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CONTACT ZONE DYNAMICS AND THE EVOLUTION OF REPRODUCTIVE
ISOLATION IN A NORTH AMERICAN TREEFROG, THE SPRING PEEPER
(PSEUDACRIS CRUCIFER)
by
Kathryn Anne Stewart
A thesis submitted to the Department of Biology
in conformity with the requirements for
the degree of Doctor of Philosophy
Queen’s University
Kingston, Ontario, Canada
(February, 2013)
Copyright © Kathryn Anne Stewart, 2013
Abstract
Despite over seven decades of speciation research and 25 years of phylogeographic
studies, a comprehensive understanding of mechanisms that generate biological species remains
elusive. In temperate zones, the pervasiveness of range fragmentation and subsequent range
expansions suggests that secondary contact between diverging lineages may be important in the
evolution of species. Thus, such contact zones provide compelling opportunities to investigate
evolutionary processes, particularly the roles of geographical isolation in initiating, and indirect
selection against hybrids in completing (reinforcement), the evolution of reproductive isolation
and speciation.
The spring peeper (Pseudacris crucifer) has six well-supported mitochondrial lineages
many of which are now in secondary contact. Here I investigate the evolutionary consequences
of secondary contact of two such lineages (Eastern and Interior) in Southwestern Ontario using
genetic, morphological, acoustical, experimental, and behavioural evidence to show accentuated
divergence of the mate recognition system in sympatry.
Mitochondrial and microsatellite data distinguish these two lineages but also show
ongoing hybridization. Bayesian assignment tests and cline analysis imply asymmetrical
introgression of Eastern lineage nuclear markers into Interior populations. Male calls are
divergent between Eastern and Interior allopatric populations and show asymmetrical
reproductive character displacement in sympatry. Female preference of pure lineage individuals
is also exaggerated in sympatry, with hybrids showing intermediate traits and preference. I
suggest that these patterns are most consistent with secondary reinforcement.
I assessed levels of post-zygotic isolation between the Eastern and Interior lineages using
a laboratory hybridization experiment. Hybrid tadpoles showed equal to or greater fitness than
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their pure lineage counterparts, but this may be countered through competition. More deformities
and developmental anomalies in hybrid tadpoles further suggest post-zygotic isolation.
Despite evidence for pre-mating isolation between the two lineages, isolation appears
incomplete (i.e. hybridization is ongoing). I hypothesize that potentially less attractive hybrids
may circumvent female choice by adopting satellite behaviour. Although mating tactics are
related to body size, genetic status may play a role. I show that pure Eastern males almost always
engage in calling, while hybrids adopt a satellite tactic. An absence of assortative mating, despite
evidence of female preference, suggests successful satellite interception possibly facilitating
introgression.
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Co-Authorship
This thesis was formatted in the manuscript format as outlined in the guidelines provided by The
Department of Biology, Queen’s University. All data chapters are co-authored by Stephen C.
Lougheed, who contributed financially and intellectually to the design, development and editing
of the thesis. The Introduction and literature review includes material from a peer reviewed
species account (Stewart 2009) that was published on-line. The second chapter was co-authored
with James D. Austin who contributed samples collected in 2003 and 2005, and assisted in
editing the manuscript. The fourth chapter was co-authored with Cameron M. Hudson whose
fourth year honours thesis is partially encapsulated within the data. Cameron assisted in some
field collections, some laboratory analysis, and editing of the manuscript.
Publications arising from and included in this thesis:
Stewart, K.A. 2009. Species account: northern spring peeper / Rainette crucifère. Opinicon
Natural History Note. http://opinicon.wordpress.com/species-accounts/spring-peeper/
Stewart, K.A., Austin, J.D., & S.C. Lougheed. (in review). Reproductive character displacement
and isolation asymmetries between cryptic lineages of a North American treefrog,
Pseudacris crucifer. Evolution ms.# 12-1036
Stewart, K.A., Hudson, C. M., & S.C. Lougheed. (in review). Satellite male mating behaviour as
a mechanism for lower fitness matings in a hybrid zone. Proc. Roy. Soc. B ms.# RSPB2012-2978
Stewart, K.A. & S.C. Lougheed. (in prep). Intraspecific post-zygotic isolation and tadpole
competition in Pseudacris crucifer. (will be submitted to PLoS ONE)
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Publications arising from but not included in this thesis:
Stewart, K.A., Wang, R., Montgomerie, R., & S.C. Lougheed. (in prep) Postmating – prezygotic
barriers reduce hybrid fitness in a North American treefrog. (will be submitted to PLoS
ONE)
Stewart, K.A., Austin, J.D., & S.C. Lougheed. (in prep) Comparative contact zone dynamics and
the evolution of reproductive isolation barriers. (will be submitted to Molecular Ecology).
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Acknowledgements
This thesis is truly the brainchild and collaborative effort of numerous people for which I
will be forever grateful. Most importantly, I would like to acknowledge the hard work, tireless
effort and enthusiasm of my supervisor (and mentor), Dr. Stephen Lougheed, without whom I
would have never been given the opportunity to participate in such an interesting research
endeavor. Dr. Lougheed has been instrumental in intellectually seeding many aspects of this
project, and has provided me many opportunities to hone my love for research and the natural
world. His constant passion for outreach, and predilection for polysyllabic words are both
qualities that have not gone unnoticed and may have even transferred.
I would also like to thank Jim Austin for housing me and giving me a ‘crash-course’ in
peeper sampling within the first days of my PhD, and for the constant encouragement
throughout. I would like to extend a large amount of gratitude to Jim Bogart at the University of
Guelph for his intellectual contribution and expertise to the P. crucifer hybrid crosses, and to
Emily Moriarty-Lemmon for her assistance in phonotaxis design. I would also like to thank
Zhengxin Sun for his assistance in genotyping. The manuscripts in this thesis would not have
been made possible without assistance from my co-authors Cameron Hudson, and Jim Austin. I
am indebted and would like to show my appreciation to Laurene Ratcliffe, Paul Martin, and
Scott Lamoureux, for their valuable input, effort, and serving as my committee members, as well
as Kelly Zamudio for being generous enough to act as my external examiner.
This thesis was the accumulation of long months in the field collecting data, a feat not
accomplished in isolation. For this reason I would like to thank numerous field assistants who are
not only some of the most enthusiastic and hard working biologists I have encountered, but also
who show great strength-in-spirit for having to live with me, often in remote and isolating
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conditions for long periods of time without complaint, including: Michelle DiLeo, Kevin
Donmoyer, Ahdia Hasan, Surbhi Jain, Caroline Jameson, Natalie Morril, Jeff Row, Henry Pai,
Ayden Sherritt, Matt Teeter, and Rachel Wang. This field (and ensuing lab) work was partially
funded by an Natral Sciences and Engineering Research Council (NSERC) Discovery Grant to
Stephen Lougheed, by a Sigma-Xi Grant-In-Aide of Research Grant to Jim Austin (2002) and
myself (2008), a Queen’s University Graduate Dean's Travel Grant for Doctoral Field Research
(2010), as well as a Rosemary Grant Award for Graduate Student Research from the SSE (2010).
Logistical support in the field was also provided by the Bruce Peninsula National Park, Point
Pelee National Park, Long Point Research and Education Centre, and the Ontario Ministry of
Natural Resources.
I am grateful to the Natural Sciences and Engineering Research Council of Canada for
awarding me a PGS-D3 scholarship, to Queen’s University for the various funding opportunities,
to the Biology Department at Queen’s University for supporting the Core facility, and to
Genome Quebec and Robarts Research Institute.
I would like to acknowledge my friends at home and abroad for their tireless
encouragement, patience, and for taking my sarcasm in stride. To my amazing colleagues in the
Biology Department here at Queen’s University, for creating a battlefield of intellectual
discourse which was never dull or more fun. I consider myself honoured to be in such great
company. To my housemates over the years Jeremy, Erica, Scott, and everyone at 113 Bay St.,
for keeping me grounded, and for providing copious amounts of coffee in the morning and red
wine at night. A special thank you also goes out to Matt Teeter and Damon Crossfield, you both
should be sainted. I would like to highlight the importance of both past and present people
working in the Lougheed lab who have forever changed the way I define both research and
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family, including Leo, Jeff, Michelle, Amy, Amanda, Sam, Cam, Caroline, Surbhi, Rachel,
Georgia, Jayda, Mari, Aarani, among many others.
Finally, I would like to thank my family. Words cannot describe the amount of
appreciation I feel for the constant support and understanding throughout this process. First and
foremost however, I want to thank them for their willingness to let a kid get dirty, catch frogs
and snakes, and become fascinated with the natural world.
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Table of Contents
Abstract……………………………………………………………………………………………ii
Co-Authorship……………………………………………………………………………………iv
Acknowledgements………………………………………………………………………………vi
List of Figures…………………………………………………………………………………...xiii
List of Tables……………………………………………………………………………………xiv
List of Appendix Figures………………………………………………………………………...xv
List of Appendix Tables………………………………………………………………………...xvi
List of Abbreviations…………………………………………………………………………...xvii
Chapter 1: General Introduction…………………………………………………………………..1
1.1 Phylogeography & speciation…………………………………………………………...2
1.2 Consequences of secondary contact ……………………………………………………3
1.3 The spring peeper, Pseudacris crucifer…………………………………………………6
1.3.1 Distribution……………………………………………………………………..8
1.3.2 Habitat………………………………………………………………………….9
1.3.3 Feeding & activity patterns…………………………………………………….9
1.3.4 Breeding behaviour & reproduction…………………………………………..10
1.3.5 Voice…………………………………………………………………………..12
1.4 Research Questions……………………………………………………………………13
1.5 Figures…………………………………………………………………………………16
1.6 Literature Cited………………………………………………………………………..21
Chapter 2: Reproductive character displacement and isolation asymmetries between cryptic
lineages of a North American treefrog, Pseudacris crucifer…………………………………….31
ix
2.1 Abstract………………………………………………………………………………..31
2.2 Introduction……………………………………………………………………………32
2.3 Materials & Methods………………………………………………………………......36
2.3.1 Study species…………………………………………………………………..36
2.3.2 Sampling study populations…………………………………………………...37
2.3.3 DNA extraction and genotyping………………………………………………37
2.3.4 TMRCA analyses using cytochrome b………………………………………..38
2.3.5 Clinal variation, linkage disequilibrium, & quantifying hybridization……......39
2.3.6 Testing for character divergence & displacement…………………………......41
2.3.7 Pre-zygotic preference experiments…………………………………………...42
2.4 Results…………………………………………………………………………………44
2.4.1 Lineage dating & TMRCA…………………………………………………....44
2.4.2 Selection against hybrids……………………………………………………...44
2.4.3 Morphological & call character displacement………………………………...46
2.4.4 Female preference……………………………………………………………..47
2.5 Discussion……………………………………………………………………………..48
2.6 Conclusion……………………………………………………………………………..54
2.7 Acknowledgements……………………………………………………………………55
2.8 Tables & Figures……………………………………………………………………....56
2.9 Literature Cited………………………………………………………………………..63
Chapter 3: Intraspecific post-zygotic isolation and tadpole competition in Pseudacris crucifer..76
3.1 Abstract………………………………………………………………………………..76
3.2 Introduction……………………………………………………………………………77
3.3 Materials & Methods………………………………………………………………......80
3.3.1 The spring peeper……………………………………………………………...80
x
3.3.2 Populations sampling………………………………………………………….80
3.3.3 Hybrid crosses…………………………………………………………………81
3.3.4 Competition trials……………………………………………………………...82
3.3.5 Genotyping tadpoles…………………………………………………………..83
3.3.6 Tadpole fitness measurements………………………………………………...84
3.3.7 Statistical analysis……………………………………………………………..84
3.4 Results…………………………………………………………………………………84
3.4.1 Tadpole fitness………………………………………………………………...84
3.4.2 Tadpole competition trials…………………………………………………….85
3.4.3 Body deformities & developmental problems………………………………...86
3.5 Discussion……………………………………………………………………………..86
3.6 Conclusion……………………………………………………………………………..93
3.7 Acknowledgements……………………………………………………………………94
3.8 Tables & Figures……………………………………………………………………....95
3.9 Literature Cited……………………………………………………………………….101
Chapter 4: Satellite male mating behaviour as a mechanism for lower fitness matings in a hybrid
zone..............……………………………………………………………………………………113
4.1 Abstract………………………………………………………………………………113
4.2 Introduction…………………………………………………………………………..114
4.2.1 The spring peeper, Pseudacris crucifer……………………………………...116
4.3 Methods………………………………………………………………………………118
4.3.1 Specimen collection………………………………………………………….118
4.3.2 Genotyping & testing for hybridization……………………………………...119
4.3.3 Statistical analysis……………………………………………………………120
4.4 Results………………………………………………………………………………..121
xi
4.4.1 Size dependent mating tactics………………………………………………..121
4.4.2 Contact zone mating tactics………………………………………………….122
4.4.3 Successful amplexus…………………………………………………………123
4.5 Discussion……………………………………………………………………………124
4.5.1 Alternative mating tactics within the contact zone…………………………..126
4.6 Conclusion……………………………………………………………………………127
4.7 Acknowledgements…………………………………………………………………..128
4.8 Tables & Figures……………………………………………………………………..129
4.9 Literature Cited……………………………………………………………………….134
Chapter 5: General Discussion………………………………………………………………….144
5.1 Significance………………………………………………………………………......146
5.1.1 Phylogeography & reproductive isolation…………………………………...146
5.1.2 Intraspecific reinforcement…………………………………………………..147
5.1.3 Cryptic species delimitation………………………………………………….148
5.1.4 Future research……………………………………………………………….149
5.1.4.1 Costs to hybridization………………………………………………..149
5.1.4.2 Comparative contact zone dynamics………………………………...150
5.1.4.3 Genomics of reproductive isolation………………………………….152
5.2 Literature Cited……………………………………………………………………….152
Summary of Chapters…………………………………………………………………………..158
Appendix A…………………………………………………………………………………….161
Appendix B……………………………………………………………………………………..179
Appendix C……………………………………………………………………………………..184
xii
List of Figures
Figure 1.1 Calling adult male spring peeper…………………………………………………….16
Figure 1.2 Metamorphosing spring peeper tadpole …………………………………………….17
Figure 1.3 Distribution map of spring peeper mtDNA lineages………………………………...18
Figure 1.4 Dyad of spring peepers, highlighting satellite behaviour……………………………19
Figure 1.5 Spectrograph and oscillogram of spring peeper calls………………………………..20
Figure 2.1 P. crucifer distribution and sampling map…………………………………………..57
Figure 2.2 Structure of P. crucifer genome across Southwestern Ontario …………………….58
Figure 2.3 DFA of P.crucifer morphology and call…………………………………………….60
Figure 2.4 Phonotaxis response to P. crucifer stimuli…………………………………………..62
Figure 3.1 Boxplots of tadpole fitness for different genotypes…………………………………95
Figure 3.2 Cumulative P. crucifer tadpole survival curves……………………………………..96
Figure 3.3 Cumulative proportion of total P. crucifer tadpole mortality………………………..97
Figure 3.4 Cumulative P. crucifer metamorphosis curve……………………………………….98
Figure 3.5 Mean tadpole survival of P. crucifer competition treatments……………………….99
Figure 4.1 Sampling map of P. crucifer caller-satellite dyads…………………………………131
Figure 4.2 Correspondence analysis of mating tactic based on genotype……………………..132
xiii
List of Tables
Table 2.1 Frequency of pure and hybrid spring peepers in Southwestern Ontario…………….56
Table 3.1 Summary of tadpole fitness parameters……………………………………………..94
Table 4.1 Body size and body condition of P. crucifer based on mating tactic………………128
Table 4.2 Contingency table of mating tactic probability based on genotype………………..129
Table 4.3 Contingency table of mating tactic dyad associations based on genotype…………130
xiv
List of Appendix Figures
Figure A1 Phylogeny of P. crucifer cytochrome b data……………………………………….175
Figure A2 Body size of male and female P. crucifer of different genotypes………………….177
Figure B1 Mean percent cumulative survival curves for different genotypes…………………178
Figure B2 Mean percent cumulative metamorphosis curves for different genotypes…………180
Figure B3 Photo of hybrid tadpole gigantism………………………………………………….181
Figure B4 Photo of hybrid tadpole deformity…………………………………………………182
xv
List of Appendix Tables
Table A1 P. crucifer sampling information from 2003-2011…………………………………162
Table A2 Microsatellite primer information…………………………………………………..164
Table A3 Unique haplotype sampling information used for TMRCA………………………..166
Table A4 Correlations between call parameters and temperature………………………….....168
Table A5 DFA loadings for call parameters before and after correcting for body size……….169
Table A6 DFA loadings for morphological parameters……………………………………….170
Table A7 PCA factor loadings for body size…………………………………………………..171
Table A8 Divergence estimates for TMRCA………………………………………………….172
Table A9 Parameter estimates for cline analysis using CFIT………………………………….173
Table A10 Contact zone average linkage disequilibrium (LD) ……………………………….174
Table C1 Population sampling information for P. crucifer dyads……………………………..183
Table C2 Estimates of measurement error for P. crucifer morphology………………………..184
xvi
List of Abbreviations
AIC...................................................................................
Akaike information criterion
ANOVA............................................................................
Analysis of variance
AMMS..............................................................................
Alternative Male Mating Strategy
BE……………………………………………………….
bp......................................................................................
Backcross F1 hybrid with Eastern
mtDNA
Base pairs
cyt b..................................................................................
Cytochrome b
dB……………………………………………………….
Decibel
DFA..................................................................................
Discriminant Function Analysis
DNA................................................................................
Deoxyribonucleic acid
E........................................................................................
Eastern Lineage
ECD..................................................................................
Ecological Character Displacement
ESS.................................................................................... Effective sample size
F1………………………………………………………..
First generation hybrid
F2………………………………………………………..
Second generation hybrid
g........................................................................................
Gram
GPS...................................................................................
Global positioning system
HE.....................................................................................
Hybrids with Eastern mtDNA
HI......................................................................................
Hybrids with Interior mtDNA
HPD……………………………………………………..
Highest Probability Density
I.........................................................................................
Interior Lineage
kHz.................................................................................... Kilohertz
km.....................................................................................
xvii
Kilometer
LD……………………………………………………….
Linkage disequilibrium
MCMC..............................................................................
Markov chain Monte Carlo
mtDNA.............................................................................. Mitochondrial DNA
MYA.................................................................................
Million years ago
ng....................................................................................... Nanogram
nuDNA.............................................................................. Nuclear DNA
ON..................................................................................... Ontario
PCA……………………………………………………..
Principal Component Analysis
PCR...................................................................................
Polymerase Chain Reaction
RCD..................................................................................
Reproductive Character Displacement
SPL……………………………………………………… Sound Pressure Level
SVL...................................................................................
Snout Vent Length
TMRA…………………………………………………...
Time to Most Common Recent
Ancestor
UTM.................................................................................. Universe tranverse mercator
xviii
Chapter 1: General Introduction
Few topics in evolutionary biology prompt as much controversy as the causes and
consequences of speciation (Otte and Endler 1989; Coyne and Orr 1998; Howard and Berlocher
1998; Turelli et al. 2001). Consensus has proved elusive because speciation is usually slow (and
therefore not directly observable in terms of scientific research programs), myriad species
definitions have been proposed (Mayden 1997; de Quieroz 2007; Cellinese et al. 2012)
complicating studies of processes implicated in species origins, and taxa vary in both rates and
modes of speciation – both being contingent on details of geography, ecology, mating system,
and genetics (Mayr 1942). Such difficulties obviously hinder broad conclusions about the
processes involved in the emergence of new species. They also complicate studies of the
evolution of reproductive isolation – the sine qua non of biological speciation, requiring at the
most basic level research that examines the full suite of factors that shape and promote the
divergence of mate recognition systems, and how these vary over time and space.
Since the publication of The Origin of Species (Darwin 1859) but more particularly the
Modern Evolutionary Synthesis (Huxley 1942), an abundance of research has been conducted on
speciation, yet key questions remain, including: a) Which are the principle mechanisms that
drive the evolution of reproductive isolation? b) How can the study of taxa with unusual life
histories and mating systems illuminate understanding of speciation? c) What role does physical
isolation among populations play in speciation? (The Marie Curie SPECIATION Network,
2012).
1
1.1 Phylogeography & Speciation
Phylogeography has played an important role in our understanding of how historical
factors, like geographical isolation, and resulting cessation or restriction of gene flow, influence
species distributions and evolutionary trajectories (Avise 2000; Coyne and Orr 2004; Soltis et al.
2006). For example, vicariant events promoted by tectonic separation of continents, formation of
rivers or glaciers, or uplift of mountain ranges underlie the origin of many new lineages
worldwide (Riddle et al. 2000). Indeed, despite recent empirical evidence showing speciation
with ongoing gene flow (Niemiller et al. 2008; Nosil 2008; Carling et al. 2010; Storchová et al.
2010), many evolutionary biologists hold that most contemporary species began in allopatry with
little or no gene flow – at least during the initial stages of speciation (Coyne and Orr 2004;
Fitzpatrick et al. 2009; Mallet et al. 2009). This assertion is robustly supported both theoretically
and experimentally (see Knowlton 1993; Duvernell and Turner 1998, 1999; Barraclough and
Vogler 2000; Xiang et al. 2000; Coyne and Orr 2004).
Within temperate North America, innumerable studies have shown the dramatic
influences of glacial dynamics during the Pliocene and Pleistocene on species diversity and
intraspecific genealogy (Avise et al. 1998; but also see Klicka and Zink 1997). While many
phylogeographic studies survey only a proportion of the focal species global distribution
(Templeton et al. 1995; Conroy and Cook 2000) with a few exceptions (e.g. Milot et al. 2000;
Austin et al. 2002; Zamudio and Savage 2003; Hoffman and Blouin 2004), many of these data
reveal striking phylogeographic structure with geographically restricted clades that probably
reflect isolation within historical refugia, and potentially the early stages of speciation (Avise
1999; Soltis et al. 2006). However despite the accumulation of a huge array of phylogeographic
data spanning hundreds of taxa, a mechanistic link between historical range fragmentation and
2
physical isolation among populations, and the processes that shape the evolution of reproductive
isolation, remains elusive. Moreover, secondary contact zones, particularly between diverging
intraspecific lineages, provide unique opportunities to investigate the early stages of speciation.
Specifically, such contact zones allow us to investigate the roles of selection and drift in
geographic isolation in initiating, and interactions among diverging lineages after contact in
completing, the evolution of reproductive isolation (Martin et al. 2010; Weir and Wheatcroft
2011).
1.2 Consequences of Secondary Contact
One of the more contentious facets of geographic models of speciation concerns the
consequences of contact zones between conspecific populations that initially diverged in
allopatry (Liou and Price 1994). Dobzhansky (1937) originally considered three possible
outcomes of secondary contact: 1. No inter-mating as reproductive isolation is complete and the
two populations may be considered newly-emerged biological species; 2. Dissolution of genetic
differences between populations as there are no fitness consequences to hybridization; and 3.
Strengthening of reproductive barriers and continued divergence between incipient species
(‘character displacement’ sensu Brown and Wilson 1956), possibly as a by-product of selection
against maladaptive hybridization (later termed ‘reinforcement’ – Blair 1955).
Character displacement is suggested to be important in the process of speciation (Rice
and Pfennig 2010). It is a leading cause of adaptive diversification (reviewed by Pfennig and
Pfennig 2009), occurring when two incipient species that otherwise might be phenotypically and
ecologically similar, diverge in one or more traits when they are in direct contact (sympatry).
Character displacement may be ecological, driven by the competition for a limited resource
through niche partitioning (e.g. food - Rundle et al. 2000; Schluter 2000; Adams 2004; acoustic
3
space – Wilkins et al. 2012). It may also involve reproductive traits, thus producing or
accentuating barriers against costly heterospecific reproductive interactions (e.g. Marshall and
Cooley 2000). Character displacement is presumed to be widespread among interacting taxa
(Pfennig and Pfennig 2009). For recently diverged populations for which ‘mis-matings’ or
hybridization is maladaptive and frequent, the evolution of enhanced pre-zygotic isolation during
contact may result from natural selection, commonly known as ‘reinforcement’ selection (Blair
1955).
Reinforcement has classically been presented through evidence for selection against
hybrids (Butlin 1987). Yet in the ‘broad sense’ reinforcement may also occur as a response to
any manifestation of selection against heterospecific matings, such as post-mating pre-zygotic
mechanisms (e.g. genital or gametic compatibility), behavioural hybrid dysfunction, or
unattractive traits to either parental population, regardless of whether or not the hybrids
themselves are unfit (Servedio and Noor 2003). Notwithstanding original enthusiasm over this
process, reinforcement received several decades of criticism, mainly because of the restrictive
conditions under which it was thought to occur in nature (Coyne and Orr 2004), lack of
compelling empirical examples (reviewed in Marshall et al. 2002; Coyne and Orr 2004), and
presumed theoretical problems associated with the establishment of stable sympatric zones
between genetically compatible and ecologically overlapping species (Butlin 1987). To some
extent, the lack of historical evidence may simply reflect difficulties in documenting
reinforcement (Pfennig and Pfennig 2009), including distinguishing reproductive from other
forms of character displacement (Pfennig and Pfennig 2009), from an inability to deduce process
from phylogeographic pattern, and from the challenges of quantifying the specific costs
associated with heterospecific mating or the ability of individuals to discriminate (Marshall et al.
4
2002). To assist in diagnosing reinforcement from reproductive character displacement, Howard
(1993) proposed five stringent criteria that must be met for reinforcement to occur: 1)
hybridization occurs (or once occurred) in nature; 2) hybrids are selected against; 3) the trait that
has been displaced is heritable; 4) observed displacement is perceived by the opposite sex; 5)
character displacement is not caused by other processes.
Hampering our ability to diagnose the geographic and taxonomic pervasiveness of
reinforcement may be the balance required between enough gene flow for selection to act, and
the inhibiting effects of recombination (Servedio and Noor 2003). In fact most empirical
examples of reinforcement are between diagnosed species for which reproductive isolation
presumably is nearly complete (Hobel and Gerhardt 2003; Littlejohn and Loftus-Hillis 1992;
Noor 1995; Pfennig 2003; Sætre et al. 1997), or between ecologically divergent morphs for
which reinforcement is argued to lack permanency because of the potential reversal to extrinsic
post-zygotic barriers (Nosil et al. 2003; Rundle and Schluter 1998; Coyne and Orr 2004) with a
few notable exceptions (Hoskin et al. 2005; Singhal and Moritz 2012). Certainly, reinforcement
may not occur, or may be less likely detectable, once hybridization becomes rare (Bank et al
2011).
Indeed it has also been shown that reproductive isolation can evolve without obvious
morphological or ecological differentiation through less obvious niche dimension (e.g.
physiology or chemosensory cues - Janzen 1967; Smadja and Butlin 2009), or mutation order
(Mani and Clarke 1990). However, surprisingly few studies have focused on contact zones
between morphologically cryptic lineages, with recent emphasis placed on divergent,
ecologically-based targets of selection for speciation (e.g. Schluter 2009; see Singhal and Moritz
2012). In addition, non-adaptive, drift-driven divergence has garnered relatively little attention
5
(but see Wake 2006), but may well be implicated in many speciation events, particularly in areas
demonstrating dynamic glacial histories such as temperate North America.
1.3 The Spring Peeper, Pseudacris crucifer
Also known as ‘Pinkletinks’ in Martha’s Vineyard, USA, and ‘Twinkletoes’ in New
Brunswick, Canada, this enigmatic harbinger of spring is a small North American chorus frog
usually ranging in adult snout-vent length (SVL) from 1.9 to 3.2 cm; record 3.7 cm (Conant and
Collins, 1998). A mystery to many naturalists who are familiar only with its calls in spring,
spring peepers share various attributes common to both the genus Hyla and Pseudacris. Indeed,
this species was formerly placed in Hyla (Hyla crucifer; Wied-Neuwied 1838) but based on
allozyme data was reclassified as Pseudacris (Hedges 1986). Cocroft (1994) suggested that the
spring peeper might be better considered a monotypic genus based on morphological and
biochemical data, but also concluded that its inclusion in the genus Pseudacris was appropriate.
Subspecific designations have been given to the southern spring peeper (Pseudacris crucifer
bartramiana) – distributed in Florida and Georgia only – as well as the northern spring peeper
(Pseudacris crucifer crucifer) – encompassing the remainder of the species range – based
primarily on colour pattern variation (Stevenson and Crowe 1992). Phylogeographic patterns
revealed by analyses of mitochondrial DNA sequences do not relate to these subspecific
designations (Austin et al. 2002).
Characterised by a dark, usually imperfect, cross marking on their backs (hence, the Latin
name crucifer, meaning cross-bearer), this species does not usually have the distinctive mottling,
spotting or banding typical of other Pseudacris species (see Figure 1.1). It does, however, have
prominent bars on its hind legs. Spring peeper colours vary, usually with temperature or stress
(Harding 1997), spanning shades of dark brown, grey, light tan and even orange. A dark band
6
usually runs from the snout, through the nostril and eye, ending at the tympanum. Most
individuals have a dark line (or V) located between the eyes (characteristic of most species
placed within the genus Pseudacris). ‘Peepers’ also have small toe disks (or pads) typical of
Hylidae. This aids climbing trees and shrubs, although most individuals rarely ascend higher
than one metre from the ground. Slight webbing is also evident between the back toes of these
shallow-water swimmers (Conant and Collins 1998).
Sexes can easily be distinguished during the breeding season. Calling males have
pigmented vocal sacs. Even deflated vocal sacs are evident due to their elasticity, and mottled
brownish or greenish appearance (Harding 1997). Males are reported to be, on average, slightly
smaller in size and darker in colouration than females (Harding 1997); however published
evidence for such dimorphisms is lacking. Moreover, such differences between the sexes may be
obscured by age as growth continues throughout life.
Tadpoles can reach up to 3 cm in total length prior to metamorphosis (MacCulloch 2002).
They are distinct from other frog larva in the Great Lakes Regions, and have a brown or greenish
body with metallic flecks (often gold) on their dorsum, contrasted with an iridescent white belly
(Harding 1997). Tail fins are large and clear with orange, black or purplish blotches around the
edges (see Figure 1.2; MacCulloch 2002).
Species with which the spring peeper may be confused in the Canadian portion of its
range include the congeneric striped chorus frogs (P. triseriata and P. maculata). These frogs are
similar in size as spring peepers but have longitudinal dorsal stripes, instead of the characteristic
‘X’, a light lip line, and their call sounds like a fingernail running across a comb, instead of the
typical single note ‘peep’ of the spring peeper (Harding 1997). Cricket frogs (Acris crepitans
blanchardi) have rougher skin (wartier in appearance), have a dark band on the inner thigh and
7
their call resembles a sharp, quickly repeated clicking sound (similar to hitting small stones
together; Harding 1997). In Ontario, cricket frogs are known only from Pelee Island in Lake Erie
and have been neither heard nor seen in many years (McCulloch 2002). The grey treefrog (Hyla
versicolor) is substantially larger than the spring peeper (3.2 to 6.2 cm SVL) and has a distinctive
yellow or orange on the inner portion of its thighs. Grey treefrog calls are easily distinguishable
from those of the spring peeper as they are high pitched, short trills (Harding 1997).
1.3.1 Distribution
The current range of the spring peeper largely maps onto the extent of the eastern mixed
and deciduous forests with some exception (Lannoo 2005). In Canada its range includes the
Maritime Provinces (east to Labrador; Bergman 1999), west to Manitoba and extending as far
north as James Bay, Ontario (Conant and Collins 1998). Its distributions in the United States
spans much of the eastern extent of the country, including Minnesota, Iowa, Illinois, Oklahoma,
eastern Texas and as far south as Florida (although only P. crucifer bartramiana is found on the
southern peninsula of Florida and in Georgia; Conant and Collins 1998). The spring peeper is
abundant in most of Eastern North America but is considered threatened and rare in Kansas, only
occupying the extreme eastern periphery of the state. Spring peepers were initially believed to
have been introduced in Cuba (Schwartz and Henderson 1991), but this observation has yet to be
substantiated (Powell and Henderson 1999).
Phylogenetic analysis has revealed 6 major mitochondrial (mtDNA) lineages across the
spring peeper range (see Figure 1.3) with various areas of contact (Austin et al. 2002; 2004). The
deepest divergence between the Texas lineage and all others dates to approximately 5 million
years ago, with the two lineages found in Ontario (Interior and Eastern) estimated to have a
divergence time of 2.5 million years (Austin et al. 2004). Even these most recently diverged
8
lineages appear to have been isolated for a duration comparable to that required for the evolution
of two pairs of congeneric sister species (Moriarty-Lemmon et al. 2007).
1.3.2 Habitat
Commonly found during the breeding season in or near temporary or semi-permanent
ponds, peepers are more likely to be located in bodies of fish- and pollutant-free water (Hecnar
and M’Closkey 1996, and Freda and Taylor 1992, respectively). When breeding, they form
choruses near brushy growth or secondary forests, primarily where trees over-hang, or are
located within ponds, swamps or marshes (Conant and Collins 1998). In Southern Ontario spring
peepers are very common in April through late May in almost all aquatic habitats.
Although seldom observed outside of breeding choruses, peepers often call during warm
bouts of rain, and can be occasionally found hopping through damp grass and/or the forest floor.
After the breeding season they usually inhabit leaf litter in woodlands, overwintering under such
debris (MacCulloch 2002). Peepers have a unique physiology that allows for freeze and
desiccation tolerance (Schmid 1982). Freeze tolerance has only been noted in a few other
northern frogs (e.g. Lithobates sylvaticus, Hyla versicolor, and Pseudacris triseriata; Storey and
Storey 1987).
1.3.3 Feeding & Activity Patterns
Adults feed primarily on insects and other invertebrates while their tadpoles remain
completely herbivorous, feeding on plants and algae (Fisher et al. 2007). Since spring peepers
often emerge from hibernation early in the year, they frequently rely on fat reserves (Badger and
Netherton 2004). To catch prospective prey or to evade predation themselves, peepers can jump
9
nearly 17 times their body length – a feat that would be the equivalent of a human jumping
nearly 30 metres (Badger and Netherton 2004).
Spring peepers are most common in early spring (late March and early April in Ontario)
when they start to breed, although males often outnumber females 9:1 or more in these breeding
aggregations (Badger and Netherton 2004). During late fall when they can be located in
woodlands, the sex ratio reverts closer to 1:1, suspected to be a result of road mortalities,
predation and behavioural differences (i.e. prolonged chorus attendance by males; Badger and
Netherton 2004). In general, in breeding aggregations spring peepers are most active from late
afternoon and throughout most of the night (Fisher et al. 2007).
Breeding season home-range diameters have been observed between 1.2 to 5.5 metres, and
with daily movements ranging from 6.1 to 39.6 metres (Zampella and Bunnell 2000). First year
individuals travel substantially less but tend to occupy all available habitat with the exception of
the breeding ponds (Delzell 1958). Adult males restrict their movements within the breeding
ponds to small territories (0.5 to 6 metres depending on population density) for up to a month;
females are not observed to hold territories and seem to attend the breeding pond for only a day
or two (Delzell 1958).
After breeding, both sexes set up overlapping home-ranges varying in diameter from 1.1 to
6 metres within woodlands (Delzell 1958). ‘Homing’ ability has also been demonstrated to be
strongly expressed in peepers if they are moved, although site fidelity from year to year is not
observed (Delzell 1958).
1.3.4 Breeding Behaviour & Reproduction
In Canada, the spring peeper is one of the first species to emerge and begin breeding after
winter, and can often be seen congregating around breeding ponds even before full spring thaw
10
(MacCulloch 2002). Beginning to form choruses in the beginning of March in warm, wet years
in Southern Ontario, the breeding season of this species typically extends to the end of May or
the beginning of June in the northern part of their distribution (Wells 1977). In the southern
portion of their range, however, the spring peeper is often referred to as a ‘winter frog’ (Badger
and Netherton 2004) as breeding usually starts with the onset of cooler rains and chorusing starts
in November and lasts until March in places such as Kansas, Texas and Florida (Conant 1975).
Calling males occupy small territories around pond edges, usually found perched on sticks,
logs or in clumps of vegetation (MacCulloch 2002). Calling males are sometimes accompanied
by non-calling, satellite males (Forester and Lykens 1986), which position themselves in close
proximity to a calling individual (see Figure 1.4) to apparently intercept females without
incurring the cost of displaying or territory defence (Land and Wells 1993). Some researchers
have suggested that this tactic is merely used to usurp the advertiser’s calling territory during his
absence while engaging in copulation, at which time they start to call themselves (Badger and
Netherton 2004). Whether considered a form of sexual parasitism (Perrill 1984), or a legitimate
alternative mating strategy (Waltz 1982), this phenomenon has been observed across the species
range, and appears to increase in frequency with chorus density; satellite males tend not to
exceed 14% of total males present in any chorus (Forester and Lykens 1986).
Female spring peepers will enter a pond when attracted by male calls. Once in the water,
the female typically swims toward her chosen mate, touches him, and allows him to mount her
(Badger and Netherton 2004). The pair begins amplexus with the male clasping the female
around the neck (axillary amplexus), after which the female continues to swim and lays between
800-1000 eggs (each approx. 1 mm diameter; Wright and Wright 1995). The eggs are deposited
singly or in small clusters, at the bottom of a pond among grasses or other aquatic plants. Eggs
11
are laid as the female dives to the bottom of the pond where upon the male fertilizes (Wright and
Wright 1995). Eggs are half white, half black and covered with a jelly envelope (pers. obs.).
Eggs hatch in 1-2 weeks in natural populations (Ashton and Ashton 1988), or in 5-6 days if
housed at ambient temperature in a laboratory (Gosner and Rossman 1960; pers. obs.). Tadpoles
complete metamorphosis between 90-100 days (Minton 2001), although P. crucifer bartramiana
metamorphose as early as 45 days after laying at a minimum of 10.33 mm SVL (Gosner and
Rossman 1960). Tadpoles of the southern subspecies, however, average 8-10% larger than those
of P. crucifer crucifer (Gosner and Rossman 1960), and size may influence time to
metamorphosis.
Tadpoles form aggregations in the wild (Brattstrom 1962), but no sibling recognition has
been observed in laboratory tests (Fishwald et al. 1990). Once metamorphosed, froglets move
from wetlands to forest floors, or low brush (Lannoo 2005). Reproductive maturity around New
York State is attained at 23 mm SVL for females and 18 mm SVL for males observed to have
mature spermatozoa (Oplinger 1966). Bartlett and Bartlett (1999) suggest a range of 10 to 31 mm
SVL for reproductive maturity, probably achieved in the second or possibly third year. Delzell
(1958) observed that individuals remained terrestrial in their first year of life, forgoing the
opportunity to breed. Average lifespan for wild spring peepers is unknown; however captiveraised individuals live up to 3-4 years (Blaustein et al. 2001; pers. obs.).
1.3.5 Voice
Only uttered by the males, the call is produced using a single vocal sac (Figure 1.1). The
advertisement call is a single, clear whistling note (at about 3 kHz; see Figure 1.5), repeated at
intervals of approximately one second. Although seemingly simple, this call probably helps to
12
maintain species boundaries and varies significantly among mtDNA lineages (Stewart et al. in
review – Chapter 2). Some individuals also use an ‘aggressive call’, an ascending, trilling peep
usually uttered when a rival infiltrates a calling male’s territory (Harding 1997). Spring peepers
with higher call rates or lower frequency calls are more likely to be larger, heavier and/or older
individuals (Zimmitti 1999; Lance and Wells 1993, respectively). Individual males call at an
amplitude of between 80.2-92.4 dB (at 50 cm distance from source) while their chorus ambient
noise registers at an astonishing 64.7 dB (Brenowitz et al. 1984; pers. obs.). So loud are peeper
choruses that they have been likened to deafening sleigh bells (Conant and Collins 1998), and
may be heard up to a kilometre away.
1.4 Research questions
Mitochondrial DNA (mtDNA) phylogeographic studies have revealed a dynamic history
of geographical isolation, followed by post-glacial recolonization in spring peepers (Austin et al.
2004). The six divergent mtDNA lineages that have been identified throughout its range in
Eastern North America (Figure 1.3) are suggested to be a product of range fragmentation in
multiple southern refugia during the Pliocene and Pleistocene (Austin et al. 2002; 2004).
Multiple pairs of lineages are now in secondary contact at various locations throughout the
species’ range (Austin et al. 2004). Not only are these contact zones interesting because they are
geographically pervasive and occur throughout the entire range of the species (albeit in different
environments), but they also vary in ages of implicated lineages, from 2.5 million years ago
(Southern Ontario) up to as much as 5 million years ago (eastern Texas; Austin et al. 2004). The
latter divergence time well exceeds the estimated time reported for the evolution of reproductive
isolation in multiple congeneric sister species pairs (Moriarty-Lemmon et al. 2007). In the
following chapters I explore the outcome of secondary contact between two mtDNA lineages in
13
Southwestern Ontario (Figure 1.3). In my first data chapter I examine the patterns of variation in
genetic (mtDNA and microsatellite), morphometric, and acoustic traits, as well as female
preference for male vocalizations, in both allopatric populations and those that now lie within a
zone of secondary contact. I show divergence for all considered attributes between allopatric
mtDNA lineage populations. Non-coincident clines between genetic markers across the
secondary contact zone imply asymmetrical introgression/hybridization, mirrored by concordant
asymmetric acoustic character displacement and female preference. Although attributes of male
vocalizations for frogs are strongly correlated with morphology, I show that advertisement calls
are divergent in the zone of secondary contact irrespective of morphology, consistent with
reproductive character displacement. I suggest that this reproductive character displacement is a
consequence of reinforcement selection, although I explore other explanations.
In Chapter 3, I evaluate some of the fitness costs associated with hybridization by
conducting fully reciprocal hybrid crosses between the Eastern and Interior lineages. I assess
fitness correlates such as hatching success, survival and growth rates, and size at metamorphosis.
I describe significant differences between survival in pure Eastern and Interior tadpoles, and
demonstrate that hybrids have equal to or greater (mass at metamorphosis) than their pure
counterparts. Despite this apparent advantage of hybrid tadpoles, I also find some evidence that
competition between individuals from divergent genetic backgrounds detrimentally affects the
survival of juveniles, and that this pattern is exaggerated for Eastern hybrids. My results
exemplify the need for more ‘naturalized’ experimental fitness measures and also further support
my previous finding of asymmetrical costs to hybridization.
Despite apparent call displacement, female discrimination of male calls, and fitness
consequences to hybridization, my genetic data show that assortative mating is imperfect. In the
14
final data chapter of my dissertation, I explore potential behavioural mechanisms that might
mitigate the effectiveness of reproductive barriers between the Eastern and Interior lineages.
Male satellite behaviour, a non-advertising ‘alternative mating tactic’, is common among frog
species, and although it is not restricted to hybrid zones it may allow disadvantaged hybrids to
circumvent female choice. I show that pure Eastern males within the contact zone almost always
adopt a calling tactic while hybrids disproportionately assume a satellite tactic, potentially
increasing reproductive success. Although I show that such alternative tactics are typically
associated with smaller males, mine is also one of the first studies to suggest a genetic
component to such satellite behaviour, as well as the only study to my knowledge to explicitly
test satellite behaviour as a conduit to gene flow across a hybrid zone.
15
1.5 Figures
Figure 1.1. Calling male (average 1.9 to 3.2 cm SVL) spring peeper, Pseudacris crucifer,
showing the distinctive cross on its back, with an inflated vocal sac in a typical calling stance.
Photo Credit S.C. Lougheed.
16
Figure 1.2. Spring peeper tadpole placed in a Petri dish, approximately 48 hours from complete
metamorphosis. Photo Credit K.A. Stewart.
17
Figure 1.3. Current range of the spring peeper in Eastern North America. Shown are the 5
mitochondrial lineages described by Austin et al. (2004), plus a more recent lineage found in
eastern Texas (‘Texas’ lineage).
18
Figure 1.4. A dyad of male spring peepers. On the right is an actively calling male with inflated
vocal sac. On the left is a smaller, non-calling satellite male. Photo credit. K.A. Stewart.
19
3
2
3
4
Figure 1.5. Spectrograph above, oscillogram below, of three spring peeper calls recorded in
Ontario, Canada in 2008.
20
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30
Chapter 2: Reproductive character displacement and isolation asymmetries
between cryptic lineages of a North American treefrog, Pseudacris crucifer
Kathryn A. Stewart, James D. Austin, and Stephen C. Lougheed
2.1 Abstract
Many temperate species have experienced a dynamic history of range fragmentation and
expansion, and secondary contact, providing compelling opportunities to investigate the
evolution of reproductive isolation at the earliest stages of speciation. One possible outcome of
secondary contact is the pattern of reproductive character displacement (RCD) - exaggerated
divergence in the mate recognition system to avoid costly reproductive interactions with
heterospecifics. RCD may be produced by processes such as reinforcement or alternative
mechanisms, especially when mating traits are entwined with morphology. The spring peeper
(Pseudacris crucifer) is a North American hylid frog with six divergent mtDNA lineages, many
of which came into secondary contact during the Holocene. We examined genetics, morphology,
male calls, and female preference for two lineages (Eastern and Interior) that diverged between
3.75 and 4.94 mya and now meet in southwestern Ontario. We conclude that these lineages
diverged phenotypically in allopatry, and that the Eastern lineage exhibits RCD in male calls and
female call preference. Hybrids were distinct morphologically, acoustically intermediate, and
female hybrids showed no preference for either parental lineage. Non-coincident clines in
mtDNA and nDNA suggest asymmetrical hybridization. Our results imply that natural selection
in this contact zone may be enhancing pre-zygotic isolation and differentiation in the mate
recognition system.
31
2.2 Introduction
A major factor in the origins of temperate species has been recurring range fragmentation
and allopatric isolation caused by Milankovitch climate cycles and concomitant glaciation
(Klicka and Zink 1997). Pliocene and Pleistocene refugial and post-glacial dynamics have been
implicated in the diversification of a wide range of temperate organisms including birds (Klicka
and Zink 1997; Weir and Schluter 2004), amphibians (Zamudio and Savage 2003; Austin 2004;
Moriarty-Lemmon et al. 2007), plants: (Soltis et al. 2006), and mammals (Good and Sullivan
2001). However, most phylogeographic studies have not explicitly examined mechanisms that
underpin biological speciation, nor how the fragmentation of species distributions may have
influenced the evolution of reproductive isolation. Rather, many phylogeographic studies have
focused on paleohydrological, paleoclimatic or topographical correlates of genetic structure (e.g.
Avise 1998 ; Kidd and Ritchie 2006). Nevertheless secondary contact zones, primarily between
intraspecific lineages, provide unique opportunities to investigate the early stages of speciation;
in particular, the roles of selection and drift in geographic isolation in initiating, and interactions
among diverging lineages after secondary contact in enhancing, the evolution of reproductive
isolation (Martin et al. 2010; Weir and Wheatcroft 2011).
One of the more contentious facets of models of geographic speciation is whether
secondary contact between conspecific populations that have diverged in allopatry may be an
important mechanism of speciation (Liou and Price 1994). For example, how likely is the
dissolution of differences between populations (fusion) or swamping of one of the two (Gause
1934; Liou and Price 1994; Hochkirch et al. 2007; Groning and Hochkirch 2008)? Is
contemporary diversity maintained through the stable coexistence of lineages with ongoing
hybridization (Dobzhansky, 1940; Bartin and Hewitt 1981; Butlin 1987)? Alternatively, if
32
reproductive isolation was initiated in allopatry, do the two lineages continue to diverge after
contact? In this last scenario, organisms can overcome a reduction in fitness stemming from
lineage interactions when selection favours the strengthening or displacement of lineage-specific
characters in sympatry (Brown and Wilson 1956).
Continued exageration of conspecific divergent lineages in sympatry can be driven by
selection to: 1) Reduce resource competition with reproductive isolation as a by-product of
niche-partitioning, termed Ecological Character Displacement (ECD, Rundle et al. 2000;
Schluter 2000); or 2) Reduce costly reproductive interactions whereby reproductive isolation is
under direct selection, termed Reproductive Character Displacement (RCD; Brown and Wilson
1956; Harrison 1993; Howard 1993). Certainly both mechanisms can work in concert leading to
reproductive isolation (Rundle and Schluter 2004; Grant and Grant 2008; Pfennig and Pfennig
2009), but in many taxa where mate recognition traits correlate with morphology (e.g. insects
and frogs; Gerhardt and Huber 2002), it is especially important to distinguish the true nature of
selection because some morphological traits may enhance RCD, while others may be under
selective pressures not associated with reproductive isolation (Jang et al. 2009).
Character displacement is presumed to be widespread, but many critics argue that the
pattern of divergence between sympatric and allopatric populations can also arise via processes
not associated with ecological or reproductive interactions (Grant 1972; Arthur 1982; Endler
1986). Studying character displacement thus requires quantification of both indirect and direct
effects to differentiate between it and alternative hypotheses (for a review see Pfennig and
Pfennig 2009). In RCD, indirect processes such as overlapping acoustic space (masking
interference) hampers the ability of species to effectively locate or communicate with one
another (Noisy Neighbour Hypothesis, Noor 1999) and is often found among distantly related
33
taxa less likely to hybridize or have direct reproductive contact (e.g. Amezquita et al. 2006; but
see facilitated RCD, Howard 1993). Alternatively, recently diverged populations with continued
gene flow (or ‘mis-matings’) may experience selection that favours the evolution of enhanced
pre-zygotic isolation in sympatry, termed reinforcement (Dobzhansky 1937; Blair 1955).
While reinforcement has classically been presented through evidence for selection against
hybrids (Butlin 1987), in the ‘broad sense’ it may also occur as a response to any manifestation
of selection against heterospecific matings (such as post-mating pre-zygotic mechanisms,
behavioural hybrid dysfunction, or unattractive traits to either parental population), regardless of
whether or not the hybrids themselves are unfit (Servedio and Noor 2003). The strength of
selection to avoid reproductive interactions may differ between hybridizing populations; the
resulting asymmetrical pattern of RCD stems from a trade-off between the benefits associated
with heterospecific avoidance and the cost of displacing mean character traits (Cooley 2007;
Pfenning and Pfennig 2009). Reinforcement is often viewed as merely a potential process
leading to RCD (Blair 1974; Pfennig and Pfennig 2009), one requiring a balance between
enough gene flow for selection to act, against the inhibiting effects of recombination (Servedio
and Noor 2003).
For more than three decades ECD has garnered much attention and empirical support
(Schluter et al. 1985; Grant and Grant 2006; Adams and Rohlf 2000; Losos 2004; Pfennig and
Murphy 2003; Schluter 2000), while RCD has until recently been largely ignored (some notable
exceptions include Gerhardt 1994; Geyer and Palumbi 2004; Kirschel et al. 2009; Russo et al.
2007; Kameda et al. 2009; Jang et al. 2009). Despite theoretical arguements that intraspecific
character displacement is probably stronger (i.e. similar resource-use results in stronger
competition) and more frequent (conspecific rather than heterospecific encounter-rates are
34
probably higher) than its interspecific counterpart (Gurevitch et al. 1992; Pfennig and Pfennig
2010), far fewer studies focus on intraspecific examples (but see Schluter and McPhail 1992;
Dayan and Simberloff 2005, for ECD; Smadja and Ganem 2005; Richards-Zawacki and
Cummings 2011, for RCD), and fewer still provide evidence for reinforcement at incipient stages
of speciation (Smadja and Ganem 2005; Richards-Zawacki and Cummings 2011). Indeed,
finding evidence for reinforcement is not trivial as it may well represent a rapid evolutionary
process (a ‘race’ between the enhancement of mate recognition behaviours and the fusion of
populations (Bossert in Wilson 1965)), forcing many to rely on inferences based on the pattern of
RCD. However, just as RCD can arise from processes other than reinforcement, reinforcement
too can lead to patterns other than RCD, especially given enough divergence during allopatry
(Lemmon et al. 2004). Confusion also arises because divergence in mating traits may not align
with female preference for those traits (Pfennig 2000; Pryke and Andersson 2008), further
illustrating a need for an integrative approach to studying character displacement and the
processes that lead to it.
Phylogeographic studies on the hylid frog, the spring peeper (Pseudacris crucifer), reveal
a history of geographic isolation, range expansion and secondary contact among six divergent
lineages in eastern North America (Fig. 2.1A; see Austin et al. 2002, 2004). Suggested to be a
result of isolation in multiple southern refugia during the Pliocene and Pleistocene, various
lineage pairs are now in secondary contact at different locations throughout the species’ range
(Figure 1). Subspecific designations have been given to the northern (Pseudacris crucifer
crucifer) and southern (Pseudacris crucifer bartramiana) spring peeper (Stevenson and Crowe
1992), based primarily on colour pattern variation, although these phenotypic patterns do not
relate to underlying genealogical history (Austin et al. 2002). Based on average pnet-distances
35
between lineages, Austin et al. (2004) estimated divergence times among the six lineages now in
secondary contact to range from 150,000 to 5 million years before present, sufficient time for
congeneric sister species pairs to have evolved reproductive isolation (Lemmon et al. 2007).
Spring peeper males produce simple, single note advertisement calls in large choruses
(Wilczynski et al. 1984), while females show strong ability to discriminate conspecific and
heterospecific calls between acoustically similar species (Gerhardt 1973). The spring peeper thus
represents a promising system to study the effects of geographic isolation and subsequent
secondary contact on the early stages of divergence, and specifically to ask whether historically
isolated lineages now in secondary contact show a pattern of pre-mating (RCD) and post-zygotic
(hybridization) isolation, potentially consistent with reinforcement during an incipient stage of
speciation.
Our study focuses on a secondary contact zone within southwestern Ontario, Canada,
between the Interior and Eastern lineages of the spring peeper (Fig. 2.1B). Secondary contact
between these lineages probably occurred about15,000 years ago, or soon thereafter, following
the most recent glacial retreat in this region, but could be more recent as northward range
expansion may have been impeded by eastward extension of dry prairie from 8-5,000 years ago
(Transeau 1935; Austin et al. 2002). Our objectives were to: 1) Quantify divergence in
morphology and male advertisement call between lineages in allopatry. 2) Test for RCD of male
traits in zones of secondary contact, and finding evidence for such, test female preference. 3)
Quantify selection against hybridization through evaluation of clines in frequency of DNA
markers and tests of linkage disequilibrium. 4) Refine previous estimates (Austin et al. 2004; Nei
and Li 1979) of divergence time using coalescent approaches.
36
2.3 Materials and Methods
2.3.1 Study Species
Spring peepers are small (20-30 mm snout-vent length), semi-terrestrial treefrogs with females
slightly larger than males. Mate selection by females is mediated via male vocalizations from
calling points within choruses of varying sizes. Calls consist of single note/pure tone whistle,
usually lasting 91 to 280 ms in duration, and ranging between 2700 and 3200 Hz (Doherty and
Gerhardt 1984). In North America, spring peepers are among the first anurans to begin breeding
in spring, and their breeding season is prolonged. In southern Ontario, Canada, male spring
peepers typically chorus from early April until early June, with peak breeding in May.
2.3.2 Sampling Study Populations
We examined geographic variation in female preference, genetic, acoustic, and morphometric
traits of spring peepers (Pseudacris crucifer) from a zone of secondary contact in southwestern
Ontario, consisting of admixture of Interior and Eastern mtDNA lineages (Austin et al. 2002): 22
populations were sampled in 2003 to obtain acoustic and mtDNA sequence data and 14 other
populations were sampled from 2008-2011 to obtain morphometric, female preference, and
mtDNA sequence and DNA microsatellite data (see Fig. 2.1B; Appendix, Table A1). We
sampled reproductively active adults between April and June (21.00 to 2.00 h local time).
Sampling included 185 male recordings from eight populations within the area of secondary
contact (sympatry), six Interior allopatric populations (i.e., an absence of Eastern mtDNA), and
eight Eastern allopatric populations, and 423 individuals (including 81 females) from four
populations within the area of secondary contact, five Interior allopatric, and five Eastern
allopatric populations for morphometric and genetic data (Appendix, Table A1). In total, 608
individuals were collected across a 580 km straight-line sampling distance.
37
2.3.3 DNA Extraction and Genotyping
Individuals were sequenced for a 692 bp segment of cytochrome b (cyt b) using primers MVZ
15-L and MVZ 18-H (Moritz et al. 1992) and methods outlined in Austin et al. (2002;
Supplementary Materials, Methods). PCR products were cleaned using the QIAquick PCR
Purification Kit (Qiagen Inc., Chatsworth, Calif.) prior to sequencing on an Applied Biosystems
3730 Analyzer (Robarts Sequencing Facility, London, ON). Sequences were aligned in BioEdit
version 7.1.3 (Hall 1999). We used a subset of 360 base pairs with 22 parsimony-informative
characters to diagnose mitochondrial lineage affiliation for each individual. Pseudacris crucifer
individuals from 2008-2011 were typed for 11 microsatellite loci, 4 of which have been
previously published and the remaining seven optimized by us for this study (Degner et al. 2009;
see Appendix A Methods & Table A2 for protocol).
2.3.4 TMRCA Analyses Using Cytochrome b.
We estimated the Time to Most Common Recent Ancestor (TMRCA) using cytochrome b
sequences (cyt b) for all possible divergence points among the 6 mtDNA lineages identified by
Austin et al. (2002, 2004) using a Bayesian approach implemented in the programs BEAUti and
BEAST v1.4.8 package (Drummond and Rambaut 2007; see Supplementary Information Table
S3 for sampling locations). All analyses were performed using a GTR + I + G model of
nucleotide substitution (Tavaré 1986) with gamma distributed rate variation among sites and four
rate categories. Assuming an uncorrelated relaxed clock (Drummond et al. 2006) and using an
expansion growth tree prior (Tavaré 1986), results from two runs (50,000,000 generations with
the first 10,000,000 discarded as burn-in and parameter values sampled every 1000 generations)
for each combination of settings were combined and the effective sample size (ESS) for
parameter estimates (exceeding 200) and convergence checked using the program Tracer version
38
1.3 (Rambaut and Drummond 2004). We employed a conservatively bound poikilothermic
standardized molecular clock of uncorrected 1% sequence divergence per million years (Avise et
al. 1998; Austin et al. 2004). To compare divergence times to original estimates from Austin et
al. (2004), we also calculated average raw p-distances between lineages using the program
MEGA5 (Tamura et al. 2011).
2.3.5 Clinal Variation, Linkage Disequilibrium, and Quantifying Hybridization
To test for introgression, we performed a cline analysis by first collapsing all locales to a single
dimension using a straight-line distance in kilometers (Seneviratne et al. 2012). According to
program specification (see below), we set population distances allowing for a decrease in allele
frequencies over geographic distance, starting from our eastern most population near Kingston,
ON (Fig. 2.1B). Using the program CFIT (Version 0.6, Gay et al. 2008) we estimated the best-fit
shape parameters for the allele and haplotypic frequencies (f(x)) as a function of geographic
distance (x). Clines were fitted for our 11 microsatellite markers and cyt b haplotypes, wherein
alleles per locus were reduced to a simple two-allele system using lineage-specific compound
alleles (Gay et al. 2008) as follows. Based on the first axis coordinates of a Multiple
Correspondence Analysis (MCA) implemented in GENETIX (Version 4.05.2; Belkhir et al
2004), we designated alleles as being either Eastern or Interior. We identified some private
alleles across loci and found most alleles to be distinct between lineages. In instances where
alleles are shared or incorrectly assigned, cline shape is typically flattened, and thus tests for the
presence of clines are conservative (Gay et al. 2008). We fit the center and slope parameters of a
logit cline for each marker and used Akaike Information Criterion (AIC, Akaike 1974) to
compare patterns of dispersal or selection. We assessed: 1) unconstrained clines, 2) cline center
coincidence (geographic centers constrained), 3) cline width concordance (slopes constrained),
39
and 4) cline coincidence and concordance. We executed these tests with a maximum center value
of 580km (maximum transect distance) implementing 1000 repetitions for 100,000 iterations
(Gay et al. 2008). Convergence was comparing the results of each test 10 times each with
different random seeds.
We used GENETIX to test for linkage disequilibrium (LD) between pairs of loci for each
population (p < 0.05). Low levels of LD are expected with free recombination between parental
genomes, while high LD are expected under instances of rare hybridization, the production of
hybrids with low fitness, and/or low recombination (Harrison 1993).
We used STRUCTURE (Version 2.3.3; Pritchard et al. 2000) to help distinguish between
mixed versus pure lineage individuals, executing a Markov Chain Monte Carlo (MCMC)
algorithm to infer population structure from multi-locus genotype data, assigning individuals to
clusters assuming Hardy-Weinberg equilibrium and linkage equilibrium within clusters, and
identifying hybrids based on allele frequencies (Hubisz et al. 2009). We assumed admixture,
correlated alleles, no prior population information, and used 10 iterations of 5x105 steps of the
Markov Chain (preceded by a burn-in period of 25 x104 steps). STRUCTURE was run with
number of populations, K, ranging from 1 to 14, the maximum number of locales sampled.
STUCTURE outputs were entered into Structure Harvester (Earl et al. 2003), implementing the
Evanno et al. (2005) method for identifying the most probable value of K.
To assess the power of 11 microsatellites to identify admixed individuals (i.e, reasonable
bounds from STRUCTURE q-values for designating ‘pure’ vs. ‘hybrid’ individuals), we
simulated four classes (F1, F2 and backcross individuals) each consisting of 100 hybrid
individuals. Simulations were conducted using HYBRIDLAB version 1.0 (Nielson et al 2006)
utilizing 30 ‘pure’ lineage individuals from each of the Interior and Eastern lineages (0.95 ≥ q40
value ≤ 0.05) sampled from allopatric populations that were located at least 100km from the
contact zone. These simulated genotypes (50 randomly chosen per category) were subsequently
used for a STRUCTURE analysis (K = 2 following the methods outlined previously), and the
proportion of correctly identified hybrid individuals were quantified.
2.3.6 Testing for Character Divergence and Displacement
We located males at night and recorded their advertisement calls using a Marantz
PMD660 and a Sennheiser ME67 directional microphone held approximately one meter from the
calling individual. We analysed at least 10 consecutively produced advertisement calls per
calling male that were clear and distinct from other proximate calling individuals. Air and water
temperature, as well as calling site details were noted. After recording, males were handcaptured, measured, toe-clipped and immediately released. We measured nine call parameters
(previously identified as being important for species recognition and/or sexual selection;
Lemmon 2009) using SYRINX (v2.6 www.syrinxpc.com). Eight of the nine parameters
(maximum frequency, minimum frequency, time to maximum frequency, time to minimum
frequency, carrier length, call duration, call rate, call interval, but not delta frequency) varied
significantly (all P<0.05) with temperature (Appendix Table A4); thus we used residuals from
linear regressions of these parameters on temperature for subsequent Discriminant Function
Analyses (DFA; Appendix Table A5). Many call parameters also vary with the body size
(McClelland et al. 1996) and body size itself may be a target of selection, thus we subsequently
ran a DFA corrected for snout-vent length (SVL) to see if acoustic parameters differed despite
possible morphometric distinction between lineages (Appendix Table A5). Calls were
characterised for allopatric locales comprised solely of pure lineage individuals based on cyt b
data and our understanding of where lineages come into secondary contact.
41
To quantify morphometric variation we measured mass using a 10 g mini-Pesola (±
0.2g), and SVL, head width, tibia length, foot length, femur length and radioulnar length (± 0.2
mm) using digital callipers. Toe clips were stored in 95% ethanol at -80ºC for later genetic
analyses. We used DFA and MANOVA (Appendix Table A6) to test for differences between
allopatric versus sympatric populations, and also among Eastern, Interior and hybrid genotypic
classes for all pooled individuals. Because body size is important in anuran sound reception and
production (Gerhardt 1994), we assessed morphometric differences for each sex separately using
a multivariate component of body size derived from principal components analysis (Appendix
Table A7).
2.3.7 Pre-zygotic Preference Experiments
We performed phonotaxis experiments using a standardized test arena (Gerhardt 1994) to
quantify female discrimination between calls of males from their own lineage versus the
alternate lineage within the contact zone. As evidence of reinforcement, we predicted that female
preference for parental lineage calls would exist and be exaggerated within the zone of secondary
contact versus preference in allopatry to reduce the chance of maladaptive hybrids.
We used natural calls sampled in allopatric populations from three males per lineage with
average lineage SVL, whose calls were recorded at 16ºC (temperature at which females were
tested; Lemmon 2009). These calls were combined into multiple digital files with all possible
contrasting pairs of stimuli to reduce biases. Stimuli were played using an audio editor (Audacity
Version 2.2.5; http://audacity.sourceforge.net/), and broadcast through two opposing speakers
(Saul Mineroff Field Speakers SME-AFS, Elmont, NY, USA). Stimuli were presented
antiphonally at 85dB SPL as measured at the center of the testing arena (see below), mimicking
natural amplitude attenuation (Brenowitz et al. 1994). Call stimuli for each lineage were
42
normalized to the same call interval (1 call/s) and amplitude (± 2 dB) during all experiments. We
randomized which lineage-specific call was presented first (Forester and Harrison 1987) and
continuously played a background chorus at 10dB below that of the stimuli.
Amplexed and/or gravid females for phonotaxis experiments were caught in allopatric
and sympatric populations and transported for less than 2 hours to a field station (one of Point
Pelee National Park, Bruce Peninsula National Park, Long Point Waterfowl Research and
Education Centre, Queen’s University Biological Station). After > 2 hours acclimation in the
dark, females were individually placed into our testing arena, a white 142 centimeter diameter
wading pool filled with approximately 6 cm of aged tap water and perching sticks (Lemmon
2009). Within a dark room, female spring peepers were placed at just over 50 cm away from
both speakers within a plastic container with stimuli played for 2 minutes prior to release.
Container lids were remotely removed and female movements were recorded using a Sony
Handycam DCR-HC21 Mini DV HD camcorder with a Sony HVL-IRM Infrared light. Approach
to within 10cm radius of a speaker emitting stimuli was considered a positive response (Lemmon
2009). Females that did not respond to either stimulus after 15 minutes or that swam around the
perimeter of the pool instead of making a directional choice were noted as unresponsive. All
females were only tested once to reduce the possibility of false positives. After trials were
completed, females were toe clipped and genotyped to designate ancestry (morphology alone
does not distinguish between lineages - or between pure and hybrids). All females were tested on
their night of capture and released at their capture site the following evening.
All data are archived in Data archived with the Queen’s University Biological Station
Data Archive, Herpetology, http://www.qubsdata.org/datasets/herp.php.
43
2.4 Results
2.4.1 Lineage Dating and TMRCA
The TMRCA relaxed clock mean estimate for coalescence of the entire species using 1%
per million years was 11 million years ago (95% highest probability densities (HPD) 6.83
million - 15.4 million). Time of this basal split based on mean raw p-distances was 8.8 million
years ago. Based on TMRCA analysis, the Interior and Eastern lineages implicated in the
southwestern Ontario contact zone diverged in the late Pliocene, approximately 4.93 million
years ago (95% HPD 3.06 million - 6.96 million) (Appendix Table A8, Figure A1). Mean raw pdistances put this divergence at 3.75 million years ago. These estimates are substantially earlier
with those produced by Austin et al. (2004) primarily because they used pnet rather than raw pdistance or TMRCA between the Eastern and Interior lineages.
2.4.2 Selection Against Hybrids
Logit cline analysis (Fig. 2.2 A) on composite alleles (GENETIX MCA first axis
eigenvalue = 1.98) indicated that a model with concordant cline widths for nDNA and mtDNA
(constrained slopes) best fit our data (Appendix Table A9). The second closest model included
the unconstrained parameters (cline centers and slopes were not similar between genetic
markers), and the worst fitting model was the one demonstrating spatially coincident clines with
concordant widths for both marker classes. The estimated cline center values with widths of 90.9
km for our 11 microsatellites ranged from 335 – 580 km east from our QUBS (Kingston, ON)
population while our cyt b marker cline center was estimated to be 395km east (Fig. 2.2 A).
LD was widespread across populations and locus pairs (Appendix Table A10). On
average, populations with mtDNA haplotypes from both lineages (hereafter contact zone)
contained the highest percent of significant LD (72% of locus pairs), although Interior
44
populations on average showed similarly high LD (70%), with Eastern populations on average
exhibiting the lowest average LD (60%). Allopatric populations furthest away from the zone of
contact show the lowest values of LD across the transect (56% and 42% for Interior and Eastern,
respectively; Appendix Table A10).
The model with the highest support in our STRUCTURE analysis reflected the presence
of two lineages in secondary contact (K = 2; Fig. 2.2B). HYBRIDLAB simulations based on
putative “pure” lineage genotypes, produced q-values (0 = pure Eastern, 1 = pure Interior)
ranging from 0.86 to 0.96 for Interior, 0.043 to 0.11 for Eastern, 0.31 to 0.68 for F1, 0.21 to 0.92
for F2, 0.49 to 0.94 for Backcross Interior (F1 hybrid X Interior), and finally 0.06 to 0.435
Backcross Eastern (F1 hybrid X Eastern). Overlap in q-values precludes distinguishing among
F1, F2, or backcross individuals; henceforth we designate pure Interior individuals with a qvalue > 0.85, pure Eastern with a q-value < 0.15, and “hybrids” as 0.15 < q-value > 0.85, (Fig.
2.2C).
Within the area defined as our contact zone, where 79 individuals had Eastern haplotypes
and 80 individuals had Interior haplotypes distributed across 4 locales, we found evidence that
hybridization and introgression were asymmetrical. Using our defined hybrid classes, our
STRUCTURE analyses demonstrate that males and females carrying the Eastern mtDNA
haplotypes within the contact zone were diagnosed as having either Eastern nDNA genotypes
(36.4% males and 29.2% females), or were hybrids (63.6% males and 70.8% females; Fig. 2.2B,
Table 2.1). No pure Interior males or females were identified within the contact zone, further
supporting our contention that clines for mtDNA and nuclear markers (nDNA) are not spatially
coincident (Fig. 2.2B, Table 2.1).
45
2.4.3 Morphological and Call Character Displacement
Independent of sex, allopatric Eastern individuals were significantly smaller (Wilks’  =
0.847, F4,369 = 2.229, p < 0.001) than allopatric Interior individuals (Fig. 2.3A). As no pure
Interior individuals were detected within the contact zone (Table 2.1) we were unable to assess
pure Interior morphology in sympatry; however pure Eastern individuals within the contact zone
were significantly smaller than their allopatric counterparts. Hybrid or admixed individuals
within the contact zone were morphologically distinct with shorter, lighter bodies, and longer
limbs, again with hybrids possessing Eastern mtDNA showing an accentuation of these
characteristics (Fig. 2.3A). Analysis of PC1 scores (body size; Appendix Table A7) revealed
hybrid females to be demonstrably smaller than pure lineage females from either allopatric or
contact zone locations (F4,75 = 4.292, p = 0.004; Appendix Figure A2A), with size diminution in
hybrid females with Eastern haplotypes. Body size in males, however, did not significantly differ
across populations or lineage (F5,301 = 1.113, p = 0.351; Appendix Figure A2B).
Allopatric males from our two lineages had significantly different advertisement calls
(Wilks’  = 0.606, F4,175 = 2.70, p < 0.001), with Allopatric Eastern males calling at higher
frequency with longer call interval and carrier length than Allopatric Interior males. Call
attributes for contact zone pure Eastern males also differed significantly from their allopatric
counterparts showing no overlap in 95% confidence ellipses, a mark of character displacement;
their calls had longer fall times and lower frequencies, but similar carrier lengths. Again, we
found no pure Interior individuals in the contact zone, but male hybrids with Interior haplotypes
had calls there were intermediate in characteristics of the two parental lineages (Fig. 2.3B);
Eastern hybrid males had calls similar to those produced by Allopatric Eastern males.
Importantly, differences in vocalizations were apparent even after controlling for body size
46
(SVL); Allopatric Eastern males showed significantly longer carrier lengths than Allopatric
Interior males, and the vocalizations of pure Eastern males in the contact zone showed
significantly shorter call rise times and longer call fall times (see Lemmon 2009 for definitions)
than their allopatric counterparts (Wilks’  = 0.611, F4,175 = 3.20, p < 0.0001; Appendix Table
A5).
2.4.4 Female Preference
We tested 75 female spring peepers for call preferences: 13 Allopatric Interior, 19
Allopatric Eastern, and 43 females from within the contact zone (classified post hoc using
mtDNA and microsatellite markers). We had a 40.0% overall response rate; 6, 5, and 19 for each
of the aforementioned categories, respectively. Response rates varied between population types,
highest in Allopatric Interior (46.2%), contact zone (44.2%), and lowest in Allopatric Eastern
(26.3%). The differential rate of response between allopatric populations is unlikely to bias our
results as we are concerned with female preference in secondary contact, with no a priori
expectation for female preference in allopatry. Within the contact zone we found little difference
in response rate between females; pure Eastern – 57.1%, Eastern hybrids – 41.2%, and Interior
hybrids – 42.1%. Pure Eastern females within the contact zone showed a quicker response time
to stimuli (85.8 sec) compared to Eastern hybrid (141.2 sec) and Interior hybrid females (171.4
sec), which showed greater lag to respond but not significantly so (F2, 16 = 0.82, p = 0.46).
Allopatric Interior females exhibited a preference for Interior stimuli (83.3%). Allopatric Eastern
females also demonstrated a slight preference for Interior stimuli (60%). Contact zone pure
Eastern females, on the other hand, consistently (100%) chose vocalizations from Eastern males
over Interior ones (2 = 10.44, p = 0.034; Fig. 2.4). Female hybrids showed near equal preference
for stimuli (53% preferred Eastern); however when separated into their respective mitochondrial
47
lineage groupings, female hybrids with Eastern haplotypes favoured the Eastern stimuli (80%)
and female hybrids with Interior haplotypes exhibited equal preference for either stimulus (50%;
Fig. 2.4).
2.5 Discussion
The history of the Northern Hemisphere, especially for North American species, has been
dynamic and invariably included secondary contact between diverging lineages. Evolutionary
biologists have been interested in identifying possible outcomes to such contact, particularly
focusing on whether sister taxa differentiated in allopatry are reproductively isolated, and the
mechanisms responsible for this isolation. However, the majority of studies to date have
involved studying reproductive isolation among recognized sister taxa. Here we quantified clinal
variation in both mitochondrial and nuclear markers, tested for differences in morphology and
male vocalizations, and conducted female phonotaxis experiments between two lineages of a
frog species widely considered to be a single taxonomic unit. Our results collectively
demonstrate reproductive character displacement, where costs of hybridization between
diverging lineages may have caused enhanced pre-mating isolation and differentiation in the
mate recognition system, ultimately suggesting secondary reinforcement as a potential process
during the early stages of divergence (i.e., before species diverge; Howard 1993; Coyne and Orr
2004). We explore this in detail below, first discussing alternate explanations for the documented
patterns.
We found that two spring peeper lineages with origins in the Pliocene differ markedly in
a suite of attributes spanning nDNA, morphology, male advertisement call, and female call
preference. More significantly we detected evidence of asymmetrical hybridization and
48
introgression, suggesting differential fitness consequences of hybridization among lineages.
Hybrids in the southwestern Ontario secondary contact zone were distinct morphologically and
female hybrids showed no obvious preference for either parental lineage. We found hybrids and
pure Eastern individuals, but no pure Interior individuals in the secondary contact zone. Eastern
males within the contact zone differed in morphology and acoustic properties of their calls
relative to their allopatric counterparts; call divergence was still apparent in the contact zone
after controlling for male body size. Pure Eastern females in the contact zone preferred male
advertisement calls of their own lineage versus calls from Interior lineage, although exhibiting
some preference for Interior calls when in allopatry.
Discordance in mtDNA, nDNA, and other taxonomic characters provide insights into the
causes and consequences of secondary contact (Endler 1977; Toews and Brelsford 2012).
Differences in the clines of maternally inherited mtDNA and biparentally inherited nuclear
markers (Fig. 2.2A) may be caused by sex differences in dispersal, non-random mating (Arnold
1993), or selection (Parsons et al. 1993). Although poorly studied, sex-biased dispersal may
cause clines of different widths for neutral or weakly selected traits (Rheindt and Edwards 2011),
and ultimately result in a moving hybrid zone. Delzell (1958) found that post-breeding home
range sizes and dispersal distances were approximately equal for both sexes in Michigan spring
peepers, and thus sex-biased dispersal appears unlikely to be the primary cause for cline
differences. Non-coincident clines could result from differences in population sizes of the two
lineages that have come into secondary contact. Females from smaller populations may evolve
stronger pre-mating isolation barriers compared to females from larger populations as a
consequence of higher encounter rate of foreign males (Hoskin et al. 2005). This so-called rarer
female effect (Coyne and Orr 1989; Coyne and Orr 1997; Noor 1999; Yukilevich 2012) may be
49
important for many taxa. Yukilevich (2012) observed smaller range sizes itself to account for
this rarer female effect when species interact in sympatry; however the Eastern lineage in spring
peepers demonstrates the strongest premating, and possibly postzygotic, isolation asymmetries
despite having a larger geographic range than the Interior lineage (see Figure 2.1). It is possible
that the Eastern lineage distribution is larger but population densities are lower, but again this
seems an unlikely explanation for spring peepers as the species is very common throughout
much of its range and certainly abundant in Ontario where we undertook our study.
Genetic drift in small populations with a patchy historical distribution may contribute to
differences in clines by fixing alleles in isolated demes (Polechova and Barton 2011). This is
plausible for the spring peeper as recent range expansion possibly progressed unevenly allowing
for sequential founder events; however, spring peepers are also a fragmentation-resistant species
with high population densities (Gibbs 1998), and probably ecologically buffered against local
extinctions, demographic stochasticity, and environmental instability.
Environmental gradients can produce clines of different widths and locations as a
consequence of differential selection (Toews and Brelsford 2012). For our study system this
would seem doubtful given that: 1) There is no obvious contemporary habitat transition in the
region where contact occurs. 2) There is no evidence for historical environmental gradients
during the time of secondary contact after glacial retreat (Williams 2009), reducing the
likelihood for local adaptation as a cause for this pattern. 3) Spring peepers exhibit a wide
ecological tolerance both regionally and across their range. 4) The markers that we use are
putatively neutral and thus should reflect neutral processes rather than selection.
Asymmetrical mtDNA introgression, or “capture”, is common for many taxa (Good et al.
2008) and is one explanation for a selective disassociation between mito-nuclear DNA. It can be
50
caused by local adaptation, nDNA- mtDNA co-evolution, or selective sweeps of universally
favoured mutations (Irwin et al. 2009). Were this to be true for the spring peeper secondary
contact zone however, we still would have no explanation for the observed geographical patterns
in morphology, male call, or female preference. Thus, even if there were pleiotropic effects of
mtDNA on morphology and acoustics in Eastern individuals, there is no expectation that these
features would be exaggerated in the zone of overlap.
We feel selection against hybrids, and thus secondary reinforcement, is the most probable
cause of non-coincident clines in mtDNA and nDNA evident. Narrower mtDNA than nDNA
clines, a common pattern in nature, suggest either nuclear introgression and/or differences in
hybrid viability or fertility between the sexes (Toews and Brelsford 2012). Indeed, Haldane’s
rule posits that the heterogametic sex suffers the highest fitness cost to hybridization. In taxa
where females are heterogametic, mtDNA clines are narrower and there should be geographical
discordance or non-coincidence between nuclear and mitochondrial markers (Toews and
Brelsford 2012). Although heterogamy has not been reported for Pseudacris, and thus Haldane’s
Rule cannot be invoked, a higher fitness cost to Eastern hybrid females would be consistent with
the pattern of asymmetrical introgression that we found (but see Lemmon and Lemmon 2010).
Indeed female but not male hybrids are smaller than their pure lineage counterparts. While we as
yet have no direct evidence that smaller size would have fitness consequences for the spring
peeper specifically, fecundity relates to body size in female anurans both within (Camargo et al.
2005) and among species (Praddo and Haddad 2005). Morphology has also been shown to
constrain reproductive investment (Vitt 1981; Semlitsch 1985). Smaller size may indicate a loss
of egg production, as clutches comprise a large proportion of body mass and thus a large
proportion of their energy budget (Zug et al. 2001). Frogs also exhibit indeterminant growth
51
(Zug et al. 2002), and smaller body size may correlate with lower life expectancy for female
hybrids.
Eastern hybrid females show a stronger preference for calls of males from their own
lineage, while Interior hybrid females exhibit equal preference, further implying asymmetrical
mating with repercussions for the stability of the hybrid zone. In effect, asymmetrical female
preference alone can cause hybrid zone movement (Shapiro 2001). For example, if one parental
population produces a greater proportion of pure offspring than another when in secondary
contact, in subsequent generations there will be a replacement of the latter’s genome, regardless
of the fitness of hybrids per se, ultimately causing a hybrid zone shift (Bella et al. 1992, Buggs
2007). Evidence of strong Eastern female preference for calls of their own lineage, and cline
patterns illustrating asymmetrical introgression of Eastern nDNA into Interior populations,
suggest possible hybrid zone movement in the Western direction towards the Interior lineage.
Interpretation, however, is difficult using molecular markers and behaviour alone, and thus
conclusions about hybrid zone movement may be more reliable with comparative studies of the
same contact zone over time.
While we recognize small sample sizes in our female preference experiments, we find a
large effect of preference for their parental lineage call within the contact zone, almost 2.5 times
higher than their preference in allopatry. Given necessary post hoc identification of female
ancestry and our testing females only once to reduce false positives, we feel these findings also
point to asymmetrical reinforcement selection.
Alternative explanations to selection against hybridization and RCD in the spring peeper
system could be 1) Differential fusion via the Templeton effect, 2) Facilitated RCD, and 3) ECD.
The Templeton effect posits that only those taxa with sufficient prezygotic divergence (trait and
52
preference) in allopatry are able to endure in sympatry compared to weakly differentiated taxa
more likely to fuse together or go extinct (Templeton 1981; Powell 1997; Coyne and Orr 2004).
In this scenario, prezygotic isolation may simply reflect differential fusion and not a response to
natural selection (Coyne and Orr 2004). Although we do in fact observe strong divergence in
male call and morphology between the two lineages in allopatry, we see stronger displacement in
sympatry suggesting that this response is not merely a subset of already existing character states
in allopatric populations (Noor 1999; Coyne and Orr 2004). Further, female discrimination in
Eastern allopatric populations is lower than that found in sympatry inconsistent with differential
fusion. Likewise, since our two lineages hybridize, it is unlikely that divergence is driven by
avoidance for signal interference (Noisy Neighbour Hypothesis) rather than selection to avoid
hybridization itself (Noor 1999). Indeed, Pseudacris congeners also show RCD in male call traits
(albeit idiosyncratically depending on heterospecific interactions) and female preference in
sympatry as a means to avoid maladaptive hybridization (Lemmon 2009).
Ecological effects may facilitate a response to reduce niche overlap between populations,
incidentally changing mate recognition signals in sympatry (ECD), particularly in species whose
mate recognition cues are correlated with morphology (Jang et al. 2009). In the spring peeper,
there is no obvious evidence of ecological differences at the zone of secondary contact presently
(pers. observation) or historically (Williams 2009). We concede that discerning such ecological
differences may be difficult or subtle, and could result in divergent call attributes as a by-product
of selection acting on niche-body size relationships (Parmelee 1999). Nevertheless, when
correcting for morphological differences between lineages we still see strong evidence for RCD
in allopatry, and displacement within sympatry in acoustic traits. Indeed the call parameters that
53
distinguish between allopatric and pure Eastern males within the contact zone are call rise and
fall time, found to be unrelated to body size in our species (Appendix Table A4).
Evidence to date demonstrates RCD between these two lineages now in secondary
contact, and we suggest a role for reinforcement selection for the process driving this pattern
based on non-coincident clines/asymmetrical introgression, morphologically distinct hybrids,
hybrid females showing intermediate preference, and hybrid males showing physiological
(aberrant sperm) and behavioural (alternative mating tactic) dysfunctions (Stewart et al. in
review – Chapter 4). Future work will include hybridization experiments where we will use fully
reciprocal hybrid crosses to evaluate the fitness costs associated with hybridization, and the
developmental stage and generation when fitness costs are manifested (F1, F2, backcross).
Ultimately such data will help us to understand why, in the face of strong, albeit asymmetrical
female preferences, hybridization still occurs in nature.
2.6 Conclusion
Teasing apart the true nature of divergence is an important step in identifying the processes that
contribute to the evolution of reproductive isolation, especially in those organisms whose mate
recognition traits are tightly linked to morphology. Here we demonstrate that reproductive trait
displacement in sympatry is probably due to selection to avoid hybridization, although we cannot
discount the possibility of ecological causes. Finding strong evidence for reinforcement in nature
has been challenging (Coyne and Orr 2004), yet Yukilevich (2012) and others (Coyne and Orr
1989; Coyne and Orr 1997; Noor 1999; Coyne and Orr 2004) suggest that ‘concordant isolation
asymmetries’, where costlier hybridization events (post-zygotic isolation) also demonstrate
greater levels of pre-zygotic isolation barriers, may provide indirect empirical support for
54
reinforcement. Here we present genetic, morphometric, acoustic and behavioural evidence that is
consistent with such isolation asymmetries. Our results imply reinforcement but ongoing work
on the specific costs associated with hybridization between the Eastern and Interior lineages will
allow us to speak more forcefully to this hypothesis. Ultimately we hope to highlight the utility
of using phylogeographic studies to provide a genealogical and historical framework for studying
processes of speciation and the impact of dynamic fragmentation histories and secondary contact
on evolutionary trajectories.
2.7 Acknowledgements
We thank funding agencies NSERC (KAS, JDA, & SCL), SSE-RGA (KAS), and Sigma-Xi
Grant-in-aid of Research (KAS), permitting agencies in both Canada and the USA, Queen’s
University Animal Care (UACC Lougheed 2008-059-R3), and the following field stations for
their support during field sampling: Queen’s University Biological Station, Bruce Peninsula
National Park, Middle Mississippi River Wetland Field Station, and Long Point Waterfowl
Research and Education Centre. We especially thank E.M. Lemmon for assistance in
experimental design and call analysis. We are grateful to numerous field assistants for their
contribution to data collection including Kevin Donmoyer, Ahdia Hassan, Cam Hudson, Surbhi
Jain, Natalie Morrill, Henry Pai, Ayden Sherritt, Matt Teeter, and Rachel Wang. The authors
declare no conflict of interest in the funding, acquisition or analyses of data used for this
manuscript.
55
2.8 Table and Figures
Table 2.1: Ancestral frequency distribution (percentage in brackets) by sex, diagnosed
STRUCTURE admixture analysis (K = 2) within southwestern Ontario contact zone. Q-value
cut-offs were Eastern Pure (q < 0.15), Hybrid (q = 0.15-0.85), and Interior Pure (q > 0.85).
Eastern
Interior
Pure
Hybrid
Totals
Pure
Hybrid
Totals
Males
20 (36.4)
35(63.6)
55
0 (0)
61 (100)
61
Females
7 (29.2)
17 (70.8)
24
0 (0)
19 (100)
19
Totals
79
80
56
A
B
Figure 2.1: A) Distribution of 6 mtDNA lineages spring peeper (P. crucifer) across North
America (from Austin et al. 2004) with focal contact zone in southwestern Ontario, Canada
indicated by triple line. B) Sites sampled from 2008-2011 for this study. Shown are 14 sites for
which we obtained genetic data (locale numbers correspond to STRUCTURE analysis), with pie
charts representing the proportions of Interior or Eastern mtDNA haplotypes for each sampled
locale: Interior (dark gray), Eastern (light gray), and sites for which we also obtained call and
morphometric data (22 populations sampled from 2003-2011): Interior (triangle), Eastern
(square) and admixed (asterisk) - see Appendix, Table A1 for more detail.
57
A
B
C
Figure 2.2: A) Frequency of 11 Eastern spring peeper compound alleles (grey diamonds) and
Eastern cyt b mtDNA haplotypes (black circles) across the contact zone from 0km (Queen’s
University Biological Station near Kingston or QUBS, Ontario) to 580km (Point Pelee National
Park/Hillman Marsh); polynomial trend lines (solid = nDNA, dashed = mtDNA) over geographic
distance. Population (Pop) numbers below x-axis correspond to STRUCTURE analysis for
geographic locale comparison. B) Bar plot of admixture coefficients resulting from
STRUCTURE analysis with K = 2. Individuals are represented as vertical bars partitioned into
two segments the length of which is proportional to the estimated membership in the two clusters
(dark grey = Interior, and light grey = Eastern). Pie charts above show proportion of Interior or
Eastern cyt b haplotypes for each sampled locale. Admixture of lineages is present in sites 6
58
through 9; see Appendix, Table A1. C) STRUCTURE bar plot (K = 2) from HYBRIDLAB
simulations: Pure Interior and Easter, F1, F2, Backcross Interior, and Backcross Eastern.
59
A
B
Figure 2.3: Discriminant function analyses with 95% confidence ellipses of pure Eastern and
Interior individuals in allopatry (AE and AI) and sympatry (SE; no SI individuals were
identified), and hybrids of different haplotypes (HE and HI) for A) external morphology, and B)
male advertisement calls. Amount of canonical variation is represented on each axes; arrow
60
highlights the direction of displacement for pure Eastern males in sympatry (SE). Males with
Interior haplotypes are shown in dark grey circles and males with Eastern haplotypes are show in
light grey circles (solid lines for genetically pure, dashed lines for hybrid, individuals).
61
1
6
15
4
5
0.9
0.8
Proportion
0.7
0.6
E
I
0.5
0.4
0.3
0.2
0.1
0
AI
SI
H
SE
AE
Figure 2.4: Response from 2-choice phonotaxis experiments of Allopatric Interior (AI),
Allopatric Eastern (AE), Sympatric Interior (SI – no individuals found), Sympatric Eastern (SE),
and hybrids (H) diagnosed using STRUCTURE to Eastern (E) and Interior (I) stimuli. Sample
sizes are indicated at the top of each bar. Dark grey portions of bars represent preference for
Interior stimuli, light grey represents preference for Eastern stimuli.
62
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Chapter 3: Intraspecific post-zygotic isolation and tadpole competition
in Pseudacris crucifer
Kathryn A. Stewart and Stephen C. Lougheed
3.1 Abstract
The consequences of secondary contact among diverging lineages depend on hybrid fitness, and
may even help to strengthen pre-zygotic isolation barriers, called reinforcement. Identifying
naturally occurring examples of reinforcement selection has been challenging, and possibly
reflects difficulties in quantifying selection against heterospecific mating under biologically
relevant scenarios. Previous research suggests that two intraspecific mitochondrial lineages of
the spring peeper, Pseudacris crucifer, meet in secondary contact in Southwestern Ontario and
show a pattern of reproductive character displacement consistent with reinforcement. Our study
quantified early life-history fitness traits for pure lineage and hybrid tadpoles, including hatching
success, tadpole survival, size at metamorphosis, and development time. Results suggest that
lineages differ in tadpole survival and that hybrids have equal fitness or higher than average
mass at metamorphosis compared to pure parental crosses. However, apparent advantages or
similarities of hybrids compared to pure lineage tadpoles may disappear when tadpoles are raised
with competitors of different genetic constitutions. We observed gigantism, failure to
metamorphose, and bent appendages in some tadpoles from hybrid families. These results
provide further evidence for reinforcement in this spring peeper secondary contact zone, and
highlight the importance of testing hybrid fitness under biologically realistic scenarios.
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3.2 Introduction
Biological species are defined by reproductive isolation – the suite of mechanisms
(genetic, physiological, or behavioural) that maintain distinct gene pools by preventing the
production of viable or fertile offspring (Mayr 1963). Thus, understanding biological speciation
requires that we examine factors associated with the evolution of pre- and post-zygotic barriers
among natural populations. Most animal sister species begin as geographically isolated
conspecific populations (Barraclough & Vogler 2000; Coyne & Orr 2004; Fitzpatrick et al.
2009), with secondary contact between diverging populations potentially important in shaping
evolutionary trajectories (Servedio 2001). Reproductive barriers evolved in allopatry are not
always impermeable and may allow gene flow between lineages upon secondary contact, with a
variety of outcomes including: i, fusion or genetic assimilation where one population
predominates (Arnold & Hodges 1995; Burke & Arnold 2001); ii, formation of a stable ‘hybrid
swarm’ (Nielsen et al. 2003; Seehausen et al. 2003); or iii, reinforcement of species boundaries
(Rundle 2002; Geyer & Palumbi 2003; Schluter 2003). Thus, in contact zones where
hybridization is maladaptive (e.g. with post-zygotic barriers) natural selection may strengthen
incipient species-recognition systems, reducing the probability of heterospecific mating and unfit
hybrids, commonly referred to as reinforcement (Dobzhansky 1937; Blair 1955). Ultimately,
then, the outcome of secondary contact depends on hybrid fitness, highlighting the potential
importance of post-zygotic isolating barriers in the origins of biological species.
The study of reinforcement has a long contentious history with often polarizing debate
(Noor 1999, Servedio and Noor 2003, Coyne and Orr 2004). Controversies encompassed
discussions of the restrictive conditions under which reinforcement may occur in nature, and
presumed problems associated with the establishment of a stable sympatric zone between
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genetically compatible and ecologically overlapping species (Butlin 1987; Marshall et al. 2002;
Coyne and Orr 2004). Past criticisms were due, in part, to a deficiency of compelling empirical
examples (reviewed in Marshall et al. 2002; Coyne & Orr 2004) and may reflect challenges in
assessing specific costs of heterospecific matings (Coyne & Orr 2004).
In the reinforcement model, geographically isolated populations gradually accumulate an
array of mutations, be they beneficial, mildly deleterious, or neutral, as a correlated response to
genetic divergence and time (Mayr 1942). Upon secondary contact, hybrids between these two
diverging populations are less fit than their pure counterparts as a result of negative epistatic
interactions (Dobzhansky 1937; Muller 1940); such mutations are referred to as DobzhanskyMuller Incompatibilities. Post-zygotic incompatibilities may be intrinsic (inviability or sterility)
or extrinsic (ecological or behavioural/sexual dysfunction), and the magnitude and direction of
effects may occur differentially over the lifespan of an individual, favourable at one life stage
and detrimental in another (Parris et al. 1999; Coyne & Orr 2004; Lemmon & Lemmon 2010).
Complicating matters further, selection against hybridization is frequently not equal between
populations, often apparent in asymmetrical fitness costs (Parris et al. 1999; Pearson 2000; Tiffin
et al. 2001; Veen et al. 2001; Pfennig & Simovich 2002). Moreover, hybrids are not always unfit
(Arnold 1997), with growing evidence for the maintenance of stable hybrid zones (Slatkin 1973;
Endler 1977; Barton 1979; Mallet 1986; Brelsford & Irwin 2009), hybrid superiority over
parental individuals (May et al. 1975; Moore 1977), and even hybrid speciation (via
allopolyploidy – Wood et al. 2009; Mable et al. 2011; homoploid hybrid speciation – Welch &
Riesenberg 2002; Lai et al. 2006; Mallet 2007; Jiggins et al. 2008; Mavárez & Linares 2008;
Hegarty et al. 2009; Hermansen et al. 2011), further highlighting the need for hybrid
assessments.
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The spring peeper (Pseudacris crucifer), a North American treefrog from the family
Hylidae, is an excellent species to study the consequences of hybridization on the evolution of
reproductive isolation. Earlier work revealed a dynamic history of range fragmentation, range
expansion, and secondary contact among mitochondrial lineages across the species range (Austin
et al. 2002; 2004). In Southwestern Ontario, Canada, two such lineages (designated Eastern and
Interior), which began diverging approximately 3.5 million years ago, now meet in secondary
contact (Austin et al. 2002, 2004; Stewart et al. in review - Chapter 2). Stewart et al. (in review Chapter 2) found evidence of asymmetric reproductive character displacement in Eastern lineage
male vocalizations and female preferences and proposed that it is most likely a by-product of
reinforcement selection. They found non-coincident clines for nuclear compared to
mitochondrial genetic markers implying differences in hybrid fitness, while distinct morphology
and intermediate male calls of hybrids suggested detrimental fitness consequences of
hybridization (Stewart et al. in review – Chapter 2). While genetic evidence indicates
hybridization between lineages in nature, and provocative evidence of reproductive character
displacement, we lack the necessary data on fitness costs of hybridization and possible postzygotic isolation barriers to diagnose reinforcement. Here we conduct reciprocal hybrid crosses
between spring peepers from these diverging lineages, and quantify hybrid fitness.
Our study focuses on tadpole hybrid fitness from hatching to metamorphosis. Secondary
contact between these lineages probably occurred at most 15,000 years ago following the most
recent glacial retreat in the Northern Unites States and Southern Ontario (Austin et al. 2002). Our
objectives for this study were to: 1) Test for intrinsic post-zygotic barriers between two lineages
by assaying tadpole F1 hybrid viability and developmental rate; and (2) Simulate a more
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biologically relevant tadpole rearing experience through competition trials, raising tadpoles from
different genetic backgrounds together to attest whether competition diminishes hybrid fitness.
3.3 Materials and Methods
3.3.1 The Spring Peeper
Spring peepers are a well-characterized species with regard to reproductive ecology. Males can
be found in aggregations (choruses) in the spring where they set up small territories and actively
call to attract females. Females enter choruses once reproductively receptive, choose a male by
approaching him, touching him on the shoulder, and then permitting amplexus (Badger &
Netherton 2004). Females then swim with males attached and external fertilization ensues, with
eggs deposited on submerged vegetation (Wright & Wright 1995).
3.3.2 Population Sampling
We hand-collected males and females on two consecutive nights (April 10th and 11th,
2011) from allopatric populations of the two focal lineages, located at least 50 km from the zone
of secondary contact (Stewart et al. in review - Chapter 2) and transported them live to the
Animal Care Facility at the University of Guelph (located within the contact zone). Only males
actively advertising, and females either caught in amplexus or gravid (eggs visible within the
abdominal cavity) were used to ensure reproductive receptivity. Pure Eastern and Interior
allopatric populations were diagnosed using data from male advertisements calls, morphology,
mtDNA sequence, and nDNA microsatellite data (Austin et al. 2004; Stewart et al. in review –
Chapter 2).
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3.3.3 Hybrid Crosses
We conducted a fully reciprocal hybridization experiment by creating the following
crosses: Eastern females and males, Interior females and males, Interior males and Eastern
females, and Eastern males and Interior females. To minimize maternal affects, we used eggs
from the same females for both pure and inter-lineage crosses and nested sires within dams
(Parris 1999). For the pure Interior crosses, 5 Interior females were crossed with 6 Interior
males. These same Interior females were crossed with 6 Eastern males to create one class of F1
hybrids with Interior haplotypes (hereafter Interior hybrids). For the pure Eastern crosses, 6
Eastern females were crossed with 5 Eastern males. These same Eastern females were crossed to
6 Interior males to create a reciprocal F1 hybrid treatment with Eastern haplotypes (hereafter
Eastern hybrids). In total, we produced 6 pure Interior, 6 Interior hybrid, 6 pure Eastern, and 7
Eastern hybrid crosses (1 Eastern female was used twice with different males).
Females were injected with LHRH and held at 4C for 8 hours and then brought to room
temperature to induce ovulation (Silla 2011). Sperm suspensions were made by macerating a
testis from each male in a small volume of aged (de-chlorinated) tap water (Parris 1999). Crosses
were performed by stripping between 40 and 100 eggs from each female in sequence and
immersing them in the sperm suspension of the relevant male in a Petri dish. We allowed 10
minutes for fertilization, after which the sperm suspension was diluted, and eggs covered in more
aged tap water. Each male was used for two crosses only. Use of left and right testes was
randomized, as was order of egg stripping, and crosses (in terms of hybrid versus pure). All
fertilizations were performed within 3 hours of each other, well within a time period known to
produce equivalent levels of fertilization success in previous experiments involving Lithobates
blairi and Lithobates sphenocephala (Parris 1999; Parris 2004). At ambient temperature within
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the facility (23ºC), all larvae hatched within seven days at which point we calculated hatching
success from total number of eggs. Upon hatching, tadpoles were chosen haphazardly and
transferred to larger glass containers of aged tap water (2 L) of between 12-14 siblings, with each
cross type replicated twice. Containers were randomized and were exposed to a 16:8 hour light
cycle within the University of Guelph, Animal Care Facilities. Containers were re-arranged
every 7 days to minimize spatial affects, such as positioning near light sources (UV radiation
may reduce hatching success and tadpole development – see Blaustein et al. 1998; Blaustein et
al. 2001). Tadpoles were fed ad libitum boiled lettuce, and aged water was continually added to
maintain a volume of 2 L per container. Once tadpoles started to develop legs (Gosner Stage 3139; Gosner 1960), containers were angled at 15º to allow for a resting surface and covered with
mesh to preclude escape. Tadpoles no longer employing an aquatic feeding regime were
switched to wingless D. melanogaster. Full metamorphosis was considered to have been
achieved at Gosner Stage 45-46 (Gosner 1960), when tail is fully absorbed, at which time
tadpoles were removed from their containers, sacrificed by immersion in tricaine
methanesulfonate (MSS222), measured, and genotyped (see below). Minton (2001) noted that
spring peeper tadpoles require between 90-100 days for metamorphosis; we thus ended the
experiment at 100 days post-hatching.
3.3.4 Competition Trials
Newly-hatched tadpoles not used for the experiment above (and demonstrating no obvious
deformities), were used in competition trials. Here, groups of 5 full sibling tadpoles were paired
with 5 other (full-sibling) tadpoles from either another family with the same lineage (pure or
hybrid) or with 5 tadpoles from the opposite lineage (pure or hybrid) in a smaller (1 L) container.
In other words, sibling tadpoles from one pure Interior full cross were combined with tadpoles
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from either another pure Interior cross or from an Interior hybrid cross or with tadpoles from a
pure Eastern or Eastern hybrid cross, and so on. For these competition trials in total we had 2
containers with pure Interior and pure Eastern tadpoles, 1 container with pure Interior and
Eastern hybrids, 1 container with pure Interior and Interior hybrids, 1 container with pure Eastern
and Eastern hybrids, 1 container with pure Eastern and Interior hybrids, and 2 containers with
Interior hybrids and Eastern hybrids, and 1 container each of tadpoles from similar genetic
backgrounds but from different crosses (Eastern with Eastern, Interior with Interior, Eastern
hybrid with Eastern hybrid, and Interior hybrid with Interior hybrid). In total we set up and
followed 12 tadpole competition trials to metamorphosis. Rearing conditions were identical to
those outlined above.
3.3.5 Genotyping Tadpoles
Spring peeper adults and tadpoles from divergent lineages cannot be distinguished through
external morphology alone (Stewart et al. in review – Chapter 2). We thus collected individuals
as they died or metamorphosed (sacrificed metamorphs as above), and placed them in 95%
ethanol with individual tracking numbers and date for later genetic identification. Tadpole DNA
was extracted using a QIAGEN DNeasy kit (Mississauga, Ontario, Canada) and sequenced for a
portion of mtDNA cytochrome b (cyt b) using primers MVZ 15L and MVZ 18H (Moritz et al.
1992) as described in Stewart et al. (in review – Chapter 2). If tadpoles originated from a
competition container comprised of tadpoles with the same haplotype (Interior with Interior
hybrids, or Eastern with Eastern hybrids) we further distinguished individuals using 5
polymorphic microsatellites, in triplex (Pcru32, Prcu 10, Pcru 05) and duplex (Pcru 11, Pcru 21)
reactions according to the methods outlined in Stewart et al. (in review - Chapter 2).
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3.3.6 Tadpole Fitness Measurements
We measured a suite of fitness-related variables for each cross treatment (Interior, Eastern,
Interior hybrids, Eastern hybrids). Hatching success was determined as the proportion of tadpoles
hatched from total eggs added to a particular sperm suspension. Survival rate was the proportion
of tadpoles that hatched relative to those that survived to metamorphose. Upon metamorphosis,
we measured Snout-Vent-Length (SVL ±0.2 mm) using digital calipers and mass (±0.02 g) using
a digital scale (TR-2102, Denver Instrument Co., USA). Cumulative averages were tallied for
rate of metamorphosis post hatch date, and cumulative rate of mortality post hatch, per cross
treatment. For our competition trials we assessed survival rate to metamorphosis (genotyping
individuals within mixed containers as above).
3.3.7 Statistical Analysis
To test for the effect of genotype on P. crucifer tadpole fitness, we compared hatching
success (proportion of eggs hatched), survival to metamorphosis, mass (g) at metamorphosis, and
size (SVL in mm) at metamorphosis, by performing one-way Analyses of Variance (ANOVA).
Tadpole mortality and metamorphosis curves were compared using Log-Rank Mantel Cox tests,
and displayed as cumulative measures over time. Competition trial sample sizes hampered the
use of statistical tests and were used for anecdotal arguments only. All analyses were conducted
using JMP (version 10, SAS Institute Inc.).
3.4 Results
3.4.1 Tadpole Fitness
For all crosses combined, we obtained a total of 1957 eggs of which 1228 (62.75%) eggs
hatched, all hatching occurring within one day among all crosses in all treatments. Tadpole
hatching success was significantly greater for Pure Interior compared to Pure Eastern
84
individuals, with hybrids of both cytotypes displaying intermediate hatching success to their pure
counterparts (ANOVA F3,25=4.06, p=0.0194; see Figure 3.1A, Table 3.1). One pure Eastern
family cross completely failed and as such was not included in any other measure of tadpole
fitness, reflected diminished sample size for this treatment.
Mean survival rate was not significantly different among P. crucifer tadpoles (ANOVA
F3,24= 1.60, p=0.22; Figure 3.1B; Figure 3.2; Appendix Figure B1); pure tadpoles had a slightly
diminished mean survival to metamorphosis compared hybrids (Table 3.1). Time of tadpole
mortality however, was significantly earlier for pure Interior tadpoles compared to Eastern
tadpoles in their developmental period (Log-Rank 2=14.24, df=3, p=0.0026); hybrid mortality
was intermediate (Table 3.1; Figure 3.3).
Pure Eastern tadpoles metamorphosed, on average, a few days earlier than pure Interior
tadpoles, and hybrids metamorphosed at an intermediate time (Table 3.1; Figure 3.4; Appedix
Figure B2); there was no significant difference between mean time to metamorphose for
P.crucifer tadpoles of different genotypes (Log-Rank 2= 0.69, df=3, p=0.88).
Hybrids with Eastern haplotypes metamorphosed into adults at a significantly heavier
mass (F3,23=3.54, p=0.033) compared to pure Eastern, pure Interior, and hybrid individuals with
Interior haplotypes (Table 3.1; Figure 3.1C). SVL at metamorphosis, however, was not
significantly different among treatments (F3,23=1.89, p=0.164; Figure 3.1D; Table 3.1).
3.4.2 Tadpole Competition Trials
Although small sample sizes preclude formal statistics, we found that tadpoles raised in
mixed family groups exhibited higher mortalities compared to those raised in isolation. Pure
Interior and pure Eastern tadpoles raised together had 40% overall mortality, most of which were
pure Eastern tadpoles. Hybrids with Interior haplotypes had 100% mortality when raised with
85
pure Interior tadpoles but 100% survival when raised with pure Eastern tadpoles. Similarly,
hybrids with Eastern haplotypes showed 100% mortality when raised with pure Eastern tadpoles
and only 40% survival when raised with pure Interior tadpoles. Hybrids with Eastern haplotypes
also showed 100% mortality when raised with other hybrids with Interior haplotypes which
showed 100% survival (see Figure 3.5).
3.4.3 Body Deformities and Developmental Problems
After 100 days post-hatching, five tadpoles remained un-metamorphosed, four of which
were hybrids with Eastern haplotypes and the other was a pure Eastern tadpole (gigantism was
apparent but development remained stunted at Gosner Stage <30 with no limb development;
Appendix Figure B3). Bent tails causing tadpoles to swim in circles was also observed
(Appendix Figure B4), ranging from 10.2% in hybrids with Eastern haplotypes, 3% in hybrids
with Interior haplotypes, and 7% in pure Eastern individuals (pooled across families). Bent tails
did not seem to restrict metamorphosis or survival, but did compromise locomotion (pers. obs).
3.5 Discussion
Our study of tadpole fitness and the consequences of hybridization between two
diverging spring peeper lineages in Southwestern Ontario reveal four striking results: 1) We
found additional evidence of marked phenotypic divergence between Eastern and Interior
lineages in key aspects of tadpole survival. 2) Hybrids from both reciprocal crosses show equal,
and sometimes higher, tadpole fitness than pure lineage crosses. 3) Apparent hybrid advantage
may be countered by competition. 4) The costs of hybridization may be asymmetrical,
exaggerated in Eastern hybrids. Below we discuss these different findings in turn.
Representing two of the most recently diverged spring peeper lineages (Austin et al.
2002; 2004), the Eastern and Interior lineages differ significantly in adult morphology, in male
86
advertisement calls, and in female preference for male calls of their own lineage (Stewart et al. in
review – Chapter 2). We found significantly diminished hatching success for pure Eastern
lineage eggs compared to the fertilized eggs of any other treatments (Fig. 3.1B) and, although
there was no significant differences in survival to metamorphosis, those tadpoles that did die
showed mortality at a significantly later time in Eastern families.
Differences in survivorship between the two spring peeper lineages may in part be a byproduct of neutral processes. For instance, within the Ontario contact zone the Eastern lineage is
the furthest from its presumed glacial refugium (Austin et al. 2002; 2004), and protracted
northerly migration after glacial retreat may have involved sequential founder events and drift in
populations with small effective size. Erosion of genetic variability through sequential founder
effects during range expansion from glacial refugia and contemporary range fragmentation can
combine to diminish population genetic diversity (Garner et al. 2004; Ficetola et al. 2007). This
may be accentuated by inbreeding with consequent fitness reductions (Hitchings & Beebee
1998). For example, genetic diversity has been shown to affect hatching success and tadpole
fitness in R. latastei (Ficetola et al. 2007), and pre-metamorphic survival in Bufo bufo (Hitchings
& Beebee 1998).
Alternatively, differential hatching success among different environments may be a
consequence of past selection. For example, environmental temperature has been shown to have
important implications for geographic distribution, time of breeding, site and mode of egg
deposition, egg and clutch size, and embryo and larval temperature tolerance and development
rate among frog species (Moore 1939; Volpe 1953; Pettus & Angleton 1967; Zweifel 1968; Licht
1971; Anderson 1972; Brown 1973), and among conspecific populations (Moore 1949; Volpe
1955; Ruibal 1955; Brown 1967; Herreid & Kinney 1967). Additionally, habitat type and canopy
87
cover have been shown to relate to thermal tolerance of larval amphibians, both among and
within species (Freidenburg & Skelly 2004). Our laboratory crosses were conducted at ambient
temperatures of 23º C consistent with temperatures used by other researchers (Gossner &
Rossman 1960; Earl et al. 2012). However, spring peeper eggs and tadpoles in Ontario
populations, at least early in their development, experience a lower range of temperatures (P.
crucifer adults are found breeding in air and water temperatures that range from 3º to 20º C
(Mean ± SD, 10.86 º C ± 3.96) in Southwestern Ontario, Stewart, unpubl. data). Thus, we may
have inadvertently favoured individuals from our more southerly distributed lineage (Interior)
where water temperatures may be higher. This assertion requires further experimentation across
a range of developmental temperatures.
Pathogens (fungi and water molds) pose an additional risk to amphibian larva and can
cause deformities and elevated mortality (Blaustein et al. 1994; Czeczuga et al. 1998). Such
effects may also be exacerbated at higher water temperatures (Gomez-Mestre et al. 2006),
although adaptive behaviours such as oviposition site choice (Green 1999) or early hatching
(Warkentin et al. 2001; Wedekind 2002; Moreira & Barata 2005; Touchon et al. 2006) may
decrease exposure.
Much evidence to date demonstrates that abiotic (temperature, UB-B radiation,
hydroperiod, pH) and biotic (competition, predators, pathogens) factors can affect both
embryonic and tadpole development, even over relatively short evolutionary times (e.g. Skelly et
al. 2002). It is not unreasonable then to anticipate that 3.5 million years of divergence and
geographical isolation in different environments would have influenced phenotypes at various
life history stages, from ontogenetic trajectories shown here to adult mating behaviour (Stewart
et al. in review – Chapter 2).
88
Our study suggests that hybrid tadpoles between lineages have equal, and sometimes
higher, fitness than either pure Eastern or pure Interior tadpoles, when raised in isolation (Table
1), despite some hybrid tadpoles demonstrating gross morphological and developmental
deformities (Appendix Figure B3 & B4).
Investigating post-zygotic isolation requires that we quantify both extrinsic and intrinsic
factors that may impact hybrid fitness (Coyne & Orr 2004). Two hypotheses have been proposed
for maladaptive hybridization: 1) Disruption of co-adapted gene complexes or negative epistatic
interactions between parental genomes affects hybrid inviability or sterility regardless of
environmental context (but see Orr & Turelli 2001; Presgraves et al. 2003; Detmman et al.
2007), known as intrinsic post-zygotic isolation (Barton & Hewitt 1985, 1989); 2) Viability or
fertility is relative and varies among environments. This would be evident in intermediate values
for traits that are relevant to ecology or mate recognition systems – called extrinsic post-zygotic
isolation (Endler 1977; Harrison & Rand 1989; Parris 2001). Hybrid inviability evolves
gradually and empirical evidence suggests that few recently diverged taxa have completely
inviable F1 hybrids (Coyne & Orr 1989; Presgraves 2002; Coyne & Orr 2004). For example,
postzygotic isolation through hybrid inviability seems to not become complete until species have
been diverged for 2 to 3 million years for mammals (Prager & Wilson 1975), 10 to 20 million
years for fish (Bolnick & Near 2005; Stelkens et al. 2010) and 20 to 30 million years for birds
and frogs (Prager & Wilson 1975). Moreover, many genes responsible for intrinsic post-zygotic
isolation are partially recessive, and thus the costs of hybridization may be manifested in
subsequent generations (e.g. as F2 or backcrossed descendents - Coyne & Orr 2004). We only
investigated the consequences of hybridization on development from fertilization to
metamorphosis so cannot speak to this directly. Indeed hybrid sterility is suggested to evolve
89
more quickly than hybrid inviability in many groups (Coyne & Orr 1997; Presgraves 2002;
Coyne & Orr 2004). For example, Sasa and colleagues (1998) found that among 116 anuran taxa
surveyed, hybrid sterility evolved faster than inviability, and for reinforcement in the “broad
sense” may have equally strong fitness consequences (Servedio & Noor 2003). Certainly, to
accurately predict hybrid fitness we not only need to understand genetic compatibility but also
the conditions under which hybrid genotypes survive and reproduce (Parris 2001).
In contrast to intrinsic Dobzhansky-Muller genetic incompatibilities, extrinsic postzygotic isolation may arise quickly (Schluter 1993; Rundle et al. 2000; Kitano et al. 2007;
Hendry et al. 2009). We have reported some indirect evidence of this in that hybrids are distinct
morphologically and may disproportionately assume a satellite mating strategy (Stewart et al. –
Chapter 4) and also male calls of hybrids are intermediate in many aspects which may have
ramifications for territory formation and mating success (Stewart et al. in review – Chapter 2).
As one final observation we note that many of the lineages within spring peepers are much more
deeply diverged than those in our focal contact zone, and as such when in contact may reveal
more pronounced intrinsic and extrinsic costs of hybridization (see Austin et al. 2002, 2004;
Stewart et al. in review – Chapter 2).
Secondary contact formed across environmental gradients may result in hybrids that are
selectively favoured because they are better adapted than either parental genotype to intermediate
habitats within the ecotone, ultimately forming a stable hybrid zone (Barton & Hewitt 1985;
Hewitt 1988). This ‘bounded hybrid superiority’ hypothesis, or simply ‘hybrid superiority’
(Moore 1977; Moore & Price 1993) has received little empirical support (but see Moore &
Buchanan 1985; Grant & Grant 1992; Arnold 1997; Good et al. 2000) with the predominant view
being that hybridization is detrimental. Southwestern Ontario shows no evidence of any obvious
90
ecological transition across the hybrid zone (Stewart et al. in review – Chapter 2), and thus
hybrid niche partitioning seems unlikely. This hybrid zone however, does represent a relatively
disturbed habitat, and since habitat modification can alter the relative fitness of different
genotypes and the likelihood of hybridization (Arnold 1997; Ellstraind & Schierenheck 2000),
hybrids may be better able to use novel habitats in the absence of parental competition.
Fitness consequences to anuran hybridization may be manifested only under stressful
environments, for example through pathogenic infection (Parris 2004) or resource competition
(but see Semlitsch 1993; Pfennig et al. 2007). Indeed when raised in mixed rearing conditions
(competition trials), apparent spring peeper hybrid equality/superiority was mitigated. What’s
more, most hybrids raised with pure lineage individuals did not survive to metamorphosis,
suggesting competitive inferiority. Although only descriptive due to low sample size, the
competition trial results presented here illustrate a pivotal consideration when looking for postzygotic isolation. Studies highlighting the importance of inter- and intra- specific competition in
wild assemblages are numerous in community ecology, with a recent increase in incorporating
such aspects into anuran hybridization studies (Semlitsch 1993; Semlitsch et al. 1997; Parris
2001; Pfennig et al. 2007). Considering that tadpole competition can cause longer development
times, smaller mass at metamorphosis, reduced survival (Griffiths 1991; Blaustein & Margalit
1996), and even cannibalism (Pfennig et al. 1993), competition between frequently interacting
individuals should be considered when testing for consequences of hybridization. All previously
described populations within the Southwestern Ontario spring peeper contact zone contained
pure and hybrid individuals (Stewart et al. in review – Chapter 2) raising the possibility of
competition at all life-history levels. Although cannibalism was observed in tadpole rearing
91
trials, it is unclear whether cannibalism itself caused mortalities or whether this occurred postmortem. Since tadpoles were fed ad-libitum, starvation due to lack of food seems unlikely.
While kin recognition (Hamilton 1964) may enhance full-sibling growth by reducing
time and energy dedicated to aggressive interactions (Hokit et al. 1996), mixed rearing
conditions may intensify aggression through resource competition. Passive interference (Maurer
1984), where subordinate individuals actively avoid aggressive interactions (Kiesecker et al.
2001), may have been adopted by hybrid tadpoles ultimately affecting their survivorship.
Alternatively, direct interference through aggressive exclusion (Christian 1970), or the release of
growth inhibitors (Steinwascher 1978) by pure parental tadpoles may also cause elevated
mortality in hybrids. Direct interference is characteristically asymmetrical, utilized by larger
individuals (Weiner 1990); however, due to size limitations of newly hatched spring peeper
tadpoles (approximately 2mm, Stewart, unpubl. data) we were unable to measure size among
tadpoles from various crosses in this experiment. If mass at metamorphosis is correlated with
tadpole size, and tadpole size relates to competitive ability, than we would have expected hybrids
with Eastern haplotypes to fare better in competition, yet these individuals were observed to
almost always die compared to their competitors. In fact, even hybrids with Interior haplotypes
exhibited greater survival than hybrid with Eastern haplotypes. Such asymmetries to the cost of
hybridization have been previously suggested through genetic evidence, and a size decrease in
female hybrids with Eastern haplotypes (Stewart et al. in review – Chapter 2).
Disentangling intrinsic versus extrinsic post-zygotic isolation can be difficult. Here
hybrid mortality at the tadpole stage may either stem from intermediate phenotypes being unable
to compete for resources or withstand aggressive interactions, or reduced feeding efficiency.
Alternatively, mortality may simply reflect an accumulation of developmental abnormalities or
92
reduced vigour caused by genetic incompatibilities (Turelli et al. 2001). Clearly future studies
are warranted to identify of the nature of tadpole competition and causes of mortality in this
system.
3.6 Conclusion
While reinforcement is a possible outcome whenever diverging lineages capable of
hybridizing come into contact, uncertainty as to its geographic and taxonomic pervasiveness
remains. Our study shows that hybrids between two spring peeper lineages exhibit equivalent, if
not better, survivorship and growth when raised in isolation; however such apparent fitness
benefits may be countered by competition. Previous research suggests reproductive character
displacement between these two lineages, with reinforcement evident in genetic signatures of
asymmetrical introgression. Mortalities at the tadpole stage of hybrid individuals further lends
support to reinforcement possibly through either extrinsic (competitive ability), or instrinsic
(gigantism and deformities) post-zygotic isolation mechanisms. We show divergence between
Eastern and Interior lineages of the spring peeper in tadpole survival, suggesting differentiation
between these two lineages affects attributes across the life cycle. While further work is needed,
our study exemplifies how important it is to incorporate ecologically relevant information when
assessing hybrid fitness. Indeed laboratory conditions themselves may contribute to
underestimations of hybrid inviability, lacking important aspects of the natural context of wild
populations (Kozak et al. 2012). Although we demonstrate only moderate post-zygotic isolation
via hybrid inviability, extrinsic, context-dependent, post-zygotic isolation mechanisms may have
equally strong fitness consequences helping to reinforce species boundaries.
93
3.7 Acknowledgments
We thank funding agencies NSERC (KAS & SCL), and SSE Rosemary Grant Award for
Graduate Student Research (KAS), OMNR for scientific collection permits, Queen’s University
Animal Care (UACC Lougheed 2008-059-R3), University of Guelph Animal Utilization
Protocol (05R054). We also thank Long Point Waterfowl Research and Education Centre for
field accommodation. We especially thank Jim Bogart for assistance in experimental design and
husbandry protocols, and Cam Hudson for assistance in tadpole rearing. We are also grateful to
our field assistants including Ahdia Hassan and Rachel Wang.
94
3.8 Tables and Figures
Table 3.1: A summary of all P. crucifer tadpole fitness measures (mean ± standard error) across
treatments. Sample size (number of families) per treatment are 6, 6, 7, 6 for I, E, HE, HI
(respectively) unless otherwise noted. Letters in superscript indicate significant differences
between groups (p<0.05).
Hatching
Success
(%)
A
82.7
± 3.87
*B40.3
± 10.23
AB
60
± 9.26
Survival to
Metamorph.
(%)
60.1
± 13.50
56.5
± 13.51
82.4
± 6.07
Interior
(I)
Eastern
(E)
Eastern
Hybrids
(HE)
Interior AB69.3
81.2
Hybrids ± 9.61
± 4.51
(HI)
*denotes sample size of 7 families.
Mean Time
of Mortality
(days)
B
13.03
± 4.2
A
53.1
± 5.6
B
13.12
± 1.5
Mean Time of
Metamorph.
(days)
54.0
± 3.1
49.34
± 3.8
51.1
± 3.3
Mass at
Metamorph.
(g)
AB
0.11
± 0.008
B
0.096
± 0.008
A
0.13
± 0.007
SVL at
Metamorph.
(mm)
10.81
± 0.32
10.9
± 0.032
11.1
± 0.3
B
52.3
± 2.2
AB
10.6
± 0.35
16.4
± 1.2
95
0.11
± 0.009
A
B
D
C
Figure 3.1: Boxplots with standard deviation bars for pure Eastern (E), hybrids with Eastern
haplotypes (HE), hybrids with Interior haplotypes (HI) and pure Interior (I) tadpoles from invitro crosses (n=6, 7, 6, 6 crosses per treatment, respectively) A) Mean hatching success (%), B)
Mean survival to metamorphosis (%), C) Mass at metamorphosis (g), and D) Snout-Vent-Length
at metamorphosis (mm). Letters above boxplots denote grouping according to Tukey-Kramer
HSD test. Dashed line represents grand mean across all families.
96
100
Percent Cumulative Survival
90
80
70
60
50
40
I
30
E
20
HI
10
HE
0
7
13
26
49
55
61
76
87
Days Post-Hatch
Figure 3.2: Cumulative survival of spring peeper tadpoles post-hatch (Day 0) from different
genetic lineages. Families were pooled by treatment types (n=6, 6, 7, 6 for I, E, HE, and HI,
respectively). Other details as in Figure 3.1.
97
Cumulative Prop. of Total Mortality
0.45
0.4
0.35
0.3
Interior
0.25
Eastern
0.2
HE
0.15
HI
0.1
0.05
0
6
14
26
33
39
47
52
58
63
70
80
Days Post-Hatch
Figure 3.3: Cumulative proportion of total mortality for P. crucifer tadpoles hatched (Day 0)
tadpoles from different genetic lineages. Families were pooled by treatment types (n=6, 6, 7, 6
for I, E, HE, and HI, respectively). Other details as in Figure 3.1.
.
98
1
Prop. of Cumulative Metamorphs
0.9
0.8
0.7
0.6
Interior
0.5
Eastern
0.4
HE
HI
0.3
0.2
0.1
0
37
42
47
52
57
63
70
79
Days Post-Hatch
Figure 3.4: Cumulative metamorphosis of spring peeper hatched (day 0) tadpoles from different
cross treatments. Families were pooled by treatment type (n = 6, 6, 7, 6, for I, E, HE, and HI,
respectively). Other details as in Figure 3.1.
99
Survival (%)
Figure 3.5: Pseudacris crucifer tadpole average survival (%) for Pure Interior (I), Pure Eastern
(E), Hybrids with Interior haplotypes (HI), and Hybrids with Eastern haplotypes (I, E, HI, HE,
respectively) when raised in mixed groups. Treatments (mixed rearing groups) denoted on Xaxis.
100
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Chapter 4: Satellite male mating behaviour as a mechanism for
lower fitness matings in a hybrid zone
Kathryn A. Stewart, Cameron M. Hudson, and Stephen C. Lougheed.
4.1 Abstract
Reproductive isolation via character displacement and divergence in species’ mate recognition
systems are precursors to major models of speciation. In secondary contact populations,
however, alternative male mating strategies may allow poorer quality individuals to gain
reproductive fitness while also facilitating introgression by circumventing female choice. In
particular, maladapted hybrids may disproportionately adopt a satellite (non-calling) tactic. To
test this hypothesis, we genotyped and measured the morphology of 80 male Pseudacris crucifer
individuals involved in caller-satellite associations from a secondary contact zone between two
deeply diverged (3.75 mya) intraspecific lineages (Eastern and Interior) in Southwestern Ontario.
Across populations, body size, but not condition, was the best predictor of mating tactics, with
satellites physically smaller than callers. Within the contact zone, pure Eastern and Eastern
backcrossed individuals showed a significantly greater probability of being callers than satellites,
while hybrids with Interior haplotypes were more likely to adopt satellite tactics; hybrids with
Eastern haplotypes were equally likely to implement either mating tactic. We suggest that
satellite behaviour in P. crucifer promotes hybridization, contributing to asymmetrical
introgression in this secondary contact zone.
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4.2 Introduction
Divergence in mate recognition systems is the foundation of biological speciation, and
understanding the evolution of reproductive isolation and barriers to gene flow has been the
major focus of speciation research for decades [1, 2]. The ability to identify and select a mate
from a pool of potential suitors has profound implications for the process of speciation [3]. In
practise, mate choice requires the simultaneous evaluation of two pieces of information, species
identity and mate quality [4]. Thus, many argue that sexually selected traits are important for
species diversification in both animals [5, 6] and plants [7, 8].
Most sexual ornaments impose great energetic demand and predation risk on their
bearers; thus the development and maintenance of energetically expensive ornaments or
behaviours should serve to honestly indicate male quality, a theory referred to as the ‘handicap
principle’ [9, 10]. In short, males increase their reproductive fitness by attracting and mating
with females, while females attempt to choose mates which are of the highest quality as they
often incur the greatest penalty of mating with a poor quality mate or failing to recognize a
heterospecific [11, 12].
Honest advertisements have been shown across an array of taxa [13, 14, 15] and may be
used by either sex to assess the quality of advertisers. However, many animal species exhibit
behaviours that, at first glance, appear to operate contrary to the expectations of sexual selection.
Although instances of an alternative male mating strategy (AMMS) often exist at lower
frequencies [16], they have been found across taxa (e.g. birds, [17]; mammals, [18]; fish, [19];
insects, [20]; reptiles, [21]; and amphibians, [22]).
While AMMS may not yield equivalent payoffs to active advertisement, they may allow
disadvantaged competitors to obtain subsidiary fitness benefits. For example, in some fish,
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‘satellite’ (female mimicry) and ‘sneaker’ (peripheral spawning) behaviour increases male
reproductive success [23]. Fitness benefits to male cuckoldry, and females acceptance of extrapair-paternity has also been widely demonstrated across bird species (reviewed in [24]). Similar
tactics can also be observed in acoustically advertising species, such as insects or anurans. Here,
‘satellite’ males typically position themselves near an advertising male, remain silent, and
attempt to intercept females which are attracted to the advertiser [20, 25]. Advertising males
often produce aggressive calls or engage in physical combat with interlopers on their territory;
however satellite males may be ignored due to their submissive posture or lack of vocalization
[26]. Though this behaviour has been observed in many species exhibiting a range of population
dynamics and life histories, in many instances the underlying mechanism that causes males to
adopt this strategy is unknown.
In anurans, it has been widely accepted that key information may be assembled by
females when assessing a male’s call during mating opportunities, such as species identity and
mate quality [27]. Evidence suggests female preference is primarily mediated by assessment of
various male advertisement call parameters such as intensity, duration, dominant frequency and
repetition rate (review in [28, 29], accurately reflecting male size, age, or quality [30, 31].
Results from female choice experiments have indeed confirmed size-based mating success for
various species [29], suggesting assortative mating should be either size and/or species
dependent. Thus low quality (small or unattractive) males may adopt satellite tactics as a means
of circumventing female choice. Since AMMS has strong repercussions for mate choice, and
conflicts between mate and quality recognition have been observed in numerous anuran species
[32, 33, 34], it is surprising how few studies have looked at the implications of AMMS for
biological speciation.
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Indeed pre-zygotic reproductive mechanisms, such as acoustic signals and mating
preferences for them, can serve as strong behavioural barriers in heterospecific interactions,
potentially leading to effective isolation when species come into secondary contact. The relative
strength of pre-zygotic isolation barriers has been argued as more important or even critical in
impeding introgression, often because they are purported to evolve earlier and are individually
stronger than post-zygotic barriers (e.g., [2, 35, 36, 37, 38]). Despite this, we often encounter
hybrid zones and gene flow across species boundaries, suggesting that many barriers are not
impermeable.
Recent evidence suggests that hybridization occurs in at least 25% of plants and 10% of
animals in nature [39]. Moreover, some have argued that between 40-70% of plant species [40]
and numerous animal species [41] have hybrid origins. Thus, irrespective of the exact
proportions of taxa with hybrid origins, apparently “good” biological species do exchange genes
to some extent [42, 43]. Differences in taxon-specific rates of hybridization are usually suggested
to be attributable to genetic or developmental mechanisms, ignoring the importance of
behavioural decisions to mate in sympatry [39]. While patterns of introgression can certainly be
attributed to population dynamics, ecological factors, or failures in species recognition,
individual mating behaviour can have equally strong implications [33]. The ability for AMMS to
contribute to introgression, slow down or impede the process of speciation, and even increase the
probability of extinction, has scarcely been considered; however recent theoretical work
highlights the importance of AMMS in speciation studies [44].
4.2.1 The spring peeper, Pseudacris crucifer
The spring peeper (Pseudacris crucifer) is a hylid tree frog that is broadly distributed in
eastern North America. Its genealogical history involves Pliocene and Pleistocene range
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fragmentation that produced a series of distinct evolutionary lineages, subsets of which are now
in secondary contact [45]. Southwestern Ontario represents one such zone of secondary contact
between two mitochondrial lineages that began to diverge at least 3.75 million years ago [46,
47]. Genetic patterns, and evidence from phonotaxis experiments, suggest that natural selection
against maladapted hybrids may reinforce reproductive isolation [47]. Moreover, within the
contact zone, male hybrids exhibit distinct vocalisations compared to their pure lineage
competitors, suggesting vocalizations could act as a cue for females to identify genetic
backgrounds [47].
Spring peepers are typically found in large aggregations in spring (late April and May in
southern Ontario) where males occupy small territories [48] and advertise using a persistent and
energetically expensive call [49]. Calling males are occasionally accompanied by non-calling
satellite males, which potentially intercept females as they swim towards the advertising suitor
without incurring the cost of displaying or territory defense [50]. In calling assemblages across
the spring peeper range, satellite behaviour is common and found at frequencies between 2 to
14%, depending on population densities [51]. Previous studies of phenotypic or fitness correlates
of satellite behaviour in P. crucifer have produced conflicting results. For example, Forester and
Lykens [51] found that satellite males were significantly smaller than the calling males that they
were found with, implying that satellite behaviour was undertaken by younger or poorer quality
males. However, Lance and Wells [50] found no significant body size or body condition
difference between individuals that adopt a satellite or calling tactic.
The juxtaposition of hybrid zones and satellite behaviour in the spring peeper system
presents a compelling opportunity to investigate factors implicated in the process of speciation,
particularly aspects of behavioural isolation in facilitating introgression. Here we hypothesize
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that: 1) mating tactics will be age- or condition-dependent, where satellites are smaller and/or in
poor condition than their calling counterparts; 2) that females will show preference for males that
are larger and of similar genetic background; and 3) that hybrids will disproportionately adopt a
satellite mating tactic to circumvent female choice and increase their fitness.
4.3 Methods
4.3.1 Specimen collection
In 2009 and 2010, 80 (40 dyad) male Pseudacris crucifer individuals involved in callersatellite associations were captured across a secondary contact zone in Southwestern Ontario
(Figure 4.1; Appendix Table C1). Dyads were designated as comprising vocalizing males with a
non-calling male within 20 centimetres of their perch site. Dyads were observed for 5 minutes to
confirm that the satellite individual was not vocalizing, rather than trying to usurp the calling
male’s perch site. From 2009-2011 we also collected 28 amplexed (mating) pairs to test for
assortative mating between size and lineage. Mating pairs in amplexus were separated and
measured individually. Upon capture, we measured 7 morphological traits for each individual:
snout-vent length (SVL), head width (HW), radioulna, tibiofibula, femur, and foot length using
digital callipers (± 0.2mm), and mass using a 10g Pesola scale (± 0.02g). To quantify
repeatability of measurements between individuals and the utility of each measurement for
subsequent statistical analyses, we measured 10 individuals three times following the protocol of
Yezerinac et al. [52]. Individuals were measured blindly in a two-person measuring team (i.e.
one person handling frog ID while another person measures) to eliminate biases. All
measurements were taken by the same observer, to minimize inter-observer bias. Following
measurements, we toe clipped each individual and stored these tissues in 95% ethanol for
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subsequent genetic analysis (see below). Sampling took place after dusk from 19.00 to 02.00hrs
(local time) during peak chorus activity between the months of April and June.
We also included 348 reproductively active spring peeper adults sampled from 20082011 within 14 Southwestern Ontario populations (characterised as allopatric Eastern and
Interior, or sympatric) for genetic analysis, assisting in the identification of hybrid versus pure
individuals in our caller-satellite dyads and to help characterise morphometric variation (see
[47], Figure 4.1). All data have been archived and can be retrieved at the Queen’s University
Biological Station Data Archive ([53]; http://www.qubsdata.org/datasets/herp.php).
4.3.2 Genotyping and testing for hybridization
Each individual was sequenced for a 692 bp segment of the mitochondrial gene
cytochrome b (cyt b), and genotyped for eleven previously published [47, 54] microsatellite loci
according to the protocol outlined in [47].
To provide a means to accurately identify hybrids, we used genotype data from 428
reproductively active spring peeper adults from 14 Southwestern Ontario populations, dyads
included (see [47], Figure 4.1). We used the assignment program STRUCTURE version 2.3.3
[55] to distinguish between mixed versus pure lineage individuals. This program uses a Markov
chain Monte Carlo algorithm to infer population structure by assigning individuals to clusters,
assuming Hardy-Weinberg equilibrium and linkage equilibrium within clusters, and identifying
hybrids based on allele frequencies [55]. Following Stewart and colleagues [47] we set a burn-in
of 2.5x105 steps followed by 5x105 iterations. STRUCTURE was run assuming admixture,
correlated allele frequencies, and no prior population information. Output from STRUCTURE
was then entered into Structure Harvester version 0.6.91 [56], which implements the Evanno et
al. [57] method for identifying the most likely value of K clusters. Based on STRUCTURE
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(K=2) and HYBIRDLAB version 1.0 [58] simulations within this contact zone, individuals were
designated as hybrids (H) if 0.15<q>0.85. Individuals with q-values between 0.0 and 0.15
represent pure Eastern (E), and individuals with q-values between 0.85 and 1.0 represented pure
Interior (I) individuals [see 47 for justification].
4.3.3 Statistical analysis
Statistical analyses were performed using JMP Version 10 (SAS Institute Inc., Cary NC).
We first did separate Model II nested ANOVAs for each variable for the subset of individuals for
which we had repeated measurements. The percentage of variation among individuals that is
attributable to measurement error was determined. We then performed a principal component
analysis (PCA) based on a correlation matrix of the seven morphological measurements for all
individuals from our dyads and those from our broader survey of the contact zone (N=428). This
was done to derive independent, orthogonal multivariate axes that best summarize morphometric
variation. We also took the residuals from a regression of body mass on a linear measure of body
length (SVL) across all individuals. We use body condition as a measure of energetic reserves
accounting for structural size wherein positive residuals depict individuals in better body
condition than those with negative residuals [59]. We used logistical regressions of mating tactic
(caller versus satellite) on body size and body condition. Since we predict satellites to be
comparatively smaller than their calling counterparts as found in previous studies, we did a onetailed paired t-test on body size and body condition using all dyads. Analyses of Variance were
used to test for difference in body size and body condition among males of difference genetic
backgrounds within the zone of sympatry.
We used one-tailed 2 tests to ascertain whether pure males were more likely to be
callers, or conversely, if hybrids were disproportionately satellites. Combined genetic categories
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(mtDNA and nuclear DNA) were tested using a one-tailed Cochran Armitage Trend Test
followed by a correspondence analysis. We further checked for pairing associations between
dyads using 2 tests.
We used a linear regression of females and their chosen males captured in amplexus to
test for positive assortative mating for both body size and body condition. Finally, we performed
a Fisher’s Exact test (due to small sample size) evaluating if females within the zone of
secondary contact were more or less likely to mate with males sharing the same genetic
background.
4.4 Results
4.4.1 Size dependent mating tactics
Estimates of measurement error, expressed as a percentage of total among individual
variation, were all less than 6% (Appendix Table C2) suggesting that such error would have a
negligible impact on our morphometric analyses. Results from our PCA of 7 morphometric
parameters showed that Principal Component Axis 1 accounted for most of the variation
(61.45%) with an Eigen value of 4.3 (no other axes values over 1). PC1, hereafter referred to as
‘body size’, had positive loadings for all morphometric variables (mass 0.791, SVL 0.819, HW
0.741, radioulna 0.675, femur 0.793, tibia 0.832, foot 0.825). All subsequent analyses requiring
estimates of body size use individual scores from PC1. Regression of body mass on SVL was
highly significant (R2=0.52, F428=467.08, p<0.001), and residuals from this were used as our
proxy of body condition.
Results from a logistical regression using individuals from all populations showed that
body size (2=15.73, df=1, p<0.0001), but not body condition (2=0.028, df=1, p=0.87), was the
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best predictor of mating tactics, with satellites having significantly smaller body size than callers
(Table 4.1). In sympatric populations alone, body size (2=15.88, df=1, p<0.0001) remained the
best predictor of mating tactic while body condition was marginally insignificant (2=2.83, df=1,
p=0.093), again with satellites having significantly smaller body size (Table 4.1). Satellites were
also significantly smaller than the caller they paired with based on our multivariate measure of
body size (Paired t=5.61, df=39, p<0.001), but again there was no significant difference in body
condition (Paired t=0.79, df=39, p=0.43). Similarly, in sympatry, satellites were smaller than
their calling counterpart (Paired t=4.83, df=24, p<0.001), but again show no significant
difference in body condition (Paired t=1.86, df=24, p=0.75). Moreover, we found no significant
difference in either body size (F5,46=0.24, p=0.94) or body condition (F5,46=0.99, p= 0.44) based
on the genotype (E, HE, HI, BE – for definitions see below) of a male within the zone of
secondary contact.
4.4.2 Contact zone mating tactics
Within the zone of secondary contact we found twice as many individuals with Interior
(I) mtDNA haplotypes (N=32) as Eastern (E) mtDNA haplotypes (N=16) in caller-satellite
dyads. STRUCTURE analysis revealed no pure I individuals, but did identify six pure E
individuals, ten putative F1 hybrids with Eastern haplotypes (HE), twenty-five putative F1
hybrids with Interior haplotypes (HI), and six individuals with Interior haplotypes but with a
greater proportion of Eastern nuclear alleles representing putative F2 offspring of an HI
backcross to Eastern individuals (BE).
Based on mtDNA alone, individuals of neither mitochondrial lineage were significantly
more likely to assume a calling or satellite tactic (21,48=1.51, p=0.22); however, analyses based
on nuclear genotypic designations suggest pure Eastern individuals are more likely to assume a
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caller tactic compared to hybrids (21,48=3.84, df=1, p=0.05). When genetic information is
combined (mtDNA and nuclear DNA), E and BE individuals show significant greater probability
of being callers than satellites (Cochran Armitage Trend Test, one-tail: Z=1.79, p=0.037; Table
4.2). Correspondence analysis further confirms E and BE individuals were more likely to assume
a calling strategy, while HI individuals are more likely to adopt a satellite tactic; HE individuals
are equally likely to assume either mating tactic (Figure 4.2).
Comparing genotypes of dyads, HI individuals were more likely to pair with other HI
males, but HI satellites were found in close to 50% of all other associated pairings (E, BE and
HE) within sympatric populations. HE were equally likely to pair with BE or other HE,
sometimes satellite E individuals, but never HI. Eastern individuals were almost never found as
satellites, with the exception of one individual that paired with another E male, although pairing
associations were not significant (2=13.52, df=9, p=0.14; Table 4.3).
4.4.3 Successful amplexus
Across all populations, females were significantly larger (PC1; t=4.29, df=394, p<0.001)
and showed significantly better body condition than males (t=2.69, df=439, p=0.007). We found
positive correlations between body size (PC1; R2=0.14, F24=3.16, p=0.092) and body condition
(R2=0.24, F247.1, p=0.014) of amplexed males and females, although only the latter was
significant. Females within the secondary contact zone were not significantly more likely to mate
with a male sharing the same mtDNA (Fisher’s exact test, 2-tail p=1.00). HE and HI females
were also not significantly more likely to mate with BE, HE or HI males (2=3.33, df=3,
p=0.77).
123
4.5 Discussion
Satellite behaviour has been documented extensively in frogs [25, 26, 50, 51, 60, 61, 62,
63] and is generally considered to not be genetically determined [22]. Two non-mutually
exclusive hypotheses have been traditionally proposed to account for this type of AMMS: 1) A
condition dependent tactic expressed by unattractive, energetically depleted males, and/or 2) agedependent behaviour employed by younger, inexperienced males (discussed below).
In frogs and toads the energetic cost of vocal production and active advertisement is
typically quite high [64], and mating success / amplexus is positively correlated with calling
persistence, chorus tenure, and body size [65]. While condition-dependence seems a plausible
explanation, some studies have found no difference in body condition [25, 50, 66], implying
energetic constraints may not be the main determinant for AMMS. Here too, we found no
evidence to suggest that body condition (as we measured it) had any influence on mating tactics
of male spring peepers, either across all populations or within the zone of secondary contact
alone.
Alternatively, younger, potentially smaller individuals may adopt a satellite tactic more
frequently compared to older / larger males [60, 66, 67]. Size for taxa with indeterminate growth
like frogs is often linked to age [68], and smaller males may have less-attractive calls because of
size-dependent call features such as frequency or amplitude [69]; however, this has not been
shown in all species [61]. Assuming individuals can switch tactics as they get older, satellite
behaviour may initially be employed by juveniles who later adopt an advertising tactic with age.
Similar to Forester and Lykens [51], we demonstrate that on average smaller spring
peeper males engage in satellite tactics across all populations, and in sympatric populations
alone. We also show that satellite males are invariably smaller than their calling counterpart. In
124
instances where males commonly engage in physical combat to defend territories from rival
males, as observed in Lithobates catesbeiana, larger presumably older males typically win
contests for calling sites [60]. Older males may also achieve mating success more quickly than
younger, inexperienced males as has been found in Pelobates fuscus [70]. Therefore, ontogenetic
limitations may mean that there is a continuous pool of smaller, younger individuals for which
satellite behaviour is the optimum strategy to enhance the chance of mating irrespective of
population dynamics.
A far less explored explanation for AMMS posits that differences in development or
growth trajectories affect behaviour or adult phenotype [71]. In this scenario, size may not reflect
condition or age, per se [61]. Rather, satellite spring peepers may be individuals that have
physiological constraints to active advertisement. For example, recent studies on the Puerto
Rican coqui frog (Eleutherodactylus coqui) show that satellite males can be produced in the
laboratory by activating the serotonergic system; a system known to influence social and
aggressive behaviours [26]. Similarly, male Woodhouse’s toads (Anaxyrus woodhousii)
exhibiting satellite behaviour in the wild had elevated levels of circulating corticosterone, a
hormone indicative of stress [72]. Leary and colleagues [61] also observed slower growth rates
in male toads engaging in satellite behaviour and suggested that males were restricted from
switching between tactics due to physiological constraints. Thus, differences in intrinsic
attributes of individuals, such as hormonal regulation, physiology, or genetic quality may explain
the likelihood of employing a particular mating tactic. In fact, previous research illustrates
raising hybrid tadpoles in potentially stressful, inter-familial competition conditions drastically
lowers survivorship (Chapter 3), suggesting stress may play a pivotal role for hybrid fitness.
125
4.5.1 Alternative mating tactics within the contact zone
Theory suggests that genetic incompatibilities between parental types may cause reduced
fitness within individuals of mixed ancestry through intrinsic (physiological inferiority) or
extrinsic (behavioural) barriers to reproductive success (reviewed in [2, 73]). As we have shown
that females strongly prefer the distinct advertisement calls of pure males of their own lineage in
P. crucifer [47], hybrids may be at a disadvantage if they were to compete directly with pure
lineage males; thus, satellite behaviour may allow hybrids to circumvent this limitation and
achieve at least some mating success.
Here we show HI spring peepers were more likely to adopt a satellite tactic within the
Southwestern Ontario zone of secondary contact, whereas E males almost always engaged in
active calling. While size obviously predicts, in part, whether a particular male will adopt
satellite behaviour, we show that hybrids status too plays an important role, with no observable
size or condition difference between males of divergent genetic backgrounds. Satellite behaviour
in spring peepers may thus act as a conduit for gene exchange across this hybrid zone, resulting
in gene flow regardless of apparently strong prezygotic preferences mediated by female choice
[47].
Supporting our contention was the fact that successful amplexus with females was
correlated to male body size and body condition, but assortative mating based on genotype was
not evident. E and HE females showed strong pre-mating preference for males of their own
parental lineage [47]. Thus, absence of evidence for assortative mating with respect to
mitochondrial lineage implies successful interception by satellite individuals with dissimilar
genetic composition, ultimately facilitating ‘leaky’ reproductive barriers.
126
Recent modelling suggests AMMS can increase introgression even when assortative
mating is high and/or prezygotic isolating mechanisms remain intact [44]. Satellite behaviour has
been proposed to be the mechanism for directional introgression within hybrid populations of
two frog species, Hyla cinerea and Hyla gratiosa [74]; however this hypothesis was not
explicitly tested. Moreover, Garant et al. [75] showed that some Atlantic salmon (Salmo salar L.)
wild/farm hybrids actually have higher reproductive success achieved through AMMS than their
pure counterparts, enhancing possibilities for backcrossing and introgression. This evidence
together implies AMMS may have far reaching repercussions for the process of speciation and
consequences to secondary contact.
Although not significant, we did observe a trend for males of similar genetic background
to pair with each other in caller-satellite dyads. For example, E, HE, and BE individuals were
always found in dyads, but never found to act as a satellite with HI individuals. Similarly HI
males were found as satellites with other HI individuals at greater than predicted frequencies
compared to any other male type, suggesting possible self-referent phenotype (reflecting
genotype) matching, similarly found in other non-anuran systems [76 and references therein].
Preferential clustering of males with conspecifics over heterospecifics has also been shown in
spadefoot toads (S. multiplicata; [77]) and may help to further facilitate reproductive isolation
through spatial segregation.
4.6 Conclusion
Limitations to introgression across hybrid zones have traditionally been explained by
genetic or developmental impediments [39]. For example, the selection-linkage hypothesis
suggests some immigrant loci are linked to deleterious alleles and thus are subject to indirect
127
selection, while other unlinked markers, such as mitochondrial or chloroplast DNA genes,
remain relatively free for introgression [78, 79]. Adaptive superiority of one species’ organellar
genome (i.e. ‘mitochondrial catch’ [80]), or positively selected nuclear genes may also enhance
introgression [81]. Sex-biased dispersal [82] or frequency-dependent reproductive isolation
barriers [43] can also cause biased, or uni-directional, introgression. Finally, Haldane’s Rule has
frequently been invoked to account for differential introgression across species boundaries [83].
We present evidence that differences in individual mating behaviour can also have significant
impacts on hybridization and introgression. Despite the pervasiveness of satellite behaviour in
anurans [22], to our knowledge only one previous study has implied AMMS as a means for gene
flow across species boundaries [74]. We hope that our study prompts further exploration of this
phenomenon and its contribution to leaky reproductive isolation barriers.
4.7 Acknowledgements
Funding was provided by the Natural Sciences and Engineering Research Council of
Canada through a Discovery grant to (SCL) and scholarship to KAS, Sigma Xi Grant-in-aid of
Research (KAS), the SSE Rosemary Grant Award (KAS), and Queen’s University Summer
Work Experience Program (CMH). The Ontario Ministry of Natural Resources, Parks Canada,
and Long Point Waterfowl provided permits, park access, and accommodations during field
seasons. We thank Ahdia Hassan, Caroline Jamison, Surbhi Jain, and Ayden Sherritt for
assistance in the field.
128
4.8 Tables and Figures
Table 4.1: Mean ± SE of body size (PC1) and body condition (residuals from a linear regression
of mass to SVL) variables (see Methods for details) between male spring peepers in
Southwestern Ontario contact zone based on mating tactic, caller or satellite. ‘Pop’ represents
callers and satellites sampled throughout Southwestern Ontario in All populations (allopatry and
sympatry) or just those sampled in sympatry. Bold mean values represent level of significance
p<0.001.
Pop
Tactic
Body Size
Body Condition
All
Caller
0.22 ±0.24
-0.19 ±0.03
Satellite -1.35 ±0.28
Sympatry Caller
-0.17 ±0.04
-0.14 ±0.24
-0.23 ±0.04
Satellite -1.79 ±0.31
-0.14 ±0.04
129
Table 4.2: Contingency table (observed (expected)) of mating tactic (caller or satellite) within
sympatric populations based on both mtDNA + nuclear DNA data. Because no pure Interior (I)
individuals were found within the zone of secondary contact (sympatry) only pure Eastern (E),
hybrid individuals (HE and HI), and Backcross Eastern (BE) were compared.
Genotypic
Category
Caller
Satellite
N
E
5 (5.62)
1 (5.38)
6
BE
4(3.06)
2 (2.94)
6
HE
5 (5.12)
5 (4.89)
10
HI
10 (12.766)
15 (12.23)
25
N
24
23
47
130
Table 4.3: Contingency table (observed (expected)) based on combined mtDNA and nuclear
DNA data of caller-satellite dyad associations found within the zone of secondary contact.
Satellites
E
BE
HE
HI
N
1 (0.22)
0 (0.43)
1 (1.1)
3 (3.26)
5
BE
0(0.17)
0 (0.35)
2 (0.87)
2 (2.61)
4
HE
0 (0.17)
0 (0.35)
2 (0.87
2 (2.61)
4
HI
0 (0.43)
2 (0.87)
0 (2.17)
8 (6.52)
10
N
1
2
5
15
23
Callers E
131
Figure 4.1: Pseudacris crucifer sampling map across the Southwestern Ontario contact zone.
Populations from which caller-satellite dyads were sampled are represented by asterisks; all other
populations contain individuals used for morphometric and genetic analysis (see Supplementary
Information Table 1 for details). Range of spring peepers with Interior mtDNA haplotypes is
represented in dark gray, and of spring peepers with Eastern mtDNA haplotypes in light gray.
The hatched area corresponds to the area of secondary contact. The Canada – USA border is
represented by a solid black line.
132
Figure 4.2: Correspondence analysis of mating tactic (Caller and Satellite; dashed lines) for
males of various genetic backgrounds within the zone of secondary contact (E=pure Eastern,
HE=hybrids with Eastern mtDNA haplotypes, HI= hybrids with Interior mtDNA haplotypes, and
BE= F2 with mainly Eastern nuclear DNA but Interior mtDNA haplotype).
133
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Chapter 5: General Discussion
Two of the most challenging goals of evolutionary biology are to reconstruct the
evolutionary history of extant species, and to understand the processes responsible for the
origination and maintenance of diversity. With the advent of phylogeography in the 1980s (Avise
et al. 1987), evolutionary biologists gained important insights into how historical factors like
geographical isolation can influence contemporary distributions and evolutionary trajectories of
focal taxa. Temperate zones, for example, have experienced a dynamic history of glacial
oscillations, and consequently range fragmentation and restrictions to gene flow among
populations. Phylogeographic data have provided much insight into species range dynamics
including the impact of isolation in refugia and subsequent range expansions (Weir & Schluter
2004; Soltis et al. 2006), and also have figured prominently in defining ‘evolutionary significant
units’ for conservation (Moritz 1994), yet have rarely been used comprehensively to illuminate
mechanisms of speciation. My research bridges the gap between phylogeography and studies of
the mechanisms of biological speciation, providing insights into how range fragmentation and
subsequent secondary contact may contribute to the origination of species diversity.
In this thesis I present work on the spring peeper (Pseudacris crucifer) for which
phylogeographic studies identified six deeply diverged lineages now in secondary contact
throughout the species range (Austin et al. 2002, 2004). My overarching goal was to evaluate
contact zone dynamics and the evolution of reproductive isolation between two diverging
conspecific populations. I focused on two relatively recently diverged lineages (Eastern and
Interior - 3.5 mya; Austin et al. 2002; Stewart et al. in review - Chapter 2) now meeting in
secondary contact in Southwestern Ontario, Canada. Although previous descriptions of the
spring peeper suggests little phenotypic differentiation across its range (Conant & Collins 1998),
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my research shows that individuals of the Eastern and Interior lineages are subtly differentiated
in a suite of attributes not just in putatively neutral genetic markers (Austin et al 2002, 2004;
Stewart et al. in review - Chapter 2). I found significant differences in external morphology, male
acoustic advertisements, female preference (Chapter 2), and in hatching success and tadpole
survival (Chapter 3). My genetic surveys showed hybridization between lineage populations but
asymmetrical introgression of Eastern nuclear markers but not mtDNA towards the Interior
lineage, implying higher costs to hybridization for the Eastern lineage (Chapter 2). Moreover,
morphology and call characteristics also show a pattern that I interpret as asymmetrical
reproductive character displacement for the Eastern lineage (Chapter 2). I proposed that this
reproductive character displacement may be a result of reinforcement, where natural selection
has favoured stronger pre-zygotic isolation barriers in the zone of contact. Hybrids had distinct
adult morphology, intermediate advertisement calls, intermediate female preference (Chapter 2),
and also disproportionately adopted the alternative satellite male mating tactic (Chapter 4).
Although hybrid tadpoles exhibited equal or superior vigour than their pure parental counterparts
when raised in isolation, survivorship was lower when reared together with pure lineage tadpoles
(Chapter 3) implying some degree of extrinsic post-zygotic isolation (diminished competitive
ability). Interestingly, I found concordance between pre- and post- zygotic isolation barriers with
both asymmetrical costs to hybridization for the Eastern lineage mirrored by call and female call
preference displacement within sympatry, further implying reinforcement (Yukilevich 2012).
Together, my work has attempted to assess every criterion for diagnosing reinforcement
put forth by Howard (1993). Pseudacris crucifer lineages meeting in secondary contact in
Southwestern Ontario are observed to currently hybridize (Criterion 1). Hybrids appear to have
diminshed fitness (Criterion 2). While I have not shown this directly, other work shows that
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variation on anuran calls is indeed heritable (Criterion 3). There is strong female preference in
the zone of secondary contact (Criterion 4). Finally, character displacement is unlikely to be the
result of other processes (Criterion 5). Another signature of reinforcement put forth by Yukilevch
(2012), that of concordant pre- and post-zygotic asymmetries, is also supported with my
research.
My work has important and broad implications for the study of speciation (explored
below), the major themes of which include: 1) The value of phylogeographic studies for
providing a spatial and temporal framework for speciation studies; 2) The value of testing for
evidence of reinforcement between diverging intraspecific lineages rather than already diagnosed
species; 3) Implications for the study of cryptic biological species, and 4) Future studies that
should be undertaken with Pseudacris crucifer.
5.1 Significance
5.1.1 Phylogeography and Reproductive Isolation
Approximately 25 years has elapsed since the term ‘phylogeography’ was coined (Avise
et al. 1987), and the literature is now rich with well over 20,000 articles in the primary literature
allowing insights into organismal evolution over time and space. Not only can genealogical data
be interpreted in a geographic context to infer patterns of historical and contemporary population
structure and demography (e.g. population bottlenecks and expansions; Avise et al. 1987; Avise
2000), but also can be used to infer the processes that generate them, including vicariant events
or differentiation with sustained migration (Richards et al. 2007). Phylogeographic studies have
proved particularly useful for understanding the biogeographical consequences of climate change
over thousands or even millions of years. For example, much of the intraspecific genealogical
diversity, or recently evolved sister species in North America, dates to events within the Pliocene
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and Pleistocene (Avise 1994). Yet even with such a compendium of articles illustrating 1) the
presence of geographically restricted clades within long-diagnosed species like the spring peeper,
often having diverged for sufficient lengths of time to fit within the ‘time course of speciation’
(Hewitt 1996); and 2) the presence of secondary contact zones between diverging lineages,
relatively little attention has been given to actual mechanisms of speciation in a phylogeographic
context. I hope my research has highlighted the importance of marrying comprehensive
phylogeographic studies to detailed genetic and phenotypic surveys of contact zones, certainly
for experiments aimed at understanding mechanisms of speciation and the evolution of
reproductive isolation. Phylogeography provides an historical and temporal framework, but the
broader phylogeographic literature itself can be used by others to identify promising systems to
further test for the consequences of contact and the possibility of secondary reinforcement.
Indeed even Southern Ontario itself is a contact zone for other taxa (e.g. fish - Dowling & Hoeh
1991; April & Turgeon 2006; butterflies - Scriber et al. 2002; Mullen et al. 2008; amphibians Green 1984; Zamudio & Savage 2003; Rissler & Smith 2010).
5.1.2 Intraspecific Reinforcement
Most studies evaluating the consequences of secondary contact tend to focus on
previously diagnosed species for which the process of biological speciation is presumably nearly
complete (e.g. Sætre et al. 1997; Cooley et al. 2001; Cicero 2004; Gay et al. 2007; Gompert et al.
2010). The likelihood that character displacement is both stronger and more frequent for intracompared to inter- specific interactions (Pfennig & Pfennig 2010) raises the important point, that
signatures of such potentially ephemeral processes (such as reinforcement) may be easier to
detect between intraspecific lineages. Indeed finding signatures of reinforcement may require
hitting a ‘sweet spot’ during the divergence of lineages. Hybridization is a prerequisite, yet too
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little divergence and hybridization may occur indefinitely (Bank et al. 2011), or gene pools may
simply merge; too much divergence and hybridization becomes rare and reproductive isolation is
complete, leaving little chance for reinforcement selection. The historical importance of
reinforcement in completing speciation may also be obscured upon the cessation of gene flow
(Bank et al. 2011). Thus, given reinforcement’s potential rapidity, at least in terms of
evolutionary time over which new species may arise, it is perplexing that only a few studies to
date have looked for the signatures of reinforcement between intraspecific lineages (e.g. Smadja
& Ganem 2005; Richards-Zawacki & Cummings 2011). My work highlights the importance of
evaluating contact zones between intraspecific lineages for studying the process of speciation,
particularly because they are in the early stages of divergence.
5.1.3 Cryptic Species Delimitation
Myriad studies have revealed the presence of cryptic species with little to no phenotypic
differentiation, but levels of genetic divergence equal to or greater than those between
morphologically defined species (Avise 2000). Although such cryptic species are common
especially in the tropics (Elmer et al.2007; Fouquet et al. 2007), they have been somewhat less
considered in North American phylogeographic and phylogenetic studies, although there are
some striking examples even among anurans (e.g. in Pseudacris; see Lemmon et al. 2008). Some
studies already show such cryptic lineages may evolve substantial barriers to gene flow, and
certainly in some cases have been evolving independently for millions of years (Phillips et al.
2004; Hoskin et al. 2005; Elmer et al. 2007; Kawakami et al. 2009; Singhal & Moritz 2011); yet
without overt phenotypic differentiation, important evolutionary questions remain regarding
mechanisms maintaining lineage boundaries or the nature of reproductive isolation (Bickford et
148
al. 2006; Singhal & Moritz 2011). In fact some ask whether these cryptic lineages are nascent or
fully independent species, or simply “evolutionary ephemera” (Avise & Wollenberg 1997).
Across the range of the spring peeper, there exist six deeply diverged lineages some of
which may well represent cryptic, fully-reproductively isolated species (Austin et al. 2004). My
research suggests that even relatively young lineages (Eastern and Interior) within this system
show marked genetic, behavioural, and subtle morphological boundaries with potentially some
extrinsic costs to hybridization. Although other lineages (and contact zones) merit study, my
research at least implies older, more deeply diverged lineages may represent even more strongly
delimitated taxa, perhaps suggesting that P. crucifer is more of a species complex. For instance,
the contact zone in Wisconsin comprising of Western, Interior, and Eastern lineages frogs,
suggests no discernible hybridization between the Western and Eastern lineages (Stewart et al.
unpublished data).
5.1.4 Future Research
5.1.4.1 Costs to hybridization
The probability of speciation depends on the cumulative strength and persistence of
prezygotic, extrinsic, and intrinsic postzygotic reproductive isolation barriers (Coyne & Orr
2004). Lacking from the research completed thus far on divergence and speciation in P. crucifer
are key aspects of the potential costs to hybridization. While I found some evidence for
maladaptive hybridization between the Eastern and Interior lineages meeting in secondary
contact, further examination is required to investigate viability, fertility and fecundity of F2
hybrids and backcross individuals, as both theory (Burke & Arnold 2001) and empirical evidence
suggest negative aspects of hybrid fitness may become evident in later generations (e.g. Dawley
1987). Hybrids may also demonstrate reduced fertility. I have conducted some (as yet)
149
unpublished studies on sperm morphology and abnormalities in hybrids versus pure lineage
males. Provocatively, these preliminary results indicate a significantly higher proportion of
tailless sperm in Eastern hybrids, implying a reduction in fertilization success. Quantifying
fertilization rate is still required and may allow for further investigations on other important
sperm fitness measures (speed and longevity).
Although we have conducted female phonotaxis experiments using Eastern and Interior
male call stimuli, we have not investigated female discrimination of hybrid calls which we know
to be different from calls of pure males (Chapter 2). It would also be important to confirm the
genetic basis of call variation. For example, call parameter variation of pure and hybrid males
recorded in the field should overlap with those reared and tested in captivity (sensu Lemmon &
Lemmon 2010).
5.1.4.2 Comparative contact zone dynamics
A difficulty in studying speciation in taxa that are at least partially sympatric, is in
understanding the origin of isolating barriers that prevent gene flow in sympatry (Coyne & Orr
2004). Identifying the reproductive barriers which were involved in the initial reduction of gene
flow is important, yet hampering this is the fact that isolating barriers often act together and their
present importance may distort our perception of their historical importance during speciation.
One approach to elucidate the tempo of isolating barriers is a comparative analysis using
different lineages within the same species at different stages of evolutionary divergence - thus
potentially evaluating speciation from its very earliest inception through to incipient,
reproductively isolated species. In this way, we can shed some light on the relative importance of
different types of isolating barriers by the sequence in which they appear over evolutionary time.
It also eliminates an obvious, yet underappreciated caveat to speciation studies: isolating barriers
150
can continue to accumulate after speciation is complete (Coyne & Orr 2004). By studying
multiple contact zones of a single species where different pairs of lineages of different ages now
interact, and determining which reproductive barriers are operating, we gain insights into their
relative importance to the cessation of gene flow and the rate at which they evolve.
The spring peeper is a promising system for comparative contact zone analysis because
the many contact zones between divergent lineage pairs may represent unique ‘snap-shots’ in the
evolution of reproductive isolation (see Figure 1.3). Currently we have data from three contact
zones in the Northern range, encompassing secondary contact zones between Eastern and
Interior (Southwestern Ontario), Interior and Western (Southern Illinois), and Western, Interior
and Eastern (Wisconsin), all differing in divergence time. Although I have only preliminary
genetic data, as I allude to above, the results imply different levels of hybridization. To some
extent we would expect responses to depend on ecological context. For example, MoriartyLemmon (2009) found that contact zones with distinct heterospecific assemblages of Pseudacris
diverged differentially in various signal traits, maximizing differences in male advertisement call
attributes that may promote reproductive displacement. Accordingly, we may also find very
different evolutionary divergence in the mate recognition system for each contact zone
depending on environmental and historical context. Alternatively, we may find that similar
isolating barriers are present, but that they vary in strength, with stronger barriers being present
in lineages with longer divergent times. Detailed quantification of phenotype, as well as field and
lab experiments, as I did for the Southwestern Ontario contact zone, should be replicated in these
other contact zones.
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5.1.4.3 Genomics of reproductive isolation
Essential for the study of origins of new species is the interpretation of genomic
divergence during speciation. During the early stages of speciation, reproductive barriers are
likely to be maintained by just a few loci which contribute directly to local adaptation, mate
choice, sexual conflict or genetic incompatibility (Butlin 2010). By identifying loci responsible
for hybrid inviability or sterility in individuals reared in controlled laboratory conditions, we will
gain some insights into the genetic underpinnings of speciation. Advances in Next Generation
Sequencing (or NGS) are also enabling new research approaches such as examining genomewide patterns of population divergence along the speciation continuum, from weakly diverged
populations through to species (Feder et al. 2012). The next decade will witness an explosion of
transcriptomic studies that will allow us to identify key genes expressed at different
developmental stages, in different tissues, and in individuals of different lineages. Thus, an
exciting future avenue of research into the evolution of reproductive isolation among spring
peeper lineages should delve into patterns of genomic divergence among the six diverging
populations.
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Summary of Data Chapters
Chapter 2: I used 11 microsatellites (4 previously published and 7 from Genbank) and 360bp of
cytochrome b DNA sequence to characterize a contact zone between the Eastern and Interior
mitochondrial lineages of the spring peeper in Southwestern Ontario, Canada. I used Bayesian
clustering to diagnose individuals (hybrid versus pure), and tested the validity and statistical
power of my methods using simulations. I documented clinal variation in both co-dominant
(microsatellite) and maternally (cytochrome b) markers and diagnosed introgression across the
hybrid zone. I also quantified variation in morphology, male advertisement call, and female
preference both in allopatric populations and in populations where both lineages were present
(sympatry). This chapter had four major findings:
1.
Individuals of the Eastern and Interior lineages are indeed hybridizing in Southwestern
Ontario.
2.
Clinal variation is non-coincident between marker classes, implying an asymmetrical cost
to hybridization for Eastern individuals. No pure Interior individuals were found within
sympatry.
3.
Eastern and Interior lineages are divergent in both morphology and in male call attributes
in allopatric populations.
4.
Patterns in morphology, male call, and female preference in sympatry all suggest
asymmetrical reproductive character displacement for Eastern individuals, implying that
reinforcement has strengthened mate recognition.
Chapter 3: I tested for post-zygotic isolation between the Eastern and Interior by generating
hybrid and pure lineage tadpoles in the laboratory. Tadpoles were raised until metamorphosis
158
and measured for fitness correlates such as hatching success, tadpole survival, development time,
and size at metamorphosis. I compared pure lineage to hybrid tadpoles. Additional treatments
studied early life-history fitness in rearing conditions where competition was present, again
comparing pure and hybrid tadpoles. Four main outcomes were reached:
1.
I found further evidence of divergence between the Eastern and Interior lineage
evident in tadpole survival.
2.
Hybrids when raised in isolation show equal or even higher early lifetime fitness
than their parental counterparts.
3.
Apparent hybrid superiority may be eliminated with competition, ultimately
reducing hybrid survival rate compared to pure individuals.
4.
I found further evidence for asymmetrical costs to hybridization, with Eastern
tadpoles showing the highest mortality in the presence of competition, as well as
the highest proportion of deformities.
Chapter 4: The objective of this chapter was to determine why, in light of apparently strong prezygotic and some post-zygotic isolation, gene flow between the Eastern and Interior lineages still
occurs. I hypothesized that apparently unattractive hybrids may circumvent female choice by
adopting an alternative mating tactic, satellite behaviour. I reached three main conclusions:
1.
Smaller males, but not those in worse body condition, were more likely to assume
satellite behaviour.
2.
Pure lineage males were almost always callers, and hybrids with Interior
haplotypes disproportionately adopted satellite, non-calling, behaviour. Hybrids
with Eastern haplotypes took on either behaviour.
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3.
Despite previous evidence for strong female preference, we did not find
assortative mating within ponds in the contact zone, suggesting satellite
interception.
4.
Asymmetrical satellite behaviour circumventing female choice may facilitate
introgression across this contact zone, maintaining hybridization.
160
Appendix A: Supplementary Information for Chapter 2
Methods
Genotyping
DNA extractions were conducted using the DNeasy® (QIAGEN, Mississauga, Ontario, Canada)
Extraction Kit following the manufacturer’s protocols. DNA concentrations were quantified
using a Nanodrop ND-1000 spectrophotometer. Individuals were sequenced for a 692bp segment
of cytochrome b (cyt b) using primers MVZ 15-L and MVZ 18-H (Moritz et al. 1992) according
to methods outlined in Austin et al. (2004). Briefly, 25μL Polymerase Chain Reactions (PCR)
cocktails contained 10x Fermentas reaction buffer (KCl), 2.5mM MgCl2, 0.5mM dNTPs, 0.5 U
of Taq Polymerase (Fermentas), and 2 μL of DNA (5-20ng/μL). Amplifications were performed
in a GeneAmp PCR System 2700 using the following cycling profile: denaturation step of 3 min
at 94°C, followed by 45 cycles of 45 sec at 94°C, 45 sec at 52°C, and 45 sec at 72°C with a final
extension of 5 min at 72°C (modified from Austin et al. 2004).
Pseudacris crucifer individuals from 2008-2011 were typed for 11 microsatellite loci, 4
of which have been previously published (Degner et al. 2009); Locus pairs Pcru11 and Pcru21,
Pcru08 and Pcru24, Pcru06 and Pcru12, and Pcru09 and Pcru14 were amplified together in
duplexes, while loci Pcru05, Pcru10, and Pcru32 were amplified in a triplex reaction. Each 11μL
PCR reaction contained: 0.2-0.5µl of forward and reverse primers (see Supplementary Materials,
Table S2 for primer quantities), 10x Fermentas reaction buffer (KCl), 1.5mM MgCl2, 10mM
dNTPs, 0.5 U of Taq Polymerase (Fermentas), and 2 μL of DNA (5- 20ng/μL). Thermocycling
conditions for Pcru09, Pcru14, Pcru06, Pcru12, Pcru24 and Pcru08 are outlined in (Degner et al.
2009). Thermocycling conditions for Pcru32, Pcru10, Pcru05, Pcru11 and Pcru21 following an
initial denaturing (94 ºC for 3 min.) were as follows: 35 cycles of 94 ºC for 15 seconds, 48 ºC
161
for 30 seconds, and 72 ºC for 40 seconds, followed by an extension of 15 minutes at 72 ºC. PCR
products were run on (2%) agarose gels (Invitrogen, Carlsbad, California, U.S.A.) at 100 volts in
1x TBE buffer, post-stained with 0.5μg/mL ethidium bromide, and visualized under UV light to
evaluate efficacy of amplification. PCR products were genotyped using a Beckman Coulter CEQ
8000 capillary automated sequencer and scored using the CEQ 8000 Genetic Analysis System
(Appendix Table A2).
162
Table A1: Sampling information of populations from 2003-2011. The type of data collected for
each population is represented by G (genetic), M (morphology), C (male call), and F (female
preference). GPS coordinates are in decimal-degrees (Latitude, Longitude).
Site Name
Collection
Years
Pop
Type
GPS coordinates
Data Type
CFIT
distance (km)
STRUCTURE
Pop #
Pelee/Hillman
2009-2010
Interior
41.971037, -82.534279
G, M, C, F
580.76
1
Kopegaron
2009-2010
Interior
42.07971, -82.49243
G, M
567.58
2
Mosa Forest
2004,
2010, 2011
Interior
42.656561, -81.807461
G, M, C
483.45
3
Fingal
2003,
2009-2011
Interior
42.671617, -81.32635
G, M, C, F
453.98
4
PondMills
2011
Interior
42.945366, -81.216774
G, M
427.04
5
ElginRd.
2009
Contact
42.961887, -81.014814
G, M, C,
413.66
6
Calton
2003,20092011
Contact
42.7251, -80.883783
G,M,C, F
420.68
7
Dereham
2003, 2011
Contact
42.90885, -80.834133
G, M, C
405.90
8
Starkey Hill
2003,
2010, 2011
Contact
43.545624, -80.156121
G, M, C, F
382.44
9
LPWREC
2011
Eastern
42.718358, -80.351729
G,M,
373.28
10
Vanessa
2009-2011
Eastern
42.960443, -80.397949
G, M, C, F
337.74
11
LaFortune
2009-2011
Eastern
43.092883, -80.001717
G, M, C, F
318.76
12
BPNP
2009, 2010
Eastern
45.209134, -81.56559
G, M, F
211.76
13
QUBS
2009
Eastern
44.496804, -76.414847
G, M
0
14
Wainfleet
2004
Eastern
42.922399, -79.374779
C
N/A
N/A
Minesing
2004
Eastern
44.410927, -79.819107
C
N/A
N/A
Sudden Tract
2003
Eastern
43.309191, -80.38044
C
N/A
N/A
Chippawa
2003
Eastern
43.053367, -79.05427
C
N/A
N/A
Alton
2003
Eastern
43.870733, -80.0588
C
N/A
N/A
163
Port Dover
2003
Eastern
42.783307, -80.230408
C
N/A
N/A
Parrot Bay
2003
Eastern
42.783307, -80.230408
C
N/A
N/A
Harlowe
2003
Eastern
44.787649, -77.121965
C
N/A
N/A
N. Guelph
2003
Contact
43.586622, -80.231512
C
N/A
N/A
Vanisttart
2004
Contact
43.16775, -80.664817
C
N/A
N/A
Elice Swamp
2004
Contact
43.4686, -80.954883
C
N/A
N/A
Stapleton
Tract
2004
Contact
43.926917, -81.355883
C
N/A
N/A
Hallet
2003
Contact
43.65615, -81.485533
C
N/A
N/A
Wildwood
2004
Contact
43.267206, -81.076355
C
N/A
N/A
Rondeau
2004
Contact
42.279089, -81.86182
C
N/A
N/A
Big Bend
2004
Contact
42.64691, -81.70833
C
N/A
N/A
Parkhill
2004
Interior
43.17065, -81.645283
C
N/A
N/A
Moore
2004
Interior
42.801447, -82.310658
C
N/A
N/A
164
Table A2: Primer ID and GenBank Accession numbers, author affiliation, allele ranges, and
PCR reaction forward and reverse primer quantities for 11 microsatellite markers used during the
genotyping of P. crucifer from 2008-2011.
Primer
ID
Forward
Sequence (5’
to 3’)
Reverse
Sequence (3’ to
5’)
Ta
GenBank
Accession
#
Reference
Allele
Range
Repeat
Motif
Quantity
PCR
Reaction
Primer
Pcru32
CCCTACAT
AGGATTG
GTACACCCAC
CAAAAGTGCT
54
EF190904
Van Buskirk,et al.
2006
(unpublished)
121-163
(CA)11
0.2 µl
Pcru10
GGGGGATG
CAGAATT
CGGTTCCTATT
GAAGAACA
54
EF190896
Degner et al. 2009
187-213
(CA)13
0.5 µl
Pcru05
CATTTATAA
GCAGTGCA
GAGAGG
TGCATTGATGT
TTCTCATGG
54
EF190892
Van Buskirk,et al.
2006
(unpublished)
320-366
(CA)13
0.5 µl
Pcru11
GGATATGC
TCACATG
CCCAGATTCC
AAGTGTTTTC
55
EF190897
Van Buskirk,et al.
2006
(unpublished)
112-168
(GT)16
0.3 µl
Pcru21
TGGAGACA
TCATTGC
CCCTTGGTCCT
GAATAGGTT
55
EF190900
Van Buskirk,et al.
2006
(unpublished)
210-264
(CA)14
0.45 µl
Pcru09
GGGGGATG
CAGAATT
CGGTTCCTATT
GAAGAACA
54
EF190896
Degner et al. 2009
125-159
(CA)15
0.2 µl
Pcru14
GATCAGAC
AGTCTACA
GTAATGAG
GAG
CATAACACAG
GGCAACCAAG
54
EF190899
Degner et al. 2009
184-232
(GT)13
0.3 µl
Pcru06
CATTTACA
AACGGCAC
TGCTC
CCCCAGTCATC
AGGAATACA
54
EF190893
Van Buskirk,et al.
2006
(unpublished)
186-236
(GT)16
0.3 µl
Pcru12
TCAAATTG
ACCATCCA
TCC
GCCAGCCCCT
ATAGGATTAG
54
EF 190898
Van Buskirk,et al.
2006
(unpublished)
114-166
(GT)13
0.5 µl
Pcru24
TGCCATGG
GGATGTTA
TATG
CGAGCTATAG
GAAAAGGCAG
AG
54
EF190902
Degner et al. 2009
96-146
(CA)17
0.35µl
165
Pcru08
CTAACCGT
AGCAGGGA
GGTTG
TAGGACTGAG
GGAGGAGAGG
54
EF 190894
166
Van Buskirk,et al.
2006
(unpublished)
222-270
(CA)12
0.5 µl
Table A3: Sampling location information for individuals with unique haplotypes (Field ID –
Genbank Accession numbers can be found in Austin et al. 2004) used for the construction of the
Bayesian tree and TMRCA analysis tree (see Table A8 and Figure A1).
Field ID
Lineage
Location
Tex1, Tex2
Southwest
Morris Co. TX, country rd 3312, near Omaha.
MHP8377
West
W311th St., Louisburg, KS N 38.5625 W 94.6691
INHS267, 269
West
Rte. 159, Jackson Co. IL 38°15 N 90°00 W
Pcru1289
West
Stone Co. AR
MHP8407
West
ca. 1 mi W Bluff City, Fayette Co. IL 38.962,-89.059
MHP8375
West
2 mi S jct St rte Y and MO Rt 215 on unnamed country road.
N37.5216 W93.7415, Dade Co. MO
MHP8379
West
4.5 mi. N Pere Marquette State Park on Ill Rt. 100, Dabbs
Study Area, IL
MHP8385
West
10 km NW Winchester, E of Ill Rt. 100 and 1.6 km S jct US Rt
67 and I 72; SE Sec 27, T14N, R13W, Scott Co. IL
MHP8380,
MHP8382
West
4.5 mi. N Pere Marquette State Park on Ill Rt. 100, Dabbs
Study Area, Jersey Co. IL
MHP8409
West
4.8 mi E Coffeen, 0.3 mi E. Oak Trail Road on Ill Rt. 185,
Montgomery Co. IL
Pcru68492
Southwest
Salem, Pearl River, MS, 30°290 N 089°360 W
Pcru1288
Southwest
White Co. AR
Pcru1864
Interior
Big Bend Conservation Area, Ontario 42.644, -81.705
Pcru1854
Interior
Melbourne R., South of Strathroy, ON (RR between Century
and Falconbridge Roads. 42°51.310 N 81°36.260 W
Pcru1164
Interior
Murfreesburo, Rutherford, TN 35°500 N 086°240 W
Pcru1876, Pcru1880,
Pcru1881, Pcru1883
Interior
Moore Habitat Management Area, Ontario 42.767, -82.336
Pcru1867, Pcru1870,
Pcru1871
Interior
Mosa Forest Conservation Area, Ontario 42.644, -81.809
167
Pcru1510
Interior
Danville, Virmillion, IL 40°110 N 087°440 W
Pcru1146
Interior
Long Point, Haldimand-Norfolk, ON 42°370 N 080°280 W
ECM60
Southeastern
Barnwell, SC 33°180 N 081°150 W
PcruFL1
Southeastern
Ocala, FL 29°100 N 082°200 W
ECM958
Southeastern
Macon Co. GA N32.44951 W85.65438
Pcru1601
Eastern
Beech Fork State Park, Wayne Co., WV
ECM81
Eastern
Pitt NC 35°350 N 077°230 W
Pcru57852
Eastern
Dawn, Caroline, VA 37°390 N 077°170 W
Pcru5575
Eastern
Ottawa, ON 45°190 N 075°480 W
Pcru1158
Eastern
Innisfil, Simcoe, ON 44°220 N 079°360 W
Pcru1010
Eastern
Sherry Township, WI 44°150 N 090°050 W
Pcru1618
Eastern
Aberfoyle, Wellington, ON 43°290 N 080°090 W
Pcru1886, 1887
Eastern
Dundas, Hamilton—Wentworth, ON 43°140 N 080°010 W
Pcru1142
Eastern
Long Point, Haldimand-Norfolk, ON 42°370 N 080°280 W
168
Table A4: Correlations between call parameters and temperature. Asterisks indicates
significance at P<0.05. Call parameter details can be found in Lemmon et al. [72].
Call Parameter
Slope
r2
P value
Max Freq
22.624
0.138
<0.001*
Min Freq
29.198
0.198
<0.001*
Delta Freq
-3.852
0.013
0.120
Call Rise Time
0.0
0.026
0.028*
Call Fall Time
-0.001
0.041
0.006*
Carrier Length
-0.004
0.083
<0.001*
Call Interval
-0.038
0.110
<0.001*
Call Duration
-0.006
0.185
<0.001*
Call Rate
0.052
0.245
<0.001*
169
Table A5: Eigen values, loadings, and scoring coefficients of Discriminant Function Analysis
(DFA) on 9 acoustic parameters (8 of which use residuals from correlations to temperature,
Table A1) taken from the calls of Pseudacris crucifer in allopatry and sympatry during 20032011. Also included is a DFA of 9 acoustic parameters after controlling for body size (SVL) and
temperature. Asterisks denotes acoustic parameters that were corrected for temperature, and
double asterisks denotes parameters corrected for temperature and SVL.
Canonical
Eigen
Value
Cum.
Percent
Scoring Coefficients
Max
Freq
Min
Freq
**
**
Delta
Freq
Call
Rise
Time
*
Call
Fall
Time
*
Carrier
Length
Call
Interval
Call
Dur
Call
Rate
**
**
**
**
1
0.35
62.0%
0.43
-0.17
0.11
0.47
0.58
0.99
0.53
-0.96
0.36
2
0.12
83.7%
-1.66
1.36
-0.18
-0.34
0.37
0.14
-0.09
0.64
-0.14
3
0.06
94.7%
0.66
-0.97
-0.88
0.05
-0.05
-0.09
1.74
-0.55
1.19
Correcting for body size
1
0.35
62.9%
-0.002
-0.002
-0.0002
-4.75
4.71
14.40
0.11
-5.46
0.14
2
0.17
29.9%
0.004
0.004
-0.0001
-29.86
29.87
4.20
0.75
5.25
-0.26
3
0.04
7.2%
0.005
0.005
0.0010
-26.87
26.88
8.68
-1.01
17.32
-0.45
170
Table A6: Eigen values, loadings, and scoring coefficients of Discriminant Function Analysis
(DFA) on 7 morphometric measurements taken from all individuals of Pseudacris crucifer in
allopatry and sympatry during 2008-2011.
Canonical
Eigen
Value
Cum.
Percent
Scoring Coefficients
SVL
Mass
Head
Width
Radioulnar
Femur
Tibia
Foot
1
0.09
57.10%
-0.32
-0.22
-0.59
1.31
0.25
0.03
-0.20
2
0.05
83.43%
0.03
1.97
-01.17
0.39
0.46
-0.37
-0.09
3
0.02
94.78%
0.66
-0.27
-0.08
0.43
0.16
0.69
-0.16
4
0.01
100%
-0.46
1.266
-0.13
-0.57
-0.28
0.55
0.27
171
Table A7: Factor loadings of Principal Components on morphometric measurements taken from
male and female Pseudacris crucifer across all populations.
Females
Males
PC1
PC2
PC3
PC4
PC1
PC2
PC3
PC4
Eigen
Value
3.69
1.93
0.6
0.4
4.59
0.63
0.50
0.42
Cum %
Variance
52.68%
80.26%
88.90%
94.64%
65.52%
74.50%
81.62%
87.65%
SVL
0.90
0.02
-0.02
-0.02
0.80
0.39
0.19
-0.18
Mass
0.88
0.08
-0.39
-0.09
0.76
0.52
-0.06
0.27
Head
Width
0.26
0.95
-0.04
-0.06
0.84
-0.12
0.06
0.11
Radioulnar
0.16
0.94
0.28
0.09
0.76
-0.33
0.43
0.27
Femur
0.87
-0.01
-0.05
0.42
0.77
-0.19
-0.51
0.20
Tibia
0.83
-0.34
0.25
0.21
0.86
-0.21
-0.04
-0.29
Foot
0.76
-0.20
0.49
-0.31
0.87
-0.02
-0.07
-0.32
172
Table A8: A. Divergence estimates (mean, standard error, median, upper and lower highest
probability densities (HPD), and effective sample size (ESS)) from BEAST analysis for Time to
Most Common Recent Ancestor (TMRCA) in substitutions. Divergence times were later
calculated by assuming a substitution rate consistent with 1% per million years. B. Average raw
p-distances calculated between lineages using MEGA5 (below diagonal) with 95% standard
errors estimated from 500 bootstrap replicates (above the diagonal). See text for details.
A.
Nodes
All
Texas
All but
Texas
I/E/SE/SW
Int/E/SE
E/SE
E
I
SE
SW
W
Mean
5.48E-2
1.50E-3
4.46E-2
2.9135E-2
2.47E-2
1.51E-2
2.87E-3
6.81E-3
1.5136E2
4.21E-3
7.48E-3
Standard
Error
1.32E-4
9.38E-6
9.05E-5
7.17E-5
5.07E-5
3.03E-5
1.15E-5
2.72E-5
3.03E-5
1.68E-5
2.87E-5
Median
5.38E-2
1.18E-3
4.33E-2
2.82E-2
2.40E-2
1.47E-2
2.68E-3
6.42E-3
1.47E-2
3.71E-3
7.09E-3
95% HPD
lower
3.42E-2
2.56E-5
2.85E-2
1.84E-2
1.53E-2
9.33E-3
9.49E-4
3.15E-3
9.33E-3
6.85E-4
3.58E-3
95% HPD
upper
7.68E-2
3.79E-3
6.37E-2
4.14E-2
3.48E-2
2.16E-2
5.17E-3
1.14E-2
2.16E-2
8.74E-3
1.20E-2
ESS
7271.67
16739.92
11004.09
7596.95
10769.35
11944.56
10184.81
7020.04
11946.13
19995.87
6777.64
B.
Interior
Interior
Southwest
Eastern
West
Southeastern
0.007
0.006
0.009
0.007
0.011
0.012
0.008
0.009
0.008
0.011
0.013
0.009
0.006
0.011
0.012
0.010
0.012
0.013
0.011
0.013
Southwest
0.038
Eastern
0.035
0.052
West
0.056
0.060
0.058
Southeastern
0.040
0.051
0.030
0.066
Texas
0.088
0.080
0.089
0.080
0.090
Outgroups
0.155
0.161
0.154
0.158
0.161
173
Texas
Outgroups
0.013
0.161
Table A9: CFIT Logit cline analysis under various run scenarios. Unconstrained (1) allows for
the width and centers of each cline to vary independently, constrained center (2) holds marker
centers at the same geographic distance, constrained slopes (3) maintains marker cline widths but
allows for non-coincident cline centers, and center and slope constrained (4) holds both
parameters constant for all markers across the cline. Width is 1/slope; N represents the number of
parameters used within each model. Smallest AIC is bolded and indicates the model of best fit.
1) Unconstrained Logit
2) Center
Constrained
3) Slope
Constrained
Centre =
551.239
Slope = -0.011
4) Center &
Slope
Constrained
Centre= 99.999
(Width=90.909)
Slope=-0.000
Genetic
Marker
Centre
Slope
Width
Likelihood
Slope
Centre
mtDNA Cyt b
408.295
-0.047
21.277
-289.362
-0.004
395.152
Pcru32
291.606
-0.006
166.667
-406.111
0.001
335.599
Pcru10
383.371
-0.006
166.667
-480.872
-0.001
394.084
Pcru05
400.440
-0.010
100.000
-460.097
-0.002
400.480
Pcru11
500.193
-0.012
83.334
-372.001
-0.008
507.766
Pcru21
466.306
-0.008
125.000
-458.006
-0.004
456.1223
Pcru09
550.235
-0.012
83.334
-309.989
-0.012
562.488
Pcru12
580.000
-0.007
142.857
-413.226
-0.008
538.088
Pcru06
524.814
-0.010
100.000
-362.929
-0.008
523.809
Pcru08
476.781
-0.011
90.909
-436.173
-0.006
477.955
Pcru24
580.000
-0.006
166.667
-387.679
-0.006
518.573
Pcru14
580.000
-0.020
50.000
-151.095
-0.02
580.000
Ln(Likelihood)
-4774.292
-5019.167
-4746.894
-6361.012
N
24
13
13
2
AIC
9596.584
10064.334
9519.788
12726.024
174
Table A10: Summary of linkage disequilibrium (LD; p<0.05) from all pair-wise comparisons for
11 microsatellite loci per population (pop; STRUCTURE population identifier in brackets).
Populations with less than 5 sampled individuals were excluded from analyses comprising of (2)
Kopegaron, (5) Pond Mills, (6) Elgin Rd., and (14) QUBS.
Pop
Pop Type
LD
% LD
(1) Pelee/Hillman
Interior
31
56.363%
(3) Mosa
Interior
45
81.818%
(4) Fingal
Interior
40
72.727%
(7) Calton
Contact
39
70.909%
(8) Dereham
Contact
37
67.273%
(9) Starkey Hill
Contact
43
78.182%
(10) LPWREC
Eastern
31
56.364%
(11) Vanessa
Eastern
42
76.364%
(12)LaFortune
Eastern
37
67.273%
(13) BPNP
Eastern
23
41.818%
175
Figure A1: Phylogeny from a Bayesian analysis of cytochrome b DNA sequence data. The
analysis was done in MrBayes Version 3.2 using a GTR+I+G model of evolution. We ran two
independent runs of 1 million Metropolis-coupled MCMC iterations (until the standard deviation
of the split frequencies was < 0.01) with 4 incrementally heated Markov chains specifying the
GTR+I+G model of evolution with parameters estimated as part of the analysis. We sampled
every 100 iterations and discarded the first 25% as burnin. We confirmed convergence by
examining a plot of the log probability versus generation to ensure stationarity, and ensuring the
176
Potential Scale Reduction Factors were all close to unity. Posterior probabilities for key nodes
are shown (typically above). Estimates of divergence times in millions of years are shown:
TMRCA from our Bayesian analysis in the program BEAST, and mean raw p-distance from an
analysis in MEGA5, both assuming a rate of divergence of 1% per million years. See main text
for details.
177
A
A
A
AB
B
AB
B
Figure A2: Boxplot illustrating allopatric (A), sympatric (S), and hybrid (H) individuals by
lineage (Eastern and Interior) A) female body size differences and B) male body size differences
according to Principle Coordinate Analysis (PC1; Supplementary Materials, Table S6). Letters
above boxplots represent individuals with significantly different body size (p<0.01).
178
Appendix B: Supplementary Information for Chapter 3
A
100
80
60
40
20
I
0
7
9
11 14 22 26 46 48 50 52 55 57 59 64 69 76 79 84 90
B 100
80
60
40
20
E
0
7
C
9
11
14
22
26
46
48
50
52
55
57
59
64
69
76
79
84
90
100
80
60
40
20
HI
0
7
D
9
11 14 22 26 46 48 50 52 55 57 59 64 69 76 79 84 90
100
80
60
40
20
HE
0
7
9
11 14 22 26 46 48 50 52 55 57 59 64 69 76 79 84 90
Figure B1: Mean percent cumulative survival over 100 days post-hatch of spring peeper
(Pseudacris crucifer) tadpoles with standard error bars for A) pure Interior families represented
by a solid black line (N=6), B) pure Eastern families represented by a solid grey line (N=6), C)
179
Hybrids with Interior haplotypes or HI represented by a dashed black line (N=6), and D) Hybrids
with Eastern haplotypes or HE represented by a dashed grey line (N=7).
180
A
B
100
100
90
90
80
80
70
70
60
60
50
50
I
40
30
30
20
20
10
10
0
0
37 40 43 46 49 52 55 58 62 65 70 76 81 97
37 40 43 46 49 52 55 58 62 65 70 76 81 97
C
D
100
100
90
90
80
80
70
70
60
60
50
E
40
HI
50
40
40
30
30
20
20
10
10
0
HE
0
37 40 43 46 49 52 55 58 62 65 70 76 81 97
37 40 43 46 49 52 55 58 62 65 70 76 81 97
Days Post-Hatch
Figure B2: Mean percent cumulative metamorphosis curves over 100 days post-hatch of spring
peeper (Pseudacris crucifer) tadpoles with standard error bars for A) pure Interior families
represented by a solid black line (N=6), B) pure Eastern families represented by a solid grey line
(N=5), C) Hybrids with Interior haplotypes or HI represented by a dashed black line (N=6), and
D) Hybrids with Eastern haplotypes or HE represented by a dashed grey line (N=7).
181
1 cm
Figure B3: Pseudacris crucifer hybrid with Eastern haplotype tadpole that did not
metamorphose by day 100 (post-hatch), exhibiting gigantism. Lower panel illustrates an index
finger beside the tadpole for size reference (Gosner stage 25-30).
182
1 mm
Figure B4: Pseudacris crucifer hybrid with Eastern haplotype tadpole pictured in a Petri dish
(mm grid marks for size reference). Photo illustrates a bent and deformed tail (Gosner stage 2530).
183
Appendix C – Supplementary Information for Chapter 4
Table C1: Details for sampled populations from 2003-2011. The type of data collected for each
population is represented by G (genetic), M (morphology), and S (caller-satellite dyads). GPS
coordinates are in decimal-degrees (Northing, Easting).
Site Names
Collection
Years
Pop
Type
GPS coordinates
Data Type
Pelee/Hillman
2008-2010
Interior
41.971037, -82.534279
G, M, S
1
Kopegaron
2008-2010
Interior
42.07971, -82.49243
G, M
2
Mosa Forest
2010, 2011
Interior
42.656561, -81.807461
G, M, S
3
Fingal
2009-2011
Interior
42.671617, -81.32635
G, M, S
4
PondMills
2011
Interior
42.945366, -81.216774
G, M
5
ElginRd.
2009
Contact
42.961887, -81.014814
G, M
6
Calton
2009-2011
Contact
42.7251, -80.883783
G,M, S
7
Dereham
2011
Contact
42.90885, -80.834133
G, M
8
Starkey Hill
2010, 2011
Contact
43.545624, -80.156121
G, M, S
9
LPWREC
2011
Eastern
42.718358, -80.351729
G,M,
10
Vanessa
2009-2011
Eastern
42.960443, -80.397949
G, M, S
11
LaFortune
2009-2011
Eastern
43.092883, -80.001717
G, M, S
12
BPNP
2009, 2010
Eastern
45.209134, -81.56559
G, M, S
13
QUBS
2009
Eastern
44.496804, -76.414847
G, M
14
184
STRUCTURE
Pop #
Table C2: Model II nested ANOVA results (Measurement Error - %ME) for 10 randomly
selected Pseudacris crucifer individuals within Ontario
Character
Mean
Range
Residual Mean Sq Total Mean Sq
SVL
24.06 mm
21.5 - 28.8 mm
0.121
4.806
2.51
Femur
10.29 mm
7.90 - 11.9 mm
0.0024
1.12
0.212
Tibia
12.26 mm
11.1 - 14.4 mm
0.006
1.14
0.528
Foot
16.73 mm
15.1 – 21.0 mm
0.043
2.68
1.60
Radioulna
6.00 mm
5.10 - 7.20 mm
0.020
0.35
5.61
Head
7.98 mm
7.20 - 10.3 mm
0.0094
0.64
1.47
Mass
1.06 g
0.70 - 1.75 g
0.00055
0.084
0.653
185
%ME