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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 ii 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. iii 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) iv 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). v 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 vi 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 vii 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. viii 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 1.6 Literature Cited Adams, D.C. 2004. Character displacement via aggressive interference in Appalachian salamanders. Ecology 85: 2664-2670. Ashton, R.E., Jr., & P.S. Ashton. 1988. Handbook of Reptiles and Amphibians of Florida. Part Three. The Amphibians. Windward Publishing, Inc., Miami. Austin, J.D., Lougheed, S.C., Neidrauer, L., Check, A.A., & P.T. Boag.2002. Cryptic lineages in a small frog: post-glacial history of the spring peeper, Pseudacris crucifer (Anura: Hylidae). Mol. Phylogenet. Evol. 25: 316-325. Austin, J.D., Lougheed, S.C., & P.T. Boag. 2004. Discordant temporal and geographic patterns in maternal lineages of eastern North American frogs, Rana catesbeiana and Pseudacris crucifer. Mol. Phylogenet. Evol. 32: 799–816. Avise , J.C. 1999. Phylogeography: The History and Formation of Species. Cambridge, MA: Harvard University Press. Avise, J.C., Walker, D., & G.C. Johns. 1998. Speciation durations and Pleistocene effects on vertebrate phylogeography. Proc. Roy. Soc. Lond. B Biol. 265: 1707–1712. Badger, D.P. & J. Netherton. 2004. Frogs. Voyageur Press. MN. Bank, C., Hermisson, J., & M. Kirkpatrick. 2011. Can reinforcement complete speciation? Evolution 66: 1229-1239. Barraclough, T.G., & A.P. Vogler. 2000. Detecting the geographical pattern of speciation from species-level phylogenies. Am. Nat. 155: 419-434. Bartlett, R.D., & Bartlett, P. P. 1999. A Field Guide to Florida Reptiles and Amphibians. Gulf Publishing Company, Houston, Texas. 21 Bergman, C.M. 1999. Range extension of spring peepers, Pseudacris crucifer, in Labrador. Can. F. Nat. 113: 309-310. Blair, W.F. 1955. Mating call and stage of speciation in the Microhyla olivacea-M. carolinensis complex. Evolution 9: 469–480. Blaustein, A., L. Belden, D. Olson, D. Green, T. Root, & J. Kiesecker. 2001. Amphibian breeding and climate change. Cons. Biol. 15: 1804-1809. Brattstrom, B. H. 1962. Thermal control of aggregation behavior in tadpoles. Herpetologica 18:38-46. Brenowitz, E.A., Wilczynski, W., & H.H. Zakon. 1984. Acoustic communication in spring peepers. Environmental and behavioural aspects. J. Comp. Phys. A 155:585-592. Brown, W.L., & E. O. Wilson. 1956. Character displacement. Syst. Zool. 5: 49-65. Butlin, R. K. 1987. Speciation by reinforcement. Trends Ecol. Evol. 2:8-13. Carling, M.D. Lovette, I.J. & R.T. Brumfield. 2010. Historical divergence and gene flow: coalescent analyses of mitochondrial, autosomal and sex-linked loci in Passerina buntings. Evolution 64: 1762-1772. Cellinese, N., Baum D.A. & B.D. Mishler. 2012. Species and phylogenetic nomenclature. Syst. Biol. 61: 885-891. Cocroft, R.B. 1994. A cladistic analysis of chorus frog phylogeny (Hylidae: Pseudacris). Herpetologica 50:420-437. Conant, R., & J.T. Collins. 1998. A Field Guide to Reptiles and Amphibians: Eastern / Central North America. Third Ed. Houghton Mifflin Company, NY. Conroy, C.J., & J.A. Cook. 2000. Phylogeography of a post-glacial colonizer: Microtus longicaudus (Rodentia: Muridae). Mol. Ecol. 9: 165-175. 22 Coyne, J.A., & H.A. Orr. 2004. Speciation. Sinauer Associates, Sunderland, MA. Darwin, C. 1859. On the Origin of Species by Means of Natural Selection. London: Murray. de Queiroz K. 2007. Species concepts and species delimitation. Syst. Biol. 56:879-886. Delzell, D.E. 1958. Spatial movements and growth of Hyla cruficer. Ph.D. Dissertation, Univ. Michigan, Ann Arbor, MI. Dobzhansky, Th. 1937 Genetics and the Origin of Species. Columbia University Press, New York. Duvernell, D.D., & B.J. Turner. 1998. Evolution genetics of Death Valley pupfish populations: mitochondrial DNA sequence variation and population structure. Mol. Ecol. 7: 279-288. Duvernell, D.D., & B.J. Turner. 1999. Variation and divergence of Death Valley pupfish populations at retrotransposon-defined loci. Mol. Biol. Evol. 16: 363-371. Fisher, C., Joynt, A., & R.J. Brooks. 2007. Reptiles and Amphibians of Canada. Lone Pine Pub. Edmonton, AB. Fishwald, T.G., R.A. Schemidt, K.M. Jankens, K.A. Berven, A.J. Gamboa, & C.M. Richards. 1990. Sibling recognition by larval frogs (Rana pipiens, R. sylvatica, and Pseudacris crucifer). J. Herp. 24:40-44. Fitzpatrick, B.M., Fordyce, J.A., & S. Gavrilets. 2009. Pattern, process and geographic modes of speciation. J. Evol. Biol. 22: 2342-2347. Forester, D.C., & D.V. Lykens. 1986. Significance of satellite males in a population of spring peepers (Hyla crucifer). Copeia 3:719-724. Freda, J., & D.H. Taylor. 1992. Behavioural response of amphibian larvae to acidic water. J. Herp. 26: 429-433. 23 Good, T. P., Ellis, J.C., Annett, C.A., & R. Pierotti. 2000. Bounded hybrid superiority in an avian hybrid zone: effects of mate, diet, and habitat choice. Evolution 54: 1774-1783. Gosner, K. L., & D. A. Rossman. 1960. Eggs and larval development of the treefrogs Hyla crucifer and Hyla ocularis. Herpetologica 16:225-232. Hammerson, G. 2004. Pseudacris crucifer. In: IUCN 2009. IUCN Red List of Threatened Species. Version 2009.1. http://www.iucnredlist.org. Downloaded on 28 July 2009. Harding, J.H. 1997. Amphibians and reptiles of the Great Lakes region. University of Michigan Press. Michigan, USA. Hecnar, S. J., & R.T. M’Closkey. 1996. The effect of predatory fish on amphibian species richness and distribution. Biol. Cons. 79:123-131 Hedges, S. B. 1986. An electrophoretic analysis of holarctic hylid frog evolution. Syst. Zool. 35:1-21. Hobel, G. & H.C. Gerhardt. 2003. Reproductive character displacement in the acoustic system of green treefrogs (Hyla cinerea). Evolution 57:894–904 Hoffman, E.A., & M. S. Blouin. 2004. Evolutionary history of the northern leopard frog: Reconstruction of phylogeny, phylogeography, and historical changes in population demography from mitochondrial DNA. Evolution 58:145-159. Hoskin, C. J., M. Higgie, K. R. McDonald, & C. Moritz. 2005 Reinforcement drives rapid allopatric speciation. Nature 437:1353–1356. Howard, D. J. 1993. Reinforcement: origin, dynamics, and fate of an evolutionary hypothesis. Pp. 46–69 in R. G. Harrison, ed. Hybrid Zones and the Evolutionary Process. Oxford Univ. Press, New York 24 Howard, D. J. & S. H. Berlocher. 1998. Endless Forms: Species and Speciation. Oxford Univ. Press, New York. Huxley J. 1942. Evolution: The Modern Synthesis Allen & Unwin, London. Janzen, D. H. 1967. Why mountain passes are higher in the tropics. Am. Nat. 101:233–249. Klicka, J., & R. M. Zink. 1997. The importance of recent ice ages in speciation, a failed paradigm. Science 277:1666–1669. Knowlton, N. 1993. Sibling species in the sea. Ann. Rev. Ecol. Syst. 24: 189-216. Lance, S. L, & K. D. Wells. 1993. Are spring peeper satellite males physiologically inferior to calling males? Copeia. 1993:1162-1166. Lannoo, M. 2005. Amphibian Declines: The Conservation Status of United States Species. University of California Press. Liou, L. W., & T. D. Price. 1994. Speciation by reinforcement of premating isolation. Evolution 48:1451–1459. Loftus-Hills, J.J., & M.L. Littlejohn. 1992. Reinforcement and reproductive character displacement in Gastrophryne carolinensis and G. olivacea (Anura: Microhylidae): A reexamination. Evolution 46:896–906 MacCulloch, R. D. 2002. The ROM field guide to amphibians and reptiles of Ontario. Royal Ontario Museum and McClelland & Stewart Ltd. Mallet, J., Meyer, A., Nosil, P., & L. Feder. 2009. Space, sympatry and speciation. J. Evol. Biol. 22:2332-2341. Mani, G. S., & B. C. Clarke. 1990. Mutation order - A major stochastic process in evolution. Proc. Roy. Soc. Lond. B. 240: 29–37. 25 Marshall, J. L., Arnold, M. L., & D. J. Howard. 2002. Reinforcement: The road not taken. Trends Ecol. Evol. 17: 558–563. Marshall, D. C., & J. R. Cooley. 2000. Reproductive character displacement and speciation in periodical cicadas, and a new 13- year species, Magicicada neotredecim. Evolution 54: 1313-1325. Martin, P.R., Montgomerie, R.D., & S.C. Lougheed. 2010. Divergent selection in sympatry causes rapid plumage evolution in high latitude birds. Evolution 64: 336-347. Mayden R.L. 1997. A hierarchy of species concepts: The denouement in the saga of the species problem. In: Claridge M.F., Dawah H.A., Wilson M.R., editors. Species: The Units of Biodiversity. London: Chapman and Hall, p. 381-424. Mayr, E. 1942. Systematics and the Origin of Species from the Viewpoint of a Zoologist. New York: Columbia University Press. Minton, S. A., Jr. 2001. Amphibians & Reptiles of Indiana. Revised 2nd Edition. Indiana Academy of Science, Indianapolis. Milot, E. Gibbs, H.L., & K.A. Hobson. 2000. Phylogeography and genetic structure of northern populations of the yellow warbler (Dendroica petechia). Mol. Ecol. 9: 677–681. Moriarty-Lemmon, E., Lemmon, A. R. & D. C. Cannatella. 2007. Geological and climatic forces driving speciation in the continentally distributed trilling chorus frogs (Pseudacris). Evolution 61:2086-2103. Niemiller, M.L. Fitzpatrick, B.M. & B.T. Miller. 2008. Recent divergence with gene flow in Tennessee cave salamanders (Plethodontidae: Gyrinophilus) inferred from gene genealogies. Mol. Ecol. 17: 2258-2275 Noor, M.A.F. 1995. Speciation driven by natural selection in Drosophila. Nature 375: 674–75. 26 Nosil, P. 2008. Speciation with gene flow could be common. Mol. Ecol. 17: 2103-2106. Nosil, P., Crespi, B.J., & C.P. Sandoval. 2003. Reproductive isolation driven by the combined effects of ecological adaptation and reinforcement. Proc. R. Soc. Lond.. B. 270: 1911-1918. Oplinger,C . S. 1966. Sex ratio, reproductive cycles, and time of ovulation in Hyla crucifer crucifer. Herpetologica 22:276-283. Otte, D. & J. A. Endler. 1989. Speciation and its consequences. Sinauer, Sunderland, MA. Parris, K. M. 2002. More bang for your buck: the effect of caller position, habitat and chorus noise on the efficiency of calling in the spring peeper. Ecol. Mod. 156: 213-224. Perrill, S. A. 1984. Male mating behavior in Hyla regilla. Copeia. 1984:727-732. Pfennig, K. S. 2003. A test of alternative hypotheses for the evolution of reproductive isolation between spadefoot toads: support for the reinforcement hypothesis. Evolution 57: 2842– 2851. Pfennig, K. S. & D. W. Pfennig. 2009. Character displacement: ecological and reproductive responses to a common evolutionary problem. Quart. Rev. Biol. 84: 253-276. Powell, R., & R. W. Henderson. 1999. Addenda to the checklist of West Indian amphibians and reptiles. Herp. Rev. 30:137-139. Rice, A. M. & D.W. Pfennig. 2010. Does character displacement initiate speciation? Evidence of reduced gene flow between populations experiencing divergent selection. J. Evol. Biol. 23: 854-865. Riddle, B.R., Hafner, D. J., & L.F. Alexander. 2000. Comparative phylogeography of Baileys’ Pocket Mouse (Chaetodipus baileyi) and the Peromyscus eremicus species group historical vicariance of the Baja California Peninsula desert. Mol. Phylogen. Evol. 17: 161-172. 27 Rosen M., & R. E. Lemon.1974. The vocal behavior of spring peepers, Hyla crucifer. Copeia 1974:940–950. Rundle, H.D., Nagel, L., Boughman, J.W., & D. Schluter. 2000. Natural selection and parallel speciation in sympatric sticklebacks. Science 287:306-308. Rundle, H.D., & D. Schluter. 1998. Reinforcement of stickleback mating preferences: Sympatry breeds contempt. Evolution 52:200-208. Sætre, G.-P., Moum, T., Bures, S., Kral, M., Adamjan, M., & J. Moreno. 1997. A sexually selected character displacement in flycatchers reinforces premating isolation. Nature 387: 589-592. Schmid, W. D. 1982. Survival of frogs in low temperature. Science 215:697-698. Schluter, D. 2000. The Ecology of Adaptive Radiation. Oxford, U. K: Oxford University Press. Schluter, D. 2009. Evidence for ecological speciation and its alternative. Science 323: 737–741. Schwartz, A., & R. W. Henderson. 1991. Amphibians and Reptiles of the West Indies: Descriptions, Distributions, and Natural History. University of Florida Press, Gainesville, Florida. xvi + 720 pp. Smadja C., & R.K. Butlin 2009. On the scent of speciation: the chemosensory system and its role in premating isolation. Heredity 102: 77-97. Servedio M.R., & M.A. Noor. 2003. The role of reinforcement in speciation: theory and data. Annu. Rev. Ecol. Evol. Syst. 34: 339–364. Singhal, S., & C. Moritz. 2012. Strong selection against hybrids maintains a narrow contact zone between morphologically cryptic lineages in a rainforest lizard. Evolution 66: 1474-1489. Soltis, D.E., Morris, A.B., McLachlan, J.S., Manos, P.S., & P.S. Soltis. 2006. Comparative phylogeography of unglaciated eastern North America. Mol. Ecol. 15: 4261-4293. 28 Stevenson, D., & D. Crowe. 1992. Geographic Distribution Note. Pseudacris crucifer. Herp. Rev. 23:86. Storchová, R., Reif, J. & M.W. Nachman. 2010. Female heterogamety and speciation: reduced introgression of the z chromosome between two species of nightingales. Evolution 64: 456471. Storey, K. B., & J. M. Storey. 1987. Persistance of freeze tolerance in terrestrial hybernating frogs after spring emergence. Copeia 1987: 720-726. Templeton, A.R., Routman, E., & C.A. Phillips. 1995. Separating population structure from population history: a cladistic analysis of the geographic distribution of mitochondrial DNA haplotypes in the Tiger Salamander, Ambystoma tigrinum. Genetics. 140: 767–782. The Marie Curie SPECIATION Network. 2012. What do we know about speciation? Trends Ecol. Evol. 27: 27-39. Turelli, M., Barton, N.H., & Coyne, J.A. 2001. Theory and speciation. Trends Ecol. Evol. 16: 330-343. Wake, D.B. 2006. Problems with species: patterns and processes of species formation in salamanders. Ann. Miss. Bot. Gard. 93:8-23. Waltz, E.C. 1982. Alternative mating tactics and the law of diminishing returns: the satellite threshold model. Behav. Eco. Sociobio. 10:75-83. Weir, J.T., & D. Wheatcroft. 2011. A latitudinal gradient in rates of evolution of avian syllable diversity and song length. Proc. Roy. Soc. Lond. B. 278:1713-1720. Wells, K. D., 1977. The social behaviour of anuran amphibians. Anim. Behav. 25: 666-693. Wilkins, M.R., Seddon, N., & R.J. Safran. 2012. Evolutionary divergence in acoustic signals: causes and consequences. Trends Ecol. Evol. 28: 156-166 29 Wright, A.H. & A. A. Wright. 1995. Handbook of Frogs and Toads of the United States and Canada. Comstock publishing Co. Inc., Cornell University, Ithaca, NY. Xiang, Q.Y., Soltis, D.E., Soltis, P.S. & S.R. Manchester. 2000. Timing the eastern Asianeastern North American floristic disjunction: molecular clocks confirm paleontological estimates. Mol. Phylogen. Evol. 15: 462-472. Zampella, R., & J. Bunnell. 2000. The distribution of anurans in two river systems of a coastal plain watershed. J. Herp. 34: 210-221. Zamudio, K.R., & W.K. Savage. 2003. Historical isolation, range expansion, and secondary contact of two highly divergent mitochondrial lineages in spotted salamanders (Ambystoma maculatum). Evolution 57: 1631–1652. Zimmitti, S. J. 1999. Individual variation in morphological, physiological and biochemical features associated with calling in spring peepers (Pseudacris crucifer). Physiol. Biochem. Zool. 72: 666-676. 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 2.9 Literature Cited Adams, D. C. and F. J. Rohlf. 2000. Ecological character displacement in Plethodon: Biomechanical differences found from a geometric morphometric study. Proc. Natl. Acad. Sci. USA 97:4106-4111. Akaike, H. 1974. A new look at the statistical model identification. IEEE Transactions on Automatic Control 19: 716-723. Amezquita, A., Hodl, W., Lima, A. P., Castellanos, L., Erdtmann, L. and M.C. De Araujo. 2006. Masking interference and the evolution of the acoustic communication system in the Amazonian dendrobatid frog Allobates femoralis. Evolution 60: 1874–1887. Arnold, A. 1993. Cytonuclear disequilibria in hybrid zones. Annu. Rev. Ecol. Syst. 24, 521–554. Arthur, W. 1982. The evolutionary consequences of interspecific competition. Advan. Ecol. Res. 12:127–187. Austin, J. D., Lougheed, S. C., and P.T. Boag. 2004. Discordant temporal and geographic patterns in maternal lineages of eastern North American frogs, Rana catesbeiana and Pseudacris crucifer. Mol. Phylogenet. Evol. 32: 799–816. Austin, J. D., Lougheed, S. C., Neidreauer, L., Chek, A. A., and P. T. Boag. 2002. Cryptic lineages of a small frog: the post-glacial history of the spring peeper, Pseudacris crucifer (Anura: Hylidae). Mol. Phylogenet. Evol. 25: 316-329. Avise, J.C. 1998. The history and purview of phylogeography: a personal reflection. Mol. Ecol. 7: 371–379. Avise, J. C., Walker, D., and G. C. Johns. 1998. Speciation durations and Pleistocene effects on vertebrate phylogeography. Proc. Roy. Soc. Lond. B Biol. 265: 1707–1712 63 Bank, C., Hermisson, J., and M. Kirkpatrick. 2011. Can reinforcement complete speciation? Evolution 66: 1229-239. Barton, N. H., and G. M. Hewitt. 1981. The genetic basis of hybrid inviability in the grasshopper Podisma pedestris. Heredity 47: 367–383. Bella J. L., Butlin R. K., Ferris C., & G. M. Hewitt. 1992. Asymmetrical homogamy and unequal sex ratio from reciprocal mating-order crosses between Chorthippus parallelus subspecies. Heredity 68: 345-352. Belkhir, K., Borsa P., Chikhi L., Raufaste N. and F. Bonhomme. 1996-2004 GENETIX 4.05, logiciel sous Windows TM pour la génétique des populations. Laboratoire Génome, Populations, Interactions, CNRS UMR 5171, Université de Montpellier II, Montpellier (France). Brenowitz, E. A., Wilczynski, W. and H. H. Zakon. 1984. Acoustic communication in spring peepers. Environmental and behavioral aspects. J. Comp. Physiol. A. 155: 585-592. Blair, W. F. 1955. Mating call and stage of speciation in the Microhyla olivacea-M. carolinensis complex. Evolution 9:469–480. Blair, W.F. 1974. Character displacement in frogs. Am. Zool. 14:1119–1125. Brown, W. L., and E. O. Wilson. 1956. Character displacement. Syst. Zool. 5: 49-65. Buggs R. J. A. 2007. Empirical study of hybrid zone movement. Heredity 99: 301-312. Butlin, R. K. 1987. Speciation by reinforcement. Trends Ecol. Evol. 2:8-13. 1989. Reinforcement of pre-mating isolation. Pp. 158-179 in D. Otte and J. A. Endler, eds. Speciation and its consequences. Sunderland, Sinauer, Mass. 64 Camargo, A., Naya, D. A., Canavero, A., Rosa, I. and R. Maneyro. 2005. Seasonal activity and the body size-fecundity relationship in a population of Physalaemus gracilis (Boulenger, 1883) (Anura, Leptodactylidae) from Uruguay. Ann. Zool. Fenn. 42: 513-521. Cooley, J.R. 2007. Decoding asymmetries in reproductive character displacement. Proc. Acad. Nat. Sci. Phil. 156:89–96. Coyne, J. A., and H. A. Orr. 1989. Patterns of speciation in Drosophila. Evolution 43:362-381. Coyne, J. A., and H. A. Orr. 1997. “Patterns of speciation in Drosophila” revisited. Evolution 51:295–303. Coyne, J. A. and H. A. Orr. 2004. Speciation. Sinauer Associates, Sunderland, MA. Dayan, T., and D. Simberloff . 2005. Ecological and community-wide character displacement: the next generation. Ecol. Lett. 8:875–894 Degner, J. F., Hether, T. D., and E. A. Hoffman. 2009. Eight novel tetranucleotide and five cross-species dinucleotide microsatellite loci for the ornate chorus frog (Pseudacris ornata). Mol. Ecol. Res. 9: 622-624. Delzell, D. E. 1958 Spatial movement and growth of Hyla crucifer. PhD Dissertation University of Michigan, USA. Dessauer, H. C., Cole, C. J., and C. R. Townsend. 2000. Hybridization among western whiptail lizards (Cnemidophorus tigris) in southwestern New Mexico: population genetics, morphology, and ecology in three contact zones. Bull. Am. Mus. Nat. Hist. 246: 4–148. Devitt, T. J., Baird, S.J.E., and C. Moritz. 2011 Asymmetrical reproductive isolation between terminal forms of the salamander ring species Ensatina eschscholtzii revealed by fine-scale genetic analysis of a hybrid zone. BMC Evol. Biol. 11: 245. 65 Dobzhansky, Th. 1937 Genetics and the Origin of Species. Columbia University Press, New York. Dobzhansky, Th. 1940. Speciation as a stage in evolutionary divergence. Am. Nat. 74:312- 321. Doherty, J. A. and Gerhardt, H. C. 1984 Evolutionary and neurobiological implications of selective phonotaxis in the spring peeper (Hyla crucifer). Anim. Behav. 32: 875–881. Drummond A. J., Ho, S. Y. W., Phillips, M. J., and A. Rambaut. 2006. Relaxed phylogenetics and dating with confidence. PLoS Biol. 4, e88. Drummond, A. J. and A. Rambaut. 2007. BEAST: Bayesian evolutionary analysis by sampling trees. BMC Evol. Biol. 7: 214. Earl, Dent A. and B. M. vonHoldt. 2011. STRUCTURE HARVESTER: a website and program for visualizing STRUCTURE output and implementing the Evanno method. Cons. Gen. Resour. 4: 359-361. Endler, J. A. 1977. Geographic variation, speciation, and clines. Princeton Univ. Press, Princeton, NJ. Endler, J.A. 1986. Natural selection in the wild. Princeton Univ. Press, Princeton, NJ. Evanno, G., Regnaut, S., and J. Goudet. 2005 Detecting the number of clusters of individuals using the software STRUCTURE: a simulation study. Mol. Ecol. 14: 2611-2620. Forbes, S. H., and F. W. Allendorf. 1991. Associations between mitochondrial and nuclear genotypes in cutthroat trout hybrid swarms. Evolution 45: 1332–1349. Forester, D. C., and W. K. Harrison. 1987. The significance of antiphonal vocalization by the spring peeper, Pseudacris crucifer (Amphibia, Anura). Behaviour 103: 1-15. Gause, G.F. 1934.The struggle for existence. Williams and Wilkins; Baltimore, MD. 66 Gay, L., Crochet., P-A., Bell, D. A. and T. Lenormand. 2008. Comparing clines on molecular and phenotypic traits in hybrid zones: a window on tension zone models. Evolution 62: 2789-2806. Gerhardt, H. C. 1973 Reproductive interactions between Hyla crucifer and Pseudacris ornata, Anura: Hylidae). Am. Midl. Nat. 89: 81-88. Gerhardt, H. C. 1994. The evolution of vocalization in frogs and toads. Annu. Rev. Ecol. Syst. 25: 293-324. Gerhardt, H.C., and F. Huber. 2002. Acoustic communication in insects and anurans: common problems and diverse solutions. Univ. Chicago Press, Chicago, IL. Geyer, L.B. and S.R. Palumbi. 2004. Reproductive character displacement and the genetics of gamete recognition in tropical sea urchins. Evolution 57: 1049-1060. Gibbs, J.P. 1998 Distribution of woodland amphibians along a forest fragmentation gradient. Land. Ecol. 13:263-268. Good, J. M., Hird, S., Reid, N., Demboski, J. R., Steppan, S. J., Martin-Nims, T. R., and J. Sullivan. 2008. Ancient hybridization and mitochondrial capture between two species of chipmunks. Mol. Ecol. 17: 1313–1327. Good, J. M., and J. Sullivan. 2001. Phylogeography of the red-tailed chipmunk (Tamias ruficaudus), a northern Rocky Mountain endemic. Mol. Ecol. 10: 2683–2695. Grant, P.R., 1972. Convergent and divergent character displacement. Biol. J. Linn. Soc. 4:39–68. Grant, P.R., and B.R. Grant. 2006. Evolution of character displacement in Darwin's finches. Science 313:224–226. Grant, P.R., and B.R. Grant. 2008. How and why species multiply: the radiation of Darwin's finches. Princeton University Press; Princeton, NJ. 67 Groning J, and A. Hochkirch 2008. Reproductive interference between animal species. Quart. Rev. Biol. 83:257–282. Gurevitch, J, Morrow, L.L., Wallace, A., and J.S. Walsh. 1992. A meta-analysis of competition in field experiments. Am. Nat. 140:539–572. Hall, T. A. 1999 Bioedit: a user friendly biological sequence alignment editor and analysis program for Windows95/98/NT. Nucleic Acids Symp. Ser. 41: 95–98. Harrison, R. (ed.). 1993 Hybrid Zones and the Evolutionary Process. Oxford Univ. Press. Hobel, G. and H. C. Gerhardt. 2003 Reproductive character displacement in the acoustic communication system of green tree frogs (Hyla cinerea). Evolution 57: 894-904. Hochkirch, A., Groning, J., and A. Bucker. 2007. Sympatry with the devil: reproductive interference could hamper species coexistence. J. Anim. Ecol. 76:633–642. Hoskin, C. J., M. Higgie, K. R. McDonald, and C. Moritz. 2005 Reinforcement drives rapid allopatric speciation. Nature 437: 1353–1356. Howard, D. J. 1993 Reinforcement: origin, dynamics and fate of an evolutionary hypothesis. In Hybrid zones and the evolutionary process (ed. R. G. Harrison), pp. 46–69. New York, NY: Oxford University Press. Hubisz, M. J., Falush, D., Stephens, M., and J. K. Pritchard. 2009. Inferring weak population structure with the assistance of sample group information. Mol. Ecol. Resour. 9: 1322-1332. 68 Irwin, D., Robtsov, A. S., and E. N. Panov. 2009 Mitochondrial introgression and replacement between yellowhammers (Emberiza citrinella) and pine buntings (Emberiza leucocephalos) (Aves: Passeriformes). Biol. J. Linn. Soc. 98: 422-438. Jang, Y., Won, Y. J., and J. C. Choe. 2009. Convergent and divergent patterns of morphological differentiation provide more evidence for reproductive character displacement in a wood cricket Gryllus fultoni (Orthoptera: Gryllidae). BMC Evol. Biol. 9: 27. Kameda, Y., Kawakita, A., and M Kato. 2009. Reproductive character displacement in genital morphology in Satsuma land snails. Am. Nat. 173:689-697 Kidd, D.M., and M. G. Ritchie. 2006. Phylogeographic information systems: putting the geography into phylogeography. J. Biogeogr. 33: 1851-1865 Kirschel, A. N. G., Blumstein, D. T. and T. B. Smith. 2009 Character displacement of song and morphology in African tinkerbirds. Proc. Natl Acad. Sci. USA 106: 8256–8261. Klicka, J., and R. M. Zink. 1997. The importance of recent ice ages in speciation, a failed paradigm. Science 277:1666–1669. Kolbe, J. J., Larson, A., Losos, J. B.,and K. de Queiroz. 2008 Admixture determines genetic diversity and population differentiation in the biological invasion of a lizard species. Biol. Lett. 4: 434-437. Lamb, T., and J. Avise. 1986. Directional introgression of mitochondrial DNA in a hybrid population of tree frogs: the influence of mating behavior. Proc. Natl. Acad. Sci. USA. 83: 2526–2530. Lemmon, E. M. 2009. Diversification of conspecific signals in sympatry: geographic overlap drives multidimensional reproductive character displacement in frogs. Evolution 63: 1155– 1170. 69 Lemmon, E. M., A. R. Lemmon, J. T. Collins, J. A. Lee-Yaw, and D. C. Cannatella. 2007. Phylogeny-based delimitation of species boundaries and contact zones in the trilling chorus frogs (Pseudacris). Mol. Phylogenet. Evol. 44:1068–1082. Lemmon, A. R., Smadja, C., and M. Kirkpatrick. 2004. Reproductive character displacement is not the only possible outcome of reinforcement. J. Evol. Biol. 17: 177–183. Liou, L. W., and T. D. Price. 1994. Speciation by reinforcement of premating isolation. Evolution 48:1451–1459. Losos, J.B. 2004. Adaptation and speciation in Greater Antillean anoles. Pp. 335-343 in U. Dieckmann, M. Doebeli, J.A.J. Metz, and D. Tautz, eds. Adaptive Speciation. Cambridge University Press, Cambridge, U.K. Marshall, J. L., Arnold, M. L., and D. J. Howard. 2002. Reinforcement: The road not taken. Trends Ecol. Evol. 17: 558–563. Martin, P. R., Montgomerie, R. and S. C. Lougheed. 2010. Rapid sympatry explains greater color pattern divergence in high latitude birds. Evolution 64: 336–347. McClelland, B. E., Wilczynski, W., and M. J. Ryan. 1996. Correlations between call characteristics and morphology in male cricket frogs (Acris crepitans). J. Exp. Biol. 199: 1907-1919. Monsen, K. J., Honchak, B. M., Locke, S. E., and M. A. Peterson. 2007. Cytonuclear disequilibrium in Chrysochus hybrids is not due to patterns of mate choice. J. Hered. 98: 325-330. Moriarty-Lemmon, E., Lemmon, A. R., and D. C. Cannatella. 2007 Geological and climatic forces driving speciation in the continentally distributed trilling chorus frogs (Pseudacris). Evolution 61: 2086-2103. 70 Moritz, C., Schneider, C. J., and D. B.Wake. 1992. Evolutionary relationships within the Ensatina eschscholtzii complex confirm the ring species interpretation. Syst. Biol. 41: 273– 291. Nei, M., and W. H. Li. 1979. Mathematical model for studying genetic variation in terms of restriction endonucleases. Proc. Natl. Acad. Sci. USA 76: 5269–5273. Nielson, E. E. G., Bach, L. A., and P. Kotlicki. 2006. HYBRIDLAB (Version 1.0): a program for generating simulated hybrids from population samples. Mol. Ecol. Resour. 6: 971-973. Noor, M. A. 1999. Reinforcement and other consequences of sympatry. Heredity 83:503–508. Nosil, P., B. J. Crespi, and C. P. Sandoval. 2003. Reproductive isolation driven by the combined effects of ecological adaptation and reinforcement. Proc. R. Soc. Lond. B 270: 1911–1918. Parmelee, J. R. 1999. Trophic ecology of a tropical anuran assemblage. Scientific Papers, Natural History Museum, Univ. Kansas 11:1–59. Parsons, T. J., Olson, S. L., and M. J. Braun. 1993. Unidirectional spread of secondary sexual plumage traits across an avian hybrid zone. Science 260: 1643-1646. Pfennig, K.S. 2000. Female spadefoot toads compromise on mate quality to ensure conspecific matings. Behav. Ecol. 11:220–227. Pfennig, K. S. 2003. A test of alternative hypotheses for the evolution of reproductive isolation between spadefoot toads: support for the reinforcement hypothesis. Evolution 57: 2842– 2851. Pfennig, D.W. and P.J. Murphy. 2003. A test of alternative hypotheses for character divergence between coexisting species. Ecology 84: 1288-1297. Pfennig, K. S. and D. W. Pfennig. 2009. Character displacement: ecological and reproductive responses to a common evolutionary problem. Q. Rev. Biol. 84: 253-276. 71 Pfennign D.W., and K.S. Pfennig. 2010. Character displacement and the origins of diversity. Am. Nat. 176: S26-S44 Polechova, J. and N. Barton. 2011 Genetic drift widens the expected cline but narrows the expected cline width. Genetics 189: 227-235. Prado, C. P. A., and C. F. B. Haddad. 2005. Size–fecundity relationships and reproductive investment in female frogs in the Pantanal, southwestern Brazil. Herpetol. J. 15: 181–189. Pritchard, J. K., Stephens, M., and P. Donnelly. 2000. Inference of population structure using multilocus genotype data. Genetics 155: 945-959. Pryke S. R., and S. Andersson. 2008. Female preferences for long tails constrained by species recognition in short-tailed red bishops. Behav. Ecol. 19: 1116–1121. Rambaut, A., and A. J. Drummond. 2004. Tracer v1.3, Available from http://evolve.zoo.ox.ac.uk/software.html Rheindt F. E., and S. V. Edwards. 2011. Genetic introgression: an integral but neglected component of speciation in birds. Auk 128: 620–632. Richards-Zawacki, C.L. and M. E. Cummings. 2011. Intraspecific reproductive character displacement in a polymorphic poison dart frog, Dendrobates pumilio. Evolution. 65: 259-267. Rundle, H. D., and D. Schluter. 1998. Reinforcement of stickleback mate preferences: sympatry breeds contempt. Evolution 52: 200–208. Rundle, H.D., and D. Schluter. 2004. Natural selection and ecological speciation in sticklebacks. In: Dieckmann U, Doebeli M, Metz JAJ, Tautz D, editors. Adaptive speciation. Cambridge, UK: Cambridge University Press; pp. 192–209. 72 Rundle, H.D., Nagel, L., Boughman, J.W., and D. Schluter. 2000. Natural selection and parallel speciation in sympatric sticklebacks. Science 287:306-308. Russo, D., Mucedda, M., Bello, M., Biscardi, S., Pidinchedda, E., and G. Jones. 2007. Divergent echolocation call frequencies in insular rhinolophids (Chiroptera): a case of character displacement? J Biogeogr. 34:2129–2138. Sætre, G.-P., Moum, T., Bures, S., Kral, M., Adamjan, M., and J. Moreno. 1997. A sexually selected character displacement in flycatchers reinforces premating isolation. Nature 387: 589-592. Schluter, D. 2000. The ecology of adaptive radiation. Oxford Univ. Press, Oxford, U.K. Schluter, D., and J.D. McPhail.1992. Ecological character displacement and speciation in sticklebacks. Am. Nat. 140:85–108. Schluter, D., Price, T.D., and P.R. Grant. 1985. Ecological character displacement in Darwin’s finches. Science 227:1056–1059. Semlitsch, R.D. 1985. Reproductive strategy of a facultatively paedomorphic salamander Ambystoma talpoideum. Oecologia 65: 305-313. Seneviratne, S. S., Toews, D. P. L., Brelsford, A., and D. E. Irwin. 2012 Concordance of genetic and phenotypic characters across a sapsucker hybrid zone. J. Avian Biol. 43: 119-130. Servedio M. R., and M. A. Noor. 2003. The role of reinforcement in speciation: theory and data. Annu. Rev. Ecol. Evol. Syst. 34: 339–364. Shapiro L. H. 2001. Asymmetric assortative mating between two hybridizing Orchelimum Katydids (Orthoptera: Tettigoniidae). Am. Midl. Nat. 145: 423-427. 73 Singhal, S., and C. Moritz. 2012. Strong selection against hybrids maintains a narrow contact zone between morphologically cryptic lineages in a rainforest lizard. Evolution 66: 14741489. Smadja, C., and G. Ganem. 2005. Asymmetrical reproductive character displacement in the house mouse. J. Evol. Biol. 18:1485-1493 Soltis, D.E., Morris, A. B., McLachlan, J. S., Manos, P. S., and P. S. Soltis. 2006. Comparative phylogeography of unglaciated eastern North America. Mol. Ecol. 15: 4261-4293. Stevenson D. and D. Crowe. 1992. Geographic Distribution Note. Pseudacris crucifer. Herp. Rev. 23: 86. Stewart, K.A., Hudson, C.M., and S.C. Lougheed. In review. Do alternative male mating tactics facilitate introgression across a Pseudacric crucifer hybrid zone? Proc. Roy. Soc. B. RSPB2012-2978. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., and S. Kumar. 2011. MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 28: 2731-2739. Tavaré, S. 1986. Some probabilistic and statistical problems on the analysis of DNA sequences. Pp. 57–86 in Lectures in Mathematics in the Life Sciences, vol. 17. Toews, D. P. L., and A. Brelsford. 2012. The biogeography of mitochondrial and nuclear discordance in animals. Mol. Ecol. 21: 3907-3930. Transeau, E. N., 1935. The prairie peninsula. Ecology 16: 423–437. Vitt, L. J. 1981. Lizard reproduction: Habitat specificity and constraints on relative clutch mass. Am. Nat.117: 506-514. 74 Wilczynski, W., Zakon, H. H., and E. A. Brenowitz. 1984 Acoustic communication in spring peepers. Call characteristics and neurophysiological aspects. J. Comp. Physiol. A 155: 577584. Williams, J. W. 2009. Quaternary vegetation distributions. Kluwer Academic Publishers. Wilson, E. O. 1965. The challenge from related species. In Baker H. G. and Ledyard Stebbins G., eds. The genetics of colonizing species. Academic Press, New York. Weir, J. T., and D. Schluter. 2004 Ice sheets promote speciation in boreal birds. Proc. Roy. Soc. Lond. B Biol. 271: 1881–1887. Weir, J. T., and D. Wheatcroft. 2011. A latitudinal gradient in rates of evolution of avian syllable diversity and song length. Proc. R. Soc. B.Biol. 278: 1713-1720 Yukilevich, R. 2012. Asymmetrical patterns of speciation uniquely support reinforcement in Drosophila. Evolution 66: 1430-1446. Zamudio, K. R., and W. K. Savage. 2003 Historical isolation, range expansion, and secondary contact of two highly divergent mitochondrial lineages in spotted salamanders (Ambystoma maculatum). Evolution 57: 1631–1652. Zug, G.R., Vitt, L.J., and J.P. Caldwell. 2001. Herpetology: An introductory biology of amphibians and reptiles. 2nd ed. Academic Press. California, USA. 75 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. 76 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 77 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. 78 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 79 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). 80 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 4C 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 81 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 82 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). 83 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 3.9 Literature Cited Anderson J.D (1972) Embryonic temperature tolerance and rate of development in some salamanders of the genus Ambystoma. Herpetologica 28: 126–130 Arnold M.L. (1997) Natural Hybridization and Evolution. Oxford University Press, Oxford, UK. Arnold M. L., & S. A. Hodges (1995) Are natural hybrids fit or unfit relative to their parents? Trends Ecol. Evol. 10: 67–71. Austin J. D., Lougheed S. C. & P.T. Boag (2004) Discordant temporal and geographic patterns in maternal lineages of eastern North American frogs, Rana catesbeiana and Pseudacris crucifer. Mol. Phylogenet. Evol. 32: 799–816. Austin J. D., Lougheed S. C., Neidreauer L., et al. (2002) Cryptic lineages of a small frog: the post-glacial history of the spring peeper, Pseudacris crucifer (Anura: Hylidae). Mol. Phylogenet. Evol. 25: 316-329. Badger D. P. & J. Netherton. (2004) Frogs. Voyageur Press. MN. Barraclough .T. G. & A.P. Vogler (2000) Detecting the geographical pattern of speciation from species-level phylogenies. Am. Nat. 155: 419-434. Barton N. H. (1979) The dynamics of hybrid zones. Heredity 43: 341-359. Barton N. H., & G. M. Hewitt (1985) Analysis of hybrid zones. Annu. Rev. Ecol. Syst. 16:113– 148. Barton N.H.& G.M. Hewitt (1989) Adaptation, speciation and hybrid zones. Nature 341:497– 503. Blair W. F. (1955) Mating call and stage of speciation in the Microhyla olivacea-M. carolinensis complex. Evolution 9:469–480. 101 Blaustein A.R., Belden L.K., Hatch A.C., et al.(2001) Ultraviolet radiation and amphibians. Ecosystems, evolutionand ultraviolet radiation (ed. by C.S. Cockell andA.R. Blaustein), pp. 63–79. Springer, New York. Blaustein A.R., Hoffman P.D., Hokit D.G., et al. (1994) UV repair and resistance to solar UV-B in amphibian eggs: a link to population declines? Proc. Nat. Acad. Sci. USA 91: 1791–1795. Blaustein A.R., & J. Margalit (1996) Priority effects in temporary pools: nature and outcome of mosquito larva-toad tadpole interactions depend on order of entrance. J. Anim. Ecol. 65: 7784. Blaustein A.R., Kiesecker, J.M., Chivers, D.P., et al. (1998) Effects of ultraviolet radiation on amphibians: field experiments. Am. Zool. 38: 799–812. Bolnick D.I. & T. J. Near (2005) Tempo of hybrid inviability in centrarchid fishes (Teleostei: Centrarchidae). Evolution 59: 1754–1767 Brown H.A. (1967) Embryonic temperature adaptations and genetic compatibility in two allopatric populations of the spadefoot toad, Scaphiopus hammondi. Evolution 21: 742–761 Browning H.C. (1973) The evolutionary history of the corpus luteum Biol. Reprod. 8: 128–157 Brelford A. & D.E. Irwin. (2009) Incipient speciation despite little assortative mating: the yellow-rumped warbler hybrid zone. Evolution 63: 3050-3060. Burke J. M., & M. L. Arnold ( 2001) Genetics and the fitness of hybrids. Annu. Rev. Gen. 35:31–52. Butlin R. K. (1987) Speciation by reinforcement. Trends Ecol. Evol. 2:8-13. Christian J. J. (1970) Social subordination, population density, and mammalian evolution. Science 168: 85–90. Coyne J. A. & H. A. Orr (1989) Patterns of speciation in Drosophila. Evolution 43:362–381. 102 Coyne J. A. & H. A. Orr (1997) ‘‘Patterns of speciation in Drosophila’’ revisited. Evolution 51:295–303. Coyne J.A. & H. A. Orr (2004) Speciation. Sinauer Associates, Sunderland, MA. Czeczuga B., Muszynska E. & A. Krzeminska (1998) Aquatic fungi growing on the spawn of certain amphibians. Amphibia-Reptilia 19:239–251. Dettman J. R., Sirjusingh C., Kohn L. M.,& J. B. Anderson (2007) Incipient speciation by divergent adaptation and antagonistic epistasis in yeast. Nature 447: 585–588. Dobzhansky Th. (1937) Genetics and the Origin of Species. Columbia University Press, New York. Earl J.E., Cohagen K.E., & R.D. Semiltsch (2012) Effects of leachate from tree leaves and grass litter on tadpoles. Environ. Tox. Chem. 31: 1511-1517. Ellstrand N.C., & K.A. Schierenbeck (2000) Hybridization as a stimulus for the evolution of invasiveness in plants? Proc. Natl. Acad. Sci. USA 97:7043-7050. Endler J. A. (1977) Geographic Variation, Speciation, and Clines. Princeton Univ. Press, Princeton, NJ. Ficetola G.F., Garner T.W.J., & F. De Bernardi (2007) Genetic diversity, but not hatching success, is jointly affected by post glacial colonization and isolation in the threatened frog, Rana latastei. Mol. Ecol. 16: 1787-1797. Fitzpatrick B.M., Fordyce J.A. & S. Gavrilets (2009) Pattern, process and geographic modes of speciation. J. Evol. Biol. 22: 2342-2347. Freidenburg L.K. & D.K. Skelly (2004) Microgeographical variation in thermal preference by an amphibian. Ecol. Lett. 7: 369-373. 103 Garner T.W.J, Pearman P.B., & S. Angelone (2004) Genetic diversity across a vertebrate species’ range. A test of the central-peripheral hypothesis. Mol. Ecol. 13: 1047-1053. Geyer L., & S. R. Palumbi (2003) Reproductive character displacement and the genetics of gamete recognition in tropical sea urchins. Evolution 57:1049–1060. Gomez-Mestre I., Touchon J. C. & K. M. Warkentin (2006) How amphibian eggs survive with pathogenic water molds: parental and embryo defenses and the egg-stage benefit of a larval predator. Ecology 87: 2570–2581. Good T. P., Ellis J.C., Annett C.A., & R. Pierotti (2000) Bounded hybrid superiority in an avian hybrid zone: effects of mate, diet, and habitat choice. Evolution 54: 1774-1783. Gosner K. L. (1960) A simplified table for staging anuran embryos and larvae with notes on identification. Herpetologica. 16: 183-190. Griffiths R.A. (1991) Competition between common frog, Rana temporaria, and natterjack toad, Bufo calamita, tadpoles: the effect of competitor density and interaction level on tadpole development. Oikos 61: 187-196. Gosner K. L., & D. A. Rossman (1960) Eggs and larval development of the treefrogs Hyla crucifer and Hyla ocularis. Herpetologica. 16:225-232. Grant P.R., & B.R. Grant (1992) Hybridization of bird species. Science 256: 193-197. Green A.J. (1999) Implication of pathogenic fungi for life-history evolution in amphibians. Funct. Ecol. 13: 573-575. Hamilton W. D. (1964) The genetical evolution of social behaviour I & II. J. Theor. Biol. 7: 1– 52. Harrison R. G., & D. M. Rand. (1989) Mosaic hybrid zones and the nature of species boundaries. Pp. 111–133 in J. A. Otte and D. Endler, ed. Speciation and Its Consequences. Sinauer 104 Hegarty M.J., Barker G.L., Brennan A.C. et al. (2009) Extreme changes to gene expression associated with homoploid hybrid speciation. Mol. Ecol. 18: 877–889. Hendry A.P., Bolnick D.I., Berner D. et al. (2009) Along the speciation continuum in sticklebacks. J. Fish Biol. 75: 2000–2036. Herreid C.F., & S.Kinney. (1967) Temperature and development of the wood frog, Rana sylvatica, in Alaska. Ecology 48: 579–590. Hermansen J.S., Saether S.A., Elgvin T.O. et al. (2011) Hybrid speciation in sparrows I: phenotypic intermediacy, genetic admixture and barriers to gene flow. Mol. Ecol. 20: 3812– 3822. Hewitt G.M. (1988) Hybrid zones — natural laboratories for evolutionary studies. Trends Ecol. Evol. 3: 158–167. Hitchings S.P., & T.J.C. Beebee (1998) Loss of genetic diversity and fitness in common toad (Bufo bufo) populations isolated by inimical habitat. J. Evol. Biol. 11: 269–283. Hokit D. G., Walls S. C. & A.R. Blaustein A. R. (1996) Context dependent kin discrimination in larvae of the marbled salamander, Ambystoma opacum. Anim. Behav. 52: 17–31. Jiggins C.D., Salazar C., Linares M. et al. (2008) Hybrid trait speciation and Heliconius butterflies. Phil. Trans. Roy. Soc. Lond. B 363: 3047–3054. Kiesecker J.M., Blaustein A.R. & L.K. Belden (2001) Complex causes of amphibian population declines. Nature 410: 681–684. Kitano J., Mori S. & C. L. Peichel (2007) Phenotypic divergence and reproductive isolation between sympatric forms of Japanese threespine sticklebacks Biol. J. Linn. Soc. 91: 671– 685. 105 Kozak G. M., Rudolph A.B., Colon B.L., & R. C. Fuller (2012) Postzygotic isolation evolves before prezygotic isolation between fresh and saltwater population of rainwater killifish, Lucania parva. Int. J. Evol. Biol. doi:10.1155/2012/523967 Lai Z. Gross B.L. Zou Y. et al. (2006) Microarray analysis reveals differential gene expression in hybrid sunflower species. Mol. Ecol. 15: 1213–1227. Lemmon E. M., & A. R. Lemmon (2010) Reinforcement in chorus frogs: lifetime fitness estimates including intrinsic natural selection and sexual selection against hybrids. Evolution 64:1748-1761. Licht L.E. (1971) Breeding habits and embryonic thermal requirements of the frogs, Rana aurora aurora and Rana pretiosa pretiosa, in the Pacific Northwest. Ecology 52: 116–124. Nielsen E. E., Hansen, M. M., Ruzzante, D. E., et al. (2003) Evidence of a hybrid-zone in Atlantic cod (Gadus morhua) in the Baltic and the Danish Belt Sea revealed by individual admixture analysis. Mol. Ecol. 12:1497–1508. Noor M. A. (1999) Reinforcement and other consequences of sympatry. Heredity 83:503–508. Mable B.K., Alexandrou M.A. & M.I. Taylor (2011) Genome duplications in amphibians and fish: an extended synthesis. J. Zool. 284: 151–182. Mallet J. (1986) Hybrid zones in Heliconius butterflies in Panama, and the stability and movement of warning colour clines. Heredity 56: 191-202. Mallet J. (2007) Hybrid speciation. Nature 446: 279-283. Maurer B.A. (1984) Interference and exploitation in bird communities. Wilson Bull. 96: 380395. Marshall J. L., Arnold M. L., & D. J. Howard (2002) Reinforcement: The road not taken. Trends Ecol Evol 17: 558–563. 106 Mavárez J., & M. Linares (2008) Homoploid hybrid speciation in animals. Mol. Ecol. 17:4181– 4185. May R.M., Endler J.A., & R. E. McMurtrie (1975) Gene frequency clines in the presence of selection opposed by gene flow. Am. Nat. 109:659-676. Mayr E. (1942) Systematics and The Origin of Species From The Viewpoint of a Zoologist. New York: Columbia University Press. Mayr E. (1963). Animal Species and Evolution. Belknap Press, Harvard. Minton S. A., Jr. (2001) Amphibians & Reptiles of Indiana. Revised 2nd Edition. Indiana Academy of Science, Indianapolis. Moore J.A. (1939) Temperature tolerance and rates of development in the eggs of Amphibia Ecology 20: 459–478 Moore J.A. (1949) Geographical variation of adaptive characters in Rana pipiens Schreber. Evolution 3: 1–24 Moore W. S. (1977) An evaluation of narrow hybrid zones in vertebrates. Quart. Rev. Biol., 52: 263—277. Moore W.S. & D. B. Buchanan (1985) Stability of the Northern Flicker hybrid zone in historical times: implications for adaptive speciation theory. Evolution 39: 135-151. Moore W.S., & J. T. Price (1993) Nature of selection in the northern flicker hybrid zone and its implications for speciation theory. In R. G. Harrison [ed.], Hybrid Zones and The Eevolutionary Process, 196- 225. Oxford University Press, New York, NY. Moreira P. L., & M. Barata (2005) Egg mortality and early embryo hatching caused by fungal infection of Iberian rock lizard (Lacerta monticola) clutches. Herp. J. 15:265–272. 107 Moritz C., Schneider C. J., & D. B.Wake (1992) Evolutionary relationships within the Ensatina eschscholtzii complex confirm the ring species interpretation. Syst. Biol. 41: 273–291. Muller H. J. (1940) Bearing of the Drosophila work on systematics. Pp. 185–268 in J. Huxley, ed. The New Systematics. Clarendon Press, Oxford, U.K. Orr H.A. & M. Turelli (2001) The evolution of postzygotic isolation: accumulating Dobzhansky-Muller incompatibilities Evolution 55: 1085–1094 Parris M. J. (1999) Hybridization in leopard frogs (Rana pipiens complex): larval fitness components in single-genotype populations and mixtures. Evolution 53:1872–1883. Parris M. J. (2001) Hybridization in leopard frogs (Rana pipiens complex): terrestrial performance of newly metamorphosed hybrid and parental genotypes in field enclosures. Can. J. Zool. 79:1552–1558. Parris M. J. (2004) Hybrid response to pathogen infection in interspecific crosses between two amphibian species (Anura: Ranidae). Evol. Ecol. Res. 6: 457–471. Pearson S. F. (2000) Behavioural asymmetries in a moving hybrid zone. Behav. Ecol. 11: 84-92. Pettus P. & G.M. Angleton (1967) Comparative reproductive biology of montane and piedmont chorus frogs. Evolution 21: 500–507 Pfennig D.W., Rice A.M., & R.A. Martin (2007) Field and experimental evidence for competition's role in phenotypic divergence. Evolution 61:257–271. Pfennig D.W., Reeve H.K., & P.W. Sherman (1993) Kin recognition and cannibalism in spadefoot toad tadpoles. Anim. Beh. 46: 87-94. Pfennig K. S., & M. A. Simovich (2002) Differential selection to avoid hybridization in two toad species. Evolution 56: 1840–1848. 108 Prager E. M., & A. C. Wilson (1975) Slow evolutionary loss of the potential for interspecific hybridization in birds: a manifestation of slow regulatory evolution. Proc. Natl. Acad. Sci. USA 72:200–204. Presgraves D. C. (2002) Patterns of postzygotic isolation in Lepidoptera. Evolution 56:1168– 1183. Presgraves D. C., Balagopalan L., Abmayr S.M. et al. (2003) Adaptive evolution drives divergence of a hybrid inviability gene between two species of Drosophila,” Nature 423: 715–719. Ruibal R. (1955) A study of altitudinal races in Rana pipiens Evolution 9: 322–338. Rundle H. D. (2002) A test of ecologically dependent postmating isolation between sympatric sticklebacks. Evolution 56:322–329. Rundle H. D., Nagel L. Boughman J. W. et al. (2000) Natural selection and parallel speciation in sympatric sticklebacks. Science 287: 306–308. Sasa M. M. Chippindale P. T. & N. A. Johnson (1998) Patterns of postzygotic isolation in frogs. Evolution 52:1811–1820. Schluter D. (1993) Adaptive radiation in sticklebacks: size, shape, and habitat use efficiency Ecology 74: 699–709. Schluter D. (2003) Frequency dependent natural selection during character displacement in sticklebacks. Evolution 57:1142–1150. Seehausen O., Koetsier E., Schneider M.V. et al. (2003) Nuclear markers reveal unexpected genetic variation and a Congolese-Nilotic origin of the Lake Victoria cichlid species flock. Proc. Roy. Soc. Lond. B 270: 129–137. 109 Semlitsch R. D (1993) Asymmetric competition in mixed populations of tadpoles of the hybridogenetic Rana esculenta complex. Evolution 47: 510-519. Semlitsch R.D., Hotz H., & G-D. Guex (1997) Competition among tadpoles of coexisting hemiclones of hybridogenetic Rana esculenta: support for the frozen niche variation model. Evolution 51: 1249-1261. Servedio M. R. (2001) Beyond reinforcement: the evolution of premating isolation by direct selection on preferences and postmating, prezygotic incompatibilities. Evolution 55:1909– 1920. Servedio M. R., & M. A. Noor (2003) The role of reinforcement in speciation: theory and data. Annu. Rev. Ecol. Evol. Syst. 34: 339–364. Silla A.J. (2011) Effect of priming injections of luteinizing hormone-releasing hormone on spermiation and ovulation in Günther’s toadlet, Pseudophryne guentheri. Repro. Biol. Endo. 8: 68. Skelly D. K., Freidenburg,L.K., & J.M. Kiesecker (2002) Forest canopy cover and the performance of larval amphibians. Ecology 83: 983-992. Slatkin M. (1973) Gene flow and selection in a cline. Genetics75: 733-756. Steinwascher K. (1978) Interference and exploitation competition among tadpoles of Rana utricularia. Ecology 59: 1039-1046. Stelkens R.B., Young K.A., &O. Seehausen (2010) The accumulation of reproductive incompatibilities in African cichlid fish. Evolution 64: 617–633. 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. 110 Tiffin P., Olson M.S., & L.C. Moyle (2001) Asymmetrical crossing barriers in angiosperms. Proc. Roy. Soc. Lond. B. 268: 861-867. Touchon J. C., Gomez-Mestre I. & K. M. Warkentin (2006) Hatching plasticity in two temperate anurans: responses to a pathogen and predation cues. Can. J. Zool. 84:556–563. Turelli M., Barton N.H. & J. A. Coyne (2001) Theory and speciation. Trends Ecol. Evol. 16: 330-343. Veen T., Borge T., Griffith S.C., et al. (2001) Hybridization and adaptive mate choice in flycatchers. Nature 411: 45–50. Volpe E.P. (1953) Embryonic temperature adaptations and relationships in toads Physiol. Zool. 26: 344–354. Volpe E.P. (1955) Intensity of reproductive isolation between sympatric and allopatric populations of Bufo americanus and Bufo fowleri Am. Nat. 89: 303–317. Warkentin K. M., Currie C. R. & S. A. Rehner (2001) Egg killing fungus induces early hatching of red-eyed treefrog eggs. Ecology 82:2860–2869. Wedekind C. (2002) Induced hatching to avoid infectious egg disease in whitefish. Curr. Biol. 12:69–71. Weiner J. (1990) Asymmetric competition in plant populations. Trends Ecol. Evol. 5: 360-364. Welch M.E., & L.H. Rieseberg (2002) Habitat divergence between a homoploid hybrid sunflower species, Helianthus paradoxus (Asteraceae), and its progenitors. Am. J. Bot. 89:472-478. Wood T.E., Takebayashi N., Barker M.S. et al. (2009) The frequency of polyploid speciation in vascular plants. Proc. Nat. Acad. Sci. USA 106: 13875-13879. 111 Wright A. H. & A. A. Wright (1995) Handbook of Frogs and Toads of The United States and Canada. Comstock publishing Co. Inc., Cornell University, Ithaca, NY. Zweifel R.G. (1968) Reproductive biology of anurans of the arid southwest, with emphasis on adaptation of embryos to temperature Bull. Am. Mus. Nat. Hist. 140: 1–64 112 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. 113 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, 114 ‘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. 115 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 116 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 117 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 118 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 119 (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 120 (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 121 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 122 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 4.9 Literature Cited 1) Mayr, E. 1963 Animal species and evolution. Harvard Univ. Press. Cambridge, MA. (DOI: 10.1002/ajpa.1330210315) 2) Coyne, J. A., & H.A. Orr. 2004 Speciation. Sinauer Associates, Sunderland, M.A. 3) Lande, R. 1981. Models of speciation by sexual selection on polygenic traits. P. Natl. Acad. Sci. U.S.A., 78, 3721-3725. (DOI: 10.1073/pnas.78.6.3721) 4) Sherman, P.W., Reeve, H.K. & D.W. Pfennig. 1997. Recognition systems. In Behavioural ecology: an evolutionary approach, 4th edn (ed Krebs J.R. and N. B. Davies), pp. 69-96. Blackwell Scientific Publications. London, U.K. 5) Elmer, K.R., Lehtonen, T.K., & A. Meyer. 2009 Color assortative mating contributes to sympatric divergence of neotropical cichlid fish. Evolution 63, 2750-2757. (DOI: 10.1111/j.1558-5646.2009.00736.x) 6) Irestedt, M., Jønsson, K.A., Fjeldså, J., Christidis, L., & P.G.P. Ericson 2009 An unexpectedly long history of sexual selection in birds-of-paradise. BMC Evol. Biol. 9, 235. (DOI: 10.1186/1471-2148-9-235) 7) Scopece, G., Cozzolino, S., Johnson, S.D., & F.P. Schiestl. 2010 Pollination efficiency and the evolution of specialized deceptive pollination systems. Am. Nat. 175, 98-105. (DOI: doi:10.1086/648555) 8) Waelti, M.O., Page, P.A., Widmer, A., & F.P. Schiestl, 2009 How to be an attractive male: floral dimorphism and attractiveness to pollinators in a dioecious plant. BMC Evo. Biol. 9,190. (DOI: doi:10.1186/1471-2148-9-190) 9) Zahavi, A. 1975. Mate selection – a selection for a handicap. J. Theor. Biol. 53, 205-214. (DOI: doi:10.1016/0022-5193(75)90111-3) 134 10) Zahavi, A. 1977. The cost of honesty (Further remarks on the handicap principle). J. Theor. Biol. 67, 603-605. (DOI: 10.1016/0022-5193(77)90061-3) 11) van der Sluijs, I., Van Dooren, T.J.M., Hofker, K.D., van Alphen, J.J.M., Stelkens, R.B. & O. Seehausen. 2008 Female mating preference functions predict sexual selection against hybrids between sibling species of cichlid fish. Phil. Trans. R. Soc. B. 363, 2871-2877. (DOI: 10.1098/rstb.2008.0045) 12) Bridle, J.R., Salamando, C.I., Koning, W., & R.K. Butlin. 2006 Assortative preferences and discrimination by females against hybrid male song in the grasshoppers Chorthippus brunneus and Chorthippus jacobsi (Orthoptera: Acrididae). Euro. Soc. Evo. Biol. 19, 12481256. (DOI: doi:10.1111/j.1420-9101.2006.01080.x) 13) Hill, G.E. 1990 Female house finches prefer colourful males: sexual selection for a condition-dependent trait. Anim. Behav. 40, 563-572. (DOI: 10.1016/S0003-3472(05)8053714) Fitch, W.T., & M.D. Hauser. 1995 Vocal production in nonhuman primates: Acoustics, physiology, and functional constraints on “honest” advertisement. Am. J. Primatol. 37, 191219. (DOI: 10.1002/ajp.1350370303) 15) Wagner, W.E. 1992 Deceptive or honest signalling of fighting ability? A test of alternative hypotheses for the function of changes in call dominant frequency by male cricket frogs. Anim. Behav. 44, 449-462. (DOI: 10.1016/0003-3472(92)90055-E) 16) Dominey, W.J. 1984 Alternative mating tactics and evolutionarily stable strategies. Am. Zool. 24, 385-396. (DOI: 10.1093/icb/24.2.385) 17) Lank, D.B., Smith, C.M., Hanotte, O., Burke, T., & F. Cooke. 1995 Genetic polymorphism for alternative mating behaviour in lekking male ruff Philomachus pugnax. Nature. 378, 5962. (DOI: 10.1038/378059a0) 135 18) Younge, A.J., Spong, G., & T. Clutton-Brock. 2007 Subordinate male meerkats prospect for extra-group paternity: alternative reproductive tactics in a cooperative mammal. Proc. R. Soc. Lond. B. Biol. Sci. 274, 1603-1609. (DOI: 10.1098/rspb.2007.0316) 19) Gross, M.R. 1982 Sneakers, satellites and parental males: polymorphic mating strategies in North American sunfishes. Z. Tierpsychol. 60, 1-26. (DOI: 10.1111/j.14390310.1982.tb01073.x) 20) Rowell, G.A. & W.H. Cade. 1993 Simulation of alternative male reproductive behavior: calling and satellite behaviour in field crickets. Ecol. Model. 65, 265-280. (DOI: 10.1016/0304-3800(93)90083-5) 21) Sinervo, B., & C.M. Lively. 1996. The rock-paper-scissors game and the evolution of alternative male strategies. Nature. 380, 240-243. (DOI: 10.1038/380240a0) 22) Zamudio, K.R., & L.M. Chan. 2008 Alternative reproductive tactics in amphibians. In Alternative Reproductive Tactics, ed. Oliveria., R.F., Taborsky., M.,& H. J. Brockmann, Cambridge Univ. Press. Cambridge, U.K. (DOI: 10.1017/CBO9780511542602.012) 23) Gross, M.R. 1991. Evolution of alternative reproductive strategies: frequency-dependent sexual selection in male bluegill sunfish, Philos. Trans. R. Sot. London Ser. B. 332, 59-66. (DOI: 10.1098/rstb.1991.0033) 24) Petrie, M., & B. Kempenaers. 1998. Extra-pair paternity in birds: explaining variation between species and populations. Trends Ecol. Evol. 13, 52-58. (DOI: 10.1016/S01695347(97)01232-9) 25) Krupa, J.J. 1989 Alternative mating tactics in the Great Plains toad. Anim. Behav. 37, 10351043. (DOI: 10.1016/0003-3472(89)90147-4) 136 26) Ten Eyck, G.R. 2008 Seratonin modulates vocalizations and territorial behaviour in an amphibian. Behav. Brain. Res. 193, 144-147. (DOI: 10.1016/j.bbr.2008.05.001) 27) Gerhardt, H.C. 1991 Female mate choice in treefrogs: static and dynamic acoustic criteria. Anim. Behav. 42, 615-635. (DOI: 10.1016/S0003-3472(05)80245-3) 28) Andersson, M. 1994. Sexual selection. Princeton Univ. Press. Princeton, NJ. 29) Sullivan, B.K., Ryan, M.J., & P.A. Verrell. 1995 Female choice and mating system structure. In Amphibian Biology: Social Behaviour, ed. Heatwole., H., and B.K. Sullivan. pp. 469-517. Surrey Beatty. Chipping-Norton, U.K. 30) Ryan, M. J. 1988 Energy, calling, and selection. Am. Zool. 28, 885-898. (DOI: 10.1093/icb/28.3.885) 31) Gerhardt, H.C. 1994 Phonotaxis in female frogs and toads: Execution and design of experiments. Biomethods; Methods in comparative psychoacoustics, pp. 209-220. Birkhaeuser Verlag. Basel, Switzerland. 32) Gerhardt, H.C. 198. Sound pattern recognition in some North American treefrogs (Anura: Hylidae): implications for mate choice. Am. Zool. 22, 581–595. (DOI: 10.1093/icb/22.3.581) 33) Pfennig, K.S. 1998 The evolution of mate choice and the potential for conflict between species and mate-quality recognition. Proc. R. Soc. Lond. B. 265, 1743-1748. (DOI: 10.1098/rspb.1998.0497) 34) Pfennig, K.S. 2000 Female spadefoot toads compromise on mate quality to ensure conspecific matings. Behav. Ecol. 11, 220-227. (DOI: 10.1093/beheco/11.2.220) 35) Lowry, D. B., Modliszewski, J. L., Wright, K. M., Wu, C. A., & J. H. Willis. 2008 The strength and genetic basis of reproductive isolating barriers in flowering plants. Phil. Trans. R. Soc. B 363, 3009-3021. (DOI: 10.1098/rstb.2008.0064) 137 36) Nosil, P., Vines, T. H., & D.J. Funk. 2005 Perspective: reproductive isolation caused by natural selection against immigrants from divergent habitats. Evolution. 59, 705-719. (DOI: 10.1554/04-428) 37) Kirkpatrick, M. & V. Ravigne. 2002 Speciation by natural and sexual selection: models and experiments. Am. Nat. 159, 22-35. (DOI: 10.1086/338370) 38) Ramsey, J., Bradshaw, H.D. Jr., & D.W. Schemske. 2003 Components of reproductive isolation between the monkey flowers Mimulus lewisii and M. cardinalis (Phrymaceae). Evolution. 57, 1520–1534. (DOI: 10.1554/01-352) 39) Mallet, J. 2005 Hybridization as an invasion of the genome. Trend Ecol. Evol. 20, 229-237. (DOI: 10.1016/j.tree.2005.02.010) 40) Stace, C.A. 1987 Hybridization and the plant species. In Differentiation patterns in higher plants ed. Urbanska., K.M, pp. 115-127. Academic Press. New York, U.S.A. 41) Arnold, M.L. 1997 Natural hybridization and evolution. Oxford Univ. Press, Oxford, U.K. 42) Harrison, R.G. 1986 Pattern and process in a narrow hybrid zone. Heredity. 56, 337-349. (DOI: 10.1038/hdy.1986.55) 43) Chan, K.M.A., & S.A. Levin. 2005 Leaky prezygotic isolation and porous genomes: rapid introgression of maternally inherited DNA. Evolution. 59, 720-729. (DOI: 10.1554/04-534) 44) Hartman, P.J., Wetzel, D.P., Crowley, P.H., & D.F. Westneat. 2012 The impact of extra-pair mating behaviour on hybridization and genetic introgression. Theor. Ecol. (DOI: 10.1007/s12080-011-0117-1) 45) Austin, J.D., Lougheed, S.C., Neidrauer, L., Chek, A., & P.T. Boag. 2002 Cryptic lineages in a small frog: the post-glacial history of the spring peeper, Pseudacris crucifer (Anura: Hylidae). Mol. Phylogenet. Evol. 25, 316-329. (DOI: 10.1016/S1055-7903(02)00260-9) 138 46) Austin, J.D., Lougheed, S.C., & P.T. Boag. 2004 Discordant temporal and geographic patterns in maternal lineages of eastern North American frogs, Rana catesbeiana (Ranidae) and Pseudacris crucifer (Hylidae). Mol. Phylogenet. Evol. 32, 799-816. (DOI: 10.1016/j.ympev.2004.03.006) 47) 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 12-1036 48) MacCulloch, R.D. 2002 The ROM field guide to amphibians and reptiles of Ontario. Royal Ontario Museum and McClelland & Stewart Ltd. Toronto, Canada. 49) Wells, K.D. & C.R. Bevier. 1997 Contrasting patterns of energy substrate use in two species of frogs that breed in cold weather. Herpetologica. 53, 70-80. 50) Lance, S.L., & D. K. Wells. 1993 Are spring peeper satellite males physiologically inferior to calling males? Copeia. 1993, 1162-1166. (DOI: 10.2307/1447103) 51) Forester, D. C., & D.V. Lykens. 1986 Significance of satellite males in a population of spring peepers (Hyla crucifer). Copeia. 1986, 719-724. (DOI: 10.2307/1444955) 52) Yezerinac, S.M., Lougheed., S.C., & P. Handford. 1992 Measurement error and morphometric studies: an assessment of statistical power and the effect of observer experience using avian skeletons. Syst. Biol. 41, 471-482. 53) Stewart, K.A., Hudson, C.M., & S.C. Lougheed. 2012 Data from “Do alternative male mating tactics facilitate introgression across a Pseudacris crucifer hybrid zone?” Queen’s University Biological Station Data Archive, Herpetology. http://www.qubsdata.org/datasets/herp.php. 139 54) Degner, J. F., Hether., T.D., & E.A. Hoffman. 2009 Eight novel tetranucleotide and five cross-species dinucleotide microsatellite loci for the ornate chorus frog (Pseudacris ornata). Mol. Ecol. Res. 9, 622-624. (DOI: 10.1111/j.1755-0998.2008.02478.x) 55) Pritchard, J.K., Stephens, M., & P. Donnelly. 2000 Inference of population structure using multilocus genotype data. Genetics 155, 945-959 56) Earl, D. A. & B.M. vonHoldt. 2011 STRUCTURE HARVESTER: a website and program for visualizing STRUCTURE output and implementing the Evanno method. Cons. Gen. Resour. 4, 359-361. (DOI: 10.1007/s12686-011-9548-7) 57) Evanno, G., Regnaut, S., & J. Goudet. 2005 Detecting the number of clusters of individuals using the software STRUCTURE: a simulation study. Mol. Ecol. 14, 2611-2620. (DOI: 10.1111/j.1365-294X.2005.02553.x) 58) Nielson, E. E. G., Bach, L. A., & P. Kotlicki. 2006. HYBRIDLAB (Version 1.0): a program for generating simulated hybrids from population samples. Mol. Ecol. Resour. 6, 971-973. (DOI: 10.1111/j.1471-8286.2006.01433.x) 59) Schulte-Hostedde, A. I., Zinner, B., Miller, J. S., & G. J. Hickling. 2005 Restitution of masssize residuals: validating body condition indices. Ecology. 86, 155-163. (DOI: 10.1890/040232) 60) Howard, R.D. 1978 The evolution of mating strategies in bullfrogs, Rana catesbiana. Evolution. 32, 850-871. (DOI: 10.2307/2407499) 61) Leary, C.J., Fox., D.J., Shepard., D.B. & A.M. Garcia. 2005. Body size, age, growth and alternative mating tactics in toads: satellite males are smaller but not younger than calling males. Anim. Behav. 70, 663-671. (DOI: 10.1016/j.anbehav.2004.12.013) 62) Humfeld, S.C. 2007 Intersexual dynamics mediate the expression of satellite mating 140 tactics: unattractive males and parallel preferences. Anim. Behav. 75, 205-215. (DOI: 10.1016/j.anbehav.2007.05.015) 63) Meuche, I. & H. Pröhl 2011. Alternative mating tactics in the strawberry poison frog (Oophaga pumilio). Herpetol. J. 21: 275-277. 64) Wells, K.D., Taigen T.L., Rusch S.W., & C.C. Robb. 1995 Seasonal and nightly variation in glycogen reserves of calling gray treefrogs (Hyla versicolor) Herpetologica. 51, 359-368. 65) Gerhardt, H. C., & F. Huber. 2002 Acoustic communication in insects and anurans; common problems and diverse solutions. Univ. of Chicago Press. Chicago, IL. (DOI: 10.1121/1.1591773) 66) Brepson, L., Troïanowski, M., Voituron, Y., & T. Lengagne. 2012 Cheating for sex: inherent disadvantage or energetic constraint? Anim. Behav. 84, 1253-1260. (DOI: 10.1016/j.anbehav.2012.09.001) 67) Forester, D.C., & K. J. Thompson. 1998 Gauntlet behaviour as a male sexual tactic in the American Toad (Amphibia: Bufonidae). Behaviour. 135, 99-119. (DOI: 10.1163/156853998793066375) 68) Heino, M., & V. Kaitala. 1999 Evolution of resource allocation between growth and reproduction in animals with indeterminate growth. J. Evol. Biol. 12, 423-429. (DOI: 10.1046/j.1420-9101.1999.00044.x) 69) Ryan, M. J. 1983 Sexual selection and communication in a neotropical frog, Physalaemus pustulosus. Evolution. 37, 261-272. (DOI: 10.2307/2408335) 70) Eggert, C., & R. Guyétant. 2003 Reproductive behaviour of spadefoot toads (Pelobates fuscus): daily sex ratios and males' tactics, ages, and physical condition. Can. J. Zool. 81, 4651. 141 71) West-Eberhard, M. J. 2003 Developmental plasticity and evolution. Oxford Univ. Press. New York, U.SA. (DOI: 10.1139/z02-224) 72) Leary, C.J., Garcia, A.M. & R. Knapp. 2008. Density-dependent mating tactic expression is linked to stress hormone in Woodhouse’s toad. Behav. Ecol. 19, 1103-1110. (DOI: 10.1093/beheco/arn102) 73) Burke, J. M., & M. L. Arnold. 2001 Genetics and the fitness of hybrids. Ann. Rev. Genet. 35, 31-52. (DOI: 10.1146/annurev.genet.35.102401.085719) 74) Lamb, T., & J. Avise. 1986 Directional introgression of mitochondrial DNA in a hybrid population of tree frogs: the influence of mating behavior. Proc. Natl. Acad. Sci. U.S.A. 83, 2526–2530. (DOI: 10.1073/pnas.83.8.2526) 75) Garant, D., Fleming, I. A., Einum, S., & L. Bernatchez. 2003 Alternative male life-history tactics as potential vehicles for speeding introgression of farm salmon traits into wild populations. Ecol. Lett. 6, 541-549. (DOI: 10.1046/j.1461-0248.2003.00462.x) 76) Hauber, M. E., & P.W. Sherman. 2001 Self-referent phenotype matching: theoretical considerations and empirical evidence. Trends Neuro. 24, 609-616. (DOI: 10.1016/S01662236(00)01916-0) 77) Pfennig, K.S., Rapa, K., & R. McNatt. 2000 Evolution of male mating behaviour: male spadefoot toads preferentially associate with conspecific males. Behav. Ecol. Sociobiol. 48, 69-74. (DOI: 10.1007/s002650000205) 78) Martinsen, G.D., Whitham, T.G., Turek, R. J., and P. Keim. 2001 Hybrid populations selectively filter gene introgression between species. Evolution. 55, 1325-1335. (DOI: 10.1111/j.0014-3820.2001.tb00655.x) 142 79) Funk, D. J., & K.E. Omland. 2003 Species-level paraphyly and polyphyly: frequency, causes, and consequences, with insights from animal mitochondrial DNA. Annu. Rev. Ecol. Evol. Syst. 34, 397-423. (DOI: 10.1146/annurev.ecolsys.34.011802.132421) 80) Toews, D.P L., & A. Brelsford. 2012 The biogeography of mitochondrial and nuclear discordance in animals. Mol. Ecol. 21, 3907-3930. (DOI: 10.1111/j.1365-294X.2012.05664.x) 81) Rieseberg, L. H., Sinervo, B., Linder, C. R., Ungerer, M. C., & D. M. Arias. 1996 Role of gene interactions in hybrid speciation: evidence from ancient and experimental hybrids. Science. 272, 741–744. (DOI: 10.1126/science.272.5262.741) 82) Palumbi, S. R., & C. S. Baker. 1994 Contrasting population structure from nuclear intron sequences and mtDNA of humpback whales. Mol. Biol. Evol. 11, 426-435. 83) Chan, K. M. A. 2003 The effects of slightly leaky prezygotic isolating barriers and the use of phylogenetic tree shape to study diversification. Ph.D. diss. Princeton Univ. Princeton, NJ. 143 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), 144 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 145 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 146 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 147 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. 151 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. 5.2 Literature Cited April, J., & J. Turgeon. 2006. Phylogeography of the banded killifish (Fundulus diaphanous): glacial races and secondary contact. J. Fish Biol. 69: 212-228. Austin, J. D., Lougheed, S. C., Neidrauer, L., Check, A.A., & P.T. Boag.2002. Cryptic lineages in a small frog: post-glacial history of the spring peeper, Pseudacris crucifer (Anura: Hylidae). Mol. Phylogenet. Evol. 25: 316-325. 152 Austin, J. D., Lougheed, S. C., & P. T. Boag. 2004. Discordant temporal and geographic patterns in maternal lineages of eastern North American frogs, Rana catesbeiana and Pseudacris crucifer. Mol. Phylogenet. Evol. 32: 799–816. Avise, J.C. 1994. Molecular Markers, Natural History, and Evolution. Chapman & Hall, New York Avise, J.C. 2000. Phylogeography: The History and Formation of Species. Harvard University Press, Cambridge. Avise, J.C., Arnold, J., Ball, R.M., Bermingham, E., Lamb, T., Neigel, J.E., Reeb, C.A., & N.C. Saunders. 1987. Intraspecific phylogeography: the mitochondrial DNA bridge between population genetics and systematics. Ann. Rev. Ecol. Syst. 18:489–522. Avise, J.C., & K. Wollenberg. 1997. Phylogenetics and the origin of species. Proc. Natl. Acad. Sci. 94: 7748–7755. Bank, C., Hermisson, J., & M. Kirkpatrick. 2011. Can reinforcement complete speciation? Evolution 66: 1229-239. Bickford, D., D. Lohman, N. Sodhi, P. Ng, R. Meier, K.Winker, K. Ingram, & I. Das. 2006. Cryptic species as a window on diversity and conservation. Trends Ecol. Evol. 22: 148–155. Burke, J.M. & M.L. Arnolde. 2001.Genetics and the fitness of hybrids. Annu. Rev. Genet. 35:3152. Butlin, R. K. 2010. Population genomics and speciation. Genetica 138: 409-418. Cicero, C. 2004. Barriers to sympatry between avian sibling species (Paridae: Baeolophus) in local secondary contact. Evolution 58: 1573-1587. Conant, R., & J. T. Collins. 1998. A Field Guide to Reptiles and Amphibians: Eastern / Central North America. Third Ed. Houghton Mifflin Company, NY. 153 Cooley, J.R., Simon, C., Marshall, D.C., Slon, K., & C. Ehrhardt. 2001. Allochronic speciation, secondary contact, and reproductive character displacement in periodical cicadas (Hemiptera: Magicicada spp.): genetic, morphological, and behavioural evidence. Mol. Ecol. 10: 661-671. Coyne, J.A. & H. A. Orr. 2004. Speciation. Sinauer Associates, Sunderland, MA. Dawley, R. M. 1987. Hybridization and polyploidy in a community of three sunfish species (Pices: Centrarchidae). Copeia 1987: 326–335. Dowling, T. E., & W. R Hoeh. 1991. The extent of introgression outside the contact zone between Notropis cornutus and Notropis chrysocephalus (Teleostei: cyprinidae). Evolution 45: 944-956. Elmer, K.R., J.A. Davila & S.C. Lougheed. 2007. Cryptic diversity, deep divergence, and Pleistocene expansion in an upper Amazonian frog, Eleutherodactylus ockendeni. BMC Evol. Biol. 7:247. Fouquet, A., Gilles, A., Vences, M., Marty, C. Blanc, M., & N.J. Gemmell. 2007. Underestimation of species richness in neotropical frogs revealed by mtDNA analysis. PLoS ONE 2: e1109 17971872 Feder, J.L., Egan, S.P., & P. Nosil. 2012. The genomics of speciation with gene-flow. Trends Genet. 28: 342-350. Gay, L., Neubauer, G., Zagalska-Neubauer, M., Debain, C., Pons, J.M., David, P., & P.A. Crochet. 2007. Molecular and morphological patterns of introgression between two large white-headed gull species in a zone of recent secondary contact. Mol. Ecol. 16: 3215-3227. 154 Gombert, A., Lucas, L.K., Fordyce, J.A., Forister, M.L. & C.C. Nice. 2010. Secondary contact between Lycaeides idas and L. melissa in the Rocky Mountains: extensive admixture and a patchy hybrid zone. Mol. Ecol. 19: 3171 – 3192. Green, D. M. 1984. Sympatric hybridization and allozyme variation in the toads Bufo americanus and B. fowleri in southern Ontario. Copeia 1984: 18-26. Hewitt, G.M. 1996. Some genetic consequences of ice ages, and their role in divergence and speciation. Biol. J. Linn. Soc. 58: 247–276. Hoskin, C., M. Higgie, K. McDonald, & C. Moritz. 2005. Reinforcement drives rapid allopatric speciation. Nature 437: 1353–1356. Howard, D. J. 1993. Reinforcement: origin, dynamics, and fate of an evolutionary hypothesis. Pp. 46–69 in R. G. Harrison, ed. Hybrid Zones and the Evolutionary Process. Oxford Univ. Press, New York. Kawakami, T., R. Butlin, M. Adams, D. Paull, & S. Cooper. 2009. Genetic analysis of a chromosomal hybrid zone in the Australian grasshoppers (Vandiemenella, viatica species group). Evolution 63: 139–152. Lemmon, E.A. & A. R. Lemmon. 2010. Reinforcement in chorus frogs: lifetime fitness estimates including intrinsic natural selection and sexual selection against hybrids. Evolution 64:1748–1761. Lemmon, E.A., A. R. Lemmon, J. T. Collins, & D. C. Cannatella. 2008. A new North American chorus frog species (Amphibia: Hylidae: Pseudacris) from the south-central United States. Zootaxa 1675:1–30. 155 Moriarty-Lemmon, E. 2009. Diversification of conspecific signals in sympatry: geographic overlap drives multi-dimensional reproductive character displacement in frogs. Evolution 63:1155–1170. Moritz, C.C. 1994. Defining ‘evolutionary significant units’ for conservation. Trends Ecol. Evol. 9: 373-375. Mullen, S. P., Dopman, E.B., & R. G. Harrison. 2008. Hybrid zone origins, species boundaries, and the evolution of wing-pattern diversity in a polytypic species complex of North American admiral butterflies (Nymphalidae: Limentis). Evolution 62: 1400-1417. Pfennig, D.W., & K.S. Pfennig. 2010. Character displacement and the origins of diversity. Am. Nat. 176: S26-S44. Phillips, B., S. Baird, & C.Moritz. 2004. When vicarsmeet: a narrowcontact zone between morphologically cryptic phylogeographic lineages of the rainforest skink, Carlia rubrigularis. Evolution 58: 1536–1548. Richards, C.L., Carstens, B.C., & L.L. Knowles. 2007. Distribution modeling and statistical phylogeography: an integrative framework for generating and testing alternative biogeographic hypotheses. J. Biogeogr. 34: 1833–1845. Richards-Zawacki, C.L. & M. E. Cummings. 2011. Intraspecific reproductive character displacement in a polymorphic poison dart frog, Dendrobates pumilio. Evolution. 65: 259-267. Rissler, L.J., & W. H. Smith. 2010. Mapping amphibian contact zones and phylogeographical break hotspots across the United States. Mol. Ecol. 19: 5404-5416. 156 Sætre, G-P., Moum, T., Burez, S., Kràl, M., Adamjan, M., & J. Moreno.1997. A sexually selected character displacement in flycatchers reinforces premating isolation. Nature 387: 589–592. Scriber, J.M., Keefover, K., & S. Nelson. 2002. Hot summer temperatures may stop movement of Papilo Canadensis butterflies and genetic introgression south of the hybrid zone in the North American Great Lakes region. Ecography 25: 184-192. Singhal, S., & C. Moritz. 2012. Strong selection against hybrids maintains a narrow contact zone between morphologically cryptic lineages in a rainforest lizard. Evolution 66: 1474-1489. Smadja, C., & G. Ganem. 2005. Asymmetrical reproductive character displacement in the house mouse. J. Evol. Biol. 18:1485-1493. Soltis, D.E., Morris, A.B., McLachlan, J.S., Manos, P.S., & P.S. Soltis. 2006. Comparative phylogeography of unglaciated eastern North America. Mol. Ecol. 15: 4261-4293. Weir, J. T. & D. Schluter. 2004. Ice sheets promote speciation in boreal birds. Proc. Roy. Soc. Lond. B 271: 1881–1887. Yukilevich, R. 2012. Asymmetrical patterns of speciation uniquely support reinforcement in Drosophila. Evolution 66: 1430-1446. Zamudio, K. R., & W. K. Savage. 2003. Historical isolation, range expansion, and secondary contact of two highly divergent mitochondrial lineages in spotted salamanders (Ambystoma maculatum). Evolution 57: 1631-1652. 157 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. 159 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