The Drivers of Mitochondrial DNA Divergences

Home , Achalinus rufescens, Achalinus spinalis, Agaminae, Causus defilippii, Chamaeleo monachus, Elaphe bimaculata

Pedro Henrique Bernardo

A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy

Department of Ecology and Evolutionary Biology

University of Toronto

The Drivers of Mitochondrial DNA Divergences

Pedro Henrique Bernardo

Doctor of Philosophy

Department of Ecology and Evolutionary Biology

University of Toronto

2018

Abstract

Mitochondrial phylogeography uses the relationship between matrilineal genealogy and geography to explain speciation, phylogeny and population structure. Several hypotheses on what maintains the parapatric matrilines have been proposed: philopatry, incomplete lineage sorting, vicariance and natural selection on female-linked traits. The small lizard

Urosaurus nigricaudus of the peninsula of Baja California, Mexico and southern

California, USA has six parapatric matrilines that differ by substantial mtDNA divergence.

In this study, I investigate what drives and maintains the deep mtDNA discontinuities in this species. I precisely locate the mtDNA lineage breaks and investigate the presence of physical barriers in those areas. I sequence the entire mtDNA genome and then test the following hypotheses: unrestricted nDNA gene flow occurs across the species; mtDNA divergences correspond to the geological events; and that functional diversifying selection is involved in the maintenance of mtDNA discordances. I use thousands of nDNA SNP loci to test the first and last hypotheses. The time-calibrated genealogy cannot reject the

hypothesis that the mtDNA discordances originated due to a series of vicariant events that happened on the peninsula starting in the late Miocene. For millions of years, females have dispersed beyond their place of birth, and yet, they do not cross the mtDNA contact zones.

Thus, I reject the hypothesis of female philopatry as an explanation for the mtDNA discontinuities. In the mtDNA contact zones, females from two lineages co-exist in a small area with no barriers forbidding dispersal beyond the mtDNA breaks. The analysis with nDNA SNPs fails to reject the hypothesis of unrestricted nDNA gene flow throughout the peninsula (K=1). Thus, analyses also reject the hypotheses of vicariance and incomplete lineage sorting as explaining the continuing mtDNA discontinuities. Selective pressure analyses point to purifying selection as the evolutionary force acting in the mitogenome of this lizard, thus rejecting the hypothesis that habitat adaptation maintains the mtDNA discontinuities. My results reject most of the proposed hypothesis for the maintenance of mtDNA discontinuities and create exciting opportunities for further investigation on the role of other female-linked traits in the maintenance of mtDNA discontinuities, such as mito-nuclear functional compensation and female behavior.

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Acknowledgments

I believe that no meaningful and pivotal transition in life, comes easily. My move to

Canada from Brazil in 2010 was one of the most significant undertakings of my life.

Learning a new language and adjusting to a new culture and all its nuances, has been quite an endeavor. What has made this entire mission worthwhile is the reason for my move and the most reliable and solid foundation I could have ever asked for: my precious wife, Aya

Refaeli-Bernardo. I thank Aya for always standing by my side during the difficult times and for her continuous support during my Ph.D. degree. I thank my parents Maria da Graça and Jorge, my brothers João Paulo and Luiz Fernando, and Aya’s family, for their continued support and understanding during graduate school.

I would like to thank my Canadian father and Ph.D. Supervisor, Prof. Robert W. Murphy.

Bob has received me with open arms to his lab when I arrived in Canada, and I thank him for his guidance, financial support, friendship and for continually challenging me to become better. I also thank Bob for understanding the challenges I faced during graduate school, for supporting my new career as a Police Officer, and for allowing me to conclude my Ph.D. degree.

I thank professors Deborah McLennan and Marie-Joseé Fortin who were members of my committee, for their continuous support and advice that significantly improved this thesis.

I further thank professors Nathan Lovejoy, Doug Currie, Sebastian Kvist, Helen Rodd and

Fernando Marques who kindly accepted the invitation to participate in my appraisal and final examinations.

A special thanks to the lab technicians Amy Lathrop and Kristen Choffe; my fellow lab- mates Christina Davy, Christopher Blair, Kevin Kong, Santiago Sánchez-Pacheco,

Santiago Sánchez-Ramírez, Mateus Pepinelli and Hollis Dahn. Tulio Soares and Olivera

Joksimovic volunteered to assist with my lab work. They all contributed immensely to my project and made the lab a pleasant and fun place to work.

To my Mexican colleagues Prof. Sergio Ticul Álvarez-Castañeda, Prof. Fausto Roberto

Mendez-de-la-Cruz, Dr. Eduardo Felipe Aguilera-Miller, Carmen Izmene Gutiérrez-Rojas

(Mene), Cintya Segura-Trujillo and Griselda Gallegos-Simental, thank you for the invaluable help in acquiring the material necessary for this thesis. I am also thankful to Dr.

Yessica Rico, Jonathan R. Galina-Mehlman and Dr. Taylor Edwards for the valuable assistance during the ddRAD-seq process.

I am grateful to the Royal Ontario Museum and the Department of Ecology and

Evolutionary Biology at the University of Toronto (EEB) for providing an intelligent and stimulating environment during my years as a Ph.D. student.

I thank York Regional Police Chief Eric Jolliffe and all my supervisors for authorizing my leave of absence to complete this thesis. A special thanks to my YRP friends Paulo

Ferreira, Brad Weick and Alvaro Almeida for the continuous support and for inspiring me as examples of excellence.

During my Ph.D. I was supported by a Canada Graduate Scholarship from the National

Science and Engineering Research Council of Canada (NSERC), and a number of EEB grants. My research was funded through a NSERC Discovery Grant to Prof. Robert W.

Murphy and field equipment was generously donated by IdeaWild.

Table of Contents

Acknowledgments ...... iv

List of Tables ...... xi

List of Figures ...... xiv

Chapter 1 General Introduction ...... 1

1.1 Mitochondrial DNA Discontinuities and Research Objectives ...... 1

1.2 The Peninsula of Baja California ...... 2

1.3 Model Organism ...... 3

1.4 Research Outline ...... 4

1.4.1 Chapter 2: Using Maternal Ancestry Monophyly Analysis (MAMA) in the

field to detect contact zones between parapatric populations ...... 6

1.4.2 Chapter 3. The complete mitochondrial genome of the black-tailed brush

lizard Urosaurus nigricaudus (Reptilia, Squamata, Phrynosomatidae) ...... 6

1.4.3 Chapter 4. When mitochondrial phylogeography fails: female genealogy is

only part of the story ...... 7

1.4.4 Chapter 5. The drivers of deep mitochondrial DNA divergence of black-

tailed brush lizard (Urosaurus nigricaudus) ...... 8

Chapter 2 Using Maternal Ancestry Monophyly Analysis (MAMA) in the field to detect contact zones between parapatric populations ...... 10

Abstract ...... 11

2.1 Introduction ...... 12

2.2 Material and Methods ...... 13

2.2.1 Sampling ...... 13

2.2.2 DNA Extraction, Amplification and Sequencing ...... 14

2.2.3 Development of Lineage-Specific Primers ...... 16

2.3 Results and Discussion ...... 17

2.4 Figures ...... 20

2.5 Tables ...... 25

Chapter 3 The complete mitochondrial genome of the black-tailed brush lizard

Urosaurus nigricaudus (Reptilia, Squamata, Phrynosomatidae)...... 28

Abstract ...... 29

3.1 Main text ...... 30

3.2 Figures ...... 34

3.3 Tables ...... 37

Chapter 4 When mitochondrial phylogeography fails: female genealogy is only part of the story ...... 48

Abstract ...... 49

4.1 Introduction ...... 50

4.2 Material and Methods ...... 52

4.2.1 Sampling ...... 52

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4.2.2 MtDNA genome sequencing ...... 53

4.2.3 Genealogical tree ...... 53

4.2.4 Double digest RAD Sequencing and analyses ...... 54

4.2.5 Population structure ...... 56

4.2.6 Genetic differentiation ...... 57

4.3 Results ...... 57

4.3.1 MtDNA genome and ddRADSeq sequencing ...... 57

4.3.2 Population structure and genetic differentiation using mtDNA ...... 58

4.3.3 Population structure and genetic differentiation using nDNA ...... 59

4.4 Discussion ...... 59

4.5 Acknowledgements ...... 63

4.6 Data accessibility ...... 63

4.7 Figures ...... 64

4.8 Tables ...... 65

Chapter 5 The drivers of deep mitochondrial DNA divergence in the black-tailed brush lizard (Urosaurus nigricaudus) ...... 91

Abstract ...... 92

5.1 Introduction ...... 93

5.2 Material and Methods ...... 96

5.2.1 Sampling ...... 96

5.2.2 MtDNA genome sequencing ...... 97

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5.2.3 MtDNA genome tree and divergence times ...... 97

5.2.4 Molecular evolution analyses ...... 98

5.3 Results ...... 101

5.3.1 Mitogenome tree and divergence times ...... 101

5.3.2 Selective pressure analyses ...... 101

5.4 Discussion ...... 103

5.4.1 The matrilineal genealogy and divergence times ...... 103

5.4.2 Selection on the mitogenome ...... 105

5.4.3 The drivers of mtDNA discordances ...... 107

5.4.4 Conclusion ...... 111

5.5 Acknowledgements ...... 111

5.6 Data accessibility ...... 111

5.7 Figures ...... 112

5.8 Tables ...... 115

Chapter 6 CONCLUDING DISCUSSION ...... 124

6.1 Summary of thesis chapters ...... 125

6.1.1 Chapter 2: Using Maternal Ancestry Monophyly Analysis (MAMA) in the

field to detect contact zones between parapatric populations ...... 125

6.1.2 Chapter 3. The complete mitochondrial genome of the black-tailed brush

lizard Urosaurus nigricaudus (Reptilia, Squamata, Phrynosomatidae) ...... 125

6.1.3 Chapter 4. When mitochondrial phylogeography fails: female genealogy

does not match speciation history...... 126

6.1.4 Chapter 5. What drives the deep mitochondrial DNA divergence of black-

tailed brush lizard (Urosaurus nigricaudus) ...... 126

6.2 Opportunities for future research ...... 127

References ...... 131

List of Tables

Table 2.1: Samples of Urosaurus nigricaudus deposited on the herpetological collection of the Royal Ontario Museum used to design the lineage-selective primers. Clades S2, C1 and C2 follow Lindell et al. (2008)...... 25

Table 3.1: List of the 22 pairs of primers used to sequence the complete mitochondrial genome of Urosaurus nigricaudus. Pair ID was used for lab work and to illustrate the position of the primers in the mtDNA genome (see Figure 1). Authors: 1 – This study; 2 –

Green et al. 2010; 3 – Kumazawa & Endo 2004...... 37

Table 3.2: mtDNA genome organization and features in Urosaurus nigricaudus...... 39

Table 3.3: List of squamate species for which a complete mtDNA genome has been sequenced...... 41

Table 4.1: List of the 196 specimens of Urosaurus nigricaudus collected in the Peninsula of Baja California, Mexico in the month of August/2013. ROM#: Voucher number for the tissue deposited in the herpetological collection of the Royal Ontario Museum, Canada.

Sample ID is the ID used in the laboratory work and displayed in the figures of this paper.

...... 65

Table 4.2: Localities for the 26 individuals used for mtDNA genome sequencing and analyses...... 80

Table 4.3: Localities for the 73 individuals used for ddRAD-seq sequencing and analyses.

...... 83

Table 4.4: AMOVA results based on the mitochondrial DNA genome and nuclear SNPs different populations of Urosaurus nigricaudus...... 89

Table 4.5: Pairwise ΦPT based on the mitochondrial DNA genome (below diagonal) and nuclear SNPs (above diagonal) of Urosaurus nigricaudus...... 90

Table 5.1: Samples of Urosaurus nigricaudus used on this study. ROM voucher represents the deposit number of the tissue in the herpetological collection of the Royal Ontario

Museum...... 115

Table 5.2: Results of the LRT comparison to evaluate whether the codons evolved with one average ω (null model M0 - lnL0) or with different values of ω (alternative Model M1- lnL1). ω0 represents null model M0. Level of significance determined by P-value with degree of freedom k=1...... 117

Table 5.3: Results of the statistical tests of adaptive evolution among codon sites using site models. LRT comparisons between models M1 x M2a and M7 and M8. Significant level determined by p-value with degree of freedom k=2...... 118

Table 5.4: Results of the Bayes Empirical Bayes (BEB) analysis calculated using Models

M2a and M8 (beta and ω) to identify positively selected sites. BEB posterior probability that ω>1 (Pr) was considered significant if greater than 0.95...... 119

Table 5.5: Results of the statistical tests of ω variation among lineages using Branch models. LRT results are from the comparison between null model M0, which assumes the same ω for lineages, and the alternative Two-Ratio model that assumes a different ω for a specific ...... 121

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Table 5.6: Results of the statistical tests of adaptive evolution among sites and lineages using Branch-site models. LRTs were used to compare null model A1 (lnL0), which does not allow the specific branch on the tree (foreground branch) to evolve under positive selection to alternative model A (lnL1), which allows for positive selection...... 123

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List of Figures

Figure 2.1: Map of the southern Peninsula of Baja California, Mexico. Circles represent samples used in this study (multiple samples from the same locality represented by one circle). Red shading represents matriline S2; green shading denotes matriline C1; yellow shading shows matriline C2. Map scale in kilometers. Source: Landsat/Google Earth. .. 20

Figure 2.2: Primer design for the northern mtDNA break: nucleotide variation within and between the two mtDNA lineages. Green bars represent the lineage-selective primer designed. A: Reverse primer CytbRC1 matches all specimens from matriline C1. B:

Reverse primer CytbRS2 matches all specimens from matriline S2...... 21

Figure 2.3: Results of the survey for the mtDNA discontinuity for the northern mtDNA sympatric lineage. Red dots: matriline S2. Green dots: matriline C1. A: First surveyed transect: 60km. B: Second surveyed transect: 25km. C: Third surveyed transect: 10km. D:

Forth surveyed transect: yellow arrow shows the exact location of the mtDNA sympatric lineages...... 22

Figure 2.4: Agarose gel showing the different size bands produced by PCR using the lineage-selective primers for the northern break (S2-C1)...... 23

Figure 2.5: Divergent mtDNA populations of Urosaurus nigricaudus at the Isthmus of La

Paz mtDNA break (S2-C1). Red circle highlights northern lineage (S2) and green circle highlights central lineage (C1). The mtSL is located in the lineage overlap area. Black circles represent the isolated sampled populations...... 24

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Figure 3.1: Position of primers used on this study. Primers numbers correspond to the primer pair ID (Table 1). The mtDNA genome organization corresponds to that of

Urosaurus nigricaudus...... 34

Figure 3.2: Mitochondrial genome of Urosaurus nigricaudus with details of gene organization...... 35

Figure 3.3: Number of squamate species with an entire mtDNA genome sequenced over the past 20 years. Data collected from the Web of Science...... 36

Figure 4.1: A: Bayesian Inference matrilineal genealogy of Urosaurus nigricaudus based on the mitochondrial genes ATP6, ATP8 and CytB (totaling a 1981bp alignment) built using MRBAYES. Node values are the Bayesian posterior probability. Matrilines S2, C1 and C2 are highly supported (BPB=1). B: Map of southern Peninsula of Baja California,

Mexico showing area occupied by the three matrilines...... 64

Figure 5.1: Map of the southern Peninsula of Baja California, Mexico showing sampling localities. Different shades represent matrilines: S2, C1 and C2. Map scale in kilometers.

Source: Landsat/Google Earth...... 112

Figure 5.2: Bayesian Inference matrilineal genealogy of Urosaurus nigricaudus based on the entire mitogenome (excluding D-loop) using MRBAYES. Numbers on the nodes represent the Bayesian posterior probabilities. Scale bar represents percent genetic divergence...... 113

Figure 5.3: Time-calibrated genealogical tree of U. nigricaudus based on the mitogenome built using the molecular clock rooting method of BEAST. Grey bars represent the 95% highest posterior density...... 114

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Chapter 1 General Introduction

1.1 Mitochondrial DNA Discontinuities and Research Objectives

Mitochondrial phylogeography was a term proposed by Avise et al. (1987) to describe “the study of the relationship between genealogy and geography”. Zink & Barrowclough

(2008), in a more direct statement, described phylogeography as the “superposition of a gene tree onto geography” and defended the use of the mtDNA as a reliable tool to investigate geographical population structure, phylogeographic patterns, and species limits. The use of mtDNA as a genetic marker has its advantages: it has a simple genetic structure that is transmitted only by one parent without recombination; it has a high mutation rate and nucleotide diversity; and the sequencing process is easy and inexpensive

(Avise et al. 1987; Edwards et al. 2005; Zink & Barrowclough 2008; Dool et al. 2016). In spite of such advantages, the use of mtDNA alone, without nuclear DNA (nDNA) genes, has several limitations that can negatively influence the results. These limitations include homoplasy, introgression and maternal mode of inheritance (Avise et al. 1987; Edwards

& Bensch 2009; Dool et al. 2016; Haenel 2017; Ivanov et al. 2018). Although its use as the only genetic marker in studies has been declining (Edwards et al. 2005), hundreds of scientists continue to rely solely on mtDNA and its discordances to address a wide range of evolutionary issues from phylogenetic inference (Abiadh et al. 2010; Byrne et al. 2010;

Cameron 2014; Pozzi et al. 2014; Andújar et al. 2015), species delimitation (Kozak et al.

2006; Thomé et al. 2010; Pesarakloo et al. 2017) and the description of new species

(Streicher et al. 2014; Morin et al. 2016; Borsa et al. 2017), to population structure and

2 gene flow (Zhou et al. 2016b; Men et al. 2017; Hu et al. 2018), and to determining conservation priorities (Banguera-Hinestroza et al. 2002; Teixeira et al. 2018). The problem lies in the evolutionary assumptions resulting from such studies, which are possibly biased and inaccurate. Analyses that include nDNA markers have the potential to improve resolution and reduce the effects of mtDNA biases. Several studies, however, have shown that reciprocal monophyly of mtDNA is often not supported by nDNA genes

(known as cytonuclear discordance) (Toews & Brelsford 2012; Pavlova et al. 2013; Bar

Yaacov et al. 2015). For this reason, some authors defend the use of mtDNA instead of nDNA (Zink & Barrowclough 2008; Malukiewicz et al. 2017), while others criticize this approach (Ballard & Whitlock 2004; Bazin et al. 2006; Edwards & Bensch 2009; Fisher-

Reid & Wiens 2011; Dool et al. 2016). While this discussion continues, an emergent consideration needs attention: what causes and maintains the deep mtDNA discontinuities observed in some species? In this dissertation, I will test a set of hypotheses about the drivers of mtDNA discontinuities and investigate what processes maintain such divergences.

1.2 The Peninsula of Baja California

On the peninsula of Baja California, Mexico, deeply divergent female lineages (matrilines) are commonplace and widely distributed. Congruent patterns have been reported in several animal species in the region, including, but not limited to, lizards (Lindell et al. 2005;

2008), snakes (Rodrı́guez-Robles & De Jesús-Escobar 2000; Harrington et al. 2017), mammals (Lawlor et al. 2002; Álvarez-Castañeda & Murphy 2014), birds (Riddle et al.

2000) and scorpions (Gantenbein et al. 2001). The origin of these discordances dates back

3 millions of years and no functional explanation exists for their persistence. Paleontological and sedimentary data suggest that the Gulf of California was created during the middle

Miocene (14–16 Ma; Smith 1991) and, since then, the lineages in the peninsula have undergone several temporary vicariant events (Smith 1991; Riddle et al. 2000) that likely involved a series of ephemeral islands (Murphy & Aguirre-Léon 2002). The mtDNA discordances in the peninsula occur near the location of historical vicariant events and in particular temporary seaways that divided the peninsula in three main locations: the region of the Isthmus of La Paz during the late Miocene between 11 and 7 Ma (Lindell et al.

2008), in the Cape Region between 6.5 and 3.2 Ma (McCloy 1984; Carreño 1992; Murphy

& Aguirre-Léon 2002; Lindell et al. 2008) and a more recent midpeninsular seaway that divided the peninsula around 1 Ma (Upton & Murphy 1997; Lindell et al. 2006). This geological history is recorded in the mtDNA patterns of the black-tailed brush lizard

Urosaurus nigricaudus. The isolation of U. nigricaudus in these ephemeral islands should have prevented gene flow, and thus the distinguishing mtDNA substitutions would have become fixed within each population by genetic drift. Intriguingly though, even after secondary contact female lineages remain genetically distinct. The retention of the discontinuities forms the basis of phylogeography itself.

1.3 Model Organism

As a model organism, I use the black-tailed brush lizard Urosaurus nigricaudus (Cope,

1864) because of its distinct geographic patterns between mtDNA and nDNA (Lindell et al. 2008). Urosaurus nigricaudus is a common phrynosomatid lizard on the Peninsula of

Baja California, Mexico and it ranges continuously from the southern tip in the Cape

Region, northward into southern California, USA. It is an arboreal species that can be found in urban areas and in a wide variety of natural environments, where it mostly occupies mesquite trees (Prosopis palmeri S. Watson) and smaller shrubs in the “arroyos”

(depressions in the landscape that form temporary rivers during the rainy season). The climate in the peninsula is hot, dry and covered by cacti and desert, and the arroyos are the only areas that can hold water for a short period of time, with the exception of the oases where the lizards can be found in palm trees (Washingtonia spp. and Brahea spp.). Thus, they are the only areas where the trees occur. Female U. nigricaudus rarely leave their trees and are extremely aggressive towards intrusion by other females. Males are constantly moving and are not as territorial as the females, except during the mating season from

April to August, when males are aggressive towards one another and often defend territories (Munguia-Vega et al. 2013). The female genealogy of U. nigricaudus presented by Lindell et al. (2008) using two mtDNA genes revealed six deeply divergent mitochondrial lineages (sequence divergence of up to 11.02% between mtDNA lineages), and yet Aguirre-Léon et al. (1999) could not reject the hypothesis of unrestricted nDNA gene flow based on allozyme data. Today, females from two mtDNA lineages coexist in the mtDNA break zones as close as 2 meters from each other and they do not cross those mitochondrial DNA boundaries.

1.4 Research Outline

Mitochondrial phylogeography only exists because of mtDNA divergences and discontinuities. Parapatric matrilines are essential to any phylogeographic study and yet a fundamental question remains unanswered: what drives and maintains those

5 discontinuities in areas where there is no obvious driver of isolation? Several hypotheses have been proposed: philopatry and sex-biased dispersal, incomplete lineage sorting, vicariance, and natural selection on female-linked traits (Morales et al. 2015; Pavlova et al. 2017). In this study, I test the main hypothesis that female-linked traits maintain the mtDNA divergences and investigate what other natural processes may also be involved.

Furthermore, I test the efficacy of mtDNA phylogeography and the use of mtDNA data alone to assess gene flow and population structure.

To test my main hypothesis and elucidate both the historical and contemporary forces influencing the genetic structure of U. nigricaudus, I use a set of hypotheses and analyses that include: precisely locating the mtDNA lineage breaks using lineage-selective primers and investigate the presence of physical barriers on those contact zones (Chapter 2); sequencing the entire mtDNA genome for use in further analyses (Chapter 3); testing the hypothesis of unrestricted nDNA gene flow in a species with deep mtDNA discontinuities using thousands of nDNA SNP loci (Chapter 4); testing the hypotheses that mtDNA divergences correspond to the geological events in the Peninsula of Baja California and that functional diversifying selection is involved in the maintenance of mtDNA discordances (Chapter 5).

All chapters of this dissertation are outlined below. They were all written as independent papers that have been published or are currently being prepared for submission to peer- reviewed journals.

1.4.1 Chapter 2: Using Maternal Ancestry Monophyly Analysis (MAMA) in

the field to detect contact zones between parapatric populations

The divergence of mtDNA populations in species with unrestricted nDNA gene flow places the focus on the role of the populations that live in the contact zones. In this first chapter, I adapt a molecular assay to effectively locate the mitochondrial contact zones of

U. nigricaudus in the southern portion of the Peninsula of Baja California. I develop lineage-specific primers that amplify DNA fragments of differing lengths for each matriline, and I employ this technique in the field for the first time. The use of lineage- selective primers enables a quick and straightforward identification of matrilines in U. nigricaudus with 100% success, making the field expedition more time- and cost-efficient.

The discovery of the specific location of the mitochondrial sympatric lineages (mtSL) where two matrilines co-exist, reveals that today no physical barriers separate those matrilines.

1.4.2 Chapter 3. The complete mitochondrial genome of the black-tailed

brush lizard Urosaurus nigricaudus (Reptilia, Squamata,

Phrynosomatidae)

Having successfully collected almost 200 samples of U. nigricaudus on its natural environment and identified the mitochondrial sympatric lineages, I then sequence the entire mtDNA genome of U. nigricaudus to investigate the evolutionary implications of these parapatric mtDNA discordances. In Chapter 3, I use some universal primers for

7 reptiles, design a new set of primers, and develop new laboratory protocols especially for

U. nigricaudus to sequence its complete mitochondrial genome.

This chapter was published in Mitochondrial DNA Part A (Bernardo, P.H., Sánchez-

Ramírez, S., Aguilera-Miller, E.F., Álvarez-Castañeda, S.T., Méndez-de la Cruz, F.R., and

R.W. Murphy. 2016. The complete mitochondrial genome of the black-tailed brush lizard

Urosaurus nigricaudus (Reptilia, Squamata, Phrynosomatidae). Mitochondrial DNA Part

A, 27 (6): 4023-4025).

1.4.3 Chapter 4. When mitochondrial phylogeography fails: female

genealogy is only part of the story

Hundreds of studies have relied only on mtDNA gene trees to infer phylogenies, delimit species, infer population structure and estimate gene flow. In Chapter 4, I use the mtDNA genome sequences acquired in the previous chapter and generate RAD loci for thousands of nuclear SNPs to compare the power of both markers to infer population structure, gene flow, and molecular variance. My results confirm that U. nigricaudus has three parapatric and deeply divergent maternal lineages in southern Baja California, but analyses of nDNA do not detect genetic structuring (k=1). The results fail to reject the hypothesis of unabated gene flow between sampling sites. Based on my results, I reject the hypothesis that mtDNA gene trees can serve as the sole evidence for evolutionary inference. Furthermore, the failure to reject of unrestricted nDNA gene flow throughout the peninsula rejects the hypothesis that coalescent time and incomplete lineage sorting are involved in the maintenance of mtDNA discontinuities.

This chapter is currently being prepared for submission to Molecular Ecology (Bernardo,

P.H., Aguilera-Miller, E.F., Álvarez-Castañeda, S.T., Méndez-de la Cruz, F.R., and R.W.

Murphy. When mitochondrial phylogeography fails: female genealogy is only part of the story).

1.4.4 Chapter 5. The drivers of deep mitochondrial DNA divergence of

black-tailed brush lizard (Urosaurus nigricaudus)

Because vicariance (Chapter 2) and incomplete lineage sorting (Chapter 4) do not explain the maintenance of mtDNA discordances, in Chapter 5, I use the mtDNA genome sequences from Chapter 3 to test if one female-linked trait, selection on the mitogenome, is distinctly shaping each lineage and contributing to the maintenance of lineage isolation.

My results show that functional diversifying selection on the mtDNA is not involved in the maintenance of mtDNA discordances. Also, I use a time-calibrated Bayesian tree to date the divergence time between matrilines and the results indicate that females have been dispersing beyond their place of birth for millions of years but only within their mtDNA boundaries. Thus, the results reject the hypothesis that philopatry is involved in the maintenance of mtDNA discordances.

The results of my study reject the hypotheses that philopatry, vicariance, and incomplete lineage sorting are involved in the maintenance of the mtDNA discordance. Because diversifying selection is also not playing a role in the maintenance of these mtDNA discontinuities, my results open exciting opportunities for further investigations of other female-linked traits in this species, such as mito-nuclear functional compensation and female biology, including but not limited to, female behavior.

This chapter is currently being prepared for submission to Mitochondrion (Bernardo, P.H.,

Sánchez-Ramírez, S., Aguilera-Miller, E.F., Álvarez-Castañeda, S.T., Méndez-de la Cruz,

F.R., and R.W. Murphy. What drives the deep mitochondrial DNA divergence of black- tailed brush lizard (Urosaurus nigricaudus)).

Chapter 2 Using Maternal Ancestry Monophyly

Analysis (MAMA) in the field to detect contact zones

between parapatric populations

Abstract

Natural systems with discordances between the geographic pattern of mitochondrial DNA

(mtDNA) and nuclear DNA (nDNA) provide valuable opportunities to investigate evolutionary hypotheses on an intraspecific level. The divergence of mtDNA populations

(matrilines) in species in which no barriers to nDNA gene flow exist among them, place the focus on the role of the populations that live in the contact zones. In the present study,

I adapt a molecular assay based on the Maternal Ancestry Monophyly Analysis (MAMA) to effectively locate two mitochondrial sympatric lineages (mtSL) of Urosaurus nigricaudus on the Peninsula of Baja California, Mexico. I develop lineage-specific primers that amplify DNA fragments of different sizes specific for each matriline and for the first time employ this technique in the field. The use of lineage-selective primers enables a simple and quick identification of matrilines in U. nigricaudus with 100% success. The two mtSL located in the southern peninsula coexist in a narrow contact zone.

This approach is time- and cost-efficient when studying mitochondrial DNA contact zones and can achieve even better results when used in conjunction with new DNA amplification technologies.

2.1 Introduction

Mitochondrial phylogeography was developed by Avise et al. (1987) to study the relationship between genealogy and geography based on mitochondrial DNA (mtDNA) divergences and discontinuities. Intraspecific mtDNA discontinuities occur commonly in animals (Toews & Brelsford 2012) and are particularly interesting in species that show divergences in the presence of nuclear DNA (nDNA) gene flow (Pavlova et al. 2013).

Cytonuclear or mitonuclear discordance are terms that describe the incongruence between mtDNA and nDNA geographic patterns. Hypothesis testing in species with cytonuclear discordance is the key to understanding the dynamics involved in the maintenance of such mtDNA discontinuities (Lindell & Murphy 2008; Morales et al. 2015), especially when looking at matrilines that occupy the contact zones (mitochondrial sympatric lineages - mtSL).

The peninsula of Baja California, Mexico, contains several examples of species with deeply divergent matrilines and secondary contact zones (Aguirre-Léon et al. 1999;

Gantenbein et al. 2001; Lawlor et al. 2002; Rodriguez-Robles 2002; Lindell et al. 2005).

Those divergences originated millions of years ago (Ma) and involve several geological events that happened in the peninsula starting on the late Miocene (Chapter 5). One of those species is the arboreal black-tailed brush lizard Urosaurus nigricaudus. This small lizard, which lives on tree-islands and has six parapatric matrilines with up to 11% mtDNA sequence divergence (Lindell et al. 2008), is a good model to investigate the drivers of such deep mtDNA divergence in the presence of nDNA gene flow (Aguirre-Léon et al.

1999). Several hypotheses have been proposed to explain such cytonuclear discordances

13 including incomplete lineage sorting, vicariance, sex-biased dispersal and natural selection on female-linked traits (Morales et al. 2015; Pavlova et al. 2017). Further explorations into these hypotheses requires precisely locating the mtSL.

Lindell & Murphy (2008) developed a simple and quick polymerase chain reaction assay for identification of divergent mtDNA lineages in areas of secondary contact. This technique, named Maternal Ancestry Monophyly Analysis (MAMA), uses lineage- selective primers to amplify a lineage-diagnostic product. Their method proved to be efficient in laboratory settings, however, the success of this technique in the field has yet to be assessed. In this study, I present the results of the first use of MAMA in the field. I develop a new set of primers and PCR protocols that allow the effective identification of the mtSL in mtDNA contact zones of U. nigricaudus at the Isthmus of La Paz break

(mtDNA divergence of 11.02%) and the Cape Region break (mtDNA divergence of

7.41%), both in the southern part of the Peninsula of Baja California. The location of those mtSL will provide DNA samples for individuals that may be directly involved with the maintenance of mtDNA divergences and will provide the necessary information for further evolutionary analyses of U. nigricaudus.

2.2 Material and Methods

2.2.1 Sampling

In June 2013, prior to the first field expedition, I selected 46 tissue samples (Table 2.1) of

Urosaurus nigricaudus deposited in the Royal Ontario Museum herpetological collection, from the three southernmost matrilines on the peninsula (Figure 2.1): 13 individuals from

14 the northern matriline (hereafter referred to as S2), 32 individuals from the central matriline (hereafter referred to as C1) and one individual from the southern matriline

(hereafter referred to as C2) based on the results of Lindell et al. (2008).

For the northern contact zone (S2-C1), I chose samples from an area 30 to 110 kilometers north of La Paz, Baja California Sur. For the southern boundary (C1-C2), I chose samples from San Bartolo in the north to Cabo San Lucas in the south and the only available sample from matriline C2.

2.2.2 DNA Extraction, Amplification and Sequencing

Total genomic DNA was extracted from ethanol-preserved muscle and liver tissue using the standard phenol-chloroform protocol proposed by Sambrook et al. (1989). For the S2-

C1 break, I sequenced part of the gene encoding Cytochrome b gene (Cytb; 1085bp) and for the C1-C2 break I sequenced part of the ATP6 gene (859bp). I chose to sequence one gene per break to further test the reliability of MAMA on two different regions of the mitogenome. The primers used to amplify and to sequence the Cytb fragment were L16355

(5′-CCA TCC AAC ATC TCA GCA TGA TGA AA-3′) and H17415 (5′-GTC TTC AGT

TTT TGG TTT ACA AGA C-3′). For the ATP6 fragment, I used the primers L9839 (5′-

AGC ACT AGC CTT TTA AGY T-3′) and H10710 (5′-GTG TGC TTG GTG TGY CAT

T-3′). The PCR mix (25μl) contained: 18.55 μl of ddH20, 2.5 μl of 1.5 mM MgCl2 buffer,

0.8 μl of 10mM dNTPs, 1 μl of 10 mM of each primer, 0.15 μl of 5 U Taq DNA Polymerase

(Boehringer Mannheim), 1 μl (10 ng) of template DNA. Amplification reactions were performed on a Perkin Elmer GeneAmp 9700 and Eppendorf AG 5345 thermal cycler

(Applied Biosystems) using the follow program: initial denaturation of 94 °C for 2 minutes

15 followed by 39 cycles of 94 °C for 30 seconds, 50-52 °C for 45 seconds, 72 °C 45 for seconds, with a final extension temperature of 72 °C for 5 min. At the end of the program the temperature dropped to 4°C indefinitely. Amplified DNA was separated by electrophoresis on a 1% agarose gel and stained with SYBR Safe DNA Gel Stain

(ThermoFisher). DNA bands were visualized on Safe Imager 2.0 Blue Light

Transilluminator (ThermoFisher) and individually extracted and centrifuged through a filter pipette tip for 10 min at 3500 RPM. All samples were then sequenced for both directions (forward and reverse) using the BigDye Terminator v 3.1 Cycle Sequencing Kit

(Applied Biosystems). The sequencing reaction mix (10 μl) contained: 1 μl of BigDye, 2

μl of 5x BigDye Terminator Buffer, 2 μl of ddH20, 1 μl of 10 mM of primer, and 4 μl of the PCR product. I then ran the sequencing reaction on an Eppendorf AG 5345 thermal cycler (Applied Biosystems) using the following program: initial denaturation at 96 °C for

1 minute, 25 cycles of 96 °C for 10 seconds, 50 °C for 5 seconds and 60 °C for 4 minutes.

After the last cycle the temperature was set to 4°C indefinitely. The reactions were then cleaned and precipitated with sodium acetate and ethanol and sequenced using a 3730

DNA Analyzer (Applied Bio-Systems).

The final sequences were aligned using GENEIOUS 6.1 (Kearse et al. 2012). To verify to which lineage each sample belonged, I used MRBAYES 3.2.6 (Huelsenbeck & Ronquist

2001; Ronquist et al. 2012) to build a maternal genealogical tree. Using the topology of the consensus tree, the samples were assigned to matrilines.

2.2.3 Development of Lineage-Specific Primers

Once the samples were separated according to their matrilines, I used nucleotide variation in the sequence alignments to develop lineage-specific primers based on regions that were unique to each lineage. Those regions had at least a 4bp difference and were at least 20bp long (Figure 2.2).

When combined with the forward primer, the reverse lineage-specific primers would amplify fragments of different sizes (at least a 300bp difference). The PCR reactions followed the same protocols as described above with one variation: instead of 1 μl of 10 mM primer solution of each primer (forward and reverse), I added 0.4 μl of 10 mM primer solution of three primers to the mix (one conserved forward primer and the two lineage- specific primers). The PCR product was then separated by electrophoresis on a 1% agarose gel and stained with SYBR Safe DNA Gel Stain (ThermoFisher). The amplified fragment bands were visualized on Safe Imager 2.0 Blue Light Transilluminator (ThermoFisher).

Lineage identification was possible due to the difference in size between the lineage- specific amplified fragments.

To identify the sympatric population on the northern mtDNA discontinuity (S2-C1 break),

I used a fragment of Cytb. As a forward primer, I used the same conserved forward primer

L16355 (5′-CCATCCAACATCTCAGCATGATGAAA-3′) and developed two reverse lineage-specific primers for each of the lineages. Reverse primer CytbRC1 (5′-

TGGATCCTGTTTCGTGAAGG-3′) would only amplify a 491bp long fragment of samples belonging to matriline C1. Reverse primer CytbRS2 (5′-

GTAGAAGGGGAACTAGCATTAAG-3′) amplified a fragment 794bp long fragment of

17 samples belonging to matriline S2. The same methodology was used for the southern mtDNA discontinuity (C1-C2 break) using fragments of ATP6. I used the conserved forward primer ATP9839L (5′-AGCACTAGCCTTTTAAGYT-3′) and developed two reverse lineage-selective primers specific for each of the lineages: ATP6RC1(5′-

TGAGTCGTTTTGAGGGACTTAAG-3′) for the C1 lineage sequences, and ATP6RC2

(5′-GATGAGTAAGTGACCTGCCGTTAA-3′) for the C2 lineage sequences. These primers amplified fragments that were 281bp and 683bp long, respectively.

2.3 Results and Discussion

A field expedition to the southern portion of the Peninsula of Baja California took place in

August 2013. I started the fieldwork surveying the northern mtDNA break using the locality of the samples previously used to develop the lineage-specific primers. On the first day I sampled in a 60km transect where I believed the mtDNA break could be located

(Figure 2.3A).

After collecting 25 samples on this transect I returned to the Laboratory of Genetics at the

Centro de Investigaciones Biológicas del Noroeste (CEBINOR) in La Paz and ran PCR reactions with the lineage-specific primers previously designed. Those PCR reactions produced clear products that were very conspicuous and easy to be visualized on the agarose gel (Figure 2.4) based on differences of over 300bp between lineage-specific fragments.

The first PCR results narrowed the search for the contact zone to approximately 25km. I then repeated this methodology three more times until the mtDNA sympatric population

18 was located (Figures 2.3B-D) and all individuals were assigned to a matriline. After the mitochondrial sympatric population was located, I surveyed the area surrounding the mtSL two more times to verify the distribution of this sympatric population (Figure 2.5). I followed the same methodology for the Cape Region break (C1-C2) and was also successful in locating the mtSL.

The use of MAMA proved to be a highly efficient, cost and labor effective technique to identify mtSL in the field. The use of lineage-selective primers enabled a simple and quick identification of individuals from parapatric matrilines living in sympatry. DNA amplifications in the field worked in 100% of the samples and I was able to locate the mtDNA lineage boundaries within 6 days. For each trip, I narrowed the search area and, in the end, I was able to identify the lineage to which every individual belonged. The mtDNA breaks discovered in this study ranged between 50 to 180 meters in width, or the width of an “arroyo”. The arroyos are depressions in the landscape that form temporary rivers during the rainy season and they are they house shrubs and trees like mesquite trees

(Prosopis palmeri S. Watson), which U. nigricaudus occupy. In both mtDNA contact zones, individual females of two matrilines were found in trees as close to each other as 2 meters.

New technologies on PCR machines and amplification techniques like the miniPCRTM thermal cycler and integrated systems of electrophoresis and visualization like Bluegel (by miniPCRTM) have the potential to increase the reach of MAMA methods. Such innovations allow for basic laboratory work under field conditions, without access to temperature control, an electrical grid or permanent shelter (Guevara et al. 2017), and even on the

International Space Station (Boguraev et al. 2018).

2.4 Figures

Figure 2.1: Map of the southern region of the Peninsula of Baja California, Mexico.

Circles represent samples used in this study (some samples were collected in the same locality; thus, they are represented by one circle). Red shading represents matriline S2; green shading denotes matriline C1; and yellow shading shows matriline C2. Map scale in kilometers. Source: Landsat/Google Earth.

20 21

Figure 2.2: Primer design for the northern mtDNA break based on Cytb: nucleotide variation within and between the two mtDNA lineages. Green bars show the lineage- selective primer design. A: Reverse primer CytbRC1 matches all specimens from C1 matriline. B: Reverse primer CytbRS2 matches all specimens from S2 matriline.

Forth surveyed transect: exact location of the mtDNA sympatric lineage marked with a yellow arrow.

Figure 2.4: Agarose gel showing the different size bands produced by PCR using the lineage-selective primers for the northern break (S2-C1).

Figure 2.5: Divergent mtDNA populations of Urosaurus nigricaudus at the Isthmus of La

2.5 Tables

Table 2.1: Samples of Urosaurus nigricaudus deposited on the herpetological collection of the Royal Ontario Museum used to design the lineage-selective primers. Matrilines S2,

C1 and C2 follow Lindell et al. (2008).

CLADE ROM # LOCALITY LAT LON C1 ROM La Paz, 75.3 km N of town on Hwy 1 24.16 -110.91 37024 C1 ROM La Paz, 75.3 km N of town on Hwy 1 24.16 -110.91 37025 C1 ROM La Paz, 75.3 km N of town on Hwy 1 24.16 -110.91 37026 C1 ROM La Paz, 75.3 km N of town on Hwy 1 24.16 -110.91 37176 C1 ROM La Paz, 75.3 km N of town on Hwy 1 24.16 -110.91 37179 C1 ROM La Paz, 75.3 km N of town on Hwy 1 24.16 -110.91 37180 C1 ROM La Paz, 75.3 km N of town on Hwy 1 24.16 -110.91 37184 C1 ROM La Paz, 75.3 km N of town on Hwy 1 24.16 -110.91 37185 C1 ROM Vicinity of San Agustin, 76 km NW of La Paz 24.16 -110.92 37311 C1 ROM Vicinity of San Agustin, 76 km NW of La Paz 24.16 -110.92 37306 C1 ROM Vicinity of San Agustin, 76 km NW of La Paz 24.16 -110.92 37309 C1 ROM Vicinity of San Agustin, 76 km NW of La Paz 24.16 -110.92 37307 C1 ROM Vicinity of San Agustin, 76 km NW of La Paz 24.16 -110.92 37305

C1 ROM Vicinity of San Agustin, 76 km NW of La Paz 24.16 -110.92 37308 C1 ROM Vicinity of San Agustin, 76 km NW of La Paz 24.16 -110.92 37304 C1 ROM Arroyo de Canejo 24.16 -110.93 37406 C1 ROM Arroyo Conejo (just N of San Agustin which is ~76 km 24.16 -110.92 37224 N of La Paz on Hwy 1) C1 ROM La Paz, 67.2 km N of town on Hwy 1 24.15 -110.84 35337 C1 RWM La Paz, El Sombrero Trailer Park, 0.2 mi W of Hwy 1 24.14 -110.31 583 C1 RWM La Paz, El Sombrero Trailer Park, 0.2 mi W of Hwy 1 24.14 -110.31 572 C1 ROM La Paz, 59 km N of town on Hwy 1 24.14 -110.77 37020 C1 ROM La Paz, 59 km N of town on Hwy 1 24.14 -110.77 35336 C1 ROM23 La Paz 24.14 -110.32 100 C1 ROM23 La Paz 24.14 -110.32 105 C1 ROM23 La Paz 24.14 -110.32 112 C1 ROM La Paz, 10 km W of La Paz on Hwy 1 24.11 -110.38 35321 C1 ROM La Paz, 43 km N of town on Hwy 1 24.09 -110.64 35332 C1 ROM La Paz, 43 km N of town on Hwy 1 24.09 -110.64 35331 C1 RWM San Antonio, 7.3 mi S of town on Hwy 1 23.76 -109.98 652 C1 RWM San Bartolo, below microwave tower 23.74 -109.85 2315 C1 ROM23 Boca de La Sierra 23.39 -109.82 134

C1 RWM Hwy 1, 6.9 mi S of Todos Santos turnoff 23.36 -110.17 632 C2 RWM Cabo San Lucas, 4.0 mi E of town and 0.3 mi N of Hwy 22.91 -109.86 736 1 on road to microondas S2 ROM La Paz, 91 km N of town on Hwy 1 24.28 -110.96 35340 S2 ROM La Paz, 91 km N of town on Hwy 1 24.28 -110.96 37011 S2 ROM La Paz, 88.3 km N of town on Hwy 1 24.26 -110.96 37203 S2 ROM La Paz, 86 km N of town on Hwy 1 24.24 -110.95 37206 S2 ROM La Paz, 81.8 km N of town on Hwy 1 24.20 -110.95 37207 S2 ROM La Paz, 78.2 km N of town on Hwy 1 24.17 -110.93 37208 S2 ROM La Paz, 77 km N of town on Hwy 1 24.17 -110.92 37028 S2 ROM La Paz, 75.3 km N of town on Hwy 1 24.16 -110.91 37001 S2 ROM La Paz, 75.3 km N of town on Hwy 1 24.16 -110.91 37175 S2 ROM La Paz, 75.3 km N of town on Hwy 1 24.16 -110.91 37177 S2 ROM Vicinity of San Agustin, 76 km NW of La Paz 24.16 -110.92 37310 S2 ROM Arroyo de Canejo 24.16 -110.92 37403 S2 ROM La Paz Urosaurus break (vicinity of San Agustin, 76 km 24.15 -110.92 37312 NW of La Paz)

Chapter 3 The complete mitochondrial genome of

the black-tailed brush lizard Urosaurus nigricaudus

(Reptilia, Squamata, Phrynosomatidae).

A modified version of this chapter was published in Mitochondrial DNA Part A (Bernardo,

P.H., Aguilera-Miller, E.F., Álvarez-Castañeda, S.T., Méndez-de la Cruz, F.R., and R.W.

Murphy. 2016. The complete mitochondrial genome of the black-tailed brush lizard

Urosaurus nigricaudus (Reptilia, Squamata, Phrynosomatidae). Mitochondrial DNA Part

A, 27 (6): 4023–4025).

Abstract

The black-tailed brush lizard Urosaurus nigricaudus is a small arboreal lizard that can be found throughout the Peninsula of Baja California, from the Cape Region in the south into southern California, USA, in the north. Previous studies using the mitochondrial DNA

(mtDNA) genes CytB and ATP6 suggested that this species has 6 deeply divergent mtDNA lineages (matrilines) with sequence divergence ranging from 4% to 11.2%. In this study I used some universal primers for reptiles and developed a new set of primers, as well as new laboratory protocols to sequence the complete mitochondrial genome of U. nigricaudus. This genome is 17,298 bp long and comprises two rRNAs, 22 tRNAs, 13 protein-coding genes, one L-strand origin of replication and one control region. The overall nucleotide content is A= 34.2%; C= 26.8%; G= 13.5%; T= 25.5%. The gene organization and features agree with the general vertebrate organization and that found in other lizards.

The control region is 1,909 bp long and is located between tRNAPro and tRNAPhe.

3.1 Main text

The black-tailed brush lizard, Urosaurus nigricaudus, is a small, diurnal and arboreal phrynosomatid lizard endemic to the Baja California Peninsula, Mexico and Southern

California, USA. Using CytB and APT6 gene sequences, Lindell et al. (2008) found deeply divergent mtDNA lineages differing from 4% to 11.2% of nucleotides. These deep mtDNA divergences originated in a scenario of allopatric differentiation followed by secondary lineage contact. Multiple events of temporary seaways in the Peninsula of Baja California starting in the late Miocene, isolated U. nigricaudus populations. This ephemeral isolation should have prevented gene flow, and the distinguishing mtDNA substitutions would thus have become fixed within each population by genetic drift (Chapter 5).

To investigate the evolutionary implications of these parapatric mtDNA discordances, I sequenced the entire mitochondrial genome of U. nigricaudus. The specimen sequenced was collected in the Oasis San Pedrito (62 Km N of Cabo San Lucas), Baja California Sur,

Mexico (ROM tissue #53168). Polymerase chain reaction (PCR) amplifications were performed using universal mtDNA primers for reptiles (Kumazawa & Endo 2004; Green et al. 2010) and specific primers for U. nigricaudus (Table 2.1) were developed to amplify the remaining parts of the genome and to assure all sequences overlapped before assembly

(Figure 2.1).

Total genomic DNA was extracted from ethanol-preserved muscle tissue using the standard phenol-chloroform protocol proposed by Sambrook et al. (1989). The PCR mix

(25μl) contained: 18.55 μl of ddH20, 2.5 μl of 1.5 mM MgCl2 buffer, 0.8 μl of 10mM

31 dNTPs, 1 μl of 10 mM of each primer, 0.15 μl of 5 U Taq DNA Polymerase (Boehringer

Mannheim), 1 μl (10 ng) of template DNA. Amplification reactions were performed on a

Perkin Elmer GeneAmp 9700 and Eppendorf AG 5345 thermal cycler (Applied

Biosystems) using the follow program: initial denaturation of 94 °C for 2 minutes followed by 39 cycles of 94 °C for 30 seconds, 48-52 °C for 45 seconds, 72 °C 45 for seconds, with a final extension temperature of 72 °C for 5 min. Amplified DNA was separated by electrophoresis on a 1% agarose gel and stained with SYBR Safe DNA Gel Stain

(ThermoFisher). DNA bands were visualized on Safe Imager 2.0 Blue Light

(Applied Biosystems). The sequencing reaction mix (10 μl) contained: 1 μl of BigDye, 2

1 minute, 25 cycles of 96 °C for 10 seconds, 50 °C for 5 seconds and 60 °C for 4 minutes.

After the last cycle the temperature was set to 4°C indefinitely. The reactions were then cleaned and precipitated with sodium acetate and ethanol and sequenced using a 3730

DNA Analyzer (Applied Bio-Systems).

The mitogenome assembly, gene annotation and alignments were done in GENEIOUS 6.1

(Kearse et al. 2012) using the mitochondrial genome of the phrynosomatid Sceloporus occidentalis (GenBank Accession number AB079242) as a template.

The complete mitochondrial genome of U. nigricaudus is 17,298 bp long (GenBank

Accession No. KP091282). This genome has a typical vertebrate mitochondrial gene arrangement with two rRNAs, 22 tRNAs, 13 protein-coding genes and one control region

(CR) (Figure 2.2 and Table 2.2). The overall nucleotide content is A= 34.2%; C= 26.8%;

G= 13.5%; T= 25.5%. Most of the genes are encoded on the H-strand except for ND6 and the tRNAs: tRNAGln, tRNAAla, tRNAAsn, tRNACys, tRNATyr, tRNASer, tRNAGlu and tRNAPro. All protein-coding genes start with ATG, except for COI (GTG). Stop codons are more variable. Seven genes have complete stop codon (TAA=6/13; AGA= 1/13) and six genes have incomplete stop codons (TA- = 1/13; T-- = 5/13).

The incomplete stop codons occur in genes that immediately precede a tRNA and are supposed to be completed with post-transcriptional polyadenylation (Ojala et al. 1981). A

33 bp fragment located in the WANCY cluster, between tRNAAsn and tRNACys, was considered the putative L-strand origin of replication. This mitochondrial genome comprises eight regions with spacers and nine with overlaps. Uda et al. (2011) suggested that overlapping could help to prevent rearrangements and loss of genes during replication of the mitochondrial genome. The CR is 1,909 bp long and is similar to other vertebrates in having conserved regions (CSB1, CSB2 and CSB3) and an array of tandem repeats.

These repeats are 37 bp long and are rich in G + C content. The CR region is located between tRNAPro and tRNAPhe.

Mitochondria are essential elements of eukaryotic cells as they are directly involved with oxygen use, metabolism, and energy production via oxidative phosphorylation (OXPHOS)

(Saraste 1999). All genes in the mitogenome work in conjunction with 72 nuclear DNA genes on the OXPHOS system (Saraste 1999; Morales et al. 2015; Wai & Langer 2016).

The use of mtDNA genomes in evolutionary studies has its advantages (i.e. simple genetic structure, easy sequence process, no recombination, and rapid evolution) and its caveats

(i.e. homoplasy, introgression and biased inheritance mode) (Edwards et al. 2005; Zink &

Barrowclough 2008; Dool et al. 2016; Haenel 2017). But when used in combination with nuclear genes, the mtDNA genome can provide evolutionary evidence and specific information about female population history and biogeographical events. This study adds one more species to the dataset of mitogenomes for squamates that have, in general, been increasing moderately over the past eight years (Figure 2.3). Despite this interest, only 176 of 10,336 squamate species (http://www.reptile-database.org as of 23 May 2018, Uetz et al. 2018) have had their mitogenome sequenced (Table 3.2), falling well behind data accumulated for fish and mammals (Kumazawa et al. 2014). Concerning my particular research interest, information from mtDNA, in conjunction with nuclear DNA data can help to understand the evolutionary processes involved in the maintenance of the deep mtDNA lineage discordances found in Urosaurus nigricaudus on the peninsula of Baja

California, Mexico. Given the importance of mtDNA on so many biological levels and the advances in sequencing technology, it is critical for researchers to focus on sequencing the whole mtDNA genome for more squamate species, as it proved to be an essential tool in investigating evolutionary processes.

3.2 Figures

Figure 3.1: Position of primers used on this study. Primers numbers correspond to the primer pair ID (Table 1). The mtDNA genome organization corresponds to that of

Urosaurus nigricaudus.

Figure 3.2: Mitochondrial genome of Urosaurus nigricaudus with details of gene organization.

25 Number of species of Number

1996/7 1998/99 2000/1 2002/3 2004/5 2006/7 2008/9 2010/11 2012/13 2014/15 2016/17 Date

Figure 3.3: Number of squamate species with an entire mtDNA genome sequenced over the past 20 years. Data collected from the Web of Science on May 23, 2018.

3.3 Tables

Table 3.1: List of the 22 pairs of primers used to sequence the complete mitochondrial

genome of Urosaurus nigricaudus. Pair ID was used for lab work and to illustrate the

position of the primers in the mtDNA genome (see Figure 1). Authors: 1 – This study; 2 –

Green et al. 2010; 3 – Kumazawa & Endo 2004.

Pair FORWARD REVERSE Author ID Primer Primer Sequence Sequence name name CCAACTGGGATTAGA GTAGCTCACTTGATT 1 12S1L 16S-3H 2 TACCCCACTAT TCGGG CCGGATCCCCGGCCG ACACACCGCCCGTCA 2 12S2LM 16S2H GTCTGAACTCAGATC 2 CCCT ACG 16S_ND AGTAAAACTGATCAC 16S_ND ATGTCAGTTATTGCG 3 1 1F1 CGAACC 1R2 TGTGG ND1_N ATGCCTATGACATAC ND1_N TCTTCTAGGATTAGT 4 1 D2F2 AGCCC D2R1 CATTTTGG ND2- CTCAAACACGAAAAA ND2- GAAATGATGGGGGTA 5 1 COIF2 TCATAGCC COIR1 GCAG COI- ACAGACCGMAACCT COI- GGGGTTTAGTTGTGG 6 1 ATP6F2 AAACAC ATP6R2 CATRTCACTG COII- GCCCTACCATCCCTA COII- TAGTACTGTTGCTAG 7 1 ATP6F1 CGAATCC ATP6R1 TCATATTGG AGCACTAGCCTTTTA uCO3- AAYGTCTCGTCATCA 8 uLys-lL 3 AGC 1H TTG

uCO3- ATAGTWGACCCMAG uND3- GGGTCRAAKCCRCAT 9 3 1L CCCATGACC 2H TCRTA uCO3- GAAGCMGCWGCCTG rND4L- GCTAGGCCAGTRCYT 10 3 3L ATACTGACA 2H GCTTCRCA rND4L- TGCATTGAARGYATA uND4- CTACRTGKGCTTTTG 11 3 1L ATACT 2H GKARTCA rND4L- TAACCTTCTCMGCMT rND4- GATGTTAAKCCGTGG 12 3 2L GYGAAGC 2H GCRATTAT CCAAAAGCCCAYGTA rCUN- CTTTTACTTGGADTT 13 rND4-3L 3 GARGC (20) 1H GCACC AACAAAAACAYTAG rND5- ACWACTATTGTGCTK 14 rHis-2L 3 RCTGTG 1H GAGTG TCCAAGCMATYATCT rND5- ATWGYGTCTTTTGAG 15 rND5-1L 3 AYAACCG 2H TARAAKCC GAACARGACATYCGA rND6- ATGTTAGTGGTDTTT 16 rND5-2L 3 AAAATRGG 4H GCKTATTC YACMYMAACGCCTG ATTACAACGGYGGTT 17 rND5-3L rGlu-1H 3 AGCCCT TTTC GCAACWGAATAHGC ucytb- GCCCCTCAGAATGAT 18 rND6-3L 3 AAATAC 1H ATTTGTCCTCA GAAAAACCRCCGTTG GCGTAGGCRAATAGG 19 rGlu-1L rcytb-1H 3 TWATTCAACTA AAGTATCA TGAGGACAAATATC TTAAAATKCTAGTTT 20 rcytb-2L rPro-1H 3 MTTCTGAGG TGG YAAAGCMTTGRTCTT rCONT- CTCGKTTTWGGGGTT 21 rThr-2L 3 GTAA 4H TGRCGA rCONT- TCGYCAAACCCCWA TRTAACCGCGGTKGC 22 r12S-1H 3 4L AAMCGAG TGGCAC

Table 3.2: mtDNA genome organization and features in Urosaurus nigricaudus.

Space Gene Strand Start Stop (+) Anticodon Position 5’-3’ Anticodon (Element) (H/L) codon codon Overlap position (-) tRNAPhe 1-73 H GAA 35-37 12S 74-1014 H

Val tRNA 1015-1083 H TAC 1046-1048 16S 1084-2611 H

Leu tRNA 2612-2686 H TAA 2647-2649 ND1 2687-3655 H ATG TAA +3

Ile tRNA 3661-3731 H -1 GAT 3692-3694

Gln tRNA 3731-3800 L -1 TTG 3767-3769

Met tRNA 3800-3869 H CAT 3831-3833 ND2 3870-4902 H ATG T--

Trp tRNA 4903-4979 H TCA 4935-4937

Ala tRNA 4980-5048 L +3 TGC 5016-5018

Asn tRNA 5053-5125 L -1 GTT 5090-5092

OL 5125-5157 L -5

Cys tRNA 5153-5218 L GCA 5189-5191

Tyr tRNA 5219-5289 L +1 GTA 5255-5257 COI 5291-6838 H GTG AGA -5

Ser tRNA 6834-6904 L +3 TGA 6870-6872

Asp tRNA 6908-6975 H GTC 6938-6940 COII 6976-7663 H ATG T--

Lys tRNA 7664-7731 H +1 TTT 7694-7666 ATP8 7733-7900 H ATG TAA -10

ATP6 7891-8573 H ATG TA- COIII 8574-9357 H ATG T--

Gly tRNA 9358-9427 H TCC 9389-9391 ND3 9428-9773 H ATG T--

Arg tRNA 9774-9841 H TCG 9804-9806 ND4L 9842-10138 H ATG TAA -7 ND4 10132-11.512 H ATG T--

His 11513-11581 H GTG 11543- tRNA 11545

Ser 11582-11648 H -1 GCT 11608- tRNA 11610

Leu 11648-11718 H +1 TAG 11680- tRNA 11682 ND5 11720-13519 H ATG TAA +4 ND6 13516-14034 L ATG TAA

Glu 14035-14103 L +3 TTC 14071- tRNA 14073 Cytb 14107-15246 H ATG TAA +4

Thr 15251-15321 H TGT 15284- tRNA 15286

Pro 15322-15389 L TGG 15364- tRNA 15362 CR 15390-17298

Table 3.3: List of squamate species for which a complete mtDNA genome has been sequenced.

Species Reference

Anguimorpha Abronia graminea (Kumazawa 2004) Anguis cephallonica (Strzała et al. 2017) Anguis colchica (Strzała et al. 2017) Anguis fragilis (Albert et al. 2009) Anguis veronensis (Strzała et al. 2017) Heloderma suspectum (Kumazawa 2007) Ophisaurus attenuates (Castoe et al. 2008) Ophisaurus harti (Pan et al. 2013) Shinisaurus crocodilurus (Kumazawa 2004) Varanus niloticus (Kumazawa 2007) Varanus salvator (Castoe et al. 2008) Varanus komodensis (Kumazawa & Endo 2004)

Gekkota Aprasia parapulchella (MacDonald et al. 2015) Cnemaspis limi (Yan et al. 2013b) Coleonyx variegatus (Kumazawa 2007) Hemitheconyx caudicinctus (Jonniaux et al. 2012) Hemitheconyx taylori (Jonniaux et al. 2012) Heteronotia binoei (Fujita et al. 2007) Gekko chinensis (Hao et al. 2015) Gekko gecko (Zhou et al. 2006) Gekko hokouensis (Hao et al. 2016) Gekko japonicus (Kim et al. 2015; Hao et al. 2016) Gekko swinhonis (Li et al. 2012)

Gekko vittatus (Kumazawa 2007) Goniurosaurus luii (Li et al. 2016c) Lepidodactylus lugubris (Kumazawa et al. 2014) Paroedura picta (Starostová & Musilová 2015) Phyllodactylus unctus (Yan et al. 2013a) Phelsuma guimbeaui (Kumazawa et al. 2014) Quedenfeldtia moerens (Lyra et al. 2017) Quedenfeldtia trachyblepharus (Lyra et al. 2017) Stenodactylus petrii (Kumazawa et al. 2014) Tarentola mauritanica (Albert et al. 2009) Teratoscincus keyserlingii (Macey et al. 2005) Teratoscincus roborowski (Li et al. 2016d) Tropiocolotes steudneri (Kumazawa et al. 2014) Tropiocolotes tripolitanus (Kumazawa et al. 2014) Uroplatus ebenaui (Kumazawa et al. 2014) Uroplatus fimbriatus (Kumazawa et al. 2014)

Iguania Acanthosaura armata (Okajima & Kumazawa 2010) Acanthosaura lepidogaster (Yu et al. 2015) Anolis carolinensis (Castoe et al. 2008) Anolis cybotes* (Okajima & Kumazawa 2009) Basilicus vittatus (Okajima & Kumazawa 2009) Brookesia decaryi (Okajima & Kumazawa 2010) Calotes versicolor (Amer & Kumazawa 2007) Calumma parsonii (Okajima & Kumazawa 2010) Chalarodon madagascariensis (Okajima & Kumazawa 2009) Chamaeleo calcaricarens (Macey et al. 2008) Chamaeleo chamaeleon (Macey et al. 2008) Chamaeleo calyptratus (Macey et al. 2008) Chamaeleo dilepis (Macey et al. 2008) Chamaeleo monachus (Macey et al. 2008)

Chamaeleo zeylanicus (Macey et al. 2008) Chlamydosaurus kingii (Ujvari et al. 2007; Ujvari & Madsen 2008) Furcifer oustaleti (Kumazawa 2007) Gambelia wislizenil (Okajima & Kumazawa 2009) Holbrookia lacerata (Roelke et al. 2018) Holbrookia maculata (Roelke et al. 2018) Holbrookia propinqua (Roelke et al. 2018) Hydrosaurus amboinensis (Okajima & Kumazawa 2010) Iguana iguana (Okajima & Kumazawa 2009) Kinyongia fischeri* (Okajima & Kumazawa 2010) Leiocephalus personatus (Okajima & Kumazawa 2009) Leiolepis guttata (Okajima & Kumazawa 2010) Leiolepis reevesii (Tong et al. 2014a) Oplurus grandidieri (Okajima & Kumazawa 2009) Phrynocephalus albolineatus (Shao et al. 2016b) Phrynocephalus axillaris (Li et al. 2013b) Phrynocephalus erythrurus erythrus (Jin et al. 2018) Phrynocephalus frontalis (Jin et al. 2018) Phrynocephalus erythrurus parva (Zhu et al. 2014) Phrynocephalus forsythii (Shao et al. 2016a) Phrynocephalus guinanensis (Fu et al. 2014) Phrynocephalus grumgrizimailoi (Shuang et al. 2016) Phrynocephalus helioscopus (Li et al. 2016a) Phrynocephalus mystaceus (Chen et al. 2013) Phrynocephalus przewalskii (Li et al. 2013a) Phrynocephalus putjatia (Tong & Jin 2014) Phrynocephalus theobaldi orientalis (Liao & Jin 2014) Phrynocephalus theobaldi theobaldi (Jin et al. 2018) Phrynocephalus versicolor (Song et al. 2014a) Phrynocephalus vlangalii (Jin et al. 2018)

Phrynosoma blainvillii (Ayala et al. 2017) Plica plica* (Okajima & Kumazawa 2009) Pogona vitticeps (Amer & Kumazawa 2005) Polychrus marmoratus (Okajima & Kumazawa 2009) Pseudotrapelus sinaitus (Okajima & Kumazawa 2010) Rieppeleon kerstenii* (Okajima & Kumazawa 2010) Sceloporus occidentalis (Kumazawa 2004; Okajima & Kumazawa 2010) Trioceros melleri (Okajima & Kumazawa 2010) Uromastyx benti (Okajima & Kumazawa 2010) Urosaurus nigricaudus (Bernardo et al. 2016) Xenagama taylori (Macey et al. 2006)

Lacertoidea Amphisbaena schmidti (Macey et al. 2004) Bipes biporus (Macey et al. 2004) Bipes canaliculatus (Macey et al. 2004) Bipes tridactylus (Macey et al. 2004) Blanus cinereus (Albert et al. 2009) Darevskia unisexualis (Komissarov et al. 2016) Diplometapon zarudnyi (Macey et al. 2004) Eremias brenchley (Rui et al. 2009) Eremias przewalskii (Du et al. 2016) Eremias stummeri (Zhou et al. 2015) Eremias vermiculata (Tong et al. 2014b) Geocalamus acutus (Macey et al. 2004; Kumazawa 2007) Lacerta agilis (Qin et al. 2013a) Lacerta bilineata (Kolora et al. 2016) Lacerta viridis (Böhme et al. 2007) Phoenicolacerta kulzeri (Podnar et al. 2009) Podarcis muralis (Podnar et al. 2009)

Podarcis siculus (Podnar et al. 2009) Rhineura floridana (Macey et al. 2004) Takydromus amurensis (Ma et al. 2016) Takydromus sexlineatus (Qin et al. 2013b) Takydromus sylvaticus (Tang et al. 2013) Takydromus tachydromoides (Kumazawa 2007) Takydromus wolteri (Yu & Ji 2012) Zootoca vivipara (Liu et al. 2016a)

Scincomorpha Cordylus warrendi (Kumazawa 2004) Eumeces chinensis (Zhang et al. 2015b) Eumeces egregious (Kumazawa & Nishida 1999) Eumeces elegans (Song et al. 2014b) Lepidophyma flavimaculatum (Kumazawa 2007) Scincella vandenburghi (Park et al. 2016) Scincella huanrenensis (Park et al. 2016)

Serpentes Acrochordus granulatus (Dong & Kumazawa 2005) Achalinus meiguensis (Wang et al. 2009) Achalinus rufescens (Zhang et al. 2017c) Achalinus spinalis (Peng et al. 2017) Agkistrodon contortrix (Castoe et al. 2011) Agkistrodon piscivorus (Jiang et al. 2007) Anilius scytale (Douglas & Gower 2010) Boa constrictor (Dong & Kumazawa 2005) Bothrops jararaca (Almeida et al. 2016) Causus defilippii (Castoe et al. 2010) Crotalus horridus (Hall et al. 2012) Cyclophiops major (Sun et al. 2017) Cylindrophis ruffus (Dong & Kumazawa 2005) Deinagkistrodon acutus (Yan et al. 2008)

Dinodon semicarinatus (Dong & Kumazawa 2005) Elaphe anomala (Liu & Zhao 2016a) Elaphe bimaculata (Yan et al. 2014) Elaphe carinata (Ding et al. 2016) Elaphe davidi (Xu et al. 2016a) Elaphe guttata (Douglas et al. 2006) Elaphe perlacea (Wan et al. 2016) Elaphe schrenckii (Liu & Zhao 2016b) Enhydris plumbea (Yan et al. 2008) Eunectes notaeus (Douglas et al. 2006) Gloydius intermedius (Xu et al. 2016d) Gloydius saxatilis (Xu et al. 2016b) Gloydius shedaoensis (Liu et al. 2016b) Gloydius ussuriensis (Han et al. 2016) Hebius vibakari (Xu et al. 2016c) Hypsiglena unaocularus (Mulcahy et al. 2014) Leptotyphlops dulcis (Kumazawa 2004) Micrurus fulvius (Castoe et al. 2010) Naja naja (Yan et al. 2008) Oocatochus rufodorsatus (Li et al. 2014) Orthriophis taeniurus (Li et al. 2016b) Ovophis okinavensis (Dong & Kumazawa 2005) Pantherophis slowinskii (Jiang et al. 2007) Pituophis catenifer sayi (Lele et al. 2016) Protobothrops cornutus (Zhang et al. 2015c) Protobothrops dabieshanensis (Huang et al. 2014) Protobothrops jerdonii (Huang et al. 2013) Protobothrops mucrosquamatus (Zhang et al. 2013b) Protobothrops xiangchengsis (Zhang et al. 2015a) Ptyas mucosus (Zhou et al. 2016a) Python molurus morulus (Dubey et al. 2012)

Python molurus bivittatus (Castoe et al. 2013) Python regius (Dong & Kumazawa 2005) Ramphotyphlops australis (Douglas et al. 2006) Ramphotyphlops braminus (Yan et al. 2008) Rhabdophis tigrinus (Zhao et al. 2016) Sibynophis chinensis (Oh et al. 2015) Sibynophis collaris (Jang & Hwang 2011) Sinovipera sichuanensis (Zhu et al. 2016) Thermophis baileyi (Weng et al. 2016) Thermophis shangrila (Peng et al. 2016) Thermophis zhaoermii (He et al. 2010) Trimeresurus albolabris (Song et al. 2015) Tropidophis haetianus (Castoe et al. 2009) Typhlops mirus (Douglas et al. 2006) Typhlops reticulatus (Castoe et al. 2008) Xenopeltis unicolor (Dong & Kumazawa 2005)

* incomplete CR sequences.

Chapter 4 When mitochondrial phylogeography

fails: female genealogy is only part of the story

This chapter is currently being prepared for submission to Molecular Ecology (Bernardo,

P.H., Sánchez-Ramírez, S., Aguilera-Miller, E.F., Álvarez-Castañeda, S.T., Méndez-de la

Cruz, F.R., and R.W. Murphy. When mitochondrial phylogeography fails: female genealogy is only part of the story).

Abstract

Mitochondrial phylogeography surged in the late 80's as a field that made evolutionary inferences from intraspecific mitochondrial DNA (mtDNA) genealogies and geographic locations of individuals. Since then, hundreds of studies have relied only on mtDNA gene trees to infer phylogenies, delimit species, and infer population structure and gene flow.

MtDNA is widely used as a genetic marker due to its simple genetic structure, inexpensive sequencing, absence of recombination and high genetic variability. However, mtDNA has some challenges in representing the evolutionary history of a species, including homoplasy, introgression and female-only inheritance, which can cast doubt on the validity of studies based solely on this marker. Herein, I use Urosaurus nigricaudus as a model to test the hypothesis that mtDNA data alone can be used to infer unrestricted gene flow. This small species of lizard has deep mtDNA lineage divergences that are extremely restricted geographically. I sequence the whole mtDNA genome and generate RAD loci for thousands of nuclear SNPs and compare the power of both markers to infer population structure and gene flow. My results confirm that U. nigricaudus has three parapatric and deeply divergent maternal lineages in southern Baja California, but analyses of nDNA do not detect genetic structure, suggesting unabated gene flow between populations, and rejecting the hypothesis that mtDNA gene trees can serve as the sole evidence for evolutionary inference. The inclusion of nDNA markers is essential to assess the natural processes and evolution of female-linked traits that may originate and maintain cytonuclear discordances.

4.1 Introduction

The term ‘mitochondrial phylogeography’ (genogeography sensu Sérebrovsky 1928) was coined by Avise et al. (1987) to describe the study that uses the relationship between matrilineal genealogy and geography based on mitochondrial DNA (mtDNA), to explain natural history processes (i.e., speciation, phylogeny, population structure, etc.). In a more direct statement, Zink & Barrowclough (2008) defined mtDNA phylogeography as the

“superposition of a gene tree onto geography” and contented it was a reliable tool for investigating geographical population structure, phylogeographic patterns and species limits. Advantages of using mtDNA as a genetic marker include it (i) lacks repetitive DNA, pseudogenes and introns, (ii) is easy to sequence (several copies in a cell, sequencing process simple and inexpensive), (iii) has a straightforward mode of genetic transmission

(usually female inheritance only and no recombination) and (iv) has a high mutation rate and rapid evolution (Avise et al. 1987; Edwards et al. 2005; Zink & Barrowclough 2008;

Dool et al. 2016). Although the use of mtDNA as the only genetic marker in studies has been declining (Edwards et al. 2005), hundreds of scientists continue to rely solely on mtDNA to address a wide range of evolutionary issues from phylogenetic inference

(Abiadh et al. 2010; Byrne et al. 2010; Cameron 2014; Pozzi et al. 2014; Andújar et al.

2015), to population structure and gene flow (Zhou et al. 2016b; Men et al. 2017; Hu et al. 2018) and determining conservation priorities (Banguera-Hinestroza et al. 2002;

Teixeira et al. 2018). The use of mtDNA gene trees and its reciprocal monophyly is often used as the only evidence for species delimitation (Kozak et al. 2006; Thomé et al. 2010;

Pesarakloo et al. 2017) and the description of new species (Streicher et al. 2014; Morin et

51 al. 2016; Borsa et al. 2017). Some researchers have criticized this approach (e.g., Ballard

& Whitlock 2004; Bazin et al. 2006; Edwards & Bensch 2009; Fisher-Reid & Wiens 2011;

Dool et al. 2016), while others have defended it (e.g., Zink & Barrowclough 2008; Nagy et al. 2012; Malukiewicz et al. 2017) leaving the controversy unresolved.

The main goal of this study is to test the assumptions that mtDNA phylogeography is an unambiguous way to delimit species and population structure. As a model organism, I use the black-tailed brush lizard Urosaurus nigricaudus (Cope, 1864), a common arboreal phrynosomatid found on the peninsula of Baja California, Mexico from the southern tip northward into southern California, USA. It mostly occupies mesquite trees (Prosopis palmeri S. Watson) and smaller shrubs in “arroyos” (depressions in the landscape that form temporary rivers during the rainy season), but also occurs in urban areas (Munguia-Vega et al. 2013). In sparse oases, individuals inhabit palm trees (Washingtonia spp. and Brahea spp.). Females of U. nigricaudus rarely leave their trees and often display aggressive behaviour towards other females that try to climb their tree (pers. obs.). Males constantly move around and do not appear to be as territorial as the females, except during the mating season from April to August, when males are aggressive towards other males (Munguia-

Vega et al. 2013).

Aguirre-Léon et al. (1999) could not reject the hypothesis of unrestricted nDNA gene flow in this species based on allozyme data. Intriguingly, the genealogy of U. nigricaudus based on two mtDNA genes showed six deeply divergent matrilines (Lindell et al., 2008) with sequence-divergences as high as 11.02% between lineages. The deepest mtDNA divergence was believed to have been initiated during temporary geographic isolation in the late Miocene. The isolation appears to have prevented gene flow for a sufficient time

52 to allow for the fixation of substitutions via genetic drift (Chapter 5). Today, females from two matrilines coexist as close as two meters in very narrow mtDNA zones that they do not cross (Chapter 2). There are no obvious environmental or geological explanations for the maintenance of these zones.

Herein, I report analyses of the entire mtDNA genome and nDNA SNP loci using double digest restriction-site associated DNA (ddRAD-seq), which can identify thousands of

SNPs for hundreds of individuals at a reasonable cost (Blair et al. 2015). I use (i) additional mtDNA information to test Lindell et al.’s (2008) hypothesis that U. nigricaudus shows deeply divergent matrilines and (ii) the nDNA SNP data to test the hypothesis of unrestricted gene flow (panmixia) in a species with such deep mtDNA divergences. I assess how current populations are genetically structured and compare mtDNA and nDNA genetic variation. If the results reject the hypothesis of panmixia, then I must assume the absence of gene flow is driving speciation, which will result in species delimitation.

However, if my analyses fail to reject unrestricted nDNA gene flow in spite of deep mtDNA divergences, then species delimitation based on mtDNA alone is not acceptable.

Either way, the results test the efficacy of mtDNA phylogeography and the use of mtDNA data alone to assess gene flow and population structure.

4.2 Material and Methods

4.2.1 Sampling

Fieldwork in the southern portion of the Peninsula of Baja California took place in August

2013. Several areas from San Juan de la Costa on the north to Cabo San Lucas and La

Fortuna on the south were sampled (Table 4.1). The localities were chosen to maximize the coverage of the three southernmost lineages of U. nigricaudus (S2, C1 and C2 sensu

Lindell et al. 2008) and logistics. A total of 196 individuals were collected, photographed, measured, and a small portion of muscle was taken from the tail and preserved in 95% ethanol. After collecting a small muscle sample, the lizards were released back in to their environment. Samples were collected in accordance with Animal Use Protocols by the

Royal Ontario Museum Animal Care Committee and authorized by Mexican authorities.

4.2.2 MtDNA genome sequencing

The full mtDNA genome of Urosaurus nigricaudus was first described by Bernardo et al.

(2016; Chapter 3). All Polymerase chain reaction (PCR) amplifications and sequencing reactions followed their protocols and primers. The PCR products were sequenced using a

3730 DNA Analyzer (Applied Bio-Systems) at the Royal Ontario Museum. The assembly of the mtDNA genome used GENEIOUS 11 (Kearse et al. 2012), and the gene annotation was based on the mtDNA genome of the phrynosomatid Sceloporus occidentalis

(GenBank Accession number AB079242) as a template. I sequenced the mitogenomes of

26 individuals (Table 4.2) from different sampling localities comprising all three southern matrilines.

4.2.3 Genealogical tree

The maternal genealogy of U. nigricaudus was inferred using the concatenated sequences of the protein coding genes ATP6, Cytb and ATP8 (data new to this study). A total of

1981bp was obtained. Because all genes in the mitogenome are inherited as a single unit I

54 concatenated them (Zink & Barrowclough 2008; Shen et al. 2010; Dool et al. 2016). These three genes were chosen based on the availability of sequences in GenBank for the outgroup species Uta stansburiana, Sceloporus occidentalis, Urosaurus graciosus and

Urosaurus ornatus. The sequences were aligned using the MUSCLE 3.8.31 plugin in

GENEIOUS (Kearse et al. 2012). The Akaike information criteria (AIC) implemented in

JMODELTEST 2.1.1 (Darriba et al. 2012) was used to identify the preferred substitution model for the dataset, and the GTR+I+G model of DNA substitution was the model selected with best likelihood values.

I used Bayesian inference performed by MRBAYES 3.2.6 (Huelsenbeck & Ronquist 2001;

Ronquist et al. 2012) to build the genealogical tree. MRBAYES analyses consisted of two

Markov chain Monte Carlo (MCMC) runs for 10 million iterations. Trees were sampled every 500 generations for 10 million generations and the first 1 million generations were discarded as burn-in. I then used the 50% majority rule to calculate the posterior probabilities of all nodes and to construct the consensus tree.

PAUP 4.0a161 (Swofford 2002) was used to calculate a sequence divergence matrix

(uncorrected p-distances). For the genetic distance calculations between the individuals of

U. nigricaudus, we used the entire mtDNA genome (15270 bp).

4.2.4 Double digest RAD Sequencing and analyses

Genomic DNAs were sent to the University of Arizona Genetics Core for Double Digest

RAD Sequencing (ddRAD-seq). The sequencing and library protocols followed Peterson et al. (2012). Genomic DNA was enzymatically digested using two endonucleases (Sphl and MluCl). Samples were then cleaned using AMPure XP beads A63881 (Beckman

Coulter Genomics) and quantified using Quantiflour (PRE2670, Fisher) and BioTek

FLX800 flourometer (BioTek, Vermont, USA). Once digestion was completed, double- stranded adapters (barcodes) were ligated to both ends of the fragments. About 100 ng of clean digested DNA were used as input for the ligation reaction. Barcode adapters that can recognize individual samples, were added to each fragment. Libraries were then multiplex sequenced using pair-ended Illumina HiSeq 2500.

As a first step of quality control, raw ddRAD-seq Illumina sequences were masked using a PHRED score threshold of 50 (i.e., base calls with PHRED scores lower than 50

(99.999% of base call accuracy) were masked with “N” (undetermined bases)). Masking low quality base calls instead of trimming the sequence was the option chosen because masking has been demonstrated to be more effective in reducing false-positive rate in SNP calling (Yun & Yun 2014). In addition, trimming the sequences reduced the size of the reads, consequently reducing the amount of information available for the subsequent analyzes. This first filtering step was done using a modified version of the Perl script

(https://github.com/santiagosnchez/mask_fastq) made available by Yun & Yun (2014).

After this step, raw sequence reads were de-multiplexed using the process_radtags in

STACKS 1.35 (Catchen et al. 2013). Stacks assigned each read to an individual using the barcodes and restriction cut sites on the raw ddRAD-seq reads. Sequence reads that did not contain the restriction digest site or the unique barcode were discarded. Furthermore, samples with less than 5k reads were also excluded from the dataset due to low coverage.

During this step, most forward sequence reads ended up being excluded due to quality issues in the 3' start of the reads. After the reads were de-multiplexed and had passed the first steps of quality control, I used the DDOCENT pipeline for assembly, mapping, SNP

56 calling and SNP filtering (Puritz et al. 2014). DDOCENT used CD-HIT (Fu et al. 2012) to create a consensus sequence using alignment-based clustering based on overall sequence similarity (parameter set to 90% similarity). It then used the BWA-MEM alignment algorithm (Li 2013) to map the reads to a consensus sequence generated by CD-HIT. Once the reads were properly mapped, I used GATK (McKenna et al. 2010) to call the SNPs using UNIFIEDGENOTYPER. GATK was also used to filter the final VCF file and remove sites that had more than two alleles. Next, I used a custom Perl script

(https://github.com/santiagosnchez/sing_snp_vcf) to filter SNPs with minor allele frequency < 0.01, keeping only a single SNP per locus. This extra step of quality control was used to ensure that in a locus with two alleles, had the intermediate frequency alleles chosen for the following population structure analysis. Rare alleles and high frequency alleles may not have been informative due to linkage disequilibrium or strong selection pressures.

4.2.5 Population structure

The mtDNA differentiation analyses were performed using the three highly divergent southern mtDNA lineages (S2, C1 and C2) of U. nigricaudus from Lindell et al. (2008) and supported by the results from our Bayesian genealogical tree. To detect population structure on the ddRAD-seq dataset, I used ADMIXTURE 1.3 (Alexander et al. 2009) with k=1–5. ADMIXTURE, a likelihood-based program, estimated individual ancestries from multilocus SNP data. To input the SNP dataset into ADMIXTURE, the VCF file was converted into a plink format using PLINK 1.07 (Purcell et al. 2007). Cross-validation procedure in Admixture was used to select the K value that best fits the data, where the

57 best value of K exhibited the lowest cross-validation error (CVE) compared to other K values (Alexander et al. 2009).

4.2.6 Genetic differentiation

All analyses of genetic differentiation of mtDNA genome and nuclear SNP data were done using GENALEX 6.5 (Peakall & Smouse 2006; 2012). Because the main objective of this study was to test the assumptions of mtDNA analyses, I grouped mtDNA and nDNA datasets into the three matrilines: S2, C1 and C2. For the mtDNA analysis, the mtDNA genome (15270 bp) alignment was used for all 26 samples. The level of genetic differentiation between populations was estimated using population pairwise ΦPT.

According to the GENALEX manual, ΦPT constituted the best estimator for comparison between codominant data (SNP loci) and haploid data (mtDNA sequences). An analysis of molecular variance (AMOVA) was used to estimate genetic differentiation of U. nigricaudus. GENALEX transformed the mtDNA sequences and table of SNP loci into a genetic distance table, and then used that matrix for the AMOVA. Pairwise ΦPT values and

AMOVA tests performed on GENALEX were computed using 9999 permutations.

4.3 Results

4.3.1 MtDNA genome and ddRADSeq sequencing

The total length of the mtDNA genome was 17,298 bp (Bernardo et al. 2016). I was able to successfully recover 15,396 bp by combining all 13 protein-coding genes, two rRNAs and 22 tRNAs. Sequencing problems in some samples precluded resolution of the control region (CR).

I obtained a total of 197,930,934 reads from the Illumina HiSeq 2500. The average number of reads per samples was 35,737.69 ± 15,8019.41, and the average coverage per sample was 8.48 ± 3.37. After filtering, 73 samples (Table 4.3) of U. nigricaudus out of the 196 initially sequenced passed the quality control thresholds. They provided 3,963 RAD loci for downstream analyzes.

4.3.2 Population structure and genetic differentiation using mtDNA

Bayesian analysis produced a tree with highly supported branches (posterior probability of

1.0) for each matriline (Figure 4.1A). Values of pairwise proportional sequence divergence

(uncorrected p-distances) varied from 5.23% to 8.26% among lineages. More specifically, the sequence divergence for the Isthmus of La Paz break (S2:C1) ranged from 8.39 to

8.26% and for the Cape Region Break (C1:C2) it ranged from 5.41 to 5.23%. Figure 4.1B provides a visual distribution of the matrilines in southern part of the Peninsula of Baja

California.

The results of AMOVA analyses (Table 4.4) based on the mitogenome (1881 variable sites) revealed three highly differentiated populations (ΦPT = 0.949, P<0.001) that corresponded with the three divergent and highly supported matrilines recovered in the

Bayesian inference tree (Figure 1A). The pairwise ΦPT values between populations (Table

4.5) for the mitogenome ranged from 0.796 to 0.931. The statistically significant results

(P<0.001) showed that genetic variation between populations (88%) was substantially higher than variation within populations (12%).

4.3.3 Population structure and genetic differentiation using nDNA

The results from the population structure analysis using 3,963 nDNA SNP loci performed by ADMIXTURE revealed no population structure in U. nigricaudus (K=1; CVE= 0.758).

When individuals were assigned to their respective matrilines the cross-validation error value was higher and not supported (K=3; CVE= 0.954). To compare the results from both genetic markers, I assigned each individual to their respective mtDNA lineage to further investigate the nDNA molecular variances. The AMOVA results (Table 4.4) had no support for the three populations (ΦPT = 0.012, P=0.1) and showed that the genetic variation on the nuclear DNA was massively concentrated within populations (99%) with almost no variation found between the mtDNA lineages (1%). The pairwise ΦPT values between populations (Table 4.5) for the nuclear SNPs ranged from 0 to 0.023 and there was no statistical support (P>0.05).

4.4 Discussion

My analyses fail to reject the hypothesis of unrestricted nDNA gene flow in U. nigricaudus. In doing so, the data reject the hypothesis that mtDNA data alone can confidently resolve gene flow and population structure in species. Questions of gene flow should use population genetics theory including testing for Hardy-Weinberg expectations either directly, or indirectly. The matrilineal genealogy of U. nigricaudus agrees with the gene tree of Lindell et al. (2008), who reported three matrilines in the southern peninsula of Baja, California. Although slightly lower than previously reported by Lindell et al.

(2008), the level of sequence divergence between the matrilines remains remarkable. Two historical and ephemeral isolations of U. nigricaudus in the late Miocene (Isthmus of La

Paz break) and late Pleistocene (Cape Region break) explain the divergence (Chapter 5).

In contrast, SNP analyses of population structure reveal a single genetic population, and that gene flow occurs without barriers among the matrilines. The three matrilines are completely isolated (ΦPT=0.949, P<0.001) and yet nDNA analyses do not correspond with this (ΦPT=0.012, P=0.1). This significant difference between the geographic patterns and levels of differentiation between mtDNA and nDNA markers is known as cytonuclear discordance (or mito-nuclear discordance). Similar discordances have been documented in many other species (Toews & Brelsford 2012; Morales et al. 2015; Pavlova et al. 2017;

Leavitt et al. 2017; Nguyen et al. 2017; Platt et al. 2017). Indeed, in their review of 126 cases, Toews & Brelsford (2012) concluded that cytonuclear discordance is a common phenomenon in animal systems. If lineages that have been biogeographically isolated for long periods of time come back into contact with one another, discordance might arise based on a variety of processes such as introgressive hybridization, incomplete lineage sorting, female philopatry, female behavior and natural selection on female-linked traits

(Morales et al. 2015; Pavlova et al. 2017; Aguilera-Miller et al. 2018). Disentangling the influence of each of these factors on the discordant patterns is complicated and requires input from as many data sources as possible (for excellent reviews of the problem see Dool et al. 2016 and Leavitt et al. 2017). Preliminary results for U. nigricaudus rule out most of the possible explanations and focus on two female-linked traits: mito-nuclear functional compensation and female behavior (Chapter 5).

Mechanisms underlying discordances aside, my results, underscore the problematic nature of basing species identification solely on mtDNA information. MtDNA has historically been used because of its rapid attainment of reciprocal monophyly when compared to

61 nDNA and its frequent ability to diagnose populations (Edwards et al. 2005; Zink &

Barrowclough 2008). The use of mtDNA gene trees to infer phylogenetic relationships, including the relationships among higher taxa (e.g., Cameron 2014; Pozzi et al. 2014) without nDNA markers may, however, result in innacurate hypotheses of relationships

(e.g., Willis et al. 2013; Dool et al. 2016; Leavitt et al. 2017; Nguyen et al. 2017). Thus, it is important to identify cytonuclear discordance when assessing the validity of species.

Species need not be monophyletic; hybridization occurs naturally, and it does not necessarily invalidate specieshood. Studies that rely solely on mtDNA patterns cannot test for this (Edwards et al. 2005; Dool et al. 2016; Gottscho et al. 2017). Edwards & Bensch

(2009) discussed the importance of integrating phylogeographic pattern with the phylogeographic process, as first proposed by Avise et al. (1987), warning of the problems with drawing conclusions based on mtDNA patterns without questioning the processes involved in creating such patterns. But still the practice of identifying and delimiting species using only fragments of the entire mtDNA genome persists (e.g., Morin et al. 2016;

Pesarakloo et al. 2017; Borsa et al. 2017; McGuire et al. 2018; Violi et al. 2018; Zhao et al. 2018; Melo et al. 2018; Mendes et al. 2018; Marin et al. 2018), and this, in turn, has serious ramifications for setting conservation policy. For example, based on my mtDNA analysis there are three distinct southern lineages of U. nigricaudus with sequence divergences of 8.26 to 8.39% (the Isthmus of La Paz break; S2:C1) and 5.23 to 5.41% (the

Cape Region Break; C1:C2). Historically, matriline S2 was indeed recognized as the different species Urosaurus microscutatus VanDenburg 1864. Aguirre-Léon et al. (1999) found no difference between U. microscutatus (matriline S2) and U. nigricaudus (matriline

C1), based on allozyme data and synonymized the former into the latter. However, this taxonomic change has remained controversial. A recent study on the phylogeny of

Urosaurus by Feldman et al. (2011) maintained U. microscutatus. My results based on thousands of nDNA loci indicate that there is widespread panmixia within the three southern lineages, indicating that U. nigricaudus is a single species, at least in the southern part of the peninsula. Female-structured populations exist but males disperse and mate across those populations. Thus, analyses fail to reject the hypothesis of unrestricted nDNA gene flow throughout the southern portion of the peninsula. Given that this area has the greatest levels of mtDNA discordance, it is likely that unabated gene flow occurs further north, particularly at the mid-peninsula where mtDNA discontinuity is common (Lindell et al. 2006; Meik et al. 2018).

In conclusion, U. nigricaudus in southern Baja California has three deeply divergent female populations that overlap very little, and yet unrestricted nDNA gene flow occurs between the matrilines. These results cannot reject the null hypothesis that U. nigricaudus is a single species and that assumptions based only in mtDNA patterns do not represent the species’ history and population genetics structure: such patterns are nothing more than a snapshot of female populations at any given time. Choice of the most suitable genetic marker to investigate relationships between populations or taxa is not a one-size- fits-all approach and will always depend on the specific hypothesis being tested. Variables such inheritance mode, age, extent of genetic variation and introgression will determine which genes will be most useful for testing hypotheses. The choice of nDNA markers must be made with caution because different fragments carry different information. Some slowly evolving nDNA exons appear to resolve older relationships (>60 Ma) and may not be informative for intraspecific studies, and faster evolving introns are likely to better inform intraspecific studies than mtDNA data (Dool et al. 2016). Intraspecific

63 investigations must thus employ large sample sizes and multiple loci to increase the resolution power, even in the presence of incomplete lineage sorting (Maddison &

Knowles 2006), to deliver accurate and reliable inferences (Edwards & Bensch 2009).

4.5 Acknowledgements

I thank Carmen Izmene Gutiérrez-Rojas for the assistance during fieldwork and Amy

Lathrop, Kristen Choffe, Oliver Haddrath, Tulio Soares, Cintya Segura-Trujillo and

Griselda Gallegos Simental for assistance in the laboratory. I am also thankful to Yessica

Rico, Jonathan R. Galina-Mehlman and Taylor Edwards for the invaluable assistance during the ddRAD-seq processing. This work was supported by Canada Graduate

Scholarship from the National Science and Engineering Research Council of Canada

(NSERC) to PHB, NSERC Discovery Grant A3148 to RWM, CONACYT 151189 to

STAC and PAPIIT-UNAM 215011-3 to FRMC. Field equipment was generously donated by IDEA WILD.

4.6 Data accessibility

The 26 mitochondrial DNA genomes of Urosaurus nigricaudus with the exception of the control region are available at GenBank accession numbers: MH369811 to MH369835.

Demultiplexed and quality-filtered RAD-tags used in this study are currently being submitted to at the US National Center of Biotechnology Information (NCBI) Sequence

Read Archive (SRA).

4.7 Figures

Figure 4.1: A: Matrilineal genealogy of Urosaurus nigricaudus based on the mitochondrial genes ATP6, ATP8 and Cytb (totaling a 1981bp alignment) built using

Bayesian Inference on MRBAYES. Node values are the Bayesian posterior probability.

Matrilines S2, C1 and C2 are highly supported monophyletic groups (BPB=1). B: Map showing the southern Peninsula of Baja California, Mexico and the area occupied by the three matrilines.

4.8 Tables

Table 4.1: List of the 196 specimens of Urosaurus nigricaudus collected in the Peninsula

of Baja California, Mexico in the month of August/2013. ROM#: Voucher number for the

tissue deposited in the herpetological collection of the Royal Ontario Museum, Canada.

Sample ID is the ID used in the laboratory work and displayed in the figures of this paper.

ROM Sample Sex LOCALITY LAT LON # ID

53168 297 Female Oasis San Pedrito - Pescaderos 23.389066 -110.20902

54613 329 Male 2.5 Km N, 21 Km W, Santiago, 23.50078 -109.92441 BCS

54614 321 Male 1 Km N, 1 Km W, Agua Caliente, 23.451522 -109.78506 Santiago, BCS

54615 328 Male Cascada Sol de Mayo, Sierra de la 23.498592 -109.79356 Laguna

54616 331 Female 5 Km S of San Bartolo 23.736591 -109.81412

54617 330 Female 5 Km S of San Bartolo 23.73636 -109.8141

54618 397 Male Km 86 N of La Paz on Hwy 1 24.239699 -110.95002 towards Cd. Constitucion

54619 396 Male Km 86 N of La Paz on Hwy 1 24.239516 -110.95005 towards Cd. Constitucion

54620 395 Male Km 86 N of La Paz on Hwy 1 24.239183 -110.95014 towards Cd. Constitucion

54621 389 Male Km 74 N of La Paz on Hwy 1 24.169402 -110.89338 towards Cd. Constitucion

54622 305 Male Oasis San Pedrito - Pescaderos 23.389135 -110.21153

54623 306 Female Oasis San Pedrito - Pescaderos 23.389135 -110.21153

54624 303 Male Oasis San Pedrito - Pescaderos 23.389094 -110.21069

54625 286 Male Oasis San Pedrito - Pescaderos 23.388962 -110.21075

54626 289 Male Oasis San Pedrito - Pescaderos 23.388998 -110.2101

54627 287 Male Oasis San Pedrito - Pescaderos 23.38899 -110.2099

54628 290 Female Oasis San Pedrito - Pescaderos 23.38901 -110.20928

54629 298 Female Oasis San Pedrito - Pescaderos 23.389067 -110.20893

54630 294 Male Oasis San Pedrito - Pescaderos 23.389043 -110.2089

54631 291 Male Oasis San Pedrito - Pescaderos 23.389013 -110.20855

54632 300 Male Oasis San Pedrito - Pescaderos 23.389075 -110.20861

54633 213 Male Migriños - Arroyo 23.046385 -110.0677

54634 214 Male Migriños - Arroyo 23.046433 -110.06791

54635 215 Female Migriños - Arroyo 23.046516 -110.06771

54636 216 Male Migriños - Arroyo 23.046585 -110.06781

54637 212 Male Migriños - Arroyo 23.046355 -110.06756

54638 217 Female Migriños - Arroyo 23.051913 -110.07679

54640 379 Male Km 77 N of La Paz on Hwy 1 24.163514 -110.92166 towards Cd. Constitucion

54641 385 Male Km 77 N of La Paz on Hwy 1 24.163571 -110.92178 towards Cd. Constitucion

54642 373 Female Km 77 N of La Paz on Hwy 1 24.162766 -110.92177 towards Cd. Constitucion

54643 380 Male Km 77 N of La Paz on Hwy 1 24.163515 -110.92156 towards Cd. Constitucion

54644 383 Male Km 77 N of La Paz on Hwy 1 24.163544 -110.9215 towards Cd. Constitucion

54645 382 Male Km 77 N of La Paz on Hwy 1 24.163536 -110.9218 towards Cd. Constitucion

54646 384 Male Km 77 N of La Paz on Hwy 1 24.163553 -110.92134 towards Cd. Constitucion

54647 376 Male Km 77 N of La Paz on Hwy 1 24.163395 -110.92134 towards Cd. Constitucion

54648 374 Male Km 77 N of La Paz on Hwy 1 24.163265 -110.92098 towards Cd. Constitucion

54649 338 Male Km 53 N of La Paz on Hwy 1 24.100266 -110.73253 towards Cd. Constitucion

54650 337 Male Km 53 N of La Paz on Hwy 1 24.0998 -110.7335 towards Cd. Constitucion

54651 335 Male Km 53 N of La Paz on Hwy 1 24.098976 -110.73475 towards Cd. Constitucion

54652 336 Female Km 53 N of La Paz on Hwy 1 24.098976 -110.73475 towards Cd. Constitucion

54653 238 Male Puente Los Corrales II - N of Santa 23.232762 -109.73728 Anita

54654 239 Male Puente Los Corrales II - N of Santa 23.232834 -109.73797 Anita

54655 230 Male Santa Anita - Behind the Airport 23.159871 -109.7401

54656 227 Male Santa Anita - Behind the Airport 23.159426 -109.73921

54657 228 Male Santa Anita - Behind the Airport 23.159431 -109.73902

54658 229 Male Santa Anita - Behind the Airport 23.159665 -109.73851

54659 226 Female Santa Anita - Behind the Airport 23.158986 -109.73824

54660 225 Female Santa Anita - Behind the Airport 23.158942 -109.73829

54661 221 Male Santa Anita - Behind the Airport 23.158479 -109.74001

54662 223 Female Santa Anita - Behind the Airport 23.158663 -109.73797

54663 222 Male Santa Anita - Behind the Airport 23.158649 -109.73756

54664 224 Male Santa Anita - Behind the Airport 23.158762 -109.73745

54665 219 Female 1km E of Zacatitos, East of San 23.121873 -109.54298 Jose del Cabo

54666 220 Male 1km E of Zacatitos, East of San 23.122016 -109.54292 Jose del Cabo

54667 218 Male 1km E of Zacatitos, East of San 23.121823 -109.54281 Jose del Cabo

54668 211 Male Cabo San Lucas 22.916259 -109.96387

54669 406 Male Km 39 N of La Paz towards San 24.367638 -110.69576 Juan de La Costa

54670 405 Male Km 34 N of La Paz towards San 24.32873 -110.66061 Juan de La Costa

54671 392 Male Km 20 N of La Paz towards San 24.218388 -110.59275 Juan de La Costa

54672 365 Female Between Km 75 and 76 N of La 24.15828 -110.91561 Paz towards Constitucion

54673 367 Male Between Km 75 and 76 N of La 24.158375 -110.91552 Paz towards Constitucion

54674 366 Female Between Km 75 and 76 N of La 24.15837 -110.91554 Paz towards Constitucion

54675 363 Male Between Km 75 and 76 N of La 24.158206 -110.91541 Paz towards Constitucion

54676 364 Female Between Km 75 and 76 N of La 24.158206 -110.91541 Paz towards Constitucion

54677 359 Female Between Km 75 and 76 N of La 24.157954 -110.91598 Paz towards Constitucion

54678 360 Male Between Km 75 and 76 N of La 24.157954 -110.91598 Paz towards Constitucion

54679 361 Male Between Km 75 and 76 N of La 24.157954 -110.91598 Paz towards Constitucion

54680 362 Female Between Km 75 and 76 N of La 24.158143 -110.91572 Paz towards Constitucion

54681 368 Female Between Km 75 and 76 N of La 24.160153 -110.9154 Paz towards Constitucion

54682 369 Male Between Km 75 and 76 N of La 24.160153 -110.9154 Paz towards Constitucion

54684 404 Female Between Km 75 and 76 N of La 24.160231 -110.91547 Paz towards Constitucion

54685 370 Male Between Km 75 and 76 N of La 24.160829 -110.91517 Paz towards Constitucion

54686 371 Female Between Km 75 and 76 N of La 24.16097 -110.91488 Paz towards Constitucion

54687 372 Male Between Km 75 and 76 N of La 24.16097 -110.91488 Paz towards Constitucion

54688 352 Male Between Km 75 and 76 N of La 24.156662 -110.91883 Paz towards Constitucion

54689 354 Male Between Km 75 and 76 N of La 24.156866 -110.91975 Paz towards Constitucion

54690 355 Male Between Km 75 and 76 N of La 24.156866 -110.91975 Paz towards Constitucion

54691 353 Male Between Km 75 and 76 N of La 24.156834 -110.91866 Paz towards Constitucion

54692 357 Female Between Km 75 and 76 N of La 24.157351 -110.91685 Paz towards Constitucion

54693 358 Female Between Km 75 and 76 N of La 24.157351 -110.91685 Paz towards Constitucion

54694 356 Male Between Km 75 and 76 N of La 24.157083 -110.91791 Paz towards Constitucion

54695 398 Female Km 94 N of La Paz on Hwy 1 24.301827 -110.97437 towards Cd. Constitucion

54696 399 Female Km 94 N of La Paz on Hwy 1 24.3025 -110.97447 towards Cd. Constitucion

54698 346 Female Arroyo 2Km W, on Km 77 N of La 24.151897 -110.93716 Paz on Hwy 1 towards Cd. Constitucion (behind the Restaurant)

54699 350 Male Arroyo 2Km W, on Km 77 N of La 24.152609 -110.9372 Paz on Hwy 1 towards Cd. Constitucion (behind the Restaurant)

54700 351 Male Arroyo 2Km W, on Km 77 N of La 24.152609 -110.9372 Paz on Hwy 1 towards Cd. Constitucion (behind the Restaurant)

54701 348 Male Arroyo 2Km W, on Km 77 N of La 24.152362 -110.93706 Paz on Hwy 1 towards Cd.

Constitucion (behind the Restaurant)

54702 347 Male Arroyo 2Km W, on Km 77 N of La 24.15196 -110.93706 Paz on Hwy 1 towards Cd. Constitucion (behind the Restaurant)

54703 345 Female Arroyo 2Km W, on Km 77 N of La 24.151782 -110.93714 Paz on Hwy 1 towards Cd. Constitucion (behind the Restaurant)

54704 390 Female Km 58 N of La Paz on Hwy 1 24.18078 -110.75625 towards Cd. Constitucion

54705 391 Male Km 58 N of La Paz on Hwy 1 24.18078 -110.75625 towards Cd. Constitucion

54706 332 Male La Paz, Casa de Eduardo 24.098466 -110.38314

54707 333 Male La Paz, Casa de Eduardo 24.098466 -110.38314

54708 334 Female La Paz, Casa de Eduardo 24.098466 -110.38314

54709 277 Male 1 Km South of Oasis San Pedrito 23.368629 -110.20084

54710 270 Male 1 Km South of Oasis San Pedrito 23.368425 -110.20087

54711 266 Female 1 Km South of Oasis San Pedrito 23.368326 -110.20076

54712 267 Male 1 Km South of Oasis San Pedrito 23.368352 -110.20071

54713 401 Female Km 94 N of La Paz on Hwy 1 24.303126 -110.97299 towards Cd. Constitucion

54714 402 Male Km 94 N of La Paz on Hwy 1 24.303126 -110.97299 towards Cd. Constitucion

54715 403 Male Km 94 N of La Paz on Hwy 1 24.303126 -110.97299 towards Cd. Constitucion

54716 400 Female Km 94 N of La Paz on Hwy 1 24.302932 -110.97315 towards Cd. Constitucion

54717 341 Male Arroyo 2Km W, on Km 77 N of La 24.151146 -110.93793 Paz on Hwy 1 towards Cd. Constitucion (behind the Restaurant)

54718 343 Male Arroyo 2Km W, on Km 77 N of La 24.151614 -110.93718 Paz on Hwy 1 towards Cd. Constitucion (behind the Restaurant)

54719 349 Male Arroyo 2Km W, on Km 77 N of La 24.152567 -110.93731 Paz on Hwy 1 towards Cd. Constitucion (behind the Restaurant)

54720 344 Male Arroyo 2Km W, on Km 77 N of La 24.151721 -110.93728 Paz on Hwy 1 towards Cd. Constitucion (behind the Restaurant)

54721 342 Female Arroyo 2Km W, on Km 77 N of La 24.151361 -110.93732 Paz on Hwy 1 towards Cd. Constitucion (behind the Restaurant)

54722 268 Male 1 Km South of Oasis San Pedrito 23.368352 -110.20071

54723 269 Male 1 Km South of Oasis San Pedrito 23.368384 -110.2007

54724 271 Male 1 Km South of Oasis San Pedrito 23.368465 -110.20069

54725 275 Male 1 Km South of Oasis San Pedrito 23.368518 -110.20081

54726 274 Male 1 Km South of Oasis San Pedrito 23.368517 -110.20081

54727 272 Female 1 Km South of Oasis San Pedrito 23.368487 -110.20086

54728 273 Male 1 Km South of Oasis San Pedrito 23.368497 -110.20088

54729 276 Male 1 Km South of Oasis San Pedrito 23.368549 -110.20079

54730 320 Female Km 55 on Hwy 19 towards Cabo 23.419811 -110.21074 San Lucas

54731 318 Male Km 55 on Hwy 19 towards Cabo 23.419461 -110.21247 San Lucas

54732 319 Female Km 55 on Hwy 19 towards Cabo 23.419461 -110.21247 San Lucas

54733 285 Male Oasis San Pedrito - Pescaderos 23.388939 -110.20822

54734 283 Male Oasis San Pedrito - Pescaderos 23.388705 -110.2076

54735 278 Male Oasis San Pedrito - Pescaderos 23.388397 -110.20671

54736 279 Female Oasis San Pedrito - Pescaderos 23.388397 -110.20671

54737 280 Female Oasis San Pedrito - Pescaderos 23.38847 -110.20708

54738 281 Male Oasis San Pedrito - Pescaderos 23.38851 -110.20731

54739 284 Female Oasis San Pedrito - Pescaderos 23.388902 -110.20822

54740 304 Female Oasis San Pedrito - Pescaderos 23.389115 -110.20831

54741 282 Male Oasis San Pedrito - Pescaderos 23.388545 -110.20719

54742 293 Male Oasis San Pedrito - Pescaderos 23.389042 -110.20823

54743 295 Female Oasis San Pedrito - Pescaderos 23.389057 -110.20942

54744 296 Male Oasis San Pedrito - Pescaderos 23.389062 -110.2091

54745 292 Male Oasis San Pedrito - Pescaderos 23.389033 -110.20939

54746 302 Male Oasis San Pedrito - Pescaderos 23.389083 -110.20872

54747 299 Male Oasis San Pedrito - Pescaderos 23.389073 -110.20872

54748 288 Male Oasis San Pedrito - Pescaderos 23.388996 -110.2084

54749 309 Male Oasis San Pedrito - Pescaderos 23.389234 -110.21111

54750 310 Female Oasis San Pedrito - Pescaderos 23.389234 -110.21111

54751 244 Male South of Las Casitas, on Hwy 1 23.264312 -109.78231 from San Jose del Cabo to La Paz

54752 247 Female South of Las Casitas, on Hwy 1 23.264787 -109.7825 from San Jose del Cabo to La Paz

54753 246 Male South of Las Casitas, on Hwy 1 23.264773 -109.78256 from San Jose del Cabo to La Paz

54754 240 Male South of Las Casitas, on Hwy 1 23.26423 -109.78121 from San Jose del Cabo to La Paz

54755 241 Male South of Las Casitas, on Hwy 1 23.264278 -109.78124 from San Jose del Cabo to La Paz

54756 243 Female South of Las Casitas, on Hwy 1 23.264298 -109.78232 from San Jose del Cabo to La Paz

54757 242 Male South of Las Casitas, on Hwy 1 23.26429 -109.7813 from San Jose del Cabo to La Paz

54758 245 Female South of Las Casitas, on Hwy 1 23.264342 -109.78237 from San Jose del Cabo to La Paz

54759 256 Female Km 60 on Hwy 1 from San Jose 23.304266 -109.76747 del Cabo to La Paz

54760 251 Male Km 60 on Hwy 1 from San Jose 23.303812 -109.76766 del Cabo to La Paz

54761 252 Male Km 60 on Hwy 1 from San Jose 23.303812 -109.76766 del Cabo to La Paz

54762 253 Female Km 60 on Hwy 1 from San Jose 23.304158 -109.76757 del Cabo to La Paz

54763 254 Female Km 60 on Hwy 1 from San Jose 23.304158 -109.76757 del Cabo to La Paz

54764 255 Female Km 60 on Hwy 1 from San Jose 23.304158 -109.76757 del Cabo to La Paz

54765 375 Male Km 77 N of La Paz on Hwy 1 24.163388 -110.92161 towards Cd. Constitucion

54766 386 Male Km 77 N of La Paz on Hwy 1 24.163723 -110.92059 towards Cd. Constitucion

54767 378 Male Km 77 N of La Paz on Hwy 1 24.163491 -110.92188 towards Cd. Constitucion

54768 388 Male Km 77 N of La Paz on Hwy 1 24.164048 -110.92094 towards Cd. Constitucion

54769 387 Female Km 77 N of La Paz on Hwy 1 24.164033 -110.92114 towards Cd. Constitucion

54770 381 Female Km 77 N of La Paz on Hwy 1 24.163523 -110.92134 towards Cd. Constitucion

54771 340 Male Km 55 N of La Paz on Hwy 1 24.113249 -110.74653 towards Cd. Constitucion

54772 339 Male Km 55 N of La Paz on Hwy 1 24.112676 -110.7471 towards Cd. Constitucion

54773 394 Female Km 86.5 N of La Paz on Hwy 1 24.235923 -110.94821 towards Cd. Constitucion

54774 393 Female Km 85 N of La Paz on Hwy 1 24.228201 -110.9473 towards Cd. Constitucion

54775 377 Male Km 77 N of La Paz on Hwy 1 24.163484 -110.92153 towards Cd. Constitucion

54776 307 Female Oasis San Pedrito - Pescaderos 23.389032 -110.2088

54777 308 Female Oasis San Pedrito - Pescaderos 23.38919 -110.21105

54778 311 Male Oasis San Pedrito - Pescaderos 23.389566 -110.21114

54779 313 Female Oasis San Pedrito - Pescaderos 23.389632 -110.21124

54780 316 Male Oasis San Pedrito - Pescaderos 23.389922 -110.21141

54781 317 Female Oasis San Pedrito - Pescaderos 23.389922 -110.21141

54782 314 Male Oasis San Pedrito - Pescaderos 23.389846 -110.21137

54783 315 Female Oasis San Pedrito - Pescaderos 23.389846 -110.21137

54784 301 Female Oasis San Pedrito - Pescaderos 23.389076 -110.20891

54785 312 Female Oasis San Pedrito - Pescaderos 23.389582 -110.2114

54786 326 Female Todos Santos 23.454414 -110.22315

54787 259 Male 10 km South of Cabo Pulmo 23.330389 -109.43182

54788 260 Male 10 km South of Cabo Pulmo 23.330511 -109.43164

54789 261 Female 10 km South of Cabo Pulmo 23.330634 -109.43216

54790 258 Male 10 km South of Cabo Pulmo 23.330295 -109.43153

54791 257 Male 10 km South of Cabo Pulmo 23.330179 -109.43148

54792 327 Female 7 km North of Cabo Pulmo 23.493627 -109.46802

54793 324 Male 2 km North of Cabo Pulmo 23.453119 -109.43479

54794 323 Male 2 km North of Cabo Pulmo 23.453117 -109.43487

54795 325 Female 2 km North of Cabo Pulmo 23.453137 -109.43481

54796 322 Female 2 km North of Cabo Pulmo 23.453108 -109.43493

54797 236 Female 20 km S, 15 km W of Cabo Pulmo 23.221473 -109.58074

54798 232 Male 20 km S, 15 km W of Cabo Pulmo 23.221371 -109.58048

54799 233 Male 20 km S, 15 km W of Cabo Pulmo 23.221371 -109.58048

54800 237 Male 20 km S, 15 km W of Cabo Pulmo 23.221512 -109.58039

54801 234 Female 20 km S, 15 km W of Cabo Pulmo 23.221371 -109.58048

54802 231 Male 20 km S, 15 km W of Cabo Pulmo 23.221236 -109.58015

54803 235 Male 20 km S, 15 km W of Cabo Pulmo 23.221409 -109.58013

54804 249 Male 10 km S, 15 km W of Cabo Pulmo 23.286487 -109.56306

54805 248 Male 10 km S, 15 km W of Cabo Pulmo 23.286462 -109.56302

54806 250 Male 10 km S, 15 km W of Cabo Pulmo 23.286514 -109.56301

54807 263 Female 20 km N, 15 km W of Cabo Pulmo 23.345732 -109.60075

54808 264 Female 20 km N, 15 km W of Cabo Pulmo 23.345785 -109.60063

54809 265 Male 20 km N, 15 km W of Cabo Pulmo 23.345815 -109.60067

54810 262 Male 20 km N, 15 km W of Cabo Pulmo 23.345379 -109.60081

Table 4.2: Localities for the 26 individuals used for mtDNA genome sequencing and analyses. Matriline samples: S2: 21 samples; C1: 28 samples; C2: 24 samples.

mtDNA Sample Locality LAT LONG lineage ID

Km 86.5 N of La Paz on Hwy 1 towards S2 394 24.235923 -110.94821 Cd. Constitucion

Km 85 N of La Paz on Hwy 1 towards S2 393 24.228201 -110.9473 Cd. Constitucion

Km 20 N of La Paz towards San Juan S2 392 24.218388 -110.59275 de La Costa

Km 58 N of La Paz on Hwy 1 towards C1 390 24.18078 -110.75625 Cd. Constitucion

Km 77 N of La Paz on Hwy 1 towards S2 387 24.164033 -110.92114 Cd. Constitucion

Km 77 N of La Paz on Hwy 1 towards S2 385 24.163571 -110.92178 Cd. Constitucion

Km 77 N of La Paz on Hwy 1 towards S2 383 24.163544 -110.9215 Cd. Constitucion

Km 77 N of La Paz on Hwy 1 towards S2 381 24.163523 -110.92134 Cd. Constitucion

Km 77 N of La Paz on Hwy 1 towards S2 377 24.163484 -110.92153 Cd. Constitucion

Km 77 N of La Paz on Hwy 1 towards C1 375 24.163388 -110.92161 Cd. Constitucion

Km 77 N of La Paz on Hwy 1 towards S2 373 24.162766 -110.92177 Cd. Constitucion

C1 316 Oasis San Pedrito - Pescaderos 23.389922 -110.21141

C1 317 Oasis San Pedrito - Pescaderos 23.389922 -110.21141

C1 315 Oasis San Pedrito - Pescaderos 23.389846 -110.21137

C2 313 Oasis San Pedrito - Pescaderos 23.389632 -110.21124

C2 311 Oasis San Pedrito - Pescaderos 23.389566 -110.21114

C2 310 Oasis San Pedrito - Pescaderos 23.389234 -110.21111

C1 309 Oasis San Pedrito - Pescaderos 23.389234 -110.21111

C2 308 Oasis San Pedrito - Pescaderos 23.38919 -110.21105

C2 301 Oasis San Pedrito - Pescaderos 23.389076 -110.20891

C2 300 Oasis San Pedrito - Pescaderos 23.389075 -110.20861

C2 298 Oasis San Pedrito - Pescaderos 23.389067 -110.20893

C1 297 Oasis San Pedrito - Pescaderos 23.389066 -110.20902

C1 295 Oasis San Pedrito - Pescaderos 23.389057 -110.20942

C1 307 Oasis San Pedrito - Pescaderos 23.389032 -110.2088

C2 284 Oasis San Pedrito - Pescaderos 23.388902 -110.20822

Table 4.3: Localities for the 73 individuals used for ddRAD-seq sequencing and analyses.

mtDNA Sample Locality LAT LONG lineage ID

Km 39 N of La Paz towards San Juan de S2 406 24.367638 -110.69576 La Costa

Km 34 N of La Paz towards San Juan de S2 405 24.32873 -110.66061 La Costa

Km 94 N of La Paz on Hwy 1 towards Cd. S2 398 24.301827 -110.97437 Constitucion

Km 86 N of La Paz on Hwy 1 towards Cd. S2 397 24.239699 -110.95002 Constitucion

Km 86 N of La Paz on Hwy 1 towards Cd. S2 396 24.239516 -110.95005 Constitucion

Km 86 N of La Paz on Hwy 1 towards Cd. S2 395 24.239183 -110.95014 Constitucion

Km 20 N of La Paz towards San Juan de S2 392 24.218388 -110.59275 La Costa

Km 74 N of La Paz on Hwy 1 towards Cd. C1 389 24.169402 -110.89338 Constitucion

Km 77 N of La Paz on Hwy 1 towards Cd. S2 385 24.163571 -110.92178 Constitucion

Km 77 N of La Paz on Hwy 1 towards Cd. S2 383 24.163544 -110.9215 Constitucion

Km 77 N of La Paz on Hwy 1 towards Cd. C1 382 24.163536 -110.9218 Constitucion

Km 77 N of La Paz on Hwy 1 towards Cd. C1 380 24.163515 -110.92156 Constitucion

Km 77 N of La Paz on Hwy 1 towards Cd. C1 379 24.163514 -110.92166 Constitucion

Km 77 N of La Paz on Hwy 1 towards Cd. S2 376 24.163395 -110.92134 Constitucion

Km 77 N of La Paz on Hwy 1 towards Cd. C1 374 24.163265 -110.92098 Constitucion

Km 77 N of La Paz on Hwy 1 towards Cd. S2 373 24.162766 -110.92177 Constitucion

Between Km 75 and 76 N of La Paz S2 372 24.16097 -110.91488 towards Constitucion

Between Km 75 and 76 N of La Paz C1 371 24.16097 -110.91488 towards Constitucion

Between Km 75 and 76 N of La Paz S2 370 24.160829 -110.91517 towards Constitucion

Between Km 75 and 76 N of La Paz C1 404 24.160231 -110.91547 towards Constitucion

Between Km 75 and 76 N of La Paz C1 368 24.160153 -110.9154 towards Constitucion

Between Km 75 and 76 N of La Paz C1 369 24.160153 -110.9154 towards Constitucion

Between Km 75 and 76 N of La Paz S2 367 24.158375 -110.91552 towards Constitucion

Between Km 75 and 76 N of La Paz S2 366 24.15837 -110.91554 towards Constitucion

Between Km 75 and 76 N of La Paz C1 363 24.158206 -110.91541 towards Constitucion

Between Km 75 and 76 N of La Paz C1 364 24.158206 -110.91541 towards Constitucion

Between Km 75 and 76 N of La Paz S2 362 24.158143 -110.91572 towards Constitucion

Between Km 75 and 76 N of La Paz S2 359 24.157954 -110.91598 towards Constitucion

Between Km 75 and 76 N of La Paz S2 360 24.157954 -110.91598 towards Constitucion

Between Km 75 and 76 N of La Paz S2 361 24.157954 -110.91598 towards Constitucion

Between Km 75 and 76 N of La Paz C1 357 24.157351 -110.91685 towards Constitucion

Between Km 75 and 76 N of La Paz C1 358 24.157351 -110.91685 towards Constitucion

Between Km 75 and 76 N of La Paz S2 354 24.156866 -110.91975 towards Constitucion

Between Km 75 and 76 N of La Paz S2 355 24.156866 -110.91975 towards Constitucion

Between Km 75 and 76 N of La Paz C1 353 24.156834 -110.91866 towards Constitucion

Between Km 75 and 76 N of La Paz C1 352 24.156662 -110.91883 towards Constitucion

Km 53 N of La Paz on Hwy 1 towards Cd. C1 338 24.100266 -110.73253 Constitucion

Km 53 N of La Paz on Hwy 1 towards Cd. C1 337 24.0998 -110.7335 Constitucion

Km 53 N of La Paz on Hwy 1 towards Cd. C1 335 24.098976 -110.73475 Constitucion

Km 53 N of La Paz on Hwy 1 towards Cd. C1 336 24.098976 -110.73475 Constitucion

C1 331 5 Km S of San Bartolo 23.736591 -109.81412

C1 330 5 Km S of San Bartolo 23.73636 -109.8141

C1 329 2.5 Km N, 21 Km W, Santiago, BCS 23.50078 -109.92441

C1 328 Cascada Sol de Mayo, Sierra de la Laguna 23.498592 -109.79356

1 Km N, 1 Km W, Agua Caliente, C1 321 23.451522 -109.78506 Santiago, BCS

C1 306 Oasis San Pedrito - Pescaderos 23.389135 -110.21153

C2 303 Oasis San Pedrito - Pescaderos 23.389094 -110.21069

C2 300 Oasis San Pedrito - Pescaderos 23.389075 -110.20861

C1 294 Oasis San Pedrito - Pescaderos 23.389043 -110.2089

C2 291 Oasis San Pedrito - Pescaderos 23.389013 -110.20855

C1 290 Oasis San Pedrito - Pescaderos 23.38901 -110.20928

C2 289 Oasis San Pedrito - Pescaderos 23.388998 -110.2101

C1 287 Oasis San Pedrito - Pescaderos 23.38899 -110.2099

C2 286 Oasis San Pedrito - Pescaderos 23.388962 -110.21075

C2 239 Puente Los Corrales II - N of Santa Anita 23.232834 -109.73797

C2 238 Puente Los Corrales II - N of Santa Anita 23.232762 -109.73728

C2 230 Santa Anita - Behind the Airport 23.159871 -109.7401

C2 229 Santa Anita - Behind the Airport 23.159665 -109.73851

C2 228 Santa Anita - Behind the Airport 23.159431 -109.73902

C2 227 Santa Anita - Behind the Airport 23.159426 -109.73921

C2 226 Santa Anita - Behind the Airport 23.158986 -109.73824

C2 224 Santa Anita - Behind the Airport 23.158762 -109.73745

C2 223 Santa Anita - Behind the Airport 23.158663 -109.73797

C2 222 Santa Anita - Behind the Airport 23.158649 -109.73756

1km E of Zacatitos, East of San Jose del C2 220 23.122016 -109.54292 Cabo

1km E of Zacatitos, East of San Jose del C2 219 23.121873 -109.54298 Cabo

1km E of Zacatitos, East of San Jose del C2 218 23.121823 -109.54281 Cabo

C2 217 Migriños - Arroyo 23.051913 -110.07679

C2 216 Migriños - Arroyo 23.046585 -110.06781

C2 215 Migriños - Arroyo 23.046516 -110.06771

C2 214 Migriños - Arroyo 23.046433 -110.06791

C2 213 Migriños - Arroyo 23.046385 -110.0677

C2 211 Cabo San Lucas 22.916259 -109.96387

Table 4.4: AMOVA results based on the mitochondrial DNA genome and nuclear SNPs from different populations of Urosaurus nigricaudus.

Sum of Percentage DF MS ΦPT P value Squares variation

Global 0.949 P<0.001 AMOVA

Among 2 8570.080 4285.040 88% P<0.001 populations mtDNA Within 23 1463.458 63.629 12% P<0.001 populations

Total 25 10033.538 100% P<0.001

Global 0.012 P= 0.1 AMOVA

Among 2 1108.129 554.065 1% P>0.05 populations nDNA

Within 70 30009.473 428.707 99% P>0.05 populations

Total 72 31117.603 100% P>0.05

Table 4.5: Pairwise ΦPT based on the mitochondrial DNA genome (below diagonal) and nuclear SNPs (above diagonal) of Urosaurus nigricaudus.

C1 C2 S2

C1 - 0.018* 0.000*

C2 0.931** - 0.023*

S2 0.890** 0.796** -

* P > 0.05

**P < 0.01

Chapter 5 The drivers of deep mitochondrial DNA

divergence in the black-tailed brush lizard

(Urosaurus nigricaudus)

This chapter is currently being prepared for submission to Heredity (Bernardo, P.H.,

Aguilera-Miller, E.F., Álvarez-Castañeda, S.T., Méndez-de la Cruz, F.R., and R.W.

Murphy. The drivers of deep mitochondrial DNA divergence of black-tailed brush lizard

(Urosaurus nigricaudus)).

Abstract

Widespread parapatric mitochondrial lineages (matrilines) are the basis of intraspecific phylogeography. However, much speculation exists about what maintains the discontinuity. The small black-tailed brush lizard, Urosaurus nigricaudus, of the peninsula of Baja California, Mexico and southern California, USA has six parapatric matrilines that encompass substantial mtDNA divergence and yet unrestricted nDNA flow. Herein, I use the mtDNA genome of U. nigricaudus from the three southernmost matrilines to explore what drives and maintains its parapatry. Neutral processes like philopatry, vicariance and incomplete lineage sorting do not explain the discontinuity in U. nigricaudus. Historical vicariance appears to be responsible for the initial mtDNA divergences. Selective pressure analyses of codon substitution models point to purifying selection as the evolutionary force acting in the mitogenome of this lizard. This rejects the hypothesis that habitat adaptation shapes the mtDNA genes of this species. Our time-calibrated Bayesian tree corresponds with temporary seaways that initially isolated populations of U. nigricaudus on ephemeral islands. These results explain the origin of the deep mtDNA divergences and rule out positive selection playing a role in the maintenance of the discontinuity. Further studies on other female-linked traits such as mito-nuclear functional compensation and female behavior will most likely resolve the mystery of why females do not cross the matriline border when no obvious barrier exists.

5.1 Introduction

Mitochondria are vital components of eukaryotic cells as they are directly involved with oxygen use, metabolism and energy production via oxidative phosphorylation (OXPHOS)

(Saraste 1999). In vertebrates, the circular genome of mitochondria contains 13 protein- coding genes (Melo-Ferreira et al. 2014) involved in four of the five OXPHOS complexes

(I, III, IV and V) (Morales et al. 2015). Those mitochondrial DNA (mtDNA) genes work in conjunction with 72 nuclear DNA (nDNA) protein-coding genes that participate in all five complexes to produce cellular energy (Saraste 1999; Morales et al. 2015; Wai &

Langer 2016). The mtDNA genome has a higher rate of molecular evolution when compared to nDNA and is inherited only from the female, usually without recombination with the male parent’s mtDNA. In contrast, nDNA is inherited biparentally and can undergo heterologous recombination during meiosis.

Because all genes in the mitogenome are involved in energy production and metabolism, and consequently play vital roles in the organism’s life, the mitogenome is expected to evolve under purifying selection; deleterious mutations are selected against so the functionality of the protein complexes remains unchanged (Ballard & Whitlock 2004;

Pavlova et al. 2017). Although recent studies on fishes (Pavlova et al. 2017), birds

(Morales et al. 2015) and mammals (Zhang et al. 2013c) revealed widespread signatures of purifying selection in mtDNA genes, some codons have evolved under positive selection, which may indicate adaptation of specific lineages to physiological and environmental constraints (Yu et al. 2011; Morales et al. 2015; Pavlova et al. 2017; Jin et al. 2018).

Selection is expected to act in the same way on nDNA genes involved in the OXPHOS system to maintain compatibility with the mtDNA genome and preserve efficiency in energy production (Bar Yaacov et al. 2015). This constrained coevolution of mtDNA and nDNA genes is known as mito-nuclear functional compensation. The strong functional link between these genes results in natural selection against hybrids, especially in zones of mtDNA discontinuities, to avoid individuals with inefficient energy production. For years, scientists have been using mtDNA sequences to test evolutionary hypotheses of intraspecific relationships, species delimitation and phylogeny. More recently, however, nDNA markers not involved in the OXPHOS system (e.g., RAG exons, some allozymes and microsatellites) are being incorporated into molecular analyses. When a significant difference occurs between geographic patterns and genetic patterns of mtDNA and such nDNA markers, authors have hypothesized that mito-nuclear functional compensation maintains mtDNA discontinuities (Lindell et al. 2005; 2008; Yang & Kenagy 2009;

Morales et al. 2015). Still, only very few studies have tested for mito-nuclear functional compensation when analyzing the nDNA genes directly involved in the OXPHOS system

(e.g., Bar Yaacov et al. 2015).

Species that present different geographic patterns between mtDNA and nDNA offer valuable opportunities to test the roles of neutrality and selection on mitogenomes

(Morales et al. 2015), especially since selection on mtDNA genes can drive population divergence and speciation in some cases (Tieleman et al. 2009). One such species, the black-tailed brush lizard, Urosaurus nigricaudus, occurs on the Peninsular Ranges of Baja

California, Mexico and southern California, USA (Lindell et al. 2008). This common xerophilic phrynosomatid is an arboreal species that can be found in a wide variety of

95 natural environments as well as urban areas (Munguia-Vega et al. 2013). In its natural habitat, U. nigricaudus occupies mesquite trees (Prosopis palmeri S. Watson) and smaller shrubs in the “arroyos”, which are depressions in the landscape that form temporary rivers during the rainy season. Most of the environment on the peninsula consists of desert habitat, and the arroyos are the only areas that can hold water for a short period of time; making them the only areas where the trees that house U. nigricaudus occur, with the exception of the oases.

Urosaurus nigricaudus has six parapatric mtDNA lineages (matrilines) that have substantial mtDNA divergence (Lindell et al. 2008, Chapter 4). After ephemeral isolation by seaways, populations of U. nigricaudus were reunited, yet female lineages remained distinct. The three most divergent matrilines (S2, C1 and C2) occur in the Mexican state of Baja California Sur (Figure 5.1). The deepest mtDNA divergence for U. nigricaudus occurs across the Isthmus of La Paz on the southern portion of the peninsula. Here (S2:C1 break), the matrilines differ by 11.02% in their sequences, and the second deepest discontinuity (C1:C2 break) of 7.41% sequence divergence occurs further south in the region of Los Cabos (Lindell et al. 2008). In spite of these deep genealogical breaks, which far exceed most species divergences (Wu & Murphy 2015; Meik et al. 2018; Ivanov et al.

2018), no nDNA structure has been detected in either allozyme distributions (Aguirre-

Léon et al. 1999) or analysis of thousands of nDNA SNPs acquired from ddRAD-seq

(Chapter 4). Thus, the hypothesis of unrestricted nDNA gene flow (panmixia) throughout the species distribution cannot be rejected.

Geological events may explain how the mtDNA discontinuities arose, but a fundamental question remains unanswered: what maintains matriline breaks in areas where no obvious

96 barrier to dispersal exists? The origin of these discordances dates to millions of years ago but no functional explanation exists for its persistence. In this study, I use the mitogenome to investigate if one female-linked trait, selection on the mtDNA, is shaping each lineage in a distinct way and contributing to the maintenance of the mtDNA lineage isolation. In particular, I test the hypothesis that: (1) functional diversifying selection maintains the mtDNA divergences and (2) mtDNA divergences correspond to geological events in the

Peninsular Ranges of Baja California. Finally, I discuss the possible explanations behind the observed patterns, including the roles of selection, mito-nuclear functional compensation and female behavior on the maintenance of mtDNA discontinuities.

5.2 Material and Methods

5.2.1 Sampling

During the month of August 2013, 26 samples (Table 5.1) of U. nigricaudus belonging to three distinct matrilines (S2, C1 and C2) were collected from several localities on the southernmost portion of the Baja California peninsula in Mexico. Sampling localities

(Figure 5.1) were chosen to maximize coverage of the lineages’ distributions. Lizards were hand collected, photographed, measured and a small tissue sample was collected from the tail and preserved in alcohol 95%. All samples were collected in accordance with Animal

Use Protocols approved by the Royal Ontario Museum Animal Care Committee, and approved by Mexican authorities.

5.2.2 MtDNA genome sequencing

All the laboratory work was done on the Laboratory of Molecular Systematics at the Royal

Ontario Museum. The full mtDNA genome of U. nigricaudus was described by (Bernardo et al. 2016) and we followed their protocols for the polymerase chain reaction (PCR) and sequencing reactions. The mitogenome assembly, gene annotation and alignments were done in GENEIOUS 11 (Kearse et al. 2012).

5.2.3 MtDNA genome tree and divergence times

For phylogenetic reconstruction, we aligned the mtDNA genomes using the MUSCLE

3.8.31 plugin in GENEIOUS. As a primary outgroup taxon, we used the mtDNA genome sequence of the phrynosomatid Sceloporus occidentalis (GenBank Accession AB079242) and to root our Bayesian inference tree we used mtDNA genome sequences of the iguanid lizards Gambelia wislizenii (GenBank Accession NC012831) and Leiocephalus personatus (GenBank Accession NC012834). I used a concatenated mtDNA genome dataset because it provided a greater abundance of information and had a greater likelihood of fully resolving a tree (Shen et al. 2010). I combined all 13 protein-coding genes, two rDNAs and 22 tRNAs in one dataset with a total alignment length of 15,396 bp. The control region (CR) was excluded from the dataset due to sequencing problems in some samples.

The mitogenome tree was inferred using MRBAYES 3.2.6 (Huelsenbeck & Ronquist 2001;

Ronquist et al. 2012). The best substitution model (GTR+I+G) was selected using the

Akaike information criterion (AIC) in JMODELTEST 2.1.1 (Darriba et al. 2012). This analysis consisted of two independent Markov chain Monte Carlo (MCMC) runs for 10

98 million generations. Trees were sampled every 500 generations and the first 1 million generations were discarded as burnin. I then used the 50% majority rule to calculate clade posterior probabilities and to generate a consensus tree.

Divergence times for the mtDNA breaks were estimated using BEAST 2.4.7 (Bouckaert et al. 2014) and the molecular clock rooting method (Yang & Rannala 2012). The only assumption of this method was a constant rate of evolution for the sequences of interest

(Kinene et al. 2016). Thus, the nucleotide differences between two sequences were assumed to be proportional to the time elapsed since divergence (Forest 2009). This method did not require outgroup rooting. The parameters selected on BEAST mirrored the ones used for MRBAYES, with the addition of an uncorrelated lognormal relaxed clock model and Yule tree prior. The average nucleotide substitution rate for snakes and lizards of 5.29 x 10-9 substitutions per site per year (Eo & DeWoody 2010) was used to time- calibrate the tree.

I used PAUP* 4.0a161 (Swofford 2002) to calculate a sequence divergence matrix

(uncorrected p-distances) for all samples using the mtDNA genome sequences.

5.2.4 Molecular evolution analyses

To test if the three deeply divergent matrilines from the southern peninsula were evolving under diversifying selection, I analyzed the protein-coding genes using several codon substitution models of adaptive evolution, which are based on the ratio of non-synonymous to synonymous substitutions (ω=dN/dS). The ratio measured the strength and mode of natural selection acting on the protein genes such that ω>1 indicated positive (adaptive or diversifying) selection, ω=1 neutral evolution, and ω<1 negative (purifying) selection

(Yang 2007; Jeffares et al. 2015). I first tested the 13 protein-coding genes together to obtain a general ω for the mtDNA genome. Subsequent analyses were performed separately for each protein-coding gene because each of them played a different role in the

OXPHOS system and may have been subjected to different selective pressures (Zhou et al. 2014; Morales et al. 2015). The resulting tree from the BEAST analysis was used for the tests that compare different branches in the mtDNA tree (branch models and branch-site models).

Codon-based analyses were performed using the CODEML in the package PAML 4.8a (Yang

1997; 2007). CODEML used maximum likelihood to evaluate the codon substitution models and detect natural selection acting on protein-coding genes (Jeffares et al. 2015). For

CODEML, the models were divided into three classes: site models (ω can vary at different sites in the gene), branch models (ω can vary in different branches in a tree) and branch- site models (ω can vary in particular sites and in particular branches of the tree). I used the likelihood ratio test (LRT) to compare pairs of models (one model was set as a null model and the second model as the alternative model) and determine if the data had a significantly better fit to the alternative model.

The comparisons started with LRTs between the five different site models. First, null model M0 assumed one average ω for all sites and all lineages, then it was used in comparisons with alternative M1 model (nearly neutral model). This allowed for two site- classes with ω < 1 and ω = 1. If the LRT result was statistically significant, i.e., a non- neutral model fits better the data, then specific tests for positive selection were conducted.

The LRT was then used to compare two pairs of models (Models 1 and 2a, and 7 and 8), where the null model did not allow for positive selection (ω <1 and ω = 1) and the

100 alternative model allowed for ω > 1. Null models M1 (nearly neutral model) and M7 (beta) allowed for ω <1 and ω = 1. The alternative models M2a and 8, allowed for 0 < = ω < 1,

ω = 1, and ω > 1. If the LRT results for the comparison of M1 x M2a and/or M7 x M8 were statistically significant, then some sites were assumed to be evolving under positive selection. To further investigate which of those sites might have been under positive selection, models M2a and M8 were used in a Bayes Empirical Bayes (BEB) (Yang et al.

2005) analysis to calculate the Bayesian posterior probability of positively selected sites.

Positively selected sites identified with this approach were considered to be statistically significant when the BEB posterior probability was greater than 0.95 and ω > 1 (Yang et al. 2005).

After completing the site model tests, I analyzed the branch models to test the null hypothesis that all lineages (branches in the mtDNA tree) were evolving under the same evolutionary rate. I used the LRT to compare the null model M0, which assumed that all lineages shared the same average ω, to the alternative model (two-ratio model) to check if a specific branch in the tree (foreground branch) had a ω value distinct from the other branches. I performed three LRT calculations selecting each one of the matrilines (S2, C1 and C2) as the foreground branch to test if the evolutionary rate varied among the lineages.

If the results were significant, I rejected the hypothesis that all matrilines are evolving under same ω and further tested those lineages for diversifying selection using the branch- site models.

For the branch-site models, I used the LRT to compare the null hypothesis that the selected matriline (foreground branch) was not evolving under positive selection using model A1

101

(0 < ω < 1; ω = 1), to the alternative hypothesis model A (0 < ω < 1; ω = 1; ω > 1), which allowed the foreground branch to be evolving under positive selection.

5.3 Results

5.3.1 Mitogenome tree and divergence times

The mitogenome trees generated with MRBAYES (Figure 5.2) and BEAST (Figure 5.3) had very consistent topologies with high support for the separation between each matriline

(posterior probability of 1.0). Matrilines C1 and C2 occurred south of the Isthmus of La

Paz and together they formed the sister-group of lineage S2, which occurs north of the

Isthmus of La Paz (Figure 5.1). Values of pairwise proportional sequence divergence

(uncorrected p-distances) for the Isthmus of La Paz break (S2:C1) ranged from 8.26% to

8.39% and for the Cape Break (C1:C2) from 5.23% to 5.41%.

The time-calibrated tree (Figure 5.3) built using the molecular clock rooting method of

BEAST was built to infer the divergence times between the three matrilines. Assuming a divergence rate of 5.29 x 10-9 substitutions per site per year, the Isthmus of La Paz break between the matrilines S2 and C1+C2 occurred 11.2 million Ma (95% highest posterior density -HPD: 17–5.8 Ma). The divergence between the matrilines C1:C2 in the Cape

Region happened 5.6 Ma (95% HPD: 8.5–2.5 Ma).

5.3.2 Selective pressure analyses

The one ratio model (M0) in CODEML estimated a mean ω=0.033 for the 13 concatenated protein-coding genes, suggesting that overall the mtDNA genome is under strong purifying

102 selection. COI had the highest level of purifying selection with ω=0.006 and ATP8 had the lowest with ω=0.252.

The first analysis of site models found that ND1, COI, COII, COIII, ND3, ND4L and Cytb were most likely to have one average ω for all codons in those genes (Table 5.2). However,

ND2, ATP8, ATP6, ND4, ND5 and ND6 best fit model M1, which allowed some codons to evolve under a different ω (Table 5.2).

The test for positive selection on codons evolving under different values of ω (ND2, ATP8,

ATP6, ND4, ND5 and ND6) suggested purifying or neutral selection. No significant signals of positive selection were detected (Table 5.3). The BEB test suggest that some codons of

ATP8, ND2, ND4, ND5 and ND6 experienced positive selection, but none of those results were supported by Bayesian posterior probabilities (Table 5.4).

Tests using the branch and branch-site models to evaluate if lineages were evolving independently under positive selection showed strong purifying selection (Table 5.5); no signals of diversifying selection acting in the matrilines were detected (Table 5.6).

Purifying selection, however, seemed to have been relaxed in matriline S2. When branch

S2 was set as the foreground branch for ND1, ATP6, ND4, ND5 and Cytb, there was significant evidence for S2 evolving under weaker purifying selection when compared to the other lineages. The values of branch ω for the matriline S2 on those genes averaged

70% higher than the average ω for all lineages (ω0 – Table 5.2).

103

5.4 Discussion

5.4.1 The matrilineal genealogy and divergence times

The deep genetic divergence between the three matrilines of Urosaurus nigricaudus corresponds with that reported by Lindell et al. (2008). However, my results show a lower level of genetic divergence between lineages, likely owing to the inclusion of more slowly evolving genes in the mitogenome. For the mitogenome, sequence divergence at the

Isthmus of La Paz break (S2:C1) reaches 8.39% (instead of 11.02% from Lindell et al.

2008) and the discordance within the Cape Region (C1:C2) attains 5.41% sequence divergence (instead of 7.41% from Lindell et al. 2008). Shen et al. (2010) stated that using the entire mtDNA genome increases the phylogenetic power because of the significantly higher amount of data. The first genealogy of U. nigricaudus was based in 1,966 bp from the mtDNA genes ATP6 and Cytb (Lindell et al. 2008), whereas in this study I analyze

15,396 bp including the entire mtDNA genome with the exception of the control region.

The levels of genetic divergence found within matrilines of U. nigricaudus are higher than those found in most other studies (Hebert et al. 2004; Barrett & Hebert 2005; Poyarkov et al. 2017). Matrilines S2 and C1, which are parapatric at the Isthmus of La Paz, have historically been recognized as two species based on morphological characters with U. microscutatus (S2) occurring north of the Isthmus and U. nigricaudus in the Cape Region south of the Isthmus. They were recognized as separate species until Aguirre-Léon et al.

(1999) using allozymes confirmed nDNA gene flow throughout the peninsula. This resulted in U. microscutatus being relegated to the synonym of U. nigricaudus. My

104 analyses recovered the same pattern of widespread nDNA gene flow using 3963 nDNA

SNP loci (Chapter 4).

The discordance between the mtDNA gene-tree and the pattern found in nDNA studies is even more intriguing at the specific discontinuity zones (Chapter 2). Several samples collected in these zones occur near each other. For example, sample 315 (C1 lineage) and sample 313 (C2 lineage), which have 5.29% mitogenomic divergence, occur only 26 m from each other. No geographical barriers and no evidence of climate constraints occur at both mtDNA breaks and yet females are not dispersing into the other matriline’s territory.

The female genealogy of U. nigricaudus mirrors that of the lizard Callisaurus draconoides

(Lindell et al. 2005) and several other species on the Peninsula of Baja California.

Congruent patterns have been reported in several animal species in the region, including, but not limited to, lizards (Lindell et al. 2005; 2008), snakes (Rodrı́guez-Robles & De

Jesús-Escobar 2000; Harrington et al. 2017), mammals (Lawlor et al. 2002; Álvarez-

Castañeda & Murphy 2014), birds (Riddle et al. 2000) and scorpions (Gantenbein et al.

2001). The mtDNA discontinuities occur near the location of historical vicariant events and in particular temporary seaways that divided the peninsula in the region of the Isthmus of La Paz during the late Miocene between 11 and 7 Ma (Lindell et al. 2008) and in the

Cape Region between 6.5 and 3.2 Ma (McCloy 1984; Carreño 1992; Murphy & Aguirre-

Léon 2002; Lindell et al. 2008). My analyses of divergence time suggest that the biogeographical patterns with the first mtDNA isolation (matrilines S2 and C1+C2) happening in the Isthmus of La Paz and the initial isolation of matrilines C1 and C2 happening later, involve a significant signal of Miocene events. Accordingly, I cannot reject the hypothesis that vicariant events are responsible for isolating populations of U.

105 nigricaudus in the southern peninsula long enough for nucleotide substitutions in the mitogenome to become fixed by genetic drift.

The other mtDNA division in U. nigricaudus is the mid-peninsular break (Lindell et al.

2008). Many studied species in the peninsula show mtDNA discontinuities in this area

(Harrington et al. 2017). Upton & Murphy (1997) suggested such discontinuities were caused by an ephemeral Vizcaino Seaway (Moore 1973) but recent climatic fluctuations have also been suggested as being responsible (Grismer 2002; Dolby et al. 2015). The suggestion that climate is responsible for driving mtDNA discontinuities requires evidence of positive selection on mitochondrial OXPHOS genes, and my analyses find no evidence of this in the southernmost matrilines. Although this casts some doubt on the explanation of climate-induced variation at the mid-peninsular break, information from species and populations on either side of that particular break must be collected to test the hypothesis further. It is interesting to note, however, that no two species are known to have discontinuities in the precise same area, and a few species do not have the discontinuities at all. After being reunited though, the matrilines remained parapatric but nDNA gene flow appears to have occurred unabated in most species.

5.4.2 Selection on the mitogenome

Mitochondrial DNA divergences may result from isolation and genetic drift as well as positive selection (Morales et al. 2015; Pavlova et al. 2017). I tested for codon adaptive evolution by using site models and on matrilines using branch and branch-site models to ascertain if diversifying selection maintains mtDNA divergence in U. nigricaudus. The results reveal that the mitogenome of U. nigricaudus is evolving under strong purifying

106 selection, corresponding to the results for other vertebrates (e.g., Meiklejohn et al. 2007;

Zhang et al. 2013a; Zhou et al. 2014; Morales et al. 2015). This is not surprising because all protein-coding mtDNA genes play important roles in the OXPHOS system and non- synonymous substitutions in coding regions can negatively alter the structure and function of the protein (Ballard & Whitlock 2004; Jeffares et al. 2015). Functional constraints notwithstanding, it seems at least intuitively plausible that mtDNA might be subject to positive directional selection in response to conditions that place increased demands on aerobic respiration and metabolism. Studies have confirmed this intuition; adaptive evolution in mitochondrial genes has been detected in such harsh environments as caves

(Tomasco & Lessa 2011), high elevations (hypoxia, cold, intense ultraviolet radiation:

Scott et al. 2010; Yu et al. 2011; Luo et al. 2013; Zhang et al. 2013a; Zhou et al. 2014;

Morales et al. 2015; Wang et al. 2016; Pavlova et al. 2017; Zhang et al. 2017b; Jin et al.

2018) and deep, hydrothermal vents (hypoxia, increased acidity: Zhang et al. 2017b; Sun et al. 2018). This does not appear to be the case in U. nigricaudus, in which all sites are evolving under purifying selection, suggesting that habitat adaptation is not shaping specific mtDNA codons. Overall, the results allow me to reject the hypothesis that diversifying selection has influenced the mitogenomic divergences of U. nigricaudus.

Matriline S2 is evolving under weaker purifying selection than C1 and C2. The strength of purifying selection can be affected by, among other things, population size. The high mutation rate of mtDNA combined with unisexual inheritance (no recombination) and genetic drift can lead to the fixation of slightly deleterious mutations when population sizes are small (Kimura 1962; Ohta 1992; Lynch 1996; Popadin et al. 2007; Pavlova et al. 2017;

Vasemägi et al. 2017). Relaxation of selective constraints might also be due to phenotypic

107 changes affecting metabolism (e.g., flightless versus flying birds: Shen et al. 2009; high- performance versus sedentary fishes: Zhang & Broughton 2015; Strohm et al. 2015). This latter explanation does not appear to apply to my system—no obvious phenotypic differences occur between matrilines that could be linked to relaxation of purifying selection in S2. It is possible, however, that geological events in the Peninsula of Baja

California such as the so-called Vizcaino (mid-peninsula) seaway (Lindell et al. 2006;

Álvarez-Castañeda & Murphy 2014; Leavitt et al. 2017) isolated matriline S2 from the northern populations of U. nigricaudus and created a smaller population. Further studies of historical effective population sizes in U. nigricaudus can lead to a better understanding the relaxation of purifying selection on matriline S2.

In summary, the best explanation for the origin of the current pattern of mtDNA divergence in U. nigricaudus on the Peninsula of Baja California is that geological events temporarily subdivided this species. While separated, the populations appear to have experienced a founder effect and different nucleotide substitutions in the mitogenome became fixed by genetic drift.

5.4.3 The drivers of mtDNA discordances

The discordance between the mtDNA and nDNA patterns is particularly intriguing in the specific discontinuity zones. Several samples collected in the zones occur near each other.

For example, sample 377 (S2 lineage) and sample 375 (C1 lineage), which have 8.33% mitogenomic divergence, occur only 12 m apart, and samples 309 (C1) and 310 (C2), which differ by 5.32%, are only 2 meters from each other. No geographical barriers and no evidence of climate constraints occur at both mtDNA breaks and yet females are not

108 dispersing to the other matriline’s territory. This raises an intriguing question: why are females not dispersing beyond the mtDNA break zones given the millions of years of time to do so?

Three hypotheses have been proposed to address this enigma: incomplete lineage sorting, female philopatry/male-biased dispersal, and natural selection on female-linked traits

(Morales et al. 2015; Pavlova et al. 2017). Incomplete lineage sorting—the stochastic sorting of ancestral polymorphisms in diverging lineages (Maddison & Knowles 2006;

Degnan & Rosenberg 2009)—cannot explain the maintenance of mtDNA discontinuities in U. nigricaudus because there is widespread nDNA gene flow (Aguirre-Léon et al. 1999,

Chapter 4). Male-based dispersal, which has been documented in other lizards (e.g., Anolis roquet: Johansson et al. 2008; A. sargrei: Calsbeek 2009; Calsbeek et al. 2014;

Scleroporus consobrinus: Gifford et al. 2017; Podarcis muralis: Vignoli et al. 2015), might explain the widespread nDNA gene flow, although no studies demonstrate sex- biased dispersal in U. nigricaudus. The role female philopatry may play, which can be documented over the lifetime of an individual, in maintaining these discontinuities is more difficult to envision over the long time-scales that these lineages have existed on the peninsula. Assuming a dispersion rate of merely 1 meter/year and that some lineages were isolated for 3 million years, some matrilines should have dispersed 3000km, almost three times the length of the Peninsular Ranges of Baja California itself. Females are clearly dispersing beyond their place of birth yet for some reason, they do not cross the mtDNA boundaries. Field observations revealed that females from different matrilines co-occur in a buffer zone of approximately 180 meters, with no physical barrier impeding their movement (Chapter 2). And, as mentioned above, the presence of nDNA gene flow,

109 presumably by male-based dispersal, reinforces the conclusion that environmental barriers are not maintaining mtDNA discontinuities in U. nigricaudus.

My results shows that the three matrilines are evolving under purifying selection and that diversifying selection is not involved in the mtDNA divergences of U. nigricaudus. Lindell et al. (2008) suggested that mito-nuclear functional compensation (Rand et al. 2004; Osada

& Akashi 2011; van der Sluis et al. 2015; Sunnucks et al. 2017) maintained the isolated matrilines. In this scenario lineages that have diverged in allopatry come back into contact, but mtDNA introgression is selected against because it disrupts the strong functional link between mtDNA and OXPHOS-associated nDNA genes. In other words, the coevolving

OXPHOS complex is selected to maintain function, without any changes being driven by environmental adaptation, although that also could play a role under some environmental conditions as discussed previously (for some extensive discussions see Hill 2016; Sloan et al. 2017 and references therein). If this is the case, then analysis of OXPHOS-associated nDNA should produce patterns more congruent with mtDNA than with other nDNA genes

(Bar Yaacov et al. 2015). More importantly, if coadaptation between mtDNA genes and nDNA OXPHOS genes is maintaining the matrilines, then this implies that hybrids between two matrilines are at a disadvantage. Disrupting mito-nuclear compatibility, both within and across species boundaries, has been demonstrated to negatively affect

OXPHOS efficiency and reduce fitness in a variety of organisms (e.g., Sackton et al. 2003;

Ellison & Burton 2006; Bolnick et al. 2008; Meiklejohn et al. 2013; Burton et al. 2013;

Zhang et al. 2017a and references therein). This has led some authors to propose that selection against hybridization between divergent matrilines might play a role in reproductive isolation and subsequent speciation (Gershoni et al. 2009; Burton & Barreto

110

2012). This has been termed the mito-nuclear compatibility species concept (Hill &

Johnson 2013; Hill 2016; 2018). As interesting as this dynamic is, I have no evidence that selection is opposing panmixia across the three southern lineages of U. nigricaudus. In contrast, the SNP analyses indicate widespread interbreeding (Chapter 4).

Given this, an alternative explanation for the continuing existence of the three matrilines is that female behavior is somehow maintaining the breaks. Females of U. nigricaudus are extremely territorial and do not allow other females in their trees (personal observation).

Although not as well studied as the interaction between relatedness and mate choice (see e.g., Daniel & Rodd 2016 and references therein), evidence is beginning to accumulate indicating that an individual will modulate its level of intraspecific aggression depending upon the relatedness of a competitor (e.g., Thompson et al. 2017; Aguilera-Miller et al.

2018 and references therein). For example, lactating female ground squirrels were found to be significantly more aggressive towards non-kin individuals (Viblanc et al. 2016), while female common eiders were less aggressive to brood parasitism by close relatives

(Andersson et al. 2015). Behavioral experiments examining the responses by female black- tailed brush lizards from the three different matrilines to one another are necessary to test this hypothesis further. Kin-selected modifications can only happen when individuals are capable of differentiating between kin and non-kin, something that has been poorly studied in squamates (relatedness: Egerina striolata: Bull et al. 2001; Coronella austriaca:

Pernetta et al. 2009; familiarity: Lacerta vivipara: Léna & de Fraipont 1998; L. monticola:

Aragón et al. 2001; Egerina saxatilis: O'Connor & Shine 2005; Calotes versicolor:

Ammanna et al. 2013). Olfactory cues are assumed to be involved in discrimination based on changes in tongue flicking behavior, but the actual identity of those cues has not been

111 determined, although changes in pheromones and MHC alleles seems a likely place to start

(e.g., Pearson et al. 2018). Taken together behavioral studies coupled with a comparative study of genes directly involved with female behavior and kin recognition, are needed to investigate whether female interactions are responsible for female U. nigricaudus not crossing their matriline borders.

5.4.4 Conclusion

Three distinct mtDNA matrilines of U. nigricaudus are maintained in southern portion of

Peninsula of Baja California despite evidence of widespread nDNA gene flow. Because diversifying selection is not playing a role in the maintenance of these mtDNA discontinuities, our results open exciting opportunities for further investigations of other female-linked traits in this species, and in others, such as mito-nuclear functional compensation and female biology, including but not limited to, female behavior.

5.5 Acknowledgements

I thank Amy Lathrop, Kristen Choffe, Oliver Haddrath, Cintya Segura-Trujillo and

Griselda Gallegos Simental for assistance in the laboratory; and Carmen Izmene Gutiérrez-

Rojas for the assistance during fieldwork.

5.6 Data accessibility

The 26 mitochondrial DNA genomes of Urosaurus nigricaudus with the exception of the control region are available at GenBank accession numbers: MH369811 to MH369835.

112

5.7 Figures

Figure 5.1: Map of the southern Peninsula of Baja California, Mexico showing sampling localities. Different shades represent the different matrilines: S2, C1 and C2. Map scale in kilometers. Source: Landsat/Google Earth.

113

Figure 5.2: Matrilineal genealogy of Urosaurus nigricaudus based on the entire mitogenome (excluding D-loop) using Bayesian Inference performed by MRBAYES.

Numbers on the nodes represent the Bayesian posterior probabilities. Scale bar represents percent genetic divergence.

114

Figure 5.3: Time-calibrated genealogical tree of U. nigricaudus based on the mitogenome built using the molecular clock rooting method of BEAST. Grey bars represent the 95% highest posterior density.

115

5.8 Tables

Table 5.1: Samples of Urosaurus nigricaudus used on this study. ROM voucher represents

the deposit number of the tissue in the herpetological collection of the Royal Ontario

Museum.

mtDNA Sample ROM Locality LAT LONG lineage ID Voucher Km 20 N of La Paz towards San Juan de La S2 392 54671 24.218388 -110.59275 Costa Km 85 N of La Paz on Hwy 1 towards Cd. S2 393 54774 24.228201 -110.9473 Constitucion Km 77 N of La Paz on Hwy 1 towards Cd. S2 373 54642 24.162766 -110.92177 Constitucion Km 77 N of La Paz on Hwy 1 towards Cd. S2 377 54775 24.163484 -110.92153 Constitucion Km 77 N of La Paz on Hwy 1 towards Cd. S2 381 54770 24.163523 -110.92134 Constitucion Km 77 N of La Paz on Hwy 1 towards Cd. S2 383 54644 24.163544 -110.9215 Constitucion Km 77 N of La Paz on Hwy 1 towards Cd. S2 385 54641 24.163571 -110.92178 Constitucion Km 77 N of La Paz on Hwy 1 towards Cd. S2 387 54769 24.164033 -110.92114 Constitucion Km 86.5 N of La Paz on Hwy 1 towards Cd. S2 394 54773 24.235923 -110.94821 Constitucion C2 284 54739 Oasis San Pedrito - Pescaderos 23.388902 -110.20822 C2 298 54629 Oasis San Pedrito - Pescaderos 23.389067 -110.20893 C2 300 54632 Oasis San Pedrito - Pescaderos 23.389075 -110.20861 C2 308 54777 Oasis San Pedrito - Pescaderos 23.38919 -110.21105 C2 310 54750 Oasis San Pedrito - Pescaderos 23.389234 -110.21111 C2 311 54778 Oasis San Pedrito - Pescaderos 23.389566 -110.21114 C2 313 54779 Oasis San Pedrito - Pescaderos 23.389632 -110.21124

116

C2 301 54784 Oasis San Pedrito - Pescaderos 23.389076 -110.20891 C1 297 53168 Oasis San Pedrito - Pescaderos 23.389066 -110.20902 Km 77 N of La Paz on Hwy 1 towards Cd. C1 375 54765 24.163388 -110.92161 Constitucion Km 58 N of La Paz on Hwy 1 towards Cd. C1 390 54704 24.18078 -110.75625 Constitucion C1 295 54743 Oasis San Pedrito - Pescaderos 23.389057 -110.20942 C1 307 54776 Oasis San Pedrito - Pescaderos 23.389032 -110.2088 C1 309 54749 Oasis San Pedrito - Pescaderos 23.389234 -110.21111 C1 315 54783 Oasis San Pedrito - Pescaderos 23.389846 -110.21137 C1 316 54780 Oasis San Pedrito - Pescaderos 23.389922 -110.21141 C1 317 54781 Oasis San Pedrito - Pescaderos 23.389922 -110.21141

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Gene ω0 lnL0 lnL1 2△lnL P-value

ND1 0.025 -1835.808407 -1835.508491 0.6 P>0.05

ND2 0.042 -2067.001802 -2055.605821 22.79 P<0.001

COI 0.006 -2949.845390 -2949.846805 0.003 P>0.05

COII 0.021 -1309.726386 -1309.607986 0.24 P>0.05

ATP8 0.252 -312.274257 -309.151277 6.24 P<0.05

ATP6 0.038 -1358.593554 -1350.028209 17.2 P<0.001

COIII 0.025 -1493.681866 -1493.682508 0.001 P>0.05

ND3 0.055 -587.588976 -585.434041 4.31 P>0.05

ND4L 0.030 -566.289713 -564.741847 3.09 P>0.05

ND4 0.050 -2812.126577 -2800.476474 23.3 P<0.001

ND5 0.047 -3908.761557 -3881.472377 54.58 P<0.001

ND6 0.037 -958.607197 -955.473377 6.26 P<0.05

Cytb 0.017 -2416.027053 -2414.211442 3.63 P>0.05

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Table 5.3: Results of the statistical tests of adaptive evolution among codon sites using

site models. LRT comparisons between models M1 x M2a and M7 and M8. Significant

level determined by p-value with degree of freedom k=2.

Null Alternative Gene lnL0 lnL1 2△lnL P value model Model

ND2 M1 -2055.605821 M2a -2055.392457 0.42 P>0.05

M7 -2057.471946 M8 -2055.329498 4.28 P>0.05

ATP8 M1 -309.151277 M2a -308.878294 0.54 P>0.05

M7 -309.177614 M8 -308.878317 0.59 P>0.05

ATP6 M1 -1350.028209 M2a -1350.028209 0 P>0.05

M7 -1350.253373 M8 -1350.034204 0 P>0.05

ND4 M1 -2800.476474 M2a -2800.152466 0.64 P>0.05

M7 -2799.412841 M8 -2799.732305 0.63 P>0.05

ND5 M1 -3881.472377 M2a -3881.472376 0 P>0.05

M7 -3879.311547 M8 -3878.619375 1.4 P>0.05

ND6 M1 -955.473377 M2a -955.473376 0 P>0.05

M7 -955.275532 M8 -955.275532 0 P>0.05

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Table 5.4: Results of the Bayes Empirical Bayes (BEB) analysis calculated using Models

M2a and M8 (beta and ω) to identify positively selected sites. BEB posterior probability that ω>1 (Pr) was considered significant if greater than 0.95.

Site M2a M8 Gene Amino acid Number Pr Pr

ATP8 46 H 0.744 0.852

91 D 0.531 0.680

153 T - 0.533 ND2 237 S 0.821 0.930

328 S 0.669 0.753

28 M - 0.569

55 Q 0.511 0.614

139 N 0.572 0.704

ND4 189 N 0.567 0.696

198 I 0.514 0.614

424 I - 0.511

426 A - 0.507

206 D 0.648 0.843

267 S - 0.570

ND5 361 S 0.508 0.616

365 F 0.746 0.934

368 T - 0.530

120

467 L 0.595 0.761

493 T - 0.512

573 M - 0.544

597 L - 0.511

0.587 ND6 89 I 0.505

121

Branch Branch lnL Branch ω 2△lnL P-value

S2 -1832.574462 0.042 6.47 P<0.05

ND1 C1 -1835.434993 0.026 0.74 P>0.05

C2 -1835.625141 0.028 0.37 P>0.05

S2 -2067.001952 0.042 0.003 P>0.05

ND2 C1 -2066.902327 0.042 0.20 P>0.05

C2 -2066.740045 0.044 0.52 P>0.05

S2 -2949.847209 0.006 0.004 P>0.05

COI C1 -2949.719705 0.006 1.49 P>0.05

C2 -2949.100122 0.004 0.25 P>0.05

S2 -1308.825223 0.034 1.80 P>0.05

COII C1 -1309.074574 0.020 1.30 P>0.05

C2 -1310.013374 0.021 0.57 P>0.05

S2 -311.918388 0.359 0.003 P>0.05

ATP8 C1 -312.274245 0.252 0.20 P>0.05

C2 -312.238750 0.271 0.52 P>0.05

S2 -1355.770202 0.067 5.64 P<0.05 ATP6 C1 -1357.786488 0.038 1.6 P>0.05

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C2 -1358.512343 0.038 0.160 P>0.05

S2 -1492.024275 0.036 3.31 P>0.05

COIII C1 -1493.465164 0.023 0.43 P>0.05

C2 -1493.406589 0.027 0.55 P>0.05

S2 -586.772772 0.029 1.63 P>0.05

ND3 C1 -587.588975 0.055 0 P>0.05

C2 -586.342571 0.070 2.49 P>0.05

S2 -566.289797 0.030 0 P>0.05

ND4L C1 -566.030100 0.032 0.52 P>0.05

C2 -565.579814 0.035 1.41 P>0.05

S2 -2807.623388 0.075 9 P<0.05

ND4 C1 -2811.829158 0.050 0.6 P>0.05

C2 -2812.119693 0.050 0.01 P>0.05

S2 -3906.301764 0.087 4.91 P<0.05

ND5 C1 -3908.761519 0.047 0 P>0.05

C2 -3908.759494 0.047 0.004 P>0.05

S2 -958.29489 0.029 3.7 P>0.05

ND6 C1 -958.564032 0.037 0.8 P>0.05

C2 -956.709038 0.046 0.62 P>0.05

S2 -2409.038050 0.032 13.98 P<0.001

Cytb C1 -2414.791526 0.015 2.47 P>0.05

C2 -2415.712382 0.015 0.62 P>0.05

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Gene Branch lnL0 lnL1 2△lnL P value

ND1 S2 - 1835.509016 - 1835.508491 0.001 P>0.05

ATP6 S2 -1350.028385 -1350.028209 0 P>0.05

ND4 S2 -2800.477283 - 2800.476474 0.002 P>0.05

ND5 S2 -3881.472486 -3881.472375 0.001 P>0.05

S2 -2414.211442 -2414.212615 0.002 P>0.05 Cytb C1 -2412.931690 -2412.931690 0 P>0.05

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Chapter 6 CONCLUDING DISCUSSION

In this dissertation, I test a suite of hypotheses to investigate what drives and maintains the deep mtDNA discordances in a species with unabated nDNA gene flow. The time- calibrated genealogy of U. nigricaudus cannot not reject the hypothesis that the origin of mtDNA discordances in this species directly links to a series of vicariant events that happened in the Peninsula of Baja California starting in the late Miocene. Seaways temporarily isolated the populations, and upon secondary contact female lineages remained genetically distinct without mixing. This phylogeographic pattern mirrors that of several species in the peninsula, ranging taxonomically from plants to mammals. I then investigate the natural processes that could be involved in the maintenance of those mtDNA discordances: female philopatry, incomplete lineage sorting, vicariance and selection on a female-linked trait. For philopatry to be true, individuals must not disperse.

However, female U. nigricaudus have dispersed beyond their place of birth for millions of years, and yet, they do not cross the mtDNA contact zones. Thus, I reject the hypothesis that female philopatry maintains the mtDNA discordances. Using lineage-selective primers, I precisely locate the mtDNA contact zones and verify that females from two lineages co-exist in this area. No barriers forbid dispersal beyond the mtDNA breaks. In addition, my analysis using nDNA SNPs fails to reject the hypothesis of unrestricted nDNA gene flow throughout the peninsula, which reinforces previous suggestions that males mix freely beyond the mtDNA contact zones. Thus, I reject the hypothesis that vicariance and incomplete lineage sorting maintains the mtDNA discordance. I then test the hypothesis that functional diversifying selection maintains the mtDNA divergences.

My results reject this hypothesis and in doing so opens opportunities for further

125 investigations of other female-linked traits in this species such as mito-nuclear functional compensation and female behavior.

6.1 Summary of thesis chapters

6.1.1 Chapter 2: Using Maternal Ancestry Monophyly Analysis (MAMA) in

the field to detect contact zones between parapatric populations

In this chapter I present the results of the first use of MAMA in the field. I develop a new set of primers and PCR protocols that enabled a simple and quick identification of individuals from parapatric matrilines living in sympatry. I use this technique in two mtDNA contact zones of U. nigricaudus: the Isthmus of La Paz break (mtDNA divergence of 8.39%) and the Cape Region break (mtDNA divergence of 5.41%), both in the southern part of the Peninsula of Baja California. The use of lineage-specific primers for DNA amplifications in the field worked in 100% of the samples and this allowed me to locate individual females of two matrilines living only 2 meters apart within 6 days. The use of this method allowed for the collection of the samples used in the other chapters of this dissertation in a time and cost-efficient manner.

6.1.2 Chapter 3. The complete mitochondrial genome of the black-tailed

brush lizard Urosaurus nigricaudus (Reptilia, Squamata,

Phrynosomatidae)

To investigate the drivers of mtDNA discordances, I use the samples collected in Chapter

2 and sequenced the entire mtDNA genome of U. nigricaudus. For this chapter, I develop

126 a new set of primers, as well as new laboratory protocols to sequence the complete mitochondrial genome of U. nigricaudus. The mtDNA genome is 17,298 bp long and comprises two rRNAs, 22 tRNAs, 13 protein-coding genes, one L-strand origin of replication and one control region. The gene organization and features agree with the general vertebrate organization and that found in other lizards. The control region is 1,909 bp long and is located between tRNAPro and tRNAPhe. The sequences generated in this chapter form the basis of analyses in Chapters 4 and 5.

6.1.3 Chapter 4. When mitochondrial phylogeography fails: female

genealogy does not match speciation history.

Using the mtDNA genome sequences from Chapter 3, I test the hypothesis that U. nigricaudus shows deeply divergent matrilines, and in conjunction with ddRAD loci for thousands of nuclear SNPs generate for this chapter, I test the hypothesis of unrestricted gene flow (panmixia). My results confirm that U. nigricaudus in the southern part of the

Peninsula of Baja California has three deeply divergent mtDNA lineages and yet unabated nDNA gene flow. These results expose how problematic evolutionary assumptions based solely on mtDNA patterns may be. I also further discuss the power of both genetic markers to infer population structure, gene flow, and molecular variance.

6.1.4 Chapter 5. What drives the deep mitochondrial DNA divergence of

black-tailed brush lizard (Urosaurus nigricaudus)

In Chapter 5, I use the information generated in the previous three chapters to investigate what drove the deep mtDNA discordances in U. nigricaudus and if functional diversifying

127 selection maintains the discordances. The Bayesian time-calibrated mtDNA tree reveals that the Isthmus of La Paz break separated matriline S2 from C1+C2 around 11.2 Ma (95%

HPD: 17–5.8 Ma). The divergence between the matrilines C1:C2 in the Cape Region happened 5.6 Ma (95% HPD: 8.5–2.5 Ma). Based on the correspondence of this timeframe,

I cannot reject the hypothesis that geological events are responsible for originating the mtDNA discordances in U. nigricaudus. The selective pressure analyses of codon substitution models suggest that purifying selection chiefly governs the evolution of the mitogenome and rejects the hypothesis that diversifying selection shapes the mtDNA lineages in distinct ways.

6.2 Opportunities for future research

In four chapters of this thesis, I reject the hypotheses that philopatry, vicariance, incomplete lineage sorting and natural selection on the mtDNA genome are responsible for the maintenance of mtDNA discordances. My results create exciting opportunities for further investigation on the role of other female-linked traits in the maintenance of mtDNA discordances, such as mito-nuclear functional compensation and female behavior. One or both of those two processes may be responsible for females not crossing the mtDNA contact zones.

Mito-nuclear functional compensation is the constrained coevolution of mtDNA and nDNA genes involved in the OXPHOS system to maintain compatibility of nDNA genes involved in OXPHOS with the mtDNA genome to preserve efficiency in energy production. The strong evolutionary link between those genes would result in natural selection against hybrids (especially in the mtDNA break zones) to avoid individuals with

128 inefficient energy production, thus maintaining the mtDNA divergences. This natural selection against hybrids may be impeding females from one mtDNA lineage from dispersing beyond their matriline territory. To test the hypothesis that mito-nuclear functional compensation maintains the mtDNA discordances it is necessary to investigate the nDNA genes involved in the OXPHOS system and compare their patterns to the mtDNA patterns. Such experiment can be done successfully using comparative transcriptomes such as the work by Bar Yaacov et al. (2015).

It is also possible that female behavior could maintain the breaks. Females of U. nigricaudus are extremely territorial, and they do not allow a female intruder in their trees.

During the fieldwork in 2013, I observed this aggressive female behavior aimed at other females, with females chasing other females around the tree until one of them would give up and flee. Males in the other hand had more freedom of mobility, which was confirmed by unabated nuclear gene flow (K = 1), suggesting that males can freely mix with females.

It is possible that the extent of aggressiveness could vary with relatedness (as reported for mammals on the peninsula by Aguilera-Miller et al. 2018), in which aggression could be lessened within a matriline, while in the mtDNA contact zones, the aggressiveness towards individuals of the other matriline could impede females from crossing their matriline’s boundaries. In essence, this is analogous to World War One trench warfare regarding females, where the mtDNA contact zone could shift with time.

Although my field observations may reveal important aspects of female behavior in U. nigricaudus, this hypothesis is yet to be tested. Alternatively to behavioral experiments, the study of comparative transcriptomes could also investigate if genes directly involved

129 with female behavior and kin recognition (i.e., hormones, MHCs) play a role in maintaining the system.

At the beginning of this study, I had planned to use RNA-Seq to study the transcriptomes in U. nigricaudus. In the 2013 fieldwork, I collected tissue samples from 21 individuals from the three matrilines (S2, C1, and C2) and sent them to my collaborators at the State

Laboratory of Genetic Resources and Evolution, Kunming, China, to have the transcriptomes sequenced. Due to lack of funds and time, only three samples (one from each matriline) had its transcriptomes sequenced. The de novo assembly (Martin & Wang

2011) was followed by alignment of the contigs from U. nigricaudus to the reference anole lizard (Alföldi et al. 2011; Eckalbar et al. 2013). Subsequently, I calculated the sequence divergence and built gene/contig trees, which were then compared to the mtDNA tree.

Transcriptomes with sequence divergence and trees that paralleled those of the mtDNA were selected as candidate genes that could be involved in the maintenance of the mtDNA discordances (i.e., genes related to OXPHOS and/or behavior). Unfortunately, no assumptions can be made based on only one individual per matriline, because the results could be simply being due to unselected variation on the sequences. Nevertheless, the gene annotation of those transcriptomes reveals that 98 nDNA transcriptomes and the mtDNA genes might present similar discordances. Some of those genes may be involved in mito- nuclear functional compensation (e.g., ATP2a2: ATPase sarcoplasmic/endoplasmic reticulum Ca2+ transporting 2 and NAD synthetase I), while other genes could be involved in kin recognition (e.g., Themis thymocyte selection associated) and aggressive behavior

(5-hydroxytryptamine receptor 2B (htr2b) – Serotonin). This preliminary exploration of

130 comparative transcriptomes in U. nigricaudus points to a promising direction in the investigation into what maintains the mtDNA discordances.

131

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