
predation risk increases throughout the growing season due to increased gape-limited fish

78

predation, there is a poor association between fish kairomones and predation risk. Further, there
is also a poor association between fish kairomones and predation risk to which plastic responses
would be beneficial. Large size and morphological defenses would have little adaptive value
against adult fish predators early in the growing season as adult fish are a source of non-gapelimited predation. Thus, plastic morphological response to kairomones from fish would entail no
fitness advantage, and so kairomones would not serve as a useful cue for reducing predation risk.
We believe our study is among the first to show a plastic response of a prey to a correlate
of predation risk, but no response to cues released from the predator. Although previous studies
have suggested (e.g., Bosmina: Kappes and Sinsch 2002) or are consistent with (e.g., D.
lumholtzi; Yurista 2000) the use of temperature as a proxy for predation risk, the organisms
studied also showed a concordant plastic response to predator kairomones (Kappes and Sinsch
2002; Dzialowski et al. 2003). There is general support for the sole use of proxy variables as
reliable cues of correlated environmental conditions from the well-known use of photoperiod for
breaking or inducing diapause (Stross 1966) and the use of temperature for adaptive sex
determination in many ectotherms (e.g., Conover 1984; Warner and Shine 2008).
Notably, the use of temperature as an indirect, proxy cue for plasticity by Bythotrephes is
mechanistically different from other studies that have suggested the use of “indirect” cues for
plasticity. For example, Orrock et al. (2004) found that field mice (Peromyscus polionotus) use
moonlight and precipitation (which they termed indirect cues), but not cues directly released by
the predator (i.e., predator urine), as proximate cues for behaviorally plastic responses to reduce
predation risk. Although moonlight and precipitation are not directly associated with the predator
(as, comparatively, predator urine would be), moonlight and precipitation may still directly affect
predation risk through altered prey visibility to predators. Thus, the use of abiotic cues that affect

79

predation risk by field mice contrasts with the use of temperature by Bythotrephes as temperature
has no direct effect on predation risk of Bythotrephes.
Three lines of evidence suggest there are costs associated with large morphological size
early in the growing season, and support why Bythotrephes exhibit a plastic response to fish
predation risk (via temperature as a proxy cue) rather than constitutive (fixed) expression of large
size and enhanced morphological defense. First, large size increases visibility of prey to fish
predators (Tollrian 1995), thus expression of large size and enhanced morphological defense by
Bythotrephes early in the growing season during dominant non-gape-limited predation likely
would entail a cost to Bythotrephes. Second, we found that the production of larger offspring was
associated with reduced clutch size for Bythotrephes, suggesting a size-number trade-off in
offspring investment (Smith and Fretwell 1974), which would entail a cost early in the growing
season when higher fecundity would be advantageous due to low conspecific abundance and
abundant prey resources (Burkhardt 1994; Pothoven et al. 2001; 2003). Third, large size early in
the growing season, when water is colder and therefore more viscous (Atkinson 1994), could
entail an energetic cost due to increased drag during swimming. Indeed, costs associated with
drag could be an alternative explanation for Bythotrephes morphological plasticity. However, it
is unlikely that the plastic responses we observed are adaptive for reducing drag, particularly
responses involving distal spine length which is unlikely to be a significant source of drag in
comparison to body length or brood pouch size (pers. comm. C. Laforsch and Q. Herzog,
Ludwig-Maximilians-University, Munich, Germany).
Whereas we have not tested the adaptive value of Bythotrephes trait plasticity per se, we
suggest that the plastic response of Bythotrephes traits to temperature is adaptive based on the
framework of Doughty and Reznick (2004) who present an argument for adaptation based on six

80

indirect lines of evidence (criteria), as follows. Adaptive plasticity must involve production of
different phenotypes in different developmental environments (criterion 1) which must be
present in nature (i.e., environmental heterogeneity; criterion 2). We meet these criteria by
demonstrating the production of different Bythotrephes phenotypes at different temperatures
(criterion 1) that represent natural seasonal temperature variation in Lake Michigan (criterion 2;
Pothoven et al. 2001). Further, alternative phenotypes should differ in fitness between the
different environments (criterion 3) and the developmental environments must be reliable cues
for plasticity (criterion 4). We presented three logical arguments above for why we expect the
alternative Bythotrephes phenotypes to differ in fitness between the different temperature ranges
due to their correspondence to differing predation risk (criterion 3), although we have not
explicitly measured selection on spine length here. We have also presented an argument for why
temperature is a reliable predictor of fish predation risk (criterion 4). Finally, genetic variation
for plasticity should exist in the responding traits (criterion 5) and comparative evidence should
be present from other systems (criterion 6). Studies documenting genetic variation and providing
comparative evidence for Bythotrephes trait plasticity are, unfortunately, lacking (i.e., no data are
available to address criteria 5 and 6). However, it is reasonable to assume genetic variation for
plasticity (criterion 5) exists in our system, given that gene-by-environment interactions are
common across a wide variety of organisms (Pigliucci 2005). Overall, we are able to meet four
of the six criteria discussed by Doughty and Reznick (2004), with a reasonable assumption of the
presence of a fifth criterion, and consequently conclude that Bythotrephes plasticity in response
to temperature is likely adaptive.
Plasticity in response to an abiotic, proxy cue of predation risk has implications for our
understanding of the evolution of environmentally cued plasticity, as well as the success of

81

invasive species, such as Bythotrephes, specifically. Our research provides experimental
evidence that proxy cues can induce plastic responses in prey to reduce predation risk, when
direct cues from the predator are unreliable. Organismal use of proxy cues in nature may be more
common than previously realized, if proxy cues, due to their indirect and potentially surprising
relationships to the adaptive responses they elicit, are less often investigated than more obvious
potential direct cues of plastic responses. Moreover, the use of abiotic, proxy cues for reducing
predation risk may be important to species invasions, particularly the Bythotrephes invasion.
Interactions with predators have long been recognized as strong drivers of the success or failure
of species invasions (Elton 1958). Indeed, lack of natural enemies (Keane and Crawley 2002) is
often cited as evidence for the successful establishment of invasive species in nonnative
ecosystems. Conversely, when potential predators are present, defenses may promote persistence
through reducing predation risk. It is possible that Bythotrephes’ ability to plastically adjust
morphology and life history to defend against predation has contributed to their ability to
establish in nonnative ecosystems. It is important to note, however, that the use of a correlated,
proxy cue may potentially restrict which ecosystems Bythotrephes are capable of invading, if
temperature regimes must correlate with predation risk. As invasive species may be more likely
to invade ecosystems with similar abiotic conditions (e.g., temperature regime) to their native
range (Kolar and Lodge 2001), our research suggests that the success of Bythotrephes
specifically, and invasive species in general, may be due to abiotic-matching of cues for adaptive
plasticity.

82

Acknowledgements
Doran Mason, Dennis Donahue, Steven Pothoven, and the NOAA Great Lakes
Environmental Research Laboratory and NOAA Lake Michigan Field Station offered research
vessels and field support staff. Brittany Damschroder, Lydia Kramer, Monica Lucas, Ian
McCririe, Scott Miehls, Veronica Quesnell, Emily Reed, Alexandra Sookhai, Ben Staton, Marie
Stevenson, and Brandon Vieder helped to collect data. Jason Fischer, Natalie Kim, Kevin Pangle,
Kim Schulz, and Peder Yurista provided valuable help with Bythotrephes culturing protocols and
Scott Miehls and Andria Salas provided additional methodological assistance. We thank Carla
Cáceres for a rigorous discussion of the results of this study, and Christian Laforsch and Quirin
Herzog for additional experimental investigation into our findings. This work was supported by
the Great Lakes Fishery Commission, the National Science Foundation (DEB-0089809), and an
EPA Science to Achieve Results fellowship (STAR Research Assistance Agreement No.
FP91698801-0 awarded by the U.S. Environmental Protection Agency). This work has not been
formally reviewed by the EPA and the views expressed in this document are solely those of the
authors. SDP acknowledges support from the Michigan Agricultural Experiment Station.

83

Figures
a)

b)
9°C

15°C

.1.
phes longima
anus, the spi water fle a) Bythotr
iny
ea.
rephes morp
phological tr
raits
Figure 3. Bythotrep
(the indiv
vidual pictur is a third instar); b) F2 offspring representing the average morpholog of
red
d
g
e
gy
individua born to cl
als
lonal lines ra
aised at 9°C and 15°C.

84

Figure 3.2. Predator and temperature effects on Bythotrephes: a) distal spine length; b) distal
spine length versus body length of F2 offspring; c) body length; and d) the ratio of distal spine to
body length. Error bars are standard errors. C: control treatment; F: fish treatment.

85

Figure 3.
.2.
a)

c)

b)

Wild

F1

F2

d)

F1

Wild
W

86

F2

a)

b)

Wild

F1

F1

Figure 3. Predator and tempera
.3.
a
ature effects on Bythotrep
phes: a) clut size; and b) days to f
tch
d
first
reproduction. Error bars are stand
b
dard errors. C: control tr
reatment; F: fish treatme
ent.

87

APPENDICES

88

Appendices
Appendix A: Fish Kairomone Collection
We obtained predator fish kairomones from yellow perch (Perca flavescens), one of the
primary fish predators of Bythotrephes in Lake Michigan (Schneeberger 1991; Baker et al.
1992). Twenty yellow perch (50-100mm total length) were collected on July 14, 2010 from Lake
Lansing (Ingham County, MI) using a 25ft beach seine (DNRE Scientific Collector’s Permit No.
0536). Fish were placed in five 8L aquaria in the laboratory within 1hr of collection; each
aquarium received one 50-70mm, two 80-100mm, and one 100-120mm fish (four fish total per
aquarium). Five control aquaria were also set up and treated identically except for the addition of
fish. Aquaria contained oxygenated, room temperature (21°C) Lake Michigan water passed
through a 63µm sieve and were kept under low light with a 13:11 light:dark cycle. Fish were
allowed a three day acclimation period during which all aquaria (fish and control) were
inoculated with a natural assemblage of zooplankton. On the fourth day, all aquaria were
inoculated with 200 Bythotrephes collected from Lake Michigan; previous research (e.g., Stabell
et al. 2003; Bourdeau 2010) has shown that the strongest plastic responses of prey to predators
were elicited when the predator had consumed prey conspecifics. Subsequent gut content
analysis confirmed that perch in every aquarium consumed Bythotrephes. Twenty-four hours
after addition of Bythotrephes, fish were removed from their acclimation aquaria, and all aquaria
were emptied, rinsed, and re-filled with Lake Michigan water passed through GF/F Whatman
filters; fish were then returned to their original aquaria. After 24hr, all water from the fish and
control aquaria was collected and frozen in 100mL aliquots (frozen kairomone concentration of 1
fish: 2L water); previous research has shown that freezing is an effective method to preserve
predator kairomones (Bourdeau, P., unpublished; Pangle, K. unpublished).

89

Appendix B: Daphnia Behavioral Bioassay
We conducted a Daphnia mendotae behavioral bioassay using a laboratory column
experimental design (Loose and Dawidowicz 1994; Pangle and Peacor 2006) to assess whether
our procedure to produce and preserve fish kairomones was effective. Previous laboratory
research has shown that D. mendotae in Lake Michigan vertically migrate to colder, deeper water
in response to predator kairomones (Bythotrephes: Pangle and Peacor 2006; yellow perch:
Bourdeau, P., unpublished; Pangle, K., unpublished), and thus we predicted that D. mendotae
would vertically migrate when exposed to our fish kairomone water.
We collected D. mendotae from Lake Michigan on October 18, 2010 at the 110m depth
contour (sampling coordinates: 43º 12.11' N 86º 34.28' W), approximately 19km west of
Muskegon, MI. In the laboratory, we filled twenty 1.5cm diameter, 60cm length transparent
acrylic tubes (columns) with either fish kairomone or control water (both previously frozen).
There were two replicate columns for each of the five fish and five control aquaria (20 columns
total). Each 100mL aliquot of frozen control and kairomone water was diluted with an additional
200mL of Lake Michigan water passed through GF/F Whatman filters. Columns were
illuminated from above and immersed in a water bath that maintained a temperature gradient of
19°C at the surface to 14°C at the bottom. Three D. mendotae were placed in each column and
allowed to acclimate to the experimental conditions for 1hr, during which they were fed
Ankistrodesmus ad libitum. Vertical position of all D. mendotae was recorded hourly for 4hr.
Differences in D. mendotae mean depth (averaged across individuals in the same column)
between the fish kairomone and control water were tested statistically with linear mixed effects
models (LME) that included fish kairomone treatment as a fixed effect and aquaria nested within

90

observation period as random effects using the nlme package (Pinheiro et al. 2009) in R version
2.12.2 (R Development Core Team 2011).
D. mendotae exposed to fish kairomones migrated deeper in the columns (average depth
± SE: 27.7 ± 3.3cm) than D. mendotae exposed to control water (average depth: 20.6 ± 3.1cm;
t35 = -2.14; p < 0.05), confirming that our procedure to produce and preserve fish kairomones
was effective. The LRT indicated no significant effect of observation period (p > 0.05) or aquaria
(p > 0.05) on Daphnia mean depth.

91

Appendix C: Statistical Tables
Table 3.1. Effect of fish kairomones (presence / absence) on the morphology and life history of Bythotrephes longimanus raised at 9°C
and 15°C. The generation and instar of Bythotrephes used in each analysis is noted. Significance of random effects for aquaria (all
generations), clonal line (F1 and F2 generations), and clonal sub-line (F2 generation only) was assessed using likelihood ratio tests.
Gen: generation; Aq: aquaria; CL: clonal line; SL: sub-line; N/A: not applicable.

Trait

Gen

Instar

Body length

Wild 2
Wild 3
1
F
1

9°C temperature treatment
Kairomone treat
Aq
CL
SL
F (df)
p
p
p
p
0.64 (1,8) N.S.
N.S.
N/A
N/A
1.07 (1,3) N.S.
N.S.
N/A
N/A
0.04 (1,4) N.S.
N.S.
N.S.
N/A

15°C temperature treatment
Kairomone treat
Aq
CL
F (df)
p
p
p
0.12 (1,8)
N.S. N.S. N/A
0.21 (1,7)
N.S. N.S. N/A
2.66 (1,7)
N.S. N.S. N.S.

SL
p
N/A
N/A
N/A

F1

N/A

N/A

N/A

N/A

N/A

0.001 (1,4)

N.S.

N.S.

N.S.

N/A

F1
Distal spine :
body length

2
3

0.02 (1,8)

N.S.

N.S.

N.S.

N/A

0.02 (1,8)

N.S.

N.S.

*

N/A

1
F2
Wild 2
Wild 3
1
F

0.02 (1,5)

N.S.

***

N.S.

*

0.08 (1,8)

N.S.

***

***

***

0.28 (1,8)
18.39 (1,1)
1.45 (1,4)

N.S.
N.S.
N.S.

N.S.
N.S.
N.S.

N/A
N/A
N.S.

N/A
N/A
N/A

0.73 (1,8)
0.12 (1,7)
0.91 (1,7)

N.S.
N.S.
N.S.

N.S.
N.S.
N.S.

N/A
N/A
N.S.

N/A
N/A
N/A

1

F1

N/A

N/A

N/A

N/A

N/A

0.06 (1,4)

N.S.

N.S.

N.S.

N/A

F1
Distal spine
length

2
3

0.21 (1,7)

N.S.

N.S.

N.S.

N/A

0.005 (1,8)

N.S.

N.S.

**

N/A

F2
Wild

1

0.79 (1,5)

N.S.

N.S.

***

N.S.

0.01 (1,8)

N.S.

***

***

***

All
All

0.78 (1,8)
0.71 (1,8)

N.S.
N.S.

N.S.
*

N/A
*

N/A
N/A

0.07 (1,8)
0.02 (1,8)

N.S.
N.S.

N.S.
N.S.

N/A
***

N/A
N/A

1

1.87 (1,5)

N.S.

**

***

***

0.02 (1,8)

N.S.

***

***

***

F1
F2

92

Table 3.1 (con’t)
Trait

Gen

Instar

S2 length

Wild

S3 length

F1
Wild

2 and 3
2 and 3

9°C temperature treatment
Kairomone treat
Aq
CL
SL
F (df)
p
p
0.02 (1,8) N.S.
N.S.
N/A
N/A
0.85 (1,8) N.S.
N.S.
***
N/A
0.001 (1,4)
2.86 (1,7)

N.S.
N.S.

N.S.
N.S.

N/A
N.S.

N/A
N/A

0.17 (1,6)
0.08 (1,8)

N.S.
N.S.

N.S.
N.S.

N/A
***

N/A
N/A

Clutch size

F1
Wild

3
3
3
3

0.16 (1,8)
0.31 (1,5)

N.S.
N.S.

N.S.
N.S.

N/A
***

N/A
N/A

0.02 (1,8)
0.75 (1,8)

N.S.
N.S.

*
N.S.

N/A
N.S.

N/A
N/A

F1

9°C temperature treatment
Kairomone treat
Aq
CL
SL
F (df)
p
p
1.29 (1,8)
N.S. N.S. N/A
N/A
0.09 (1,8)
N.S.
*
***
N/A

Age at first
3
0.01 (1,5) N.S.
*
*
N/A
2.15 (1,8)
N.S. N.S.
*
F1
reproduction
*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; N.S.: non-significant (significance of random effects based on likelihood ratio tests).

93

N/A

Table 3.2. Effect of temperature (9°C versus 15°C) on the morphology and life history of Bythotrephes longimanus. The generation
and instar of Bythotrephes used in each analysis is noted. Significance of random effects for aquaria (all generations), clonal line (F1
and F2 generations), and clonal sub-line (F2 generation only) was assessed using likelihood ratio tests. Gen: generation; Aq: aquaria;
CL: clonal line; SL: sub-line; N/A: not applicable.
Trait

Gen

Instar

Body length

Wild
Wild

2
3
1

F1

Temperature treatment
F (df)
p
6.40 (1,107)
**
0.50 (1,16)
N.S.
3.69 (1,23)
N.S.

Aq
p
N.S.
N.S.
N.S.

CL
p
N/A
N/A
N.S.

SL
p
N/A
N/A
N/A

F1

28.27 (1,44)

***

N.S.

*

N/A

F2
Wild
Wild

1

34.55 (1,38)

***

***

***

***

2
3
1

2.92 (1,89)
0.29 (1,12)
17.12 (1,20)

N.S.
N.S.
***

N.S.
N.S.
N.S.

N/A
N/A
N.S.

N/A
N/A
N/A

F1

Distal spine : body length

3

3

1.65 (1,42)

N.S.

N.S.

**

N/A

F2
Wild

1

24.54 (1,38)

***

***

***

***

All
All

0.45 (1,121)
56.78 (1,69)

N.S.
***

N.S.
N.S.

N/A
***

N/A
N/A

1

175.77 (1,38)

***

***

***

***

2 and 3
2 and 3

1.71 (1,140)
3.03 (1,45)

N.S.
N.S.

N.S.
N.S.

N/A
***

N/A
N/A

3
3

0.88 (1,13)
1.24 (1,44)

N.S.
N.S.

N.S.
N.S.

N/A
***

N/A
N/A

F1

Distal spine length

F1
S2 length

F2
Wild

S3 length

F1
Wild
F1

94

Table 3.2 (con’t)
Trait

Gen

Instar

Clutch size

Wild

3
3

F1

Temperature treatment
F (df)
p
2.19 (1,64)
N.S.
25.15 (1,36)
***

Age at first reproduction

Aq
p
N.S.
N.S.

CL
p
N/A
N.S.

SL
p
N/A
N/A

3
900.84 (1,36)
***
*
*
N/A
F1
*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; N.S.: non-significant (significance of random effects based on likelihood ratio tests).

95

Appendi D: Supple
ix
ementary Fig
gures
a)

b)

Wild

F1

Wild

F1

Figure 3. Predator and tempera
.4.
a
ature effects on Bythotrep
phes: a) S2 l
length; and b S3 length.
b)
h
Error bar are standa errors. C: control trea
rs
ard
:
atment; F: fis treatment
sh
t.

96

LITERATURE CITED

97

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CHAPTER 4

Evolutionary stasis despite selection on a heritable trait in an invasive zooplankton

Andrea Lynn-Jaeger Miehls, Scott D. Peacor, Lindsey Valliant, and Andrew G. McAdam

Abstract
Natural selection is fundamental to evolutionary ecology, yet few studies rigorously
assess spatial and temporal variation in selection, despite evidence that selection can vary widely
in both dimensions. Accurately characterizing spatio-temporal variation in selection is important,
as it may constrain evolutionary responses to selection. Here we quantified the strength of
natural selection on the defensive morphology (distal spine) of an invasive zooplankton,
Bythotrephes longimanus, in Lake Michigan across multiple months during three growing
seasons and between four and six spatial replicates within the lake. We also used multiple lines
of evidence (historic and contemporary wild-captured individuals, paleoecology of retrieved
spines) to assess phenotypic change in distal spine length since invasion. We found that selection
varied temporally, with selection for decreased distal spine length early in the growing season,
and strengthening selection for increased distal spine length later in the growing season. This
seasonal trend in natural selection is consistent with seasonal changes in the relative strength of
non-gape-limited and gape-limited fish predation. Selection also varied spatially, which is
consistent with well-known heterogeneity of fish predators in large lakes. Yet, despite net
selection for increased distal spine length (and known genetic basis of distal spine length), we
observed little evidence of an evolutionary response to selection. We discuss multiple factors that

102

likely limited an evolutionary response to selection, including genetic correlations, trade-offs
between different components of fitness, and phenotypic plasticity.

103

Introduction
Evolution by natural selection is considered to be common in nature and numerous
studies have documented trait changes over time consistent with adaptive evolution (reviewed in
Hendry and Kinnison 1999; Kinnison and Hendry 2001; Reznick and Ghalambor 2001).
Mechanistically, the evolutionary response to selection can be predicted from univariate models
by the product of heritability and natural selection on the trait of interest (Falconer and Mackay
1996). These models of evolutionary change are well supported (Falconer and Mackay 1996;
Lynch and Walsh 1998; but see Morrissey et al. 2010), yet there are a surprising number of
examples where no evolutionary response is observed in the wild despite selection acting on
traits with a known genetic basis (discussed in Merilä et al. 2001). Thus, one of the critical
contemporary questions for evolutionary ecology is: what limits adaptation in the wild?
Many recent studies have addressed this question and have identified multiple potential
limits to adaptive evolution. First, genetic correlations may reduce evolutionary responses to
selection, even given substantial genetic variation in individual traits (Walsh and Blows 2009;
Kingsolver and Diamond 2011). Second, trade-offs between different components of fitness
(e.g., survival vs. fecundity) can constrain evolutionary responses to selection, if total selection
on a trait across all fitness components is less than selection from a single fitness component
(Roff 2002). Similarly, if the association of a trait with fitness changes with ontogeny, total
selection measured across all life stages may be less than selection measured at a particular life
stage, thus constraining evolution (Schluter et al. 1991; Kingsolver et al. 2012). Third,
phenotypic plasticity (i.e., the ability of a genotype to produce different phenotypes in response
to differing environmental conditions; Pigliucci 2001) in traits can constrain evolutionary
responses if there is a genetic correlation between the trait and its degree of plasticity

104

(Schlichting 2004; Pfennig et al. 2010), or through altered trait heritability in different
environments (Wilson et al. 2006). Finally, temporal and/or spatial fluctuations in the magnitude
and/or direction of selection can yield weaker net selection than single selective events, thus
constraining evolution (Siepielski et al. 2009; Bell 2010; Kingsolver and Diamond 2011). For
example, strong directional selection may be followed by periods of no selection (thus lowering
net selection through time), or periods of opposing directional selection (which could further
lower or even negate the effects of prior selection, yielding no net selection; Bell 2010;
Kingsolver and Diamond 2011). Thus, there is a need for temporal replication to fully
characterize selection; similarly, there may also be a need for spatial replication, if selection is
spatially heterogeneous. Yet, despite these needs, many studies lack temporal and/or spatial
replication (temporal replicates reviewed by Kingsolver et al. 2001: range = 1–10; median = 1;
spatial replicates: range = 1–12; median = 1; Kingsolver et al. 2001).
Invasive species are valuable study systems for investigating temporal and spatial
patterns of natural selection, and how variation in selection and the other constraints affect the
evolutionary response to selection (Sax et al. 2005; 2007). Selection on traits of invasive species
is often strong, and numerous studies have shown evolution in invasive species traits (reviewed
in, e.g., Mooney and Cleland 2001; Lee 2002; Lambrinos 2004). Bythotrephes longimanus (the
spiny water flea; hereafter Bythotrephes; Figure 4.1) are invasive, cladoceran zooplankton native
to Eurasia that were first found in the Laurentian Great Lakes in the early 1980s (Mills et al.
1993) and Lake Michigan, specifically, in 1986 (Evans 1988). Bythotrephes are a dominant
predator of zooplankton (Bunnell et al. 2011) and common prey for fish (Pothoven et al. 2007),
and are having widespread negative effects on food webs through trophic cascades (Hoffman et
al. 2001; Strecker and Arnott 2008). One of the key traits of Bythotrephes governing their

105

interspecific interactions is their long defensive tail spine, the longest component of which is the
distal spine (Figure 4.1), which is considered to function as a defense against fish predation
(Branstrator 2005).
Previous research (e.g., Burkhardt 1994; Sullivan and Lehman 1998; Pothoven et al.
2001; 2003) suggests Bythotrephes distal spine experiences natural selection, but heretofore, no
study has explicitly measured natural selection nor has any study investigated a potential
evolutionary response of distal spine length to selection. The most likely agents of selection on
Bythotrephes distal spine length are their fish predators (Sullivan and Lehman 1998).
Bythotrephes are consumed by both gape-limited fish (i.e., fish predators constrained by mouth
size from consuming larger size classes; typically juvenile fish) and non-gape-limited fish (i.e.,
fish predators unconstrained by mouth size; typically adult fish; Schneeberger 1991; Mills et al.
1992; Pothoven et al. 2007; 2012). Research on other taxa indicates gape-limited predation
selects for increased morphological size (e.g., Paine 1976, Case 1978, Day et al. 2002;
Zimmerman 2007; Urban 2008), whereas non-gape-limited predation selects for decreased
morphological size if larger individuals are preferentially consumed (e.g., Hambright 1991;
Urban 2007; 2008). Thus, Bythotrephes likely experience opposing selection from their gapelimited and non-gape-limited fish predators.
Research on Lake Michigan (Pothoven et al. 2012) and other lakes (Straile and Hälbich
2000; Branstrator 2005) indicates predation on Bythotrephes by gape-limited and non-gapelimited predators varies seasonally; therefore, Bythotrephes distal spine length could experience
temporal variation in selection. Gape-limited predation increases throughout the growing season
due to growth of juvenile fish into sizes capable of consuming Bythotrephes; by the end of the
growing season, gape-limited predation is typically the dominant source of predation on

106

Bythotrephes due to the abundance as well as high energetic demand of juvenile fish relative to
adult fish (Straile and Hälbich 2000; Branstrator 2005; Pothoven et al. 2012). Given this
temporal variation in predator gape-limitation, we would expect selection for decreased distal
spine length early in the growing season (due to non-gape-limited adult fish), and selection for
increased distal spine length later in the growing season (due to gape-limited juvenile fish). This
temporal variation in selection may limit an evolutionary response in distal spine length,
depending on the relative strength of selection from these two sources (i.e., net selection).
Moreover, the abundance of adult and juvenile fish predators is known to be spatially variable in
Lake Michigan and other large lakes (O’Gorman et al. 1991; Dettmers et al. 2005; Miehls and
Dettmers 2011), thus selection may also vary spatially.
We conducted a study investigating temporal and spatial patterns of natural selection on
Bythotrephes distal spine length and the potential for an evolutionary response to selection. We
collected Bythotrephes from Lake Michigan during multiple months in three growing seasons
(July-September: 2007; July-October: 2008-2009) and multiple sites (six sites: 2007; four sites:
2008-2009) along an 8.0km transect. For each month and site, we estimated natural selection on
distal spine length within a cohort through a comparison of distal spine length between the first
two developmental (instar) stages. We predicted that selection would vary temporally, with
selection for decreased distal spine length occurring early in the growing season when predators
are predominantly non-gape-limited, and selection for increased distal spine length later in the
growing season when predators are predominantly gape-limited. We also tested for spatial
variation in selection, which could be due to the patchiness of fish abundance. Further, we
looked for evidence of phenotypic change since invasion by investigating within and among
season trends in first instar distal spine length of wild-captured Bythotrephes using data from our

107

three sampling years (2007-2009) as well as previously published data from years soon after
invasion (1989, 1992-1993, 1996). Finally, we used a paleoecological approach (Jeppesen et al.
2001) to look for additional evidence of phenotypic change since invasion by retrieving remnant
distal spines from sediment cores. Previous work (Keilty 1988; Hall and Yan 1997) has shown
that Bythotrephes spines preserve effectively in sediments, with their depth distribution reflecting
time since invasion. This paleoecological approach offers a unique opportunity for us to assess
phenotypic change through time in distal spine length.

Methods
Morphology and Life History of Bythotrephes
The longest section of the Bythotrephes tail spine is the distal spine, which extends from
the tip of the spine to the first pair of instar barbs (Figure 4.1). The distal spine is present at birth
and is retained throughout instar development at its initial length, whereas total spine length
increases during the first two molts through the addition of intercalary segments separated by
paired barbs (Burkhardt 1994; Sullivan and Lehman 1998; Branstrator 2005). Other traits, such
as body size, also increase with each instar molt (Burkhardt 1994; Sullivan and Lehman 1998).
Bythotrephes reproduce by cyclic parthenogenesis, with multiple asexual generations during the
growing season culminating in sexual reproduction at the end of the season (Yurista 1992;
Pothoven et al. 2001).
Natural Selection Analysis
Field Collection. We collected Bythotrephes monthly from July through September in
2007 and July through October in 2008-2009, time periods spanning the majority of the
Bythotrephes growing season in Lake Michigan. For each month, we completed paired sampling

108

trips separated by three days, with the exception of September 2007 and October 2008, for which
we only completed the first collection (Table 4.1). We collected Bythotrephes using a mid-water
trawl with a 1m x 2m opening and 1000μm mesh size towed horizontally through the epilimnion
and metalimnion. Tows were completed along an 8.0km south-north transect (starting
coordinates: 43° 11.76' N 86° 28.10' W; ending coordinates: 43° 15.46' N 86° 31.25' W) that
encompassed six sampling sites at the 60m depth contour, approximately 12.5km west of
Muskegon, MI, USA. One tow was completed at each site; tows were approximately 0.9km long
and sites were separated by approximately 0.5km. All six sites were analyzed in the natural
selection analysis for 2007, whereas only four of the six sites (sites 1-2, 4-5) were analyzed for
2008-2009; for one time period, October 2008, collections occurred at only one site (site 1). All
Bythotrephes collected were immediately preserved in 95% ethanol. Water temperature 1m
below the water surface was recorded during each sampling trip (Table 4.1).
Measurement of Morphological Traits. We measured Bythotrephes distal spine length in
ImageJ (Abramoff et al. 2004) to the nearest 0.001mm from photographs taken with a digital
camera attached to a dissecting microscope. Distal spine length was measured from the tip of the
spine to the anterior end of the first pair of instar barbs (Figure 4.1; Burkhardt 1994; Sullivan and
Lehman 1998). Although some organisms are known to decrease in length after preservation in
ethanol (e.g., larval fish; Moku et al. 2004), a previous test (Miehls et al. 2012) of Bythotrephes
preserved in 95% ethanol revealed no significant shrinkage of Bythotrephes distal spine length.
Natural Selection. We measured natural selection on Bythotrephes distal spine length
through a comparison of distal spine length between the first two instar stages. Because distal
spine length does not change with instar development, differences in mean distal spine length
between the first and second instar stages would reflect size-specific survival as opposed to a

109

developmentally based difference in length (Sullivan and Lehman 1998). We compared distal
spine length of first instar individuals collected on the first of the paired sampling trips to distal
spine length of second instar individuals collected on the second of the paired sampling trips. We
assumed that these individuals were members of the same cohort because our trips were
separated by three days, which is the average instar development time of Bythotrephes at 21°C
(Branstrator 2005); this water temperature was similar to the average water temperature at the
time of most collections (Table 4.1). As a result, most Bythotrephes in their first instar at the time
of the first trip should have molted to the second instar at the time of the second trip. This
estimation of selection within a cohort is important, as it provides a control for the potentially
confounding effects of phenotypic plasticity. Our previous work (Chapter 3) indicated that
Bythotrephes mothers plastically adjust distal spine length of their offspring in response to water
temperature, with higher temperature inducing the production of offspring with longer distal
spines. Individuals of the same cohort should have experienced the same temperature
environment during development, and thus are free from possible temperature-induced
differences in distal spine length, which a comparison of individuals between cohorts might
entail. For example, first and second instar individuals collected on the same sampling trip might
have different distal spine lengths because they experienced different water temperatures early in
development.
Instar stage was determined by counting the number of paired barbs on the spine, which
reliably indicates instar stage for the first two instars (Yurista 1992; Martin and Cash-Clark
1995; Sullivan and Lehman 1998). Although Bythotrephes pass through up to three or more
instar stages (Yurista 1992), the comparison between first and second instars was chosen because
survival through this early life stage likely comprises a large proportion of total fitness and

110

because the distal segment for first instar individuals represents the entire spine length, which we
suspected was the target of gape-limited fish predation.
Statistical Analysis. We statistically tested whether selection on distal spine length varied
temporally and spatially with a linear model in R version 2.15.0 (R Development Core Team
2012). The model included distal spine length as a continuous response, instar (categorical for
instars one and two), time period (categorical for each month within each year), site
(categorical), and all two-way interactions and the three-way interaction as fixed effects. In this
model, a statistically significant three-way interaction between instar, time period, and site would
indicate selection (effect of instar) varies temporally (effect of time period) and spatially (effect
of site). Component two-way interactions between instar and time period or instar and site would
indicate only temporal or spatial variation in selection, respectively. Non-significant three- and
two-way interactions but a significant main effect of instar would represent consistent directional
selection.
We estimated selection differentials (Falconer and Mackay 1996) for distal spine length
at each site within each month separately as the difference in mean distal spine length between
first and second instar individuals. We assessed the statistical significance of the selection
differentials using linear models of distal spine length as a continuous response and instar as the
sole fixed effect; in these models, a statistically significant effect of instar would indicate
significant selection on distal spine length.
Phenotypic Response to Selection
Wild-Captured Individuals. We looked for evidence of phenotypic change since invasion
by assessing trends in average distal spine length of wild-captured individuals within and among
years using our monthly collections across three years (2007-2009) as well as previously

111

published data from years soon after invasion (1989: Burkhardt 1994; 1992-1993, 1996: Sullivan
and Lehman 1998; note that Sullivan and Lehman 1998 present single yearly length estimates
without corresponding sample dates). We used only first instar individuals in this investigation as
their phenotype would most directly reflect an evolutionary response to selection, whereas the
phenotype of older instars would also be influenced by within-generation selection. Although
there are other published studies presenting historical distal spine lengths of Bythotrephes (e.g.,
Pothoven et al. 2001; 2003), we used Burkhardt (1994) and Sullivan and Lehman (1998) because
of the similarity of their measurement procedure to ours for distal spine length. Using our three
sample years (2007-2009), we statistically tested for a change in distal spine length through time.
We used a two-step process in this analysis: first, we fit a linear model with distal spine length as
a continuous response and water temperature as a continuous fixed effect, and, second, we tested
the effect of Julian day (continuous covariate) and year (continuous fixed effect) on the residuals
of the first model. This process tested for an effect of year on distal spine length (which assessed
phenotypic change in distal spine length through time), while removing the confounding effect of
phenotypic plasticity in distal spine length in response to water temperature (through the first
model). Removing the effect of water temperature on distal spine length was important, as
previous research (Chapter 3) indicated that mothers produce offspring with longer distal spines
when reared in warmer water.
Remnant Spines. We looked for additional evidence of phenotypic change since invasion
by retrieving remnant distal spines from sediment cores (Keilty 1988; Hall and Yan 1997). We
collected sediment cores from Lake Michigan during 2007-2009 at the 57m depth contour (42º
52.69' N 86º 21.35' W) approximately 12km west of Grand Haven, MI. Previous work indicated
that sedimentation rates near our field collection site were approximately 0.1cm per year

112

(Robbins and Edgington 1975), although the sedimentation rate has likely increased since the
early 1990s following dreissenid mussel invasion (Thayer et al. 1997). Conservatively allowing
for an increased sedimentation rate post-dreissenid invasion, and given a Bythotrephes invasion
year of 1986 in Lake Michigan (Evans 1988), we sampled the upper 10cm of sediment during
2007. We sampled the upper 20cm of sediment during 2008-2009 to ensure the 10cm depth
during 2007 was sufficient to encompass all sedimentation since Bythotrephes invasion.
Sediment was partitioned into 1cm depth intervals on board the ship, and intact distal spines
were removed from the sediment in the lab and measured according to the methods above. We
used a linear model to assess whether distal spine length (continuous response) correlated with
depth (proxy for time since invasion; continuous fixed effect) while accounting for variation due
to our sampling years (categorical fixed effect).

Results
Natural Selection
We found net selection for increased distal spine length across all time periods (months,
years) and sites (net selection differential: S = 0.08mm, F1, 6821 = 43.3, p < 0.001; Figure 4.2).
However, selection varied both temporally and spatially (significant three-way interaction
between instar, time period, and site: F26, 5433 = 7.80, p < 0.001; Figure 4.3; see Appendix A for
mean distal spine length of each instar, selection differentials, and selection intensities for each
site and time period combination). The greatest within-season variation in selection occurred in
2007, when we observed selection for decreased distal spine length predominantly early in the
growing season (July), and selection for increased distal spine length later in the season (August

113

and September; Figure 4.3a). Moreover, we also observed the strongest spatial variation in
selection during 2007, as the direction of selection varied by site for two months (July,
September). Significant selection for decreased distal spine length was never observed in 2008 or
2009 (Figure 4.3b;c); instead, there was consistent selection for increased distal spine length for
at least one site during each time period, with the exception of October 2008 and July 2009 when
we observed no significant selection (Figure 4.3b;c). Notably, there was a trend for the strongest
episodes of selection to occur at the temporal extremes: the strongest selection for decreased
distal spine length occurred during July 2007 (our earliest sample collection across all sample
dates; Table 4.1) and the strongest selection for increased distal spine length occurred during
October 2009 (the penultimate sample collection across all sample dates; Table 4.1).
Phenotypic Response to Selection
Wild-Captured Individuals. Considering our three sampling years (2007-2009) as well as
one historical year (1989), within season variation in distal spine length was greater than among
season variation (Figure 4.4a). During 2007 and 2008, distal spine length was smallest at the start
of the growing season (July) and longest later in the season (August-October). First instar distal
spine length (statistically controlled for temperature-induced plasticity) significantly increased
throughout our three sampling years (total length increase across the three years: 0.08mm, t6265
= 11.9, p < 0.001), although the relationship between distal spine length and year was weak
2

(model adjusted R = 0.12). The 0.08mm increase over three years corresponds to an annual rate
of 0.03mm/yr. The range of historical distal spine lengths (1989, 1992-1993, 1996) almost
completely encompassed the range of distal spine lengths found in our study years (the exception
being one smaller distal spine length estimate during July 2007), indicating little phenotypic
change in Bythotrephes distal spine length since invasion (Figure 4.4a).

114

Remnant Spines. Distal spine length increased with depth interval for all three years
(length increase across all years: 0.04mm; t267 = 2.8, p < 0.01), although the relationship
2

between distal spine length and depth interval was weak (model adjusted R = 0.04; Figure 4.4b).
Note that as deeper sediments were deposited earlier in time, this relationship indicates distal
spine length decreased slightly since invasion. Given that Bythotrephes invaded Lake Michigan
in 1986 (Evans 1988), the 0.04mm decrease occurred over 23 years (using our last year of
sediment collection, 2009), corresponding to an annual rate of -0.002mm/yr. Only two spines
were found below the 10cm depth interval (one spine each at the 11 and 13cm depth intervals in
2008). Given the rarity of spines below 10cm deep (the 269 other spines were found 9cm or
shallower), and the possibility the two deep spines resulted from contamination of samples
during processing, the 2007 sediment collection (which only included the upper 10cm of
sediment) likely reflected all sedimentation since Bythotrephes invasion.

Discussion
Although adaptive evolution is generally considered ubiquitous in nature (Kinnison and
Hendry 2001), and natural selection is frequently documented in natural populations (Kingsolver
et al. 2001; Hereford et al. 2004; Kingsolver and Diamond 2011), there are a surprising number
of examples where an evolutionary response to selection on a heritable trait is not observed
(Merilä et al. 2001). Our research has demonstrated evolutionary stasis in an important trait
governing interspecific interactions that is under significant, directional selection (Figure 4.2)
and has a strong genetic basis (Miehls et al. 2012). We base this finding of evolutionary stasis on
a temporally and spatially replicated investigation of natural selection (47 total estimates of

115

natural selection) and using multiple lines of evidence (historic and contemporary wild-captured
individuals, paleoecology of retrieved spines).
Our results showed evidence of temporal variation in both the magnitude and direction of
selection on Bythotrephes distal spine length. The dominant direction of selection was for
increased distal spine length, which we observed in all time periods, although selection for
increased distal spine length was not significant in October 2008 or July 2009 (Figure 4.3). In
2009, when we had the greatest temporal resolution, there was a trend for strengthening selection
throughout the growing season, with the strongest selection occurring during October. We only
observed significant selection for decreased distal spine length during two time periods, July
2007 and September 2007. Note, however, that the selection for decreased distal spine length in
July 2007 was the strongest selection (positive or negative) we observed over all time periods
(Figure 4.3). Interestingly, the July 2007 sample collection was also the earliest collection we
completed. It is possible the July 2008 and 2009 collections, which occurred approximately 3
and 1.5 weeks later in the growing season than the July 2007 collection, respectively, may have
occurred sufficiently late to have missed seasonal episodes of selection for decreased distal spine
length. In general, the data are suggestive of a trend for strong selection for decreased distal
spine length early in the growing season (July 2007), followed by strengthening selection for
increased distal spine length that reaches the greatest magnitude late in the season (October
2009).
The temporal pattern of selection we observed is consistent with typical seasonal shifts in
gape-limitation of fish predators, suggesting fish are the likely agents of selection in our system.
Specifically, the strong selection for decreased distal spine length early in the season occurred
when non-gape-limited (adult) fish predation would be the strongest (Straile and Hälbich 2000;

116

Branstrator 2005; Pothoven et al. 2012), whereas the strengthening selection for increased distal
spine length during the remainder of the growing season occurred when gape-limited fish
predation would be increasing coincident with growth of juvenile fish (Straile and Hälbich 2000;
Branstrator 2005; Pothoven et al. 2012). The conclusion that these changes in the direction and
magnitude of selection are the result of temporally variable gape-limitation of fish predators is
supported by previous research that indicates gape-limited predators typically exert selection for
increased morphological size (e.g., Paine 1976, Case 1978, Day et al. 2002; Zimmerman 2007;
Urban 2008), whereas non-gape-limited predators typically exert selection for decreased
morphological size (e.g., Hambright 1991; Urban 2007; 2008). Further, laboratory research
(Barnhisel 1991a; 1991b) indicated that gape-limited fish selectively consume Bythotrephes
based on distal spine length, with gape-limited fish preferentially consuming Bythotrephes with
shorter distal spines. Thus, it is likely that fish predators are the agents of selection in our system,
and that temporal variation in selection is due to changes in gape-limitation, although this
assertion remains to be tested more rigorously.
We also found that selection varied spatially (Figure 4.3). During seven of our eleven
time periods, we observed opposing directions of selection by site, although we only observed
statistically significant opposing selection by site in two time periods (July and September 2007).
The spatial variation in selection is presumably due to heterogeneous distribution of fish
predators, which is known to occur in Lake Michigan and other large lakes. Both adult
(O’Gorman et al. 1991) and juvenile fish (Dettmers et al. 2005; Miehls and Dettmers 2011) are
known to be spatially patchy at the scale at which we sampled, with patchiness of adult fish
generally due to active fish movement (for, e.g., reproduction or foraging; O’Gorman et al. 1991)
and patchiness of juvenile due to passive transport by currents (Dettmers et al. 2005; Miehls and

117

Dettmers 2011). The opposing directions of selection found in July and September 2007 could
thus be due to different sites experiencing different relative strengths of predation by non-gapelimited adult and gape-limited juvenile fish. Alternatively, we note that for both July and
September 2007 there were at least two significant selection differentials of consistent direction
and only one significant selection differential of opposing direction (Figure 4.3a); it is possible
the single opposing selection differential for each time period was a statistical artifact resulting
from high power to detect a difference in means (Appendix A) and multiple comparisons that
resulted in Type I error. In general, however, our data are indicative of fairly strong spatial
variation in selection.
Despite net selection for increased distal spine length (S = 0.08mm; Figure 4.2), we found
only limited evidence of phenotypic change in Bythotrephes distal spine length since invasion.
Our wild-captured Bythotrephes exhibited a minor increase in length from 2007-2009 (absolute
length increase: 0.08mm; annual rate: 0.03mm/yr); however, the historical data (1989, 19921993, 1996) almost completely overlapped with our 2007-2009 data, indicating little phenotypic
change in distal spine length since invasion. Moreover, the sediment-retrieved distal spines
indicated little phenotypic change in length since invasion. Contrary to the wild-captured data,
the retrieved distal spines indicated a decrease in length since invasion (absolute length decrease:
-0.04mm; annual rate: -0.002mm/yr). Given the conflicting directions, small magnitudes, and
low annual rates of change from these sources of data, it is unlikely Bythotrephes distal spine
length has undergone any consistent phenotypic change since invasion.
Multiple factors can cause evolutionary stasis despite directional selection on a heritable
trait (Merilä et al. 2001). The most relevant potential factors in our system include: 1) temporal
and spatial fluctuation in selection; 2) genetic correlations with unmeasured traits; 3) trade-offs

118

between fitness components and/or selection that varies by life stage; and 4) phenotypic
plasticity. We explore each of these factors below.
First, temporal and spatial fluctuations in the magnitude and/or direction of selection can
reduce the cumulative effects of selection and constrain evolution (Merilä et al. 2001; Siepielski
et al. 2009; Bell 2010; Kingsolver and Diamond 2011). Investigations in well-studied
populations show that both the direction and magnitude of selection can vary considerably from
year-to-year (e.g., Price et al. 1984; Grant and Grant 1993; Sinervo et al. 2000; McAdam and
Boutin 2003). We found strong temporal and spatial variation in selection (Figure 4.3), but still
found net selection (S = 0.08mm) for increased distal spine length (Fig 4.2). It is possible,
however, that our net selection differential was biased due to missing early season selection in
2008 and 2009 (discussed above). Additionally, the spatial coverage of our transect (8.0km) was
small relative to the size of Lake Michigan; it is reasonable to assume that selection may vary
considerably more across the larger spatial scale of the lake. Thus, spatio-temporal variation in
selection in Lake Michigan may be even greater than the variation reflected in our study, for
which the net effect may be no significant selection. Indeed, although our net selection
differential was statistically different from zero, its low magnitude relative to the trait standard
deviation (i.e., selection intensity = 0.10) indicates only moderate net selection (Kingsolver and
Diamond 2011).
Second, genetic correlations may reduce evolutionary responses to selection, even given
substantial genetic variation in individual traits (Walsh and Blows 2009; Kingsolver and
Diamond 2011). Previous research (Miehls et al. 2012) found that Bythotrephes distal spine
2

length is highly heritable (range of H : 0.27-0.76), but they did not investigate possible genetic
correlations between distal spine length and other traits. Our work (Appendix B) and others

119

(Sullivan and Lehman 1998; Branstrator 2005) indicates Bythotrephes distal spine length is
phenotypically correlated with body size, suggesting the possibility of a genetic correlation
(Conner and Hartl 2004). If there is selection on body size in the opposite direction as selection
on distal spine length, an evolutionary response of distal spine length could be constrained.
Alternatively, body size, and not distal spine length, may be the true target of selection; in this
scenario, our selection differentials (which estimate total selection) may be overestimating direct
selection on distal spine length. Estimating selection gradients for distal spine length would
remedy this potential problem. However, our selection design, which involved a comparison of
different instar stages, does not allow for the estimation of selection gradients because our
comparisons across developmental stages do not quantify individual fitness and selection on
body size cannot be determined using our method as body size always increases with each instar
molt (Burkhardt 1994; Sullivan and Lehman 1998). Of note, however, recent reviews
(Kingsolver et al. 2001; Kingsolver and Diamond 2011; Kingsolver et al. 2012) found that
selection differentials and selection gradients were often of similar magnitude, suggesting our
selection differentials may approximate direct selection.
Third, trade-offs between fitness components (Roff 2002) and/or variable selection by
life stage (Schluter et al. 1991; Kingsolver et al. 2012) can also limit evolutionary responses.
Specifically, trait values advantageous to one component of fitness (e.g., survival) may be
disadvantageous to some other component of fitness (e.g., fecundity). Our previous work
(Chapter 3) and others (Straile and Hälbich 2000; Pothoven et al. 2003) indicates Bythotrephes
mothers face a size-number trade-off (Smith and Fretwell 1974) in their allocation of resources
to distal spine defenses of offspring versus number of offspring, which might constrain
evolutionary responses in distal spine length. In this case, the viability selection for increased

120

spine length that we have documented here might be offset by fecundity selection for reduced
distal spine length. Another related possibility is that a trade-off exists between investment in
current defense (i.e., distal spine length of younger instar individuals) and investment in future
defense (i.e., length of the segments added with future molts); however, this scenario is unlikely
as our work indicates distal spine length positively correlates with the length of the first
intercalary segment (Appendix C). Regarding the latter constraint to evolution (i.e., variable
selection by life stage), a trait that is strongly associated with an aspect of fitness in an early
developmental stage may not necessarily be as strongly associated with that aspect of fitness in a
later developmental stage (Schluter et al. 1991). Bythotrephes pass through multiple
developmental (instar) stages, but we only estimated selection on Bythotrephes distal spine
length between the first two instar stages. For older instar individuals - whose tail spines contain
up to two intercalary segments (Figure 4.1) added with the first two instar molts - selection is
likely on total spine length, which may effectively weaken direct selection on distal spine length
and therefore constrain evolution.
Finally, phenotypic plasticity may constrain evolutionary responses to selection if there is
a genetic correlation between the trait and its degree of plasticity (Schlichting 2004; Pfennig et
al. 2010), or through altered trait heritability in different environments (Wilson et al. 2006). We
found that Bythotrephes mothers plastically altered distal spine length of offspring in response to
their temperature environment (Chapter 3). Although Bythotrephes distal spine length is heritable
(Miehls et al. 2012), the genetic basis of plasticity in Bythotrephes distal spine length is
unknown, as is the potential for a genetic correlation between absolute length and plasticity in
length. If a correlation between plasticity and absolute length exists, the correlation could
constrain an evolutionary response to selection (Pfennig et al. 2010). Further, phenotypic

121

plasticity could constrain an evolutionary response if trait heritability varies by environment
(Wilson et al. 2006). Because individuals vary in their reaction norms for plastic traits (Pigliucci
2001), phenotypic variation will differ across environments even given the same genotypic
variation, which in turn causes heritabilities to be environment-dependent (Pigliucci 2001).
Miehls et al. (2012) found high distal spine length heritability in a single temperature
environment (15°C); given a plastic response to temperature, it is possible distal spine length
heritability is lower in other temperature environments. Thus, given seasonal water temperature
variation in the wild (Pothoven et al. 2001), heritability of Bythotrephes distal spine length may
vary temporally. In this manner, phenotypic plasticity can constrain an evolutionary response of
Bythotrephes distal spine length via environment-dependent trait heritabilities.
In conclusion, our research has demonstrated evolutionary stasis in a key trait for
interspecific interactions despite significant, directional selection and known genetic trait
variation. Although these findings may initially appear contradictory, multiple constraints on the
evolutionary response to selection could exist in this system that can readily explain evolutionary
stasis. These findings have two main implications to evolutionary ecology. First, our work
strongly supports the conclusions of others (e.g., Merilä et al. 2001) that selection can vary
widely in time and space, and therefore, accurate predictions of the evolutionary response to
selection necessitate greater replication in studies of natural section. Second, even though natural
selection is common in the wild (Kingsolver et al. 2001; Hereford et al. 2004; Kingsolver and
Diamond 2011), evolutionary responses to selection may be constrained, even when traits are
under selection and have a significant genetic basis. Importantly, though, not all constraints may
be viewed as detrimental to fitness, as some constraints (e.g., phenotypic plasticity) may
themselves confer a strong fitness advantage.

122

Acknowledgements
Doran Mason, Dennis Donahue, Steven Pothoven, and the NOAA Great Lakes
Environmental Research Laboratory and NOAA Lake Michigan Field Station provided research
vessels and field support staff. Donna Kashian and Kevin Pangle co-led sediment core
collections, and Keali Chambers, Brittany Damschroder, Jason Fischer, Brittany Gunther, Nicole
Hedquist, Tyler Joseph, Stefanie Jubb, Lydia Kramer, Ian McCririe, Jennifer Pellegrini, Ariel
Porty, Veronica Quesnell, Andria Salas, Ben Staton, Marie Stevenson, Mariam Teimorzadeh,
Elizabeth Throckmorton, Brandon Vieder, and Miriam Weiss assisted with field collections and
laboratory sample processing. Paul Bourdeau and Scott Miehls provided additional
methodological assistance. This work was supported by the Great Lakes Fishery Commission
and an EPA Science to Achieve Results fellowship (STAR Research Assistance Agreement No.
FP91698801-0 awarded by the U.S. Environmental Protection Agency). This work has not been
formally reviewed by the EPA and the views expressed in this document are solely those of the
authors.

123

Tables
Table 4.1. Sample dates for paired monthly collections used to estimate natural selection on
Bythotrephes distal spine length during 2007-2009 and average water temperature 1m below the
surface. Note that the October 2, 2007 and November 3, 2008 collections are referred to in the
text as September and October collections, respectively, given the approximate one-month
interval between these collections and the previous collections. Only one sampling trip was
completed for these two time periods.
Paired Sample Dates
2007
July 9, 12
August 21, 24
October 2
2008
July 28, 31
August 26, 29
September 22, 25
November 3
2009
July 20, 23
August 17, 19
September 21, 24
October 26, 29

Temperature (°C)
19.1
21.1
19.8
21.6
21.5
19.8
11.3
20.1
21.3
19.6
10.2

124

Figures

.1.
mated natural selection on distal spine length of B
l
n
e
Bythotrephes longimanus, the
s
s
Figure 4. We estim
spiny wa flea (the individual pictured is a third instar) .
ater
p

125

Distal spine length (mm)

6.2
6.1
6.0
5.9
5.8
Pre

Post

Figure 4.2. Net selection on Bythotrephes distal spine length as illustrated by the change in
average distal spine length pre-selection (first instar individuals) and post-selection (second
instar individuals) across all years and sites (net selection differential: S = 0.08mm). Error bars
are standard errors.

126

Figure 4.3. Selection differentials (S) on Bythotrephes distal spine length for multiple sites along
an 8.0km transect in Lake Michigan during: a) 2007 (six sites: July-September); b) 2008 (four
sites: July-September; 1 site: October); and c) 2009 (four sites: July-October). Note the different
y-axis in panel (a). Black bars: statistically significant (p < 0.05) selection differentials for
increased distal spine length; white bars: statistically significant (p < 0.05) selection differentials
for decreased distal spine length; gray bars: non-statistically significant selection differentials.

127

Figure 4.3.
a)

0.6

2007

0.4

S (mm)

0.2
No Data

0.0
-0.2
-0.4
-0.6
-0.8

b)

0.6

2008

S (mm)

0.4
0.2
0.0
-0.2

c)

0.6

2009

S (mm)

0.4
0.2
0.0
-0.2

1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6
July

August

September

128

October

a)
Distal spine length, Instar 1 (mm)

6.5

6.0

1989
1992
1993

5.5

5.0
175

Distal spine length (mm)

b)

2007
2008
2009

200

225

250
275
Julian Day

No
325 day

300

8.0

2007
2008
2009

7.0
6.0
5.0
4.0
0

1

2

3

4

5
6
7
8
Depth interval (cm)

9

10

11

12

13

Figure 4.4. Distal spine length of: a) first instar wild-captured Bythotrephes from this study
(2007-2009) and previously published studies (1989: Burkhardt 1994; 1992-1993, 1996: Sullivan
and Lehman 1998); and b) retrieved spines collected from sediment cores (2007-2009); note that
the 2007 and 2009 sediment core data are shown offset from the depth interval for clarity. The
2

solid line in panel (b) is the linear regression across all three years (adjusted R = 0.04).

129

APPENDICES

130

Appendices
Appendix A. Selection differentials and intensities by time period and site
Table 4.2. Natural selection on distal spine length of Bythotrephes longimanus between the first
two developmental (instar) stages in Lake Michigan for multiple sites in an 8.0km transect from
2007-2009. Ave: average distal spine length (mm); SD: standard deviation; N: sample size; SE:
standard error; S: selection differential (mm); SDp: propagated standard deviation (calculated as
the square root of the sum of the squared standard deviation of distal spine length for each
instar); i: selection intensity (calculated as the selection differential divided by the propagated
standard deviation); p: p-value (*p < 0.05, **p < 0.01; ***p < 0.001).

Month Site
2007
1
Jul
2
3
4
5
6
1
Aug
2
3
4
5
6
1
Sep
2
3
4
5
6
2008
Jul

1
2
4
5

Instar 1 length
Ave SD N SE

Instar 2 length
Ave SD N SE

5.23
4.91
5.56
4.93
5.44
4.92
6.04
5.85
5.96
6.01
6.24
5.95
6.39
6.33
6.14
6.15
5.98
5.95

0.54
0.34
0.47
0.34
0.78
0.40
0.34
0.34
0.35
0.33
0.40
0.35
0.41
0.36
0.35
0.38
0.35
0.37

36
12
36
13
32
49
62
103
134
163
71
66
114
136
114
91
112
114

0.09
0.10
0.08
0.09
0.14
0.06
0.04
0.03
0.03
0.03
0.05
0.04
0.04
0.03
0.03
0.04
0.03
0.04

5.81
4.62
4.87
4.72
4.68
4.77
6.07
6.16
6.04
5.97
6.15
6.15
6.33
6.25
6.18
6.22
6.14
6.07

0.79
0.40
0.44
0.45
0.42
0.45
0.28
0.32
0.34
0.27
0.29
0.37
0.39
0.34
0.35
0.37
0.35
0.34

71
50
45
92
139
160
63
146
63
119
99
267
182
188
203
265
239
222

0.09
0.06
0.07
0.05
0.04
0.04
0.04
0.03
0.04
0.02
0.03
0.02
0.03
0.02
0.02
0.02
0.02
0.02

0.58
-0.29
-0.68
-0.20
-0.76
-0.15
0.02
0.31
0.09
-0.05
-0.09
0.20
-0.06
-0.08
0.03
0.06
0.16
0.12

0.95
0.53
0.65
0.56
0.89
0.60
0.44
0.47
0.49
0.43
0.49
0.51
0.56
0.49
0.50
0.53
0.50
0.51

5.76
5.58
5.83
5.81

0.45
0.54
0.45
0.49

48
49
51
50

0.06
0.08
0.06
0.07

5.81
5.85
5.91
5.89

0.59
0.55
0.66
0.55

56
48
45
46

0.08
0.08
0.10
0.08

0.06
0.26
0.08
0.08

0.74
0.77
0.80
0.74

131

Natural selection
S
i
p
SDp
0.60
-0.55
-1.05
-0.36
-0.86
-0.25
0.05
0.67
0.18
-0.11
-0.18
0.39
-0.10
-0.17
0.07
0.12
0.32
0.24

***
*
***
NS
***
*
NS
***
NS
NS
NS
***
NS
*
NS
NS
***
**

0.08 NS
0.34 *
0.10 NS
0.10 NS

Table 4.2 (con’t)
Month Site
1
Aug
2
4
5
1
Sep
2
4
5
1
Oct
2009
Jul

Aug

Sep

Oct

1
2
4
5
1
2
4
5
1
2
4
5
1
2
4
5

Instar 1 length
Ave SD N SE
6.00 0.35 50 0.05
6.03 0.44 50 0.06
6.03 0.32 51 0.05
6.16 0.32 49 0.05
6.11 0.28 50 0.04
6.28 0.38 50 0.05
6.12 0.39 50 0.05
6.16 0.31 50 0.04
6.21 0.34 44 0.05

Instar 2 length
Ave SD N SE
6.10 0.29 46 0.04
6.21 0.32 40 0.05
6.15 0.29 48 0.04
6.08 0.33 43 0.05
6.42 0.30 45 0.04
6.22 0.33 49 0.05
6.14 0.28 47 0.04
6.29 0.37 49 0.05
6.31 0.39 50 0.06

Natural selection
i
p
S
SDp
0.10 0.45 0.22 NS
0.18 0.54 0.33 *
0.11 0.43 0.26 NS
-0.08 0.46 -0.17 NS
0.32 0.41 0.77 ***
-0.07 0.50 -0.13 NS
0.03 0.48 0.06 NS
0.13 0.48 0.27 NS
0.10 0.52 0.19 NS

6.23
6.15
6.07
6.27
6.14
6.20
6.27
6.30
6.20
6.12
6.28
6.39
6.01
5.96
5.92
5.85

6.25
6.25
6.21
6.32
6.35
6.19
6.40
6.30
6.37
6.22
6.29
6.34
6.28
6.41
6.23
6.29

0.02
0.09
0.14
0.06
0.21
-0.01
0.13
0.00
0.17
0.10
0.01
-0.05
0.26
0.45
0.31
0.44

0.36
0.42
0.45
0.37
0.39
0.31
0.34
0.44
0.32
0.30
0.35
0.29
0.33
0.29
0.33
0.34

21
50
50
50
49
47
48
51
49
52
48
53
53
50
47
54

0.08
0.06
0.06
0.05
0.06
0.04
0.05
0.06
0.05
0.04
0.05
0.04
0.05
0.04
0.05
0.05

132

0.25
0.31
0.36
0.37
0.28
0.25
0.35
0.26
0.37
0.24
0.35
0.30
0.23
0.32
0.37
0.30

45
48
49
50
47
49
49
50
46
49
51
49
45
47
48
50

0.04
0.04
0.05
0.05
0.04
0.04
0.05
0.04
0.05
0.03
0.05
0.04
0.03
0.05
0.05
0.04

0.44 0.05 NS
0.52 0.18 NS
0.58 0.25 NS
0.53 0.11 NS
0.48 0.44 **
0.39 -0.04 NS
0.49 0.27 NS
0.51 -0.01 NS
0.49 0.34 *
0.39 0.26 NS
0.49 0.02 NS
0.42 -0.12 NS
0.40 0.66 ***
0.44 1.04 ***
0.50 0.62 ***
0.45 0.98 ***

Appendix B. Correlation between distal spine and body length of Bythotrephes longimanus.
Many phenotypic correlations result from underlying genetic correlations (Conner and
Hartl 2004), which may constrain evolution, even given substantial genetic variation in
individual traits (Walsh and Blows 2009; Kingsolver and Diamond 2011). Previous studies
(Sullivan and Lehman 1998; Branstrator 2005) have shown a positive phenotypic correlation
between distal spine length and body size of Bythotrephes. If this phenotypic correlation has an
underlying genetic basis, then evolution of distal spine length may be constrained given
countering selective forces on body length. We, therefore, tested for the presence of a correlation
between distal spine and body length using first and second instar Bythotrephes collected from
Lake Michigan during 2007-2009 (see main text for details on sample collection).
We measured distal spine length from the tip of the spine to the anterior end of the first
set of instar barbs and body length from the anterior edge of the eye to the base of the tail spine
along the midline of the body with segments spanning the head and eye region, thorax, and
abdomen (Figure 4.1; Miehls et al. 2012). We tested for a correlation between distal spine and
body length using a linear mixed effects model (LME) with the nlme package (Pinheiro et al.
2009) in R version 2.15.0 (R Development Core Team 2012). The model included distal spine
length as a continuous response, body length (continuous), instar (categorical for instars one and
two), and their interaction as fixed effects, and month (July-October) nested within year (20072009) as random effects. Note that this random effects structure tested for a correlation between
distal spine and body length within a time period, as opposed to among time periods. We also
investigated correlations for each instar separately using linear models of distal spine length
(continuous response) predicted by body length (continuous fixed effect).

133

Distal spine length was positively correlated with body length (slope = 0.40, t14,057 =
46.2, p < 0.001) across both instars, but there was a significant interaction between body length
and instar (F1, 14,056 = 13.9, p < 0.001). Considering each instar separately, first instar
individuals exhibited a stronger correlation between distal spine and body length (slope = 0.48,
t6300 = 31.7, p < 0.001) than second instar individuals (slope = 0.33, t7746 = 31.6, p < 0.001).
Given the presence of a phenotypic correlation between distal spine and body length for first and
second instar individuals, it is possible a genetic correlation might also be present which could
constrain evolution of distal spine length.

134

ix
s
Appendi B Figures

Figure 4. Morpholo
.5.
ogical measu
urements use to test for a correction between di
ed
r
n
istal spine an
nd
body leng of Bytho
gth
otrephes long
gimanus. Th individual pictured is a third instar
he
l
r.

135

Appendix C. Correlation between spine segments of Bythotrephes longimanus.
Variable selection by life stage may constrain an evolutionary response to selection
(Schluter et al. 1991). One way in which this constraint may manifest is when a trait that is
strongly associated with an aspect of fitness in an early developmental stage trades-off with
another trait that is associated with fitness later in development (Schluter et al. 1991).
Bythotrephes longimanus are born with a long spine (the distal spine; Figure 4.1), which they
retain through instar development at its original length (Burkhardt 1994; Sullivan and Lehman
1998). Although distal spine length does not change during development, total spine length
increases with the addition of two intercalary segments (S2 and S3; Figure 4.1) during the first
two molts (Burkhardt 1994; Sullivan and Lehman 1998). Owing to its long length and presence
at birth, the distal spine is considered to be the spine segment providing the greatest protection
from gape-limited fish predation (Branstrator 2005); the additional intercalary segments might
also function as defenses against gape-limited fish predation, although their adaptive value is less
clear (Sullivan and Lehman 1998). We investigated whether distal spine length (a trait conferring
protection early in development) exhibits a trade-off with segment two length (a trait conferring
protection later in development) for second instar Bythotrephes collected from Lake Michigan
during 2007-2009 (see main text for details on sample collection).
We measured distal spine length from the tip of the spine to the anterior end of the first
set of instar barbs; we measured segment two length from the anterior end of the first set of instar
barbs to the anterior end of the second set of instar barbs (Figure 4.1; Miehls et al. 2012). We
tested for a correlation between distal spine length and segment two length using a linear mixed
effects model (LME) with the nlme package (Pinheiro et al. 2009) in R version 2.15.0 (R
Development Core Team 2012). The model included distal spine length as a continuous

136

response, segment two length as a continuous fixed effect, and month (July-October) nested
within year (2007-2009) as random effects. Note that this random effects structure tested for a
correlation between distal spine and segment two length within a time period, as opposed to
among time periods. A negative correlation between distal spine and segment two length would
suggest a trade-off between these two traits.
We found that distal spine length was positively correlated with segment two length
(slope = 0.65, t7,765 = 27.1, p < 0.001). The positive correlation indicates that protection from
predation early in development (i.e., distal spine length) does not trade-off with protection from
predation later in development (i.e., segment two length), indicating little likelihood for a
constraint on evolution owing to varying associations of these traits with fitness by life stage.

137

ix
s
Appendi C Figures

Figure 4. Spine mo
.6.
orphology of Bythotrephe longiman The dista spine is pr
f
es
nus.
al
resent at birt
th,
whereas the second (S2) and thir (S3) spine segments ar added wit the first an second in
(
rd
e
re
th
nd
nstar
molts, respectively. We tested for a trade-off between dis spine an segment tw length us
W
f
stal
nd
wo
sing
the morp
phological measurements indicated below. The in
m
s
b
ndividual pic
ctured is a th instar.
hird

138

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139

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CHAPTER 5

Gape-limited predators as agents of selection on the defensive morphology of an invasive species

Andrea Lynn-Jaeger Miehls, Scott D. Peacor, and Andrew G. McAdam

Abstract
Evolution of invasive species is causing pronounced effects on ecosystems, yet we still
have limited knowledge of the agents of natural selection on invasive species and the rate of
adaptive evolution in invasive species traits. Bythotrephes longimanus, called the spiny water
flea because of its conspicuous distal spine that serves as a morphological defense against gape
limited (GLP) fish predation, is an invasive predatory zooplankton in North America that
invaded multiple lakes in the Canadian Shield over the past several decades. We estimated
natural selection on distal spine length of Bythotrephes collected from six replicate lakes that
varied in the dominance of gape-limited (GLP) and non-gape-limited (NGLP) fish predation.
Using a between cohort analysis, we found that natural selection varied by gape-limitation, with
strong selection (selection intensity: 0.20-0.79) for increased distal spine length in lakes
dominated by GLP, and no significant selection in lakes dominated by NGLP. Further, distal
spine length was 20% longer in lakes dominated by GLP, suggesting the possibility of local
adaptation in response to selection. As all study lakes were invaded less than twenty years prior
to our collections, these results suggest rapid divergence in a defensive morphological phenotype

146

Introduction
Invasive species are one of the world’s greatest threats to biodiversity and ecosystem
function, and there is mounting evidence that evolution plays a key role in their pronounced
ecological effects (Mooney and Cleland 2001; Lambrinos 2004). Invasive species may evolve
upon introduction into new environments due to novel selection pressures, population
bottlenecks, founder effects, and hybridization in the invaded range, as well as freedom from
selection pressures in the native range (Lee 2002; Lambrinos 2004). Evolution via population
bottlenecks, founder effects, and hybridization is often rapid, frequently occurring within a few
years or decades of introduction (Lee 2002; Lambrinos 2004; Prentis et al. 2008). Comparatively
less is known about the pace of evolution by natural selection in invasive species (especially
invasive animals), although preliminary work indicates the pace is variable, involving years,
decades, or even centuries (Reznick and Ghalambor 2001; Kristjánsson et al. 2002; Lee 2002).
In addition to limited knowledge of the pace of evolution by natural selection in invasive
species, we more generally often lack explicit knowledge of the agents of selection on traits of
prey in the wild. Presumably predators are important agents of selection (Darwin 1859), but our
knowledge of predators as agents of selection in the wild is relatively limited. The work that is
available suggests one of the primary determinants of the direction of selection exerted by
predators is the size of prey relative to the mouth (i.e., gape) size of the predator. Many predators
cannot capture prey above a certain gape-size threshold (Wilson 1975; Paine 1976; Hambright
1991), which allows predators to be functionally categorized as either gape-limited (i.e., predator
is constrained by mouth size from consuming larger size classes of prey) or non-gape-limited
(i.e., predator is unconstrained by mouth size and can consume any size class of prey) with
respect to any particular prey species (Urban 2007; 2008). Gape-limited predation (GLP)

147

typically selects for increased morphological size of prey (e.g., Paine 1976; Day et al. 2002;
Urban 2008), whereas non-gape-limited predation (NGLP) may yield no selection on
morphological size (Scharf et al. 2000; Urban 2007; 2008) or select for decreased morphological
size if larger individuals are preferentially consumed (e.g., Hambright 1991; Urban 2007; 2008).
There is evidence to suggest many prey have evolved to combat GLP through morphological
defenses that provide a size refuge (e.g., Tollrian 1995; Young et al. 2004; Lescak and von
Hippel 2011). Conversely, prey experiencing NGLP are often small, lack defenses, or exhibit
reduced morphological defense compared to conspecifics experiencing GLP (Gross 1978;
Kristjánsson et al. 2002). Thus, we have reasonable expectations for the direction of selection
based on gape-limitation of potential predators, but we rarely have adequate data to test these
predictions.
Additionally, we know particularly little about selection and adaptive evolution of
invasive prey in response to predation. One of the best-studied systems documenting evolution of
an invasive prey is the threespine stickleback (Gasterosteus aculeatus). Threespine sticklebacks
evolved reduced defenses (number of armor plates, dorsal spines) after invading freshwater
habitats where predation pressure was reduced (Kristjánsson et al. 2002). Of course, this example
is not an instance of evolution in response to selection from predators, per se, but rather
evolution resulting from the relaxation of predation (i.e., reverse evolution; Porter and Crandall
2003). Examples of invasive prey evolving due to direct selection from predators are limited,
although experimental introductions of species into habitats with high predation indicate
evolution can occur (Reznick et al. 1990).
Recent species introductions into multiple lakes of the Canadian Shield in Ontario,
Canada, offer an opportunity to investigate predators as agents of selection in the wild and the

148

potential for adaptation of invasive species to predation. Bythotrephes longimanus (the spiny
water flea; hereafter Bythotrephes; Figure 5.1a) are cladoceran zooplankton native to Eurasia that
were first found in the North American Great Lakes in the early 1980s (Mills et al. 1993). Within
the following two decades, Bythotrephes spread through all of the Great Lakes and invaded more
than 90 surrounding lakes, including many Canadian Shield lakes (Strecker et al. 2006).
Bythotrephes are a dominant predator of zooplankton (Yan et al. 2002; Vanderploeg et al. 2012)
and may have widespread negative effects on food webs through trophic cascades (Hoffman et
al. 2001; Strecker and Arnott 2008). Bythotrephes are also a common prey for fish (Pothoven et
al. 2007), which might serve as a source of size-selective predation on Bythotrephes traits.
The Bythotrephes invasion in multiple lakes of differing GLP fish risk provides a natural
experiment allowing for the measurement of selection from GLP and NGLP predators in the
wild. Previous research suggests that Bythotrephes defensive distal spine (Figure 5.1a)
experiences selection for increased length (Sullivan and Lehman 1998), but the agents of
selection are unknown. Diet analysis of wild-caught fish (Schneeberger 1991; Barnhisel and
Harvey 1995) and evidence for selective feeding of GLP fish on Bythotrephes based on distal
spine length (Barnhisel 1991a; 1991b) suggest GLP fish may act as selective agents on
Bythotrephes distal spine length, but natural selection on this important trait has not yet been
measured. We, therefore, measured natural selection on Bythotrephes distal spine length in six
lakes dominated by either GLP or NGLP fish. We predicted that there would be selection for
increased distal spine length in lakes dominated by GLP fish, but no selection on distal spine
length in lakes dominated by NGLP fish.

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Methods
Methodological Overview
We collected Bythotrephes in three lakes dominated by a GLP (rainbow smelt, Osmerus
mordax) and three lakes dominated by a NGLP (cisco, Coregonus artedi) during June 2008. We
measured natural selection on distal spine length occurring between two cohorts and within a
single cohort (Figure 5.1b) using two closely spaced Bythotrephes collections (within one week).
Viability selection was estimated through a comparison of distal spine length between the first
two developmental (instar) stages.
Morphology and Life History of Bythotrephes
The longest section of the Bythotrephes tail spine is the distal spine, which extends from
the tip of the spine to the first pair of instar barbs (Figure 5.1a). The distal spine is present at
birth and does not change with instar development, although other traits, such as body size,
increase with each instar molt (Sullivan and Lehman 1998). Instead, during the first two molts,
total spine length increases through the addition of intercalary segments separated by paired
barbs (Branstrator 2005). Bythotrephes reproduce by cyclic parthenogenesis, with multiple
asexual generations (up to ten or more) during the growing season culminating in sexual
reproduction at the end of the season (Yurista 1992; Pothoven et al. 2001). Bythotrephes
typically reproduce at either the second or third instar stage (Pothoven et al. 2001); each instar
lasts approximately 3-5 days, depending on water temperature (Branstrator 2005).
Study Lakes
We sampled Bythotrephes from six lakes in the districts of Muskoka and Haliburton in
south-central Ontario, Canada that were recently (less than twenty years prior to this study)
invaded by Bythotrephes (Table 5.1). In three of these lakes (Fairy, Mary, Peninsula; hereafter

150

collectively referred to as GLP lakes) predation on Bythotrephes is dominated (in terms of
predator abundance) by rainbow smelt (Osmerus mordax), which are GLP of Bythotrephes as
juveniles (Barnhisel and Harvey 1995), but exhibit little predation on Bythotrephes as adults
(Young and Yan 2008). In the other three lakes (Harp, Boshkung, and Kashagawigamog;
hereafter collectively referred to as NGLP lakes) predation on Bythotrephes is dominated by
cisco (Coregonus artedi), which are important NGLP of Bythotrephes (Young and Yan 2008).
Although smelt are also present in two of the NGLP lakes (Boshkung: Young and Yan 2008;
Kashagawigamog: Strecker et al. 2006), the abundance of cisco in these lakes is greater than that
of smelt, and cisco are considered to be the dominant predator of Bythotrephes (Table 5.1;
Strecker et al. 2006; S.J. Sandstrom and N. Lester, Ontario Ministry of Natural Resources,
unpublished data). The physical and biological characteristics of the six lakes are otherwise
similar (Strecker et al. 2006; Young and Yan 2008).
Field Collection of Bythotrephes
We collected Bythotrephes (Ontario Ministry of Natural Resources License to Collect
Fish for Scientific Purposes No. 1046358) during June 23-28, 2008 (Table 5.1), a time period
during the middle of the Bythotrephes growing season. We completed two collections from each
lake separated by three days (hereafter, paired trips), with the exception of Kashagawigamog, for
which we only completed the first collection due to low Bythotrephes abundance. We used a
conical zooplankton net with a 0.5m diameter opening and 363μm mesh size towed horizontally
in 1m steps through the epilimnion and metalimnion. All Bythotrephes collected were
immediately preserved in 95% ethanol. Water temperature 1m below the water surface was
collected during each sampling trip. We also obtained additional temperature data for Harp Lake

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for the entire 2008 growing season (Figure 5.1c; A. Paterson, Dorset Environmental Science
Centre, Ontario Ministry of the Environment, Dorset, ON, Canada, unpublished data).
We measured Bythotrephes distal spine length (Figure 5.1a) using ImageJ (Abramoff et
al. 2004) to the nearest 0.001mm from photographs taken with a digital camera attached to a
dissecting microscope. Distal spine length was measured from the tip of the spine to the anterior
end of the first pair of instar barbs (Figure 5.1a; Miehls et al. 2012). Although some organisms
are known to decrease in length after preservation in ethanol (e.g., larval fish; Moku et al. 2004),
a previous test (Miehls et al. 2012) of Bythotrephes preserved in 95% ethanol revealed no
statistically significant shrinkage of Bythotrephes distal spine length.
Statistical Analysis: Natural Selection between Cohorts
We estimated natural selection on Bythotrephes distal spine length through a comparison
of distal spine length between the first two instar stages collected on the same trip (Figure 5.1b);
note that this method for estimating natural selection involves a comparison between different
cohorts of Bythotrephes, as individuals from different instar stages collected on the same trip
would have been born during different time periods. Because distal spine length does not change
with instar development, different distal spine length between the first and second instar stages
reflects a difference in survival as opposed to a developmentally based difference in length
(Sullivan and Lehman 1998). Instar stage was determined by counting the number of paired
barbs on the spine, which reliably indicates instar stage for the first two instars (Yurista 1992;
Sullivan and Lehman 1998). Although Bythotrephes pass through up to three or more instar
stages (Yurista 1992), the comparison between first and second instars was chosen because
survival through this early life stage likely comprises a large proportion of total fitness and

152

because the distal segment for first instar individuals represents the entire spine length, which we
suspected was the target of GLP.
We statistically tested whether selection on distal spine length differed by lakes
dominated by GLP versus NGLP with a linear mixed effects model (LME) using the nlme
package (Pinheiro et al. 2009) in R version 2.15.0 (R Development Core Team 2012). The LME
model included distal spine length as a continuous response, instar (categorical for instars one
and two), fish predator (categorical for GLP and NGLP), trip (categorical for trip one and two),
and instar-by-fish predator and fish predator-by-trip interactions as fixed effects, and lake (six
replicate lakes) as a random effect. In this model, a statistically significant effect of instar would
indicate significant selection on distal spine length irrespective of fish predator. A significant
interaction between instar and fish predator (GLP vs. NGLP) would indicate that selection
differed by fish predator, which was the primary result of interest in our study. A significant
interaction between fish predator and trip would indicate Bythotrephes distal spine length in
lakes of each fish predator type varied by trip, a result which would suggest the effect of fish
predator may be confounded by phenotypic plasticity (see Natural Selection within a Cohort).
We estimated selection differentials (Falconer and Mackay 1996) for distal spine length
as the difference in mean distal spine length between first and second instars within each lake.
To compare our estimates of selection to other published studies (Kingsolver et al. 2001;
Kingsolver and Diamond 2011), we calculated standardized selection differentials (i.e., selection
intensities) by dividing the selection differential for each lake by the pooled standard deviation of
distal spine length within the lake. The statistical significance of our selection differentials was
determined using linear models that included distal spine length as a continuous response and

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instar as a fixed effect for every lake. In these models, a statistically significant effect of instar
would indicate significant selection on distal spine length.
Statistical Analysis: Natural Selection within a Cohort
In addition to the above method for estimating natural selection between cohorts, our
paired trip design allowed for estimation of natural selection within a cohort (Figure 5.1b),
providing a control for the potentially confounding effects of phenotypic plasticity (Pigliucci
2001). Previous work (Chapter 3) indicated that Bythotrephes mothers produce offspring with
longer distal spines at higher temperature. If first and second instar Bythotrephes collected on the
same sampling trip experienced different water temperatures during development, estimates of
selection could be confounded by temperature-induced plasticity in distal spine length.
Comparing Bythotrephes between sampling trips (i.e. comparing first instar individuals collected
on trip 1 to second instar individuals collected on trip 2) allowed us to control for these potential
confounding effects of plasticity by tracking a single cohort through time. We assumed that first
instar individuals from trip one and second instar individuals from trip two were members of the
same cohort because our trips were separated by three days, which is the average instar
development time of Bythotrephes at 21°C (Branstrator 2005). This water temperature is similar
to the average water temperature in our sampled lakes at the time of collection (Table 5.1). As a
result, most Bythotrephes in their first instar at the time of the first trip should have molted to the
second instar by the time of the second trip.
We statistically tested whether selection on distal spine length within a cohort differed
between lakes dominated by GLP versus NGLP using a LME model with distal spine length as
the response, instar, fish predator, and an instar-by-fish predator interaction as fixed effects, and
lake as a random effect. This analysis included only first instar individuals collected during trip

154

one and second instar individuals collected during trip two. In this analysis, the inclusion of a
fixed effect for trip was not necessary and would have been redundant with the instar effect. As
in the LME above, the interaction between instar and fish predator was the primary result of
interest, as this interaction examines whether selection differed between GLP and NGLP lakes.
We also calculated selection differentials and selection intensities for each lake separately except
Kashagawigamog (for which we only had the single collection on trip one, prohibiting a within
cohort analysis). The statistical significance of our selection differentials was determined using
linear models identical to the single-lake models above. As above, a significant effect of instar
indicated significant selection on distal spine length.

Results
Average Distal Spine Length
Average distal spine length of first instar individuals (i.e., pre-selection phenotype) was
20% longer in the GLP lakes (average length ± 1 SD: GLP lakes: 5.94 ± 0.48mm; NGLP lakes:
4.97 ± 0.41mm; t4 = 5.20, p < 0.01) and exhibited less among-lake variation than the NGLP
lakes (Figure 5.2a). There was no overlap in average length between the GLP and NGLP lakes.
That is, the GLP lake with the shortest average distal spine length (Mary) exhibited longer mean
length than the NGLP lake with the longest average distal spine length, Boshkung (Figure 5.2a).
Natural Selection between Cohorts
Natural selection on Bythotrephes distal spine length differed significantly between GLP
and NGLP lakes (i.e., significant instar-by-fish predator interaction: Table 5.2), and lake predator
types did not differ in the effect of trip (i.e., non-significant fish predator-by-trip interaction:
Table 5.2). There was significant selection for increased distal spine length in all GLP lakes

155

(range of selection intensities: 0.20-0.79) and no significant selection in NGLP lakes (Table 5.3;
Figure 5.2b). Within the GLP lakes, selection differentials were smallest in Peninsula and
greatest in Mary (Table 5.3). Although not significantly different from zero, the strength of
selection on distal spine length in one NGLP lake, Harp, was greater than the strength of
selection in one GLP lake, Peninsula (Table 5.3). Note that the data for Kashagawigamog should
be interpreted with caution as the pre- and post-selection sample sizes were only eight and four
individuals, respectively, which we consider too low to provide reliable estimates of selection
(Hersch and Phillips 2004).
Natural Selection within a Cohort
The within-cohort analysis for individual lakes corroborated the between-cohort analysis.
We found significant selection for increased distal spine length in all GLP lakes (range of
selection intensities: 0.32-0.93) and no significant selection in NGLP lakes (Table 5.3, Figure
5.2c). The selection differentials were again smallest in Peninsula and greatest in Mary, and one
NGLP lake (Boshkung) had a non-significant selection differential higher than that of one GLP
lake (Peninsula). These selection differentials, however, did not significantly differ between GLP
and NGLP lakes (i.e., non-significant instar-by-fish predator interaction: Table 5.2), which is
likely due to the lower sample sizes for these more restrictive subsets of the data (Table 5.3).

Discussion
Our analysis identified GLP fish as an agent of selection on the defensive morphology of
an invasive species. Natural selection on Bythotrephes distal spine length was strong compared
to other published studies of selection (Kingsolver et al. 2001; Kingsolver and Diamond 2011).
A recent review of phenotypic selection measured in natural populations by Kingsolver and

156

Diamond (2011) found that the median directional selection (estimated as the absolute value of
standardized linear selection gradients, |β|) via survival was 0.08. Even the lowest standardized
selection differentials (i.e., selection intensities) on Bythotrephes distal spine length in the GLP
lakes (range: 0.20-0.79; between cohort analysis: Table 5.1) exceeded the median selection via
survival. The highest standardized selection differentials (those greater than 0.75) fell within the
top 5% previously reported (Kingsolver et al. 2001). Although Kingsolver and Diamond (2011)
reported standardized selection gradients and we reported standardized selection differentials,
their review found congruence between standardized selection gradients and differentials.
We observed a few notable differences in selection among lakes within each fish predator
type. In Peninsula, where average distal spine length (6.05mm; Figure 5.2a) was the longest
among the GLP lakes, we found the weakest selection for increased length, whereas in Mary,
where average distal spine length (5.79mm; Figure 5.2a) was the shortest among the GLP lakes,
we found the strongest selection for increased length. These results suggest that a threshold distal
spine length provides protection from GLP consistent with the concept of “hard” natural
selection (Wallace 1975). We found no significant selection in any of the NGLP lakes, but note
that in Boshkung, where average distal spine length (5.31mm; Figure 5.2a) was the greatest of all
the NGLP lakes, we found a (non-significant) selection differential (S = 0.13; Table 5.3) for the
within cohort analysis that was greater than the selection differential in Peninsula. Although the
primary predators of Bythotrephes in Boshkung (cisco) are NGLP, smelt are present at low
abundance and could be a source of GLP maintaining longer average distal spine length in this
lake as compared to the other NGLP lakes. However, we found a (non-significant) negative
selection differential (S = -0.03; Table 5.3) in Boshkung for the between cohort analysis, which
we feel is the more rigorous of the two analyses based on the greater sample size. Thus, the

157

presence of a GLP in Boshkung may explain the greater average distal spine length in this lake
than the other NGLP lakes, although evidence for selection by the GLP is weak.
Although we did not find a significant effect of fish predator on selection in the within
cohort analysis, we found a significant effect in the between cohort analysis and, further, all
within-lake analyses indicated significant selection on Bythotrephes distal spine length by GLP,
but not NGLP; thus, we are confident in our conclusion that selection differs by fish gapelimitation. The non-significant instar-by-fish predator interaction in the within cohort analysis
(Table 5.2) was likely due to poor sample size in the NGLP lakes (Hersch and Phillips 2004);
because we had considerably greater power with which to detect a difference in selection by fish
predator in the between cohort analysis (Table 5.2), we regard these results as the more reliable
of the two comparisons. Regarding the within-lake estimates, the selection differentials were
quantitatively similar for the between and within cohort analyses in all GLP lakes, providing
strong support for our conclusion of natural selection due to GLP. Whereas the selection
differentials were quantitatively similar for the between and within cohort analyses for one
NGLP lake (Harp), there was a qualitative difference in the direction of selection for one NGLP
lake (Boshkung). Despite the less consistent results in the NGLP lakes, we found no significant
selection in any of the NGLP lakes for the between (where we had strong statistical power) or
within cohort analysis, supporting our conclusion of a lack of natural selection with NGLP. Of
note, our test of GLP and NGLP as agents of selection was observational (i.e., we assumed the
effect of each fish predator type based on their presence in lakes); experimental manipulation of
fish communities would be needed to conclusively identify GLP as the agent of selection.
We completed both between and within cohort analyses due to the greater rigor of the
latter approach for assessing selection free from the potential confounding effects of phenotypic

158

plasticity (Pigliucci 2001). Whereas the former approach is not as rigorous of a control for
phenotypic plasticity, we are confident that the between cohort analysis yielded accurate
estimates of selection given three lines of evidence against plasticity. First, in our between cohort
analysis, we found no interaction between the effect of fish predator on distal spine length and
trip (Table 5.2). There was a significant difference in distal spine length between the first and
second collection trip (Table 5.2) indicating that average distal spine length increased between
the first and second collection trip. However, the non-significant interaction between fish
predator and trip indicates that plastic differences in distal spine length between trips did not
differ by fish predator. Therefore, our conclusions regarding selection by the fish predator types
(i.e., GLP vs. NGLP) should be robust to overall differences in mean distal spine length between
the first and second collection trip. Second, the only identified cue of plasticity in Bythotrephes
distal spine length, water temperature (Chapter 3), was relatively stable both within one of our
study lakes prior to collection (Harp Lake, the only lake for which we have seasonal temperature
data; Figure 5.1c) and among our study lakes at the time of collection (Table 5.1). Thus,
temperature-induced differences in distal spine length were likely minimal. Third, we (Chapter
3) also tested fish kairomones as a cue of plasticity in Bythotrephes distal spine length, but found
no effect; thus, plasticity in response to the different fish predators present in our study lakes was
unlikely to affect our results. Therefore, our estimates of natural selection were unlikely to be
biased by phenotypic plasticity.
The large difference in average distal spine length between lakes dominated by GLP and
NGLP is consistent with the direction of selection we observed and suggests rapid local
adaptation of distal spine length in response to predation. Average distal spine length was 20%
longer (almost 1mm greater in absolute length) in GLP lakes, where we found strong selection

159

for increased distal spine length. Previous work in Lake Michigan (Miehls et al. 2012) indicated
2

that Bythotrephes distal spine length is moderately to highly heritable (range of H : 0.27-0.76)
and concluded that Bythotrephes could rapidly respond to selection, especially given the multiple
asexual generations produced during the growing season (Yurista 1992; Pothoven et al. 2001).
Heritability of Bythotrephes distal spine length in the lakes that we sampled here is unknown, but
the strength of selection that we have documented in GLP lakes is sufficient to account for our
observed differences in mean spine length between lake types in only 73 generations assuming a
broad-sense heritability of 0.1 and using the average of the GLP selection differentials from the
between cohort analysis. Seventy-three generations could correspond to as few as 7 years if
Bythotrephes in these lakes are capable of completing approximately 10 generations per season,
which seems like a reasonable estimate based on a six-month growing season and a time to
maturity of 11d at moderate water temperatures (Drake et al. 2006). Indeed, as Bythotrephes
invaded the three GLP lakes in the early 1990s (almost 20 years prior to this study), sufficient
time has passed for local adaptation comparable to observed differences in mean phenotype
based on our estimates of strength of contemporary natural selection. Our results are suggestive
of local adaptation in response to predation, although additional research (e.g., common garden
experiments) is needed to confirm the genetic basis of these phenotypes (see Mooney and
Cleland 2001 for a review of local adaptation of invasive species).
In conclusion, our study found strong directional selection on the defensive morphology
of an invasive species exerted by gape-limited predators, and no selection by non-gape-limited
predators. Previous research identifying predators as agents of selection in the wild is limited,
particularly for invasive species. We also found a large difference (20%) in average distal spine
length between lakes dominated by GLP and NGLP that was consistent with local adaptation in

160

response to selection over a relatively short time scale (less than twenty years). Thus, our
findings indicate predators are capable of exerting strong selection on invasive prey, despite a
relatively short interaction history. Our findings are also suggestive that local adaptation of
invasive species can be rapid, occurring in as little as a few years or decades, providing
additional support that evolution by natural selection in invasive species can occur as quickly as
evolution by other well-documented mechanisms (e.g., population bottlenecks, founder effects,
and hybridization; Lee 2002; Lambrinos 2004; Prentis et al. 2008). Thus, as we investigate the
ongoing ecological effects of invasive species on ecosystems, it is important to view invasive
species as dynamic populations capable of rapidly adapting to selective pressures in their
invaded ecosystems.

161

Acknowledgements
Norman Yan, Joelle Young, Dallas Linley, and Natalie Kim graciously provided field
equipment and assistance in developing the study design. Keali Chambers and Jennifer Pellegrini
assisted with field collections, Dave and Gail McAdam provided lodging while in the field, and
Brittany Damschroder, Jillian Fejszes, Skye Fissette, Stefanie Jubb, Lydia Kramer, Ian McCririe,
Claire Michael, Veronica Quesnell, Ben Staton, Marie Stevenson, Elizabeth Throckmorton, Aditi
Upadhye, Brandon Vieder, and Miriam Weiss assisted with laboratory sample processing. Paul
Bourdeau and Scott Miehls provided additional methodological assistance. This work was
supported by the Great Lakes Fishery Commission and an EPA Science to Achieve Results
fellowship (STAR Research Assistance Agreement No. FP91698801-0 awarded by the U.S.
Environmental Protection Agency). This work has not been formally reviewed by the EPA and
the views expressed in this document are solely those of the authors.

162

Tables
Table 5.1. Bythotrephes invasion year, categorical fish abundance, and field information for study lakes. Smelt are considered a gapelimited predator, whereas cisco are considered a non-gape-limited predator of Bythotrephes. Temperatures were taken 1m below the
water surface at the time of Bythotrephes collection. A: absent; L: low; M: medium; H: high.
Lake
Fairy

Invasion

Mary

1990

Peninsula
Boshkung
Harp
Kashagawigamog
1

1990
1991
2003
1993
1993
2

1
1
1
1
1
2

Smelt Cisco Sample Dates Latitude
3
3
June 24, 27 45° 19.90' N
M
A
M
M

3

45° 14.39' N

79° 15.77' W

50.3

20.9

June 24, 27

45° 20.94' N

79° 6.28' W

32.9

21.8

4

June 24, 27

45° 3.06' N

78° 43.76' W

40.8

20.3

June 23, 26

45° 22.79' N

79° 7.98' W

30.5

21.3

June 25

45° 59.91' N

78° 36.07' W

33.2

20.6

A

M

3

L/M

June 25, 28

3

3

A

Depth (m) Temp (°C)
38.7
22.4

3

A

4

L

Longitude
79° 10.49' W

3

3

H

3

M/H

3

4

Young and Yan 2008; Muirhead and MacIsaac 2005; Strecker et al. 2006; S.J. Sandstrom and N. Lester, Ontario Ministry of
Natural Resources, unpublished data

163

Table 5.2. Statistical results for the between and within cohort analyses of natural selection on
Bythotrephes distal spine length. The instar-by-fish interaction assessed whether natural selection
(effect of instar) differed by fish predator. Note that the within cohort analysis did not include
trip as a fixed effect (see text). Lake (i.e., the six replicate study lakes) was modeled as a random
effect; the significance of lake was determined through a likelihood ratio test. F: F-statistic; df:
degrees of freedom; p: p-value; Var: among-lake variance; Ratio: likelihood ratio.

Fixed
Instar
Fish
Trip
Instar:Fish
Fish:Trip

Between cohorts
F
df
p
48.86 1, 1899 ***
48.32 1, 4
***
28.08 1, 1899
**
9.02 1, 1899 ***
0.18 1, 1899 N.S.

Within cohort
F
df
p
78.09 1, 1012 ***
78.81 1, 4
***
N/A N/A
N/A
0.01 1, 1012 N.S.
N/A N/A
N/A

Random
Var
Ratio
p
Var
Ratio
p
Lake
0.18
98.0
***
0.14
39.1
***
**p ≤ 0.01; ***p ≤ 0.001; N.S.: non-significant; N/A: not applicable

164

Table 5.3. Natural selection on Bythotrephes distal spine length estimated within six replicate study lakes; we found significant
selection (i.e., significant effect of instar) in all GLP lakes (Fairy, Mary, Peninsula) and no significant selection in NGLP lakes
(Boshkung, Harp, and Kashagawigamog). t and p values are for the effect of instar on distal spine length. df: degrees of freedom; S:
selection differential (mm); SD: pooled standard deviation within a lake; i: standardized selection differential (i.e., selection intensity).
Note that a second sample collection was not completed in Kashagawigamog, precluding the estimation of selection within a cohort.
Between cohorts
Lake
t
df
p
S
SD
i
Fairy
4.8 436 *** 0.14 0.26
0.53
Mary
6.9 438 *** 0.22 0.28
0.79
Peninsula
2.8 743
**
0.05 0.23
0.20
Boshkung
-0.8 186 N.S. -0.03 0.27
-0.12
Harp
1.5
84 N.S. 0.08 0.25
0.32
Kashagawigamog
-1.5
10 N.S. -0.20 0.24
-0.85
**p ≤ 0.01; ***p ≤ 0.001; N.S.: non-significant; N/A: not applicable

165

t
4.2
7.4
3.2
1.1
0.5
N/A

df
265
227
384
87
39
N/A

Within cohort
p
S
SD
*** 0.15 0.26
*** 0.28 0.30
** 0.07 0.22
N.S. 0.13 0.28
N.S. 0.06 0.22
N/A N/A N/A

i
0.58
0.93
0.32
0.46
0.26
N/A

Figures

Figure 5.1. We estimated natural selection on: a) distal spine length of Bythotrephes longimanus,
the spiny water flea (the individual pictured is a third instar); through b) a comparison between
first and second instar Bythotrephes between cohorts (solid arrows) and within a cohort (dashed
arrow) using paired sampling trips. The collection trips were separated by 3d, which was
equivalent to the duration of a single instar at the water temperature sampled (~21°C). c)
Epilimnion water temperature (°C) in Harp Lake during the 2008 growing season. The open
diamond represents the date of first sample collection in Harp Lake.

166

Figure 5.
.1.
a)

b)

28.0
Water temperature (°C)

c)

23.0
18.0
13.0
8.0
3.0
12-A
Apr

1-Jun
J

21
1-Jul

167

9-Sep

2
29-Oct

Figure 5.2. a) Average distal spine length of first instar Bythotrephes within each lake. b) and c)
Natural selection on distal spine length assessed through a comparison of first instar individuals
(pre-selection phenotype) to second instar individuals (post-selection phenotype) between
cohorts (b) and within a cohort (c). The difference between the pre- and post-selection phenotype
for each lake in panels b) and c) is equivalent to the selection differential. There are no postselection data for Kashagawigamog for the within cohort analysis (c) because a second collection
trip was not completed. Error bars are standard errors. Kash: Kashagawigamog; ***p ≤ 0.001;
N.S.: non-significant.

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Figure 5.2.
6.3
Distal spine length (mm)

a)

NGLP (cisco) lakes
GLP (smelt) lakes

5.8
5.3
4.8
4.3

b)
Distal spine length (mm)

6.3

***

***

***

5.8
5.3

N.S.
N.S.

4.8

N.S.

4.3
c)
Distal spine length (mm)

6.3

***

***

***

5.8
5.3

N.S.
N.S.

4.8

No
Data

4.3

Pre Post Pre Post Pre Post Pre Post Pre Post Pre Post
Boshkung

Harp

Kash

Fairy

169

Mary

Peninsula

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170

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CHAPTER 6

Concluding thoughts: What insight has the Bythotrephes invasion provided to
evolutionary ecology and what more can we learn?

Andrea Lynn-Jaeger Miehls

Summary of Findings
Six years of studying the evolutionary ecology of Bythotrephes and what have we
learned? I found that distal spine and body length of Bythotrephes from Lake Michigan are
highly heritable and that mothers influence the phenotype of these traits in their offspring
(Chapter 2). I also found that genetic and maternal variation in Bythotrephes distal spine length
varied among but not within years, and that distal spine length was always heritable but was not
always influenced by maternal effects. In contrast, genetic and maternal variation in body length
varied both within and among years, but likewise body length was always heritable and not
always influenced by maternal effects. Further exploring the influence of the environment on
Bythotrephes traits, my experimental research using Bythotrephes from Lake Michigan showed
that spine length, body size, and clutch size respond plastically to temperature but not to fish
predator cues, with mothers producing fewer, but larger offspring (longer distal spine and body
length) that are better defended (greater ratio of distal spine-to-body length) against predation at
higher temperature (Chapter 3). Although Bythotrephes use temperature as the proximate cue of
plasticity, the trait changes likely represent adaptations to varying non-gape-limited (dominant at
colder temperature) and gape-limited (dominant at higher temperature) fish predation risk.

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I also found temporally and spatially variable natural selection on Bythotrephes distal
spine length in Lake Michigan across multiple months during three growing seasons and
between multiple spatial replicates within the lake (Chapter 4). Temporal variation in selection is
consistent with seasonal changes in the relative strength of non-gape-limited and gape-limited
fish predation, with selection for decreased distal spine length early in the growing season when
predation is dominated by non-gape-limited fish, and strengthening selection for increased distal
spine length later in the growing season when predation is increasingly dominated by gapelimited fish. Selection also varied spatially consistent with well-known heterogeneity of fish
predators in large lakes. Yet, despite net selection for increased distal spine length occurring
across the three years of my study, I observed little evidence of an evolutionary response to
selection based on comparisons of historic and contemporary wild-captured individuals and
retrieved spines from sediment cores. Finally, using a companion study in six Canadian Shield
lakes, I was able to identify the agents of selection on Bythotrephes distal spine length (Chapter
5). I found selection for increased distal spine length in lakes dominated by a gape-limited fish
predator and no significant selection in lakes dominated by a non-gape-limited fish predator.
Average distal spine length in the lakes dominated by gape-limited predation was 20% longer
than in the lakes dominated by non-gape-limited predation, suggesting possible local adaptation
of distal spine length to gape-limited fish predation.

Contributions to our understanding of invasion biology and evolutionary ecology
Chapter 2. My investigation into the sources of phenotypic variation in Bythotrephes
distal spine and body length indicates that these traits have heritable variation that would enable
an evolutionary response to natural selection. Moreover, maternal effects in Bythotrephes distal

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spine and body length indicate mothers can be a source of phenotypic variation in offspring,
suggesting (but not directly showing) the possibility of maternally-induced phenotypic plasticity
(Mousseau and Fox 1998; Agrawal et al. 1999). Importantly, genetic and maternal variation in
traits influence phenotypic change over different temporal scales, with genetic variation allowing
for an evolutionary response to persistent environmental change across multiple generations and
maternal variation providing phenotypic variation across a single generation (Lee and Gelembiuk
2008; Svanbäck et al. 2009). Thus, these results indicate that Bythotrephes can morphologically
respond to environmental change over both the short-term (though maternal effects) and longterm (through an evolutionary response to selection). This capacity for phenotypic change could
explain, in part, the ability of Bythotrephes to invade lake ecosystems with variable biotic and
abiotic conditions (Bilkovic and Lehman 1997; Sullivan and Lehman 1998; Young and Yan
2008). More generally, this research indicates traits of invasive species may contain quantitative
genetic variation comparable to native species (Pfrender and Lynch 2000), despite evidence that
many invasive species (Dlugosch and Parker 2008), including Bythotrephes (Colautti et al.
2005), experience bottlenecks or founder effects that should reduce genetic variation. Thus,
evolution of invasive species traits may not be limited by insufficient genetic variation.
Chapter 3. My study of phenotypic plasticity in Bythotrephes morphological and life
history traits indicates that Bythotrephes can respond (through plasticity) to seasonal fluctuations
(Straile and Hälbich 2000; Branstrator 2005; Pothoven et al. 2012) in the strength of gapelimited and non-gape limited fish predation that correlate with water temperature. Of note, the
finding that plastic responses occur through maternal effects supports the conclusion of Chapter
2 that mothers can influence the phenotype of their offspring. Plasticity in Bythotrephes traits
likely confers a strong fitness advantage, as Bythotrephes are able to capitalize on abundant prey

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resources and low gape-limited predation risk early in the growing season through the production
of many, individually low-cost (i.e., morphologically small) offspring, but also provide
protection during high gape-limited predation risk later in the growing season through the
production of well-defended (i.e., high distal spine-to-body length ratio), but fewer offspring
(i.e., an adaptive size-number trade-off; Smith and Fretwell 1974). Bythotrephes’ ability to
plastically adjust morphology and life history to defend against predation has likely contributed
to their ability to establish in nonnative ecosystems, many of which have similar seasonal
variation in gape-limitation of fish predators (Straile and Hälbich 2000; Branstrator 2005). More
generally, this research provides the first experimental evidence for prey use of a proxy cue of
plasticity to reduce predation risk, when direct cues from the predator are unreliable. As proxy
cues are likely less often investigated than more obvious potential direct cues of plastic
responses, organismal use of proxy cues in nature may be more common than previously
realized. This conclusion raises an exciting prospect for investigations of phenotypic plasticity
by broadening the potential set of cues that organisms may use for adaptive plasticity.
Chapter 4. My investigation of natural selection on Bythotrephes distal spine length in
Lake Michigan showed that selection can strongly fluctuate in magnitude and direction across
time and space. Many studies of selection lack replication in one or both of these dimensions
(discussed in Kingsolver et al. 2001), which may inaccurately characterize selection, depending
on the magnitude of fluctuations. Thus, my research supports the conclusions of others (e.g.,
Merilä et al. 2001) that accurate predictions of the evolutionary response to selection necessitate
greater replication in studies of natural section. Of note, temporal fluctuation in the direction of
selection corroborates phenotypically plastic responses observed in Bythotrephes distal spine
length (Chapter 3). Selection favored shorter distal spine length early in the growing season,

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when plastic response to water temperature would induce production of smaller offspring;
conversely, selection favored longer distal spine length later in the season, when plastic response
to temperature would induce production of larger, better defended offspring. The directions of
selection occurring in these temperature environments thus provide support that the plastic
responses I observed are adaptive to seasonally fluctuating gape-limitation of fish predators.
The finding that net selection for increased distal spine length failed to result in an
evolutionary response despite the genetic basis of distal spine length (Chapter 2) is supported by
other studies (reviewed in Merilä et al. 2001) that found evolutionary stasis in the wild despite
selection on heritable traits. Given the success of the Bythotrephes invasion in Lake Michigan, as
evidenced by their persistence at high annual abundance (Pothoven et al. 2001) since invading
almost 25 years ago (Evans 1988), evolutionary stasis in Bythotrephes distal spine length – a trait
considered critical to their food web interactions (Branstrator 2005) – was initially surprising. I
expected fish predators in Lake Michigan would act as agents of strong directional selection on
Bythotrephes distal spine length, resulting in adaptive evolution since invasion. The lack of an
evolutionary response raises the possibility that Bythotrephes distal spine length was pre-adapted
(Schlaepfer et al. 2009) to fish predation in Lake Michigan. Pre-adaptation is reasonable, given
the almost immediate explosion in Bythotrephes abundance after invasion (Lehman 1987; Evans
1988; Pothoven et al. 2001). Many species that are not pre-adapted to the invaded ecosystem
experience a lag between invasion and population explosion (Facon et al. 2006); adaptive
evolution during the lag has been implicated as an important cause of subsequent population
explosion (Strayer et al. 2006). The lack of a lag between Bythotrephes invasion and population
explosion suggests adaptation may not have been necessary. Thus, the success of the
Bythotrephes invasion in Lake Michigan, in part, may be explained by pre-adaptation.

179

Chapter 5. The identification of gape-limited fish predators as agents of selection on
Bythotrephes distal spine length provides support for the conclusion of Chapter 4 that temporal
variation in selection on distal spine length in Lake Michigan is due to seasonal variation in
gape-limitation of fish predators. Previous research identifying predators as agents of selection in
the wild is limited, particularly for invasive species; thus, my research fills an important gap in
our understanding of natural selection in the wild. Moreover, my finding of longer average distal
spine length in lakes with gape-limited predators, which select for increased distal spine length,
is suggestive of local adaptation. Given that Bythotrephes invaded these lakes less than twenty
years prior to my study, local adaptation would have occurred rapidly (but note, additional work
is needed to determine the genetic basis of phenotypic differences between lakes; see Future
Directions). If the phenotypic differences between lakes are due to local adaptation to predator
gape-limitation, this finding would be among the first documenting evolution by natural
selection in an invasive species in response to predation in the wild. Moreover, this finding
would also indicate that evolution by natural selection in invasive species traits can occur as
quickly as evolution by other well-documented mechanisms for invasive species (e.g., population
bottlenecks, founder effects, and hybridization; Lee 2002; Lambrinos 2004; Prentis et al. 2008).
Notably, the findings of (possible) adaptive evolution of Bythotrephes distal spine length
in response to predation in the Canadian Shield lakes (Chapter 5) and phenotypic plasticity that
is likely adaptive to temporal variation in predation risk in Lake Michigan (Chapter 3) indicate
that the widespread success of the Bythotrephes invasion may be due to both evolution and
phenotypic plasticity. Evolution of Bythotrephes traits may be common during invasion, due to
attributes of their life cycle as cyclic parthenogens (Pothoven et al. 2001; Branstrator 2005).
Clonal reproduction allows for rapid within-season response to selection (assuming selection on

180

traits with a genetic basis), whereas sexual reproduction maintains genetic variation in traits
across seasons (Lynch 1984). The Bythotrephes life cycle can thus allow for rapid adaptation to
current environmental conditions, while maintaining sufficient genetic variation to respond to
future environmental change: a combination that likely grants high evolvability (Hansen 2006).
Thus, I would expect rapid adaptation of Bythotrephes to the ecosystems they invade, which in
turn likely promotes their invasion success. Additionally, phenotypic plasticity may promote the
invasion success of Bythotrephes in many ecosystems. My research was not designed to
determine whether Bythotrephes trait plasticity was present prior to invasion, or whether
Bythotrephes evolved phenotypic plasticity de novo in Lake Michigan. In the former situation,
plasticity in Bythotrephes traits may pre-adapt Bythotrephes for invasion in any ecosystem where
the relative risk of gape-limited and non-gape-limited predation is correlated with water
temperature; this correlation likely exists in most large lakes that Bythotrephes invade (e.g.,
Straile and Hälbich 2000; Branstrator 2005). In the latter situation, the same aspects of the
Bythotrephes life cycle that promote evolution of absolute trait magnitude would also promote
evolution of plasticity in traits. Thus, evolution of plasticity in Bythotrephes traits may occur
after invasion, which would likely promote their persistence. Together, high evolvability and
phenotypic plasticity (whether present prior to invasion or rapidly evolved after invasion) are
likely important to the widespread success of the Bythotrephes invasion.

Future Directions
As is the case for most any scientific endeavor, my dissertation research has raised many
more questions than it has answered. Below, I explore multiple directions for future work based
on the findings of my dissertation research.

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Is heritability of Bythotrephes distal spine and body length environment dependent?
I found that Bythotrephes distal spine and body length are heritable in a 15°C temperature
environment (Chapter 2). My work has shown that these traits plastically respond to temperature
(Chapter 3), and others have shown (e.g., Wilson et al. 2006; discussed in Pigliucci 2001) that
the heritability of plastic traits is environment dependent. Thus, heritability of Bythotrephes
distal spine and body length may depend on their temperature environment. Investigating
heritability of distal spine length in different temperature environments is important, as
environment-dependent heritability will affect evolutionary response to selection.

Is there a genetic correlation between Bythotrephes distal spine and body length?
I measured broad-sense heritability (Lynch and Walsh 1998) of Bythotrephes distal spine
and body length (Chapter 2), a univariate measure that does not account for possible genetic
correlations between these traits. My work (Chapter 4) and others (Sullivan and Lehman 1998;
Branstrator 2005) indicate Bythotrephes distal spine length is phenotypically correlated with
body size, suggesting the possibility of a genetic correlation (Conner and Hartl 2004).
Investigating a genetic correlation between these traits would be important, as a correlation
might constrain an evolutionary response to selection (Walsh and Blows 2009; Kingsolver and
Diamond 2011).

Is Bythotrephes phenotypic plasticity in response to temperature adaptive to fish predation?
My investigation of phenotypic plasticity in Bythotrephes life history and morphological
traits (Chapter 3) identified a plastic response to temperature that is likely adaptive for seasonal
variation in gape-limitation of fish predators. An important caveat to this finding is that I did not

182

test the adaptive value of Bythotrephes trait plasticity per se. Exposing temperature-induced
Bythotrephes to predators of variable gape-limitation would be helpful for establishing the
adaptive value of plasticity in response to temperature. Addressing the following question would
also be useful for establishing the adaptive value of plasticity…

Does Bythotrephes phenotypic plasticity have a genetic basis?
I argued (Chapter 3) that plasticity in Bythotrephes traits is adaptive based on the
framework of Doughty and Reznick (2004) who present an argument for adaptation based on six
indirect lines of evidence (criteria). One criterion of Doughty and Reznick (2004) which I was
unable to meet was establishing the genetic basis of plasticity. Thus, measuring genetic variation
in plasticity would be beneficial to establishing the adaptive value of plasticity.

Do Bythotrephes plastically respond to the presence of prey?
I investigated temperature and fish kairomones as proximate cues of plasticity in
Bythotrephes traits (Chapter 3) due to their correlation with Bythotrephes seasonal trait variation.
A third possible proximate cue of plasticity in Bythotrephes traits is the presence of a dominant
prey item, Daphnia. Previous work has demonstrated plastic response of predators to prey
(Kishida et al. 2006), and other researchers (Burkhardt 1994; Pothoven et al. 2003) have
suggested that Bythotrephes traits may plastically respond to Daphnia presence in Lake
Michigan. Thus plastic response to prey presence is a possible explanation for Bythotrephes
seasonal trait variation.

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Are Bythotrephes morphology and life history phenotypically plastic in the native range?
I identified phenotypic plasticity in Bythotrephes morphology and life history in Lake
Michigan (Chapter 3); an ensuing question was whether Bythotrephes evolved plasticity de novo
in Lake Michigan or whether plasticity was already present at invasion. Completing experiments
exploring plasticity in response to temperature (similar to those in Chapter 3) with Bythotrephes
from their native range would be valuable to determining when plasticity evolved.

Are Bythotrephes body size and fecundity under selection?
Trade-offs between different components of fitness (e.g., survival vs. fecundity; Roff
2002) and genetic correlations among traits (Walsh and Blows 2009; Kingsolver and Diamond
2011) are known to constrain evolutionary responses to selection. My finding of evolutionary
stasis in Bythotrephes distal spine length (Chapter 4) raised the possibility that opposing
selection on body size (given a positive genetic correlation with distal spine length) and
fecundity (given a size-number trade-off) could constrain an evolutionary response to selection.
Estimating selection on both of these traits would, thus, be helpful for understanding
evolutionary stasis of Bythotrephes distal spine length in Lake Michigan.

What is the genetic basis of phenotypic differences in Bythotrephes distal spine length between
Canadian Shield lakes dominated by gape-limited and non-gape-limited predation?
My finding of a large difference in average distal spine length between lakes dominated
by gape-limited versus non-gape-limited predation (Chapter 5) was suggestive of local
adaptation, but my study did not test for local adaptation per se. Key to asserting local adaptation
of distal spine length is the determination of genetic variation underlying the phenotypic

184

variation I observed. Clonal analyses similar to those I completed for Chapter 2 would readily
establish whether the observed phenotypic variation has a genetic basis.

Do Bythotrephes life history and morphological traits exhibit phenotypic plasticity in the
Canadian Shield lakes, and are there differences in phenotypic plasticity in lakes dominated by
gape-limited and non-gape-limited predation?
I found phenotypic plasticity in Bythotrephes morphological and life history traits that is
likely adaptive to seasonal variation in predator gape-limitation in Lake Michigan (Chapter 3).
An extension of this finding is that no plasticity should be observed in lakes lacking strong
temporal variation in predator gape-limitation. This prediction could be tested using Canadian
Shield lakes (Chapter 5) that experience comparatively less temporal variation in predator gapelimitation than Lake Michigan. I would expect that Bythotrephes in Shield lakes dominated by
gape-limited predation may not exhibit phenotypic plasticity, but instead, would exhibit
constitutive expression of large size and morphological defenses. Similarly, Bythotrephes in
Shield lakes dominated by non-gape-limited predation should exhibit constitutive expression of
small size and morphological defenses.

Concluding Thoughts
My study of the evolutionary ecology of Bythotrephes has uncovered how evolution and
phenotypic plasticity likely promote their invasion success in nonnative ecosystems, advancing
our understanding of the Bythotrephes invasion in North America. Yet, my goal in studying
Bythotrephes was not just to learn about the evolutionary ecology of this alien organism, but also
to provide illumination on broader concepts in evolutionary ecology. To borrow a simple, but

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insightful description from Quammen (2000), Bythotrephes are “a small organism through which
we can address big questions”. Although Quammen (2000) wrote this statement while describing
the ecology of slime molds, the statement reflects how I view my work on Bythotrephes. I would
argue that the characteristics that make any invasive species successful are the same as (or at
least are similar to) the characteristics that make any species successful. And so, by studying
invasive species, we achieve the dual purpose of understanding the impacts of species invasions
specifically, while at the same time generating knowledge that is ultimately useful to conserving
biodiversity.

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LITERATURE CITED

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Literature Cited
Agrawal, A.A., C. Laforsch, and R. Tollrian. 1999. Transgenerational induction of defences in
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