PHYLOGENYOF UCERTIDLIZARDS (SQUAMATA:LACERTIDAE) AND THE EVOLUTIONOF UNISEXUAL]CTY

Jinzhong Fu

A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Graduate Department of Zoology University of Toronto

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Doctor of Philosophy Degree, 1999 Jinzhong Fu Department of Zoology The University of Toronto

ABSTRACT

An overall phylogeny of the Family Lacertidae was inferred fiom a parsimony analysis, using DNA sequence data fiom six mitochondrial genes. A total of 3 1 lacertid species were included, which represented most of the main lineages. The iacertids grouped into two clades, which correspond to the current subfamilies Lacertinae and Gallotiinae. The Lacertinae was further divided into two groups, the African and Eurasian groups. The relationships within the two groups were largely unresolved despite the large number of informative characters. A recent explosive speciation hypothesis was invoked to explain the lack of resolution from this analysis. The ancestors of the African and Eurasian groups experienced multiple simultaneous speciation events which left few or no fixed characters on the internodes making the phylogenetic reconstruction difficult.

A special phenomenon, unisexuality, in lacertids was closely examined. A parsimony analysis of DNA sequence data from four mitochondrial genes was conducted to establish a firm phylogenetic foundation for the Caucasian rock lizards, among which the unisexual lacertids originated fiom interspecific hybridization. All bisexual species clustered into three major clades.

The hybridization that led to unisexuals were of inter-clade and only occurred between the caucasica and the rudis clades. Moreover, these hybridization events are directional: the caucasica group only contributed to maternal parents while the rudis group to paternal parents. The formation of unisexuality is clearly under phylogenetic constraints. The causative agents of the constraints are likely multi-functional complexes, although sex chromosomes may play an important role. The divergence of cytochrome-b and ATPase 6 genes in the maternal parental species,

Lacerta raddei and L. mixta, as we11 as all seven known unisexual daughter species was

investigated. All unisexuals constantly showed very little or no variations comparing to the

substantial divergence in L. raddei, but L. mixta showed little variation as well. A phylogeny of

the mtDNA haplotypes inferred from parsimony analysis suggested that some unisexual species (clones) from the same parents shared a common ancestor while others did not. The formation of unisexual species in lacertids involved single origin, multiple origin with closely related females, as well as rnuItiple origin with distantly related females. ACKNOWLEDGMENTS

The five wonderfhl years at the Royal Ontario Museum and University of Toronto put

me in deep debt to many people. My gratehlness is far beyond the ability of words.

First, I would like to thank Dr. Bob Murphy, my supervisor and friend, offered me this

great opportunity to join his team. His friendliness, accessibility, and stimulating talks make him

the best supervisor I have ever known. Without his encouragement and support over the years,

the work presented in this thesis could not have been done. I am also grateful to the other

members of my supervisory committee, Dr. Dan Brooks, and Dr. Hans Sues, for their friendly

and stimulating advice and assistance, and critical reading of drafts of the thesis. Thanks also to

Dr. Kathy Coates for her support of my first year at U of T.

There are four other people I feel greatly indebted. They are Darlene Upton, Ross

MacCulloch, Amy Lathrop, and Raoul Bain. Darlene led me into the wonders of the DNA world,

starting with what is a primer; Ross trained me in allozyme electrophoresis, and he has always been the "grammar checker" for my publications. Amy, Raoul and I have been squeezed into a small office for the last two and half years. I have benefited huge from the numerous talks and chats, not only academically but culturally as well. I will never forget the blending of phylogenetics, music, and movies. I specially thank Amy for her devotion to herpetology which will always be a model for me to follow.

Many thanks also to Oliver Haddrath for his kind help and assistance in the lab. Many other CBCB staff members, as well as other graduate students who have offered their hands to me from time to time. Thank you all. I specially thank to Dr. Ilya Darevsky, who have heIped me in many respects from teaching me how to noose a lizard to checking many data collected from Russian literature. Many ideas presented in this thesis were fostered by the discussion with him. For field assistance,

I also thank to F. Danielyan, M. Bakradze, V. Negrnedzanov, and B. Tuniyev. I thank Dr. D. Good, Dr. R. Drewes, Dr. J. V. Vindum, Dr. V. M. Cabrera, Dr. S. D.

Busack, Dr. D. J. Harris, and Dr. 0.J. Arribas for generously providing tissue samples. It is by their offer that I have been able to complete this study.

Import permits for frozen tissues and preserved specimens were issued by Agriculture

Canada. This study was supported by the Natural Sciences and Engineering Research Council

(NSERC) of Canada grant A3148 to R. W. Murphy. Laboratory work was carried out in the

Laboratory of Molecular Systematics of the ROM. TABLE OF CONTENTS .. Abstract ...... u

Acknowledgments ...... iv

List of Tables ...... x

List of Figures...... xii

List of Appendices ...... xiv

Chapter 1. Introduction...... 1

The family Lacertidae ...... 1 The Caucasian rock lizards and the unisexuality ...... 7 Objectives ...... 1 5 Methods...... 1 5 Mitochondria1 DNA genetics ...... 16 Organization of the dissertation ...... 17

Chapter 2. Toward the Phylogeny of the Family Lacertidae - Why 4. 708 Base Pairs of mtDNA Sequences Cannot Draw the Picture ...... 1 8

Abstract ...... 1 8 Introduction ...... 19 Materials and Methods ...... 2 0 Specimens examined ...... 2 0

Genes selected ...... 2 0 Amplification and sequencing protocols ...... 25 DNA alignment...... 26 Phylogenetic anatysis ...... 2 6 Results ...... 30 COI and Cyt-b ...... 30 12s and 16s ...... 43 The tree root ...... 44 FIG/FOG analysis ...... 47 Relationships within the Eurasian and Afiican groups ...... 55 Discussion ...... 57 The preferred phytogeny ...... 57 Hypothetical recent explosive speciation ...... 60 Comparison of hypotheses...... 61 Taxonomic inferences ...... 64 Historical biogeography ...... 64 Future direction of research on this topic ...... 66

Chapter 3. From a Finn Phylogenetic Foundation to the Constraints on the Evolution of Parthenogenesis in Caucasian Rock Lizards ...... 67

Abstract ...... 67 Introduction ...... 68

Material and Methods ...... 69

Specimens examined ...... 69

Genes selected...... 70 Amplification and sequencing protocols ...... 70 DNA alignment...... 71 PhyIogenetic analysis...... 72 Genetic distance analysis ...... 75

Results ...... 75 Cyt-6 gene ...... 7 5 ATPase 6 gene...... 82 16s gene ...... 83 Combined data ...... 83 The genetic divergence among the three major clades ...... 84 Discussion ...... 89 The Lanyon's consensus and the preferred phylogeny ...... 89 Taxonomic remarks ...... 92 Comparison of hypotheses ...... 93 Constraints on the origin of parthenogenesis ...... 94

Chapter 4 . Divergence of the cytochrome b gene in the Lacerta raddei complex and its parthenogenetic daughter species: evidence for recent multiple origins ...... -10 4

Abstract ...... 104 Introduction ...... 105

Material and Methods ...... 108 Results ...... 1 11 Nucleotide composition ...... 1 13 Substitution patterns ...... 1 13 Protein-level variation ...... 1 18 Phylogenetic analyses ...... 1 18 Variability within species groups and between parthenogens and their closest maternal populations ...... 120 Discussion ...... 125 Dynamics of sequence evolution ...... 125 The taxonomic validity of L. nairensis ...... 1 25 Parentage of the parthenogens ...... 126

viii The phylogenetic tree ...... 12 7 Origin of parthenogenesis ...... 1 2 8 Species concept for unisexual species ...... 13 1

Chapter 5 . Limited variation in Lacerta rnixta and its parthenogenetic daughter species: evidence fiom cytochrome b and ATPase 6 genes...... 132

Summary...... 1 3 7

French Summary ...... 139

Literature Cited ...... 1 4 1

Appendices ...... 1 6 0 LIST OF TABLES

Table 1.1 . Current classification of family Lacertidae ...... 4 Table 1.2 . Historical overview of current species names of Caucasian rock lizards ...... 9 Table 2.1 . The current classification. definition of the main lineages and the representatives used in Chapter 2 ...... 21 Table 2-2 . Primers used for amplification and sequencing in Chapter 2 ...... 27 Table 2-3 . Summary of the results of Chapter 2 ...... 31 Table 2-4 . The PTP analysis for the African and Eurasian lacertid clades ...... 56 Table 3-1 . Primers used for amplification and sequencing in Chapter 3 ...... 72 Table 3-2 . Summary of the results of Chapter 3 ...... 81 Table 3-3 . The inter- and intra-clade genetic distances among the Caucasian rock lizards...... 85 Table 3-4 . The documented inter- and intra-clade hybridization events among Caucasian rock lizards ...... 102 Table 4-1 . Primers used for amplifying and sequencing cyt-b segments in Chapter 4 ...... 123 Table 4-2 . Nucleotide composition of cyt-b gene at different codon positions for Lacerta raddei-nairensis and its parthenogenetic daughter species (%) ...... 124 Table 4-3 . Frequencies of mismatch types of cyt-b gene among populations of Lacerta raddei-nairensis and the parthenogenetic species (%) ...... I25 Table 4-4 . Average codon usage frequencies of cyt-b gene in Lacerta raddei-nairensis ...... 126 Table 4-5 . The p-distance among populations of Lacerta raddei-nairensis ...... 127 Table 4-6 . The average within species p-distance among populations of Lacerta raddei- nairensis and the parthenogens, and the average p-distance between the parthenogens and their closest maternal populations...... 128 Table 5-1 . Cyt-b and ATPase 6 diversity within and among populations of Lacerta rnixta and its parthenogenetic daughter species ...... 138 Table 5-2. Nucleotide composition of cyt-b and ATPase 6 at different codon positions for

Lacerta rnixta and its parthenogenetic daughter species (%) ...... 13 9 LIST OF FIGURES

Figure 2.1 . A phylogeny of lizard families (Estes et al.. 1988) ...... 23 Figure 2.2 . The most parsimonious trees of lacertid phylogeny from independent analyses of the COI. cyt4. 12s and 16s genes ...... 3 4 Figure 2.3 . The most parsimonious trees of lacertid phylogeny from the combined protein encoding gene data and combined rRNA encoding gene data, with primary outgroups ...... 40 Figure 2.4 . The tree root comparison - contradiction from the independent analyses ...... 45 Figure 2.5 . The most parsimonious trees of lacertid phylogeny from six-gene combined data .. 48 Figure 2.6 . The most parsimonious trees of lacertid phylogeny from combined protein encoding genes data and combined rRNA encoding gene data, without primary

outgroups ...... 52 Figure 2.7 . The preferred tree of lacertid phylogeny ...... 58 Figure 2.8 . Comparison of hypotheses about lacertid phylogeny ...... 62 Figure 3.1 . The most parsimonious trees of Caucasian rock lizard phylogeny from the

independent analyses of the cyt4. ATPase 6 and 16s gmes...... 77 Figure 3.2 . The most parsimonious tree of Caucasian rock lizard phylogeny fiorn the four

gene combined data ...... 86 Figure 3.3 . The preferred phylogeny of Caucasian rock lizards...... -. 90 Figure 3.4 . Comparison of hypotheses about Caucasian rock lizard phylogeny ...... 95 Figure 3.5 . Phylogenetic relationships among the bisexual species of Caucasian rock lizards. and the parentage of unisexual species ...... 98 Figure 4- 1. Hypothetical parentage of parthenogenetic Caucasian rock lizards ...... 106 Figure 4.2 . Map of Transcaucasus with the distribution of localities from which bisexual and unisexual species were collected ...... 109 Figure 4-3. Distribution of variable sites across cyt-b in non-overlapping windows among L. raddei-nairensis popuiations ...... 1 1 5 Figure 4-4. One of the most parsimonious trees of the phylogeny of L. raddei-nairensis populations and its parthenogenetic daughter species...... 1 2 1 LIST OF APPENDICES

Appendix I . Specimens examined in Chapter 2...... 160 Appendix I1 . Specimens examined in Chapter 3 ...... 162 Appendix III . Specimens examined in Chapter 4 ...... 164 Appendix IV . Specimens examined in Chapter 5 ...... 165 Appendix V . The haplotypes of the Cyt-b and ATPase 6 sequences of Lacerra rnixta and its parthenogenetic daughter species ...... 166

xiv Chapter 1. Introduction

Lacertid lizards (Squarnata: Lacertidae) are the most common reptiles in Europe, and

have been of great scientific interests for centuries. However, their genealogical relationships,

the foundation for comparative biology, are controversial and largely unresolved. In addition, the evolution of unisexuality in lacertids is an intriguing subject of scientific inquiry in many

respects. Centred on phylogenetics, this study evaluates an overaII phylogeny of the family Lacertidae, and explores the phylogenetic perspectives on the evolution of unisexuality in

lacertid lizards.

The Family Lacertidae The family Lacertidae Gray, 1825 was defined as "strictly pleurodont, coelodont lizards

with the tongue flat, elongate, bifid in front and behind, and covered with rhombic scale-like

papillae or overlapping oblique plicae converging forwards; with the premaxillary bone singIe, the nasal and frontal paired, the parietal single, complete post-orbital and postfionto-squarnosal arches, roofing over the supratemporal fossa, palatines and pterygoids separated on the median line, and with bony dermal plates completely hsed with the cranial bones when in contact with them; with the clavicle dilated and perforated proximally, and the interclavicle cruciform; without dermal ossifications on the body; with symmetrical shields on the upper surface of the head; with the ventral lepidosis usually well differentialted from the dorsal; and with the lower surface of the thighs usually bearing a series of pores" (Boulenger, 1920). Morphologically, lacertids are conservative. They do not present any cases of limb reduction, or reduction of the visual or auditory organs. None is nocturnal or aquatic and few appear to be food specialists. They do not have distinctive complex morphological adaptations, such as adhesive pads or specialized tongues (Arnold, 1989a). Lacertids are widely distributed throughout most of Eurasia and all of Africa. A few species occur on some islands, including the British Isles, the Canary Islands, Madeira, many Mediterranean Islands, Socotra, Sri Lanka, Sumatra, Borneo, Java, Taiwan, the Japanese

archipelago, and Sakhalin. However, their range never reaches New Guinea and Australia. They

are absent from Madagascar and the Seychelles.

Scientific studies of lacertids date back to the end of the 18th century, when Linnaeus

(1 758: Systema Naturae) described some of them. The early pioneers include Eimer (188 1), Bedriaga (1886), Boulenger (1 887, 1905, 1913, 1916), Werner (1904), M6hely (1907, 1909,

191 O), Schreiber (1 9 12), and others. These early works are mostly descriptive, regional and/or

limited on taxonomic revision on (a) particuIar group(s). A milestone work appeared with

Boulenger's "Monograph of Lacertidae" (Vol. 1, 1920; Vol. 2, 1921). The monograph is the

first complete treatment of the family. It contains descriptions of 145 species, referred to 22 genera, and numerous varieties. More importantly, Boulenger introduced his speculations about

the phylogenetic relationships of lacertids. Certainly, his vision of phylogenetics is largely

different from today's, but several prototypes of modern cladistic concepts appeared in his work,

such as derived versus primitive characters. He also pointed out that biogeographic inference

should be based on phylogenetic relationships. Nevertheless, his work was limited almost entirely

to external characters. Arnold (1973, 1983, 1984, 1986a, 1986b, 1989% 1989b, 1997) brought the study of

lacertids into the modern era. Equipped with current phylogenetic theory, Arnold revised most

of the large genera of lacertids based on morphological characters, e.g., Lacerta, Algyroides,

Psamrnodromus, AcanthodaciyZus, Adolfus, Bedriagaia, Gastropholis, Holaspis, Aporosaura,

Meroles, Pedioplanis, and Tukydromus. Furthermore, he first approached the overall phylogeny

of the family, as well as phylogenies of several genera, using morphological characters. Studies

by Arnold are the beginning of a better understanding of the interrelationships of lacertids (Estes et al., 1988). Since the late 1970's, with the development of biochemical technologies, a series of molecular works using protein electrophoresis and immunology have been carried out (Lanza and

Cei, 1977; Lanza et al., 1977; Mayer and Tiedemann, 1980, 1981; Engelmann and Schaffner, 1981; Mayer, 1981; Engelmann, 1982; Guillaume and Lanza, 1982; Lutz and Mayer, 1984, 1985; Lutz et al., 1986; Borisov and Orlova, 1986; Busack and Maxson, 1987; Mayer and Lutz,

1989, 1990; Mayer and Bischoff, 199 1; Mayer and Benyr, 1994). These works uncovered population variations, clarified species boundaries, revealed some relationships within some

groups, and provided speculations of evolutionary rate and time. However, most of these studies only included a small number of species (e-g., 4-6), which limited their scope. One exception is

Mayer and Benyr (1994). Using albumin immunological (MC'F) method, Mayer and Benyr examined 41 lacertid species representing a substantial portion of the genera, and derived an overall phylogeny of the family.

Many karyological studies have also been completed during the last two decades (Cano et aI., 1984; Rykena and Nettmann, 1986; Kupriyanova, 1986b, 2994; Odierna et al., 1987, 1990, 1995; Capriglione et al., 1991, 1994). Olmo et al. (1992) reviewed the results and concluded that the chromosome number and general morphology of tacertids are very conservative and are of little use for systematics. Most species studied possess a kayotype with 36 uniarmed macrochromosomes and 2 microchromosomes. Exceptions are rare.

Recently, DNA techniques, especially sequencing, have been introduced into lacertid systematics. Beside several published works (Thorpe et al., 1993a,b; Fu, 1998; Harris et al., 1998a,b), there are many ongoing studies. Most of these studies are centred on phylogenetic reconstruction at different levels. This may mark a new era of lacertid systematics.

Other aspects of lacertid biology have equally received considerable attention (for example, behavior: in den Bosch, 1986; Avery et al., 1993; Tosini et al., 1994; Avery and Tosini, 1995; PCrez-Quintero, 1996; Miguel and Llorente, 1997). Thus, a wide range of investigations of Iacertid biology has been accomplished, including comparative morphology, karyology, irnmunocytochernistry, ecology, population dynamics, behavior, parasitology. The lacertids are among the best studied vertebrates, especially in Europe (Valakos et al., 1993). Presently, approximately 259 species of lacertid lizards are assigned to 24 genera (Bischoff, 1990, 199 1a,b,c, l992a,b). Table 1- 1 presents the current classification and generic Table 1-1. Current classification of family Lacertidae, including number of species in each genus and the distribution.

Genera Number of species Distribution

AcanthodactyZus North Afiica, Middle East Adolfus Equatorial Africa

AZgyroide Southern Europe

Ausiralolacerta South Afiica

Eremias Palaearctic region, from eastern Europe and

Turkey to China and adjoining USSR

Gallotia Canary Islands

Gastropholis Coastal Kenya, Tanzania and Mozambique Helio bolus Africa south of the Sahara desert Holaspis Equatorial Africa

Ic hnotropis Southern Afiica

Laceria Northern Afiica, most of Europe, S-W Asia Lat asiia Southern western Arabia to Central Afiica

Meroles Southern western Africa Mesalina North Afiica to Middle East to Northern India Nucras South Africa north to Kenya Ophisops Northern Africa, southwestern Asia, India

Pedioplanis South Africa north to Angola Philochortus Southwestern Arabia to Central Africa Podarcis Northwestern Africa to Central Europe Poromera Cameroon, Rio Muni, Gabon Table 1-1. Continued.

Psarnrnodromus 4 Southwestern Europe, northwestern Afiica Pseuderemias 7 Eastern central Africa Takydrornus 16 Asia

Tropidosaura 4 South Africa distribution. The affinity of the lacertids with the Teiidae and Gymnophthalmidae has long been

recognized (e.g., Boulenger, 1920; Camp, 1923). Recent cladistic analysis further confirmed their sister group relationship (Estes et al., 1988).

In spite of this wealth of knowledge, there is no robust overall phylogeny of the family. Arnold (1989a) first attempted a phylogeny using morphological data. However, his phylogeny

is largely unresolved, especially at the base of the tree. Although a fWy resolved tree was

presented, many nodes were considered tentative. Two problems were probably responsible for

the inability of Arnold's morphological data to resolve a solid phylogeny. First, an insufficient number of informative characters left many nodes ambiguous. Arnold examined 45 taxa and used

only 84 characters in his data matrix. Using additive binary coding, 112 binary characters were obtained, and of these, 14 were uninformative. My re-evaluation of Arnold's original data set using PAUP* (version 4.01b; Swofford, 1998) resulted in 5753 most parsimonious trees (MPTs) after 40 random sequence replicates. More trees certainly would be found if the analysis was run longer. Moreover, keeping all trees one step longer than the MPTs resulted in more than one handred thousand trees. Second, the low CI (0.307) indicated much conflict in the data. In addition, Arnold used a problematic method of data analysis, i.e., compatibility analysis (Swofford et al., 1996), and dubious assumptions of character state polarity (e.g., common equals primitive). More recently, Mayer and Benyr (1994) presented an overall phylogeny of the family. Unfortunately, the albumin immunological (MC'F) method they used is a questionable method for inferring phylogeny. The method groups taxa on the basis of similarity (Maxson and

Maxson, 1990) and not on the basis of shared derived characters (synapomorphies), which violates the taxa grouping rule of phylogenetics (Hennig, 1966). The lack of a solid phylogeny of Iacertids has underscored many of their comparative studies. The strong contrast between the wealth of knowledge about many aspects of lacertid biology and the poor understanding of their genealogical relationships has made the overall phylogeny of the family one of the most desired.

To reconstruct a phylogeny of the family Lacertidae is one of my objectives in this study. The Caucasian Rock Lizards and Unisexuaiity

In the family Lacertidae, unisexuality in the Caucasian rock lizards (genus Lacerta: the saxicola complex) has intrigued biologists for decades. Unisexuality in vertebrates is rare and bizarre. At present, there are approximately 50 known "species" of unisexual vertebrates among 22 genera of fishes, amphibians and reptiles (Dawley, 1989). The unusual nature of unisexuality has led it to become one of the hottest topics in contemporary biology. By studying what is atypical, we can often better understand what is typical. As model organisms, the unisexual vertebrate species have been used for addressing many basic issues in biology (Dawley, 1989).

Unisexual vertebrates originated from interspecific hybridization. The formation of unisexuality is rare and operates under complicated constraints. Unisexuality in squamate reptiles is true parthenogenesis, in which the eggs, produced without meiosis, develop into genetically identical offspring without stimulation or input from sperm (in contrast to gynogenesis and hybridogenesis). Caucasian rock lizards are an ideal group for studying the evolution of unisexuality.

Among the advantages of this group are tts manageable size (about 16 bisexual and 7 unisexual species), high population density, concentrated distribution, well-established species boundaries, firmly identified parentage, and steriIe triploids. These lizards are characterized by their unique habitat- rocks, from which they gained their common name. Darevsky (1967:i) best described them: "Rock lizards of the subgenus Archaeolacerta (genus Lacerta) are the most common and widely distributed terrestrial vertebrates of mountainous Caucasus. All through Ciscaucasia and

Dagestan to the mountain of Transcaucasia and Talysh, from the Black Sea coast to even the glaciers of the Bolsho Kaukaz mountain range, hardly a gorge, rock outcrop, or bare rock lack lizards of this group. Everywhere, these lizards occur in large numbers and dense populations." The scientific studies of Caucasian rock lizards date back to 1834 when Eversmann first described the species Lacerta saxicola. Shortly thereafter, a few other species were described:

Podarcis defilippii from northern Iran (Camerano, 1877), P. depressa from Trapezund and

Tbilisi (Camerano, 1878), and Lacerta portschinskii fiorn Tbilisi (Kessler, 1878). Later, Bedriaga (1 886) divided L. depressa into L. depressa var. modestra and L. depressa var. rudis. Boettger

(1 892) described L. muralis var. valentini and L. muralis var. raddei fiom eastern Transcaucasus, which together with L. depressa belong to the species L. muralis. Both Boulenger (1904) and

Nikolskii (1905) synonymized all the above varieties under the name L. muralis. Mbhely (1909) restored the name Lacerta saxicola. He also described four new subspecies, L. s. armeniaca from Armenia, L. s. gracilis fiom Dagestan, L. s. brauneri from eastern Ciscaucasia, and L. s. bithynica from Asia Minor, and two new species L. mixta and L. caucasica. Lantz and Cyrkn (1936) synonymized 14 subspecies all under name of Lacerta saxicola (Table 1-2). The Caucasian rock lizards did not attract much attention until Darevsky (1957, 1958) first discovered unisexual species in this group. Since then, a considerable number of studies have been carried out (e.g., Khonyakina, 1964; Danielyan, 1965; Shcherbak, 1962). Darevsky's

(1 967) monograph "Rock lizards of the Caucasus: systematics, ecology and phylogeny of the polymorphic lizards of Caucasus of the subgenus Archaeolacerta" (the English translation of which was published in 1978), formed the most comprehensive treatment of the group to date. In his monograph, Darevsky described 21 bisexual species (subspecies) and 4 unisexual species of the rock lizards in great detail. Two forest lizards, Lacerta derjugini and L. praticola were also included. Data on general morphology, distribution, geographic variation, as well as ecological traits, of each species were presented. Furthermore, natural hybridization and its evolutionary significance were discussed. Darevsky also proposed phylogenetic relationships among the species and subspecies, although without rigorous analysis. Since then, a few new bisexual and parthenogenetic species and subspecies have been described (Darevsky and Vadmedeja, 1977: L. darkorurn; Darevsky and Danielyan, 1977: L. uzzelli; Eiselt and Darevsky,

1991: L. rudis chechenica; Schrnidtler et al., 1994: L. sapphirina and L. bendimahiensis;

Darevsky and Tuniyev, 1997: L. dryada). Darevsky (1 993) reviewed the species status and summarized the group into 33 species (subspecies) including 5 unisexual species. Table 1-2 presents a historical overview of current species names of Caucasian rock lizards. Table 1-2. Historical overview of current species names of Caucasian rock lizards - the Lacerta saxicola complex.

- - --

MChely, 1909 Lantz and Cyrdn, 1936 Darevsky, 1967 Darevsky, 1993 This study

L. saxicola complex

L. smicola saxicola L. smicola saxicola L. saxicola saxicola L. saxicola saxicola L. saxicola

L. s. szczerbaki L. s. szczerbaki

L. s. darevskii L. s. darevskii

L, s. lindholmi L. s. lindholmi L. s. lindholmi L. lindholmi

L. s, var. brauneri L. s. brauneri L. s. brauneri L. s. brauneri L. brauneri L. caucmica complex

L. caucmica L. s. caucasica L. caucasica caucasica L. caucasica L. caucasica

L. c. alpina L. caucasica alpina L. alpina

L. clarkorum L. clarkorum

L. s. gracilis L. s. daghestanica L. daghestanica L. daghestanica

L. s. var. defilippii L. s. deftlippii L. s. detfilippii L. dejlippii L. defilippii

L. mixta L. s. mixta L. mixta L. mixta L. mixta

Table 1-2. Continued.

L. r. chechenica

L. r. macrornaculata L. r. macromaculata

L. r. obscwa L. r. obscwa L. r. svanetica

?

L. s. mehelyi ? L. valentini comdex L. valentini

L. s. valentini L. s. valentini L. v. valentini L. s. lantzicireni L. v. lantzicirenii L. v. spicenbergii Unisexual s~ecies

L. s. armeniaca L. s. armeniaca L. armeniaca L. armeniaca L. armeniaca L. dahli L. dahli L. dahli L. rostornbekovi L. rostombekovi L. rostombekovi

L. unisexualis L. unisexualis L. unisexualis Table 1-2. Continued.

L. uzzelli L. sapphirina

L. bendimahiensis

Forest species

L. praticola L. praticola

L. derjugini L. derjugini Karyological research has played a significant role in the study of Caucasian rock lizards,

especially as represented by Kupriyanova's (1 969, 1973, 1981, 1985, I986a, 1989, 1992) work.

Complementing morphology, karyology helped to confirm the interspecific hybridization origin

of parthenogenesis, identified the parentage of the parthenogens in Caucasian rock lizards, and addressed other evolutionary issues.

Allozyme electrophoresis has contributed a great deal to clarifL the bisexual species

boundaries, to confirm the hybridization origin of parthenogenesis and to identifjl the parentage.

Uuell and Darevsky (1975) first used this technique in Caucasian rock lizards to test the

hybridization origin hypothesis. Fu et al. (1995) examined the population differentiation of L. caucasica, daghestanica and L. alpina using allozymes as well as morphology, and confirmed

the status of the above species. MacCulloch et al. (in press) examined populations of L. saxicola

and diagnosed three species, L. lindholmi, L. saxicola, and L. brauneri (including L. b. brauneri,

L. b. darevskii, L. b. szczerbaki). Bobyn et al. (1996) examined population differentiation of L.

raddei and L. nairensis, and concluded that they were likely one species. Other allozyme studies

(MacCulloch et al., 1995b: L. rudis, L. portschinskii, L. valentini; MacCulloch et al., 1997b: L. portschinskii; MacCulloch et al., 1997c: L. derjugini and praticola) examined population

variation as well as confirmed species boundaries. A total of 7 unisexual species and 16 bisexual

species has been recognized in this study (Table 1-2).

Phylogenetic relationships among the species have been addressed only recently (Moritz

et al., 1992a; Murphy et al., 1996; Fu et al., 1997). Moritz et al. (1992a) used restriction

fragment mitochondria1 DNA (mtDNA) data to construct a phylogenetic tree of five unisexual species and their potential maternal parents. Murphy et al. (1996) pursued a more

comprehensive study of the bisexual species using allozyme data. Thirteen bisexual species were

included in their analysis. Fu et al. (1997) used mtDNA sequences to examine relationships

among 14 bisexual species. The evolution of unisexuality in Caucasian rock lizards has been well studied. The seven parthenogens originated from interspecific hybridization of bisexual species of the L. saxicola complex (Darevsky, 1967, 1992; Darevsky et al., 1985), and the parentage of the parthenogens

was confirmed by means of morphology, karyology, allozymes, and rntDNA data (Moritz et al.,

1992a; Murphy et al., in review). Clonal variation of the parthenogens is also well documented

(MacCulloch et al., 1995% 2997a; Murphy et al., 1997; Fu et al., 1998). All parthenogens in

Caucasian rock lizards are diploid, and the triploids are sterile. However, many questions

concerning the origin and persistence of parthenogenesis remain. Among them, what are the

phylogenetic constraints on the formation of parthenogenesis, if any? Further, what levels of divergence of mtDNA occur among the parental species and the parthenogenetic daughter

species? Are individual parthenogens derived from single or multiple hybridization events?

The scarcity of unisexuality in vertebrates indicated that their formation occurs under

severe constraints. Many potential constraints on formation of unisexuals have been studied,

e.g., developmental constraints in fishes (Wetherington et a]., 1987); genetic constraints in

lizards (Moritz et al., 1989); ecological constraints in fish and lizards (Wright and Lowe, 1968;

Moore, 1984; Veijenhoek, 1989). However, phylogenetic constraints have just been touched

(Darevsky et al., 1985; Murphy et al., in review). Murphy et al. (in review) examined the

phylogenetic constraints in Caucasian rock lizards. However, two problems weakened the strength of their analysis. First, a firm phylogenetic foundation had not been established.

Second, only four out of the seven recognized parthenogens were included in their examination.

The second objective of this study was to reconstruct a firm phylogenetic foundation and subsequently examine all seven parthenogens. Using phylogenetic methods, the genealogical relationships of the mtDNA haplotypes of the maternal and daughter species can be reconstructed. Moreover, by evaluating divergence in both mtDNA and nuclear encoded characters, we can generate inferences about the hybridization events that formed successful unisexual lineages (Moritz et al., 1989). Three scenarios of hybridization were postulated by Moritz et al. (1989). First, unisexual species with low variation in mtDNA and nuclear genes probably stem from one or two hybrid females (single origin). Second, low mtDNA diversity with considerable variation at nuclear loci can be explained by multiple hybrid origins involving closely related females with typical levels of nuclear

heterozygosity. Third, parthenogens with high levels of both mtDNA and nuclear genes may arise fiom multiple hybridization events involving distantly related females (and males). The first two scenarios have been documented from cases in Cnemidophorus and Heteronotia, but the third has not been discovered in parthenogenetic vertebrates. The nuclear encoded characters of

Caucasian parthenogens have been thoroughly analysed by means of allozyme properties and

morphology, but the mtDNA divergence has not been subject to detailed research. This is my third objective of this study.

Objectives Objective 1. Construct a phylogeny of the main lineage of the family Lacertidae. Objective 2. Construct a phylogeny of the Caucasian rock lizards (genus Lacerta, the saxicola complex), and examine the constraints on the evolution of parthenogenesis.

Objective 3. Examine the mtDNA divergence in maternal parental species and parthenogenetic daughter species, and infer origin mode of parthenogenesis.

Methods The approaches to the three objectives were all centred on phylogenetics, and parsimony analysis was used to infer phylogenies. Phylogenetics provides the foundation for comparative biology and comparative studies are either based on phylogenetic relationships of the organisms or suspicious. I used parsimony analyses because, compared to other methods (i.e., maximum likelihood and distance), it has a sound philosophical base, does not require a specific model, is thoroughly studied, and therefore is the most appropriate method for historical inferences. Mitochondria1 DNA sequences are the primary data source. The failure of Arnold's morphological approach in pursuing the lacertid phylogeny indicated that morphology may not be the way to a success. Commonplace polymorphism in Caucasian rock lizards also prevents the use of morphology in the phylogenetic reconstruction. The use of DNA sequencing data for inferring phylogeny has exploded in the past decade. There are several advantages to DNA

sequence data. Among them, the enormous gene pools of organisms supply an almost unlimited number of characters for phylogeny construction. These characters have a tremendous scope of variation, ranging fiom the most conservative to the most variable (Miyamoto and Cracraft,

1991; Hillis et al., 1996b). Because of this attribute, they allow for the examination of different, diverse evolutionary problems. Indisputably, they are heritable characters. In addition, the accessibility to ethanol-preserved specimens (and formalin-fixed specimens in some cases), the small sample size required and non-destruction of wild populations make sequencing more preferable in this study to other molecular methods. Allozyme electrophoresis data, as an economical source of nuclear gene information, were also used in this study fiom time to time.

Mitochondria1 DNA Genetics

The mtDNA of vertebrates is a closed circular molecule of around 16,000 nucleotide base pairs (bp) encoding a small unit (12s) and large unit (16s) of ribosomal RNA (rRNA), 22 transfer

RNAs (tRNAs) and 13 polypeptides. All of the mtDNA-encoded polypeptides are subunits of the mitochondria1 energy-generating pathway, oxidative phosphorylation (Wallace, 1994).

In animals, mtDNA inheritance is predominately maternal. Although exceptions do exist

(e.g., leakage in Drosophila and mice; biparental transmission in blue mussel Mytilus, see Zouros et al., 1994), they are rare. As a consequence, maternal and paternal mtDNAs rarely mix in the same cytoplasm, and no recombination has been detected between different mtDNA lineages. Thus, the only way that the mtDNA sequence can change is by the sequential accumulation of mutations along radiating maternal lineages.

The rntDNA sequence evolution rate is generally high, and this high rate is the product of both a high mutation rate and a high mutation fixation rate. The high mutation rate results in part from the mtDNA7s lack of protective histones, inefficient DNA repair systems, and continuous exposure to mutagenic effects of the oxygen radicals. The high mutation fixation rate is due to the efficient intracellular sorting of mutant molecules in the female germ line and the rapid genetic drift of rntDNAs in the general population (Wallace, 1994).

Organization of the Dissertation

This chapter (chapter 1) is the general introduction. Chapters 2 through 5 are four inter- related, but largely independent projects. Each was written in publication format, and has its own abstract, introduction, materials and methods, results and discussion sections. Chapter 2 compiles the results toward the first objective, an overall phylogeny of the family Lacertidae. Chapter 3 provides the results of the second objective, a phylogeny of Caucasian rock lizards and the phylogenetic perspective of the evolution of unisexuality. Chapters 4 and 5 summarize the mtDNA divergence study of the parental species and the parthenogenetic species, which is objective 3. Chapter 4 is a hll-length paper while chapter 5 is a short note. Chapter 2. Toward the phylogeny of the family Lacertidae - Why 4,708 base pairs of mtIDNA sequences cannot draw the picture

Abstract. -A phylogeny of the family Lacertidae was derived from DNA sequences of six

mitochondria1 genes. Only a few nodes were confidently resolved using maximum parsimony,

although the data yielded a total of 1664 potentially phylogenetically informative characters.

The lacertids clustered into two clades, the Gallotiinae and the Lacertinae. The former clade

included genera Gallotia and Psammodromus, and the latter included the remaining lacertids.

The Lacertinae split into two additional groups. The African group included all African and

Arabian lacertids and two Eurasian genera, Eremias and Ophisops; the remaining Eurasian

lacertids were included in the Eurasian group. The relationships within the African and Eurasian

groups were largely unresolved. A permutation tail probability test suggested that there is very

little character covariance in the data to support these unresolved relationships. A recent explosive speciation hypothesis was invoked to explain the inconsistency of the data. The common ancestor of the Eurasian group, as well as the ancestor of the Afiican group, experienced simultaneous, or almost simultaneous, multiple speciation events, which left none or very few characters fixed on the internodes. The phylogenetic reconstruction at the family level will be very difficult, if not impossible. Future phylogenetic research should focus on lower taxonomic levels. INTRODUCTION In spite of several attempts, the phylogeny of the family Lacertidae remains controversial and largely unresolved. Lack of a solid phylogeny has undermined many of its comparative studies. The strong contrast between the wealth of knowledge about many aspects of Iacertid biology (e.g., ecology, behavior, parasitology) and the poor understanding of their genealogical relationships has made the overall phylogeny of the family one of the most wanted.

Arnold (1989a) pursued a phylogeny of the family using morphological data. Although a filly resolved tree was presented, many of its nodes were based on educated guesses. Re-analysing the data resulted in a phylogeny with a large number of unresolved nodes, especially at the base of the tree (Fu, 1998). Deficiency and inconsistency of Arnold's morphological data were probably responsible for the resistance to phylogenetic reconstruction. Morphologically, the lacertids are a conservative group. They lack many modifications common in other lizards, such as reduced

Iimbs, eyes or ears, expanded toe pads, etc. Further examination of their morphology would be unIikely to provide more information about their genealogical relationships. Mayer and Benyr

(1994) presented another phylogeny of the family. Unfortunately, the albumin immunological

(MC'F) method they employed is a questionable method for inferring phylogeny. It groups taxa on the basis of overall similarity (Maxson and Maxson, 1990), not synapomorphies, which violates the taxa grouping rule of phylogenetics (Hennig, 1966).

Fu (1998) used DNA sequence data to retrieve the phylogeny of the family. The partial sequences of the mtDNA 12s and 16s genes were informative in resolving some relationships, but the small size of the data set (total 954 bp) limited the resoIution. DNA sequence data have many advantages in phylogenetic construction. Among them are the almost unlimited number of characters and the tremendous scope of variation ranging fiom the most conservative to the most variable (Miyamoto and Cracraft, 1991; Hillis et al., 1996b). These make DNA sequence data a promising way of pursuing the phylogeny of lacertids.

The sole objective of this study is to resolve a phylogeny of the family Lacertidae.

Species were selected to represent the main lineages of the lacertids. These lineages were defined based on the current understanding of their genealogical relationships and classification (Table 2-

1; Arnold, 1973, 1989a; Bischoff, 1990, 1991 a, b, c, l992a, b; Mayer and Benyr, 1994; Fu,

1998). A total of 4,708 base pairs of mtDNA from six genes were sequenced and analysed. A variety of phylogenetic methods were employed in an attempt to answer this long-standing question.

MATERIALS AND METHODS

Specimens Examined

Thirty-one species, representing 20 of the 24 currently recognized genera (Bischoff,

1990, 2 991% b, c, l992a, b), were used to reconstruct a phylogeny of the main lineages of the family Lacertidae (Table 2-1). The genus Lacertu has long been acknowledged as a non- monophyletic group (Arnold, 1973, 1989a; Bohme, 1984; Bohrne and Corti, 1993; Mayer and

Benyr, 1994), thus 10 species were used to represent five generally accepted natural groups

(Arnold, 1973). MultipIe representatives were also used for Adolfus, Gallotia, Meroles, and

Podarcis. The families Teiidae and Gymnophthalmidae were selected as the primary outgroups based on a phylogeny of lizard families (Fig. 2-1; Estes et al., 1988). Although theoretically the family Xantusiidae is the choice for the third outgroup (Maddison et al., 1984), it is not used in this study. The affiliation of Xantusiidae with (Lacertidae, Teiidae and Gymnophthalmidae) is contradicted by several other studies (see Estes et al., 1988) and the Xantusiidae may be too distantly related to Lacertidae to be of use. Three species, Ameiva ameiva, Cnemidophorus tigris maximus (Teiidae) and Neusticurus sp. (Gymnophthalmidae) were examined in this study.

Voucher specimens and locality data are listed in Appendix I.

Genes Selected

Two ribosomal RNA (rRNA) encoding genes from the mitochondria1 genome, the small unit (12s) and the large unit (16S), were selected to reconstruct the phylogeny. The ~RNA"~' gene, which is located between 12s and 16s genes, was also sequenced. Ribosomal RNAs are Table 2-1. The current classification, the definition of the main lineages of family Lacertidae,

and the representatives used in this study.

Classification and main lineages Representatives used in this study

- -

Subfamily Gallotiinae

Gallot ia G. galloti, G. stehlini Psammodromus P. algirus

Subfamily Lacertinae Eurasian Iacertid group Algyro ides A. fitzingeri Lacerta

Archaeolacerta (sensu Arnold 1973 Part 11-a) L. bedriagae, L. monticola L. valentini

Lacerta pama and its relations (Arnold 1973 Part 11-e) -

Lacerta (s. str.) (sensu part of Arnold 1973 Part I) L. media, L. schreiberi Omanosaura, Apathya (sensu Arnold 1973 Part 11-c) - Teira (sensu ArnoId 1973 Part 11-d) L. andreanszkyi L. perspicillata

Timon (sensu part of Arnold 1973 Part I) L. lepida, L. pater Zootoca (sensu Arnold 1973 Part IT-b) L. vivipara Podarcis P. hispanica, P. muralis P. sicula

Takydrornus T. sexlineatus

African lacertid group

Acanthodactylus A. erythrum Table 2- I. Continued.

Adolfis A. vauereselli, A. jacksoni A ustralolacerta Eremias E. velox Gastropholis - Holaspis - Helio bolus H. spekii Ichnotropis I. squamulosa Latastia L. longicaudata Meroles M ctenoductylus kt suborbitalis

Mesalina M brevirosntis Nucras N. tessellata Ophis ops 0. eleguns Pedioplanis P, namaquensis Philochortus - Poromera P. fordii Pseuderemias - Tropidosaura T. gularis Figure 2- 1. A phylogeny of lizard families (Estes et al., 1988).

functionally important in protein synthesis, which makes them relatively resistant to

evolutionary change, and the 12s and 165 genes seem best suited for divergence of about I50 MYA or less (Mindell and Honeycutt, 1990). The divergence of lacertids is thought to be within

this range (Estes, 1983a). Partial sequences of these two genes have been published in a previous

study (Fu, 1998).

Another two protein encoding genes, cytochrome-b (cyt-b) and cytochrorne-c oxidase

subunit I (COI), were also selected. The tl3.N~~~'gene, which is adjacent to cyt-Q, was also

partially sequenced. Cyt-b has been used for a wide range of phylogenetic construction up to 80

MYr (Irwin et al., 1991) or older (Meyer and Wilson, 1990). Because of its functional

importance and structural limitations, the variability in cyt-b is relatively low. COI comparatively is a less frequently used gene in phylogenetic reconstruction. Zaroya and Meyer

(1996) evaluated the performance of mitochondria1 protein-coding genes in resolving

relationships among vertebrates, and found COI, cyt-b and ND2 to be the best.

Amplification and Sequencing Protocols

Standard phenol-chloroform methods were used to extract DNA from tail muscle or liver

tissues. Laboratory protocols follow Palumbi (1996) and Hillis et a1. (I996a). Polymerase Chain

Reaction (PCR) with tag DNA polymerase (Boehringer Mannheim) was used to amplify the DNA

sample. PCR reactions were performed on a DNA engine P200 (MJ Research) with the following

parameters: cycle 1, denaturation at 94OC for 30 seconds, annealing at 42-55OC for 30 seconds,

and extension at 72OC for 90 seconds, cycle 2-33, denaturation at 92OC for 30 seconds, annealing

at 42-55°C for 30 seconds, and extension at 72OC for 90 seconds, the last cycle with extra 5 minutes for extension. PCR products were isolated by electrophoresis on 1.5% agarose gels followed by purification using Geneclean I1 kit (Bio 10 1). Double stranded DNA was sequenced directly using "P labelled terminator cycle sequencing protocol (hersham). The reactions were accomplished with a denaturation at 95OC for 30 seconds, annealing at 55OC for 30 seconds and extension at 72OC for 1 minute for 30 cycles. Sequencing reaction products were run on 6% Long Ranger sequencing gels (JT Baker) for

2-12 hours at 45-50 OC.

Primers used for PCR and sequencing are presented in Table 2-2. Both heavy and light

strands were sequenced for most regions.

DNA Alignment

The alignment of RNA genes were accomplished by computer program ClustalW with the

following paremeters: Gap.opening penalty= 5.00; gap extension penalty= 0.05 (version 1.6,

Thompson et a]., 1994). The aligned sequences were subsequentIy edited in ESEE (version 3.0, Cabot and Beckenbach, 1989). Minor modifications of the computer output alignments were

made by eye. Sites with ambiguous alignment were excluded from the phylogenetic analysis,

because the homology cannot be assumed confidentIy (Hillis and Dixon, 1991).

Phylogenetic Analysis The computer programs PAUP (version 3.1.1, Swofford, 1993) and PAUP* (version

4.Ob 1; Swofford, 1998) were used for tree search, bootstrapping, gl calculation, permutation, and decay analysis. All tree searches were accomplished with heuristic search and 50 random addition sequence replicates. MacClade (version 3.04; Maddison and Maddison, 1992) was used for data and tree manipulation.

Initially, the four major genes were analysed independently. Different genes may have experienced different evo1utionar-y pathways and different selection pressure. If they do, a combined data analysis may not be appropriate. Further, the corroboration from independent data sets provides strong evidence for the reliability of phylogenetic trees (Hillis, 1987;

Miyamoto and Fitch, 1995). In the case where the well-supported elements of the tree topologies were not conflicting, combined data analyses were conducted. Only the shared taxa among independent data sets were pooled into the combined data sets. Table 2-2. Primers used for amplification and sequencing in Chapter 2.

Human position1 Gene Sequence Reference

12s 5' TAC ACA TGC AAG TAT CCG CAC ACC AGT G 3' This study

12s 5' CAA ACT GGG ATT AGA TAC CCC ACT AT 3' Kocher et al. (1989)

12s 5' ATC GAT TAT AGA ACA GGC TCC TCT A 3' This study

12s 5" AGG GTG ACG GGC GGT GTG T 3' Kocher et al. (1989) 12s 5' ACA CAC CGC CCG TCA CCC TC 3' This study '

16s 5' CCC GAA ACC AAA CGA GCA A 3' This study

16s 5' CCA GCT ATC ACC AAG TTC GGT AGG CTT TTC 3' This study

16s 5' CCG ACT GTT TAC CAA AAA CAT 3' This study

16s 5' CTA CCT TTG CAC GGT TAG GAT ACC GCG GC 3' This study

16s 5' CCG GAT CCC CGG CCG GTC TGA ACT CAG ATC ACG 3' Polumbi (1996)

COI 5' GCC CAT GCA TTC GTA ATA ATT TTC IT 3' This study

COI 5' TTC CCG CGA ATA AAT AAC ATA AGC TI' 3' This study

COI 5' GAA PCCCT GCA GGA GGA GGA GAC CC 3' Wiister et al. (1995)

COI 5' GAA TTC CCA GAG ATT AGA GGG AAT CAG TG 3' Wiister et al. (1995) Table 2-2. Continued.

H73 19 COI S'ACT TCT CGT TTA GCT GCG AAG GCT TCT CA 3' This study

L14841 cyt-b 5' CCA TCC AAC ATC TCA GCA TGA TGA AA 3' Kocher et al. (1989)

HI5149 cyt-b 5' GCC CCT CAG AAT GAT ATT TGT CCT CA 3' Kocher et al. (1989)

L15153 cyt-b 5' TGA GGA CAA ATA TCC TTC TGA GG 3' This study

HI5488 cyt-b 5' TTG CTG GGG TGA AGT TIT CTG GGT C 3' Haddrath (pers. comm.) L153 69 cyt-b 5' CAT GAA ACT GGA TCA AAC AAC CC 3' This study

HI5915 ~RI?A~'"5' GTC TTC AGT ?TT TGG 'ITT ACA AGA C 3' Haddrath (pers. comm.)

-

' Primers are designed by their 3' ends, which correspond to the position in the human mitochondria1 genome (Anderson et al., 1981) by convenience. H and L designate heavy- and light-strand primers, respectively. * Complementary of Hl478. Modified from Polumbi (1996). Complementary of HI 5 149. Tree length distribution skewness (g, statistics; Huelsenbeck, 1991; Hillis and Huelsenbeck, 1992), permutation tail probability (PTP; Archie, 1989a; Faith and Cranston,

1991), and homoplasy excess ratio (HER;Archie, 1989b; Fu and Murphy, 1999) were used for assessing character covariance in the data sets. In all occasions, PTPs and HERS were calculated from 1000 replicates and without randomizing the outgroups. The maximum parsimony criterion was used for inferring phylogeny. Each nucleotide site was treated as a non-additive

(=unordered) character. Alignment gaps were treated as missing data. The initial analyses were conducted with equal weights to all characters. A weighting scheme was also employed which used only transversion substitutions for RNA encoding genes and only first and second codon position substitutions for protein encoding genes. Transversion changes are less likely to occur than transitions because of the structural difference of purines and pyrimidines. They are also less frequently observed in nature. Giving high weight to transversion change would more accurately reflect the genealogical relationships, especially in the cases of deep divergence (Hillis et al.,

1994). The same treatments were applied to first and second codon position changes in protein encoding genes.

Because DNA sequence data are typically highly homoplastic, it is advisable to examine the suboptimal trees as well (Swofford, 1991; Cracraft and Helm-Bychowski, 1991). Therefore, decay analysis (Bremer, 1988) was conducted. The decay index (DI) was used to assess nodal support, which is defined as the number of additional tree steps required to collapse nodes on the strict consensus tree when all trees equal to or less than the additional length were kept. Another more commonly used nodal support evaluation, bootstrap proportion (BSP; Felsenstein, 1985), was also applied. All BSPs were calculated fiom 100 replicates, and for each bootstrapping replicate, 10 random addition sequence replicates were used. In this study, the cut-off value of

0.70 suggested by Hillis and Bull (1993) 'was used. A node wit!! BSP over 0.70 is regarded as well- supported. However, the BSP should not be taken as an absolute measurement for confidence estimation. Its always-violated IID (identical independent distribution) presumption and other statistic-related problems may compromise its strength (Kluge and Wolf, 1993; Swofford, 1993;

Sanderson, 1989; Murphy and Doyle, 1998; but see Sanderson, 1995).

Based on the results of the nodal support assessment, functional ingroup and outgroup analysis (FIGfFOG; Watrous and Wheeler, 1981) were used for fbrther examining the recovered

nodes. A recent observation by Fu and Lathrop (unpublished data) indicated that a distantly

related outgroup could misroot the resulting tree and produce a less reliable tree topology.

RESULTS

COI and Cyt-b

The results are summarized in Table 2-3. A 1080 base pair (bp) fi-agment of COI gene was

sequenced and aligned for 33 species. For three species, Acanthodaclylus erythrurus, Eremias

velox, and Lacerta (7eira) andreanszbi, a shorter fragment was sequenced. No

insertions/deletions were found. The pairwise differences ranged fiom 20.5% to 24.0% between

ingroup and outgroup members and 1 1.O% to 2 1.9% among ingroup members.

A 1045 bp fragment of cyt-b gene was sequenced and aligned for 24 species. For five species, AdolJirs vauereselli, Eremias velox, Lacerta (Archaeolacerta) bedriagae, L. (Timon) parer, and Nucrus tessellata, a shorter fiagrnent was sequenced. The sequence of Lacerta

(Zootoca) vivipara was obtained from GenBank (accession number U69834). No insertionsldeletions were found. A 47 bp fragment of adjacent tl3.N~~~'gene was also resolved.

No separate analysis of ~RNA~~'was conducted because of its small size. These data were only used in the six-gene combined data set. The pairwise differences of cyt-b ranged from 29.7% to

33.8% between ingroup and outgroup members and 17.3% to 27.4% among ingroup members.

Uninformative characters, defined as those that contribute exactly the same length to every possible tree topology (Swofford, 1993), were excluded fiom the phylogenetic analysis, because they contribute no information to taxa grouping. COI yielded 434 potentially phylogenetically informative characters. Among these, 87 were fkom first and second codon positions and 347 fiom third position. Cyt-b yielded 492 potentially phylogenetically Table 2-3. Summary of the results of Chapter 2. The primary outgroups are included unless specified. POG= primary outgroup, bp= base pair, PIC= phylogenetically informative character, MPT= most parsimonious tree, TL= tree length, CI= consistency index, RT= retention index, TS= transitional substitution, TV= transversional substitution, PTP= permutation tail probability, HER= homoplasy excess ratio. All

PTPs and HERS were calculated from 1000 replicates without randomizing primary outgroups. All g,s were calculated from 10,000 random trees. Numbers in parentheses are PICs from 1st & 2nd codon positions, and 3rd codon positions.

Gene bpaligned bp included number number g, number TL CI RI TSfTV PTP HER

in analyses of PICs of taxa of MPTs ratio

COI 434(87-347) 33 -0.3590 3665 1.83 0.073 cyt-b 492(163-329) 25 -0.1845 3319 1.42 0.046

COI+cyt-b 9 1 7(244-673) 28 -0.3 106 6505 1.55 0.050

(no POGs) 88O(2 12-668) 25 -0.30 12 6005 1.62 n/a

12s 290 35 -0.75 13 1444 1.76 0.168

16s 446 35 -0.9883 2338 1.72 0.179

12S+16S 73 6 35 -0.9883 3834 1.89 0.185

(no POGs) 614 32 -0.39 12 3179 1.82 nla

~RNA"~' nla nla nla nla nla n/a

informative characters; 163 were fiom first and second codon positions and 329 from third

position. The COI and cyt-b sequence data resuIted in g, of -0.3590 and -0.1845, respectively

(1 0,000 replicates), and in a significant PTP of 0.00 1. Both measurements indicated the

presence of significant character covariance in the data sets (Faith and Cranston, 1991; Hillis and

Huelsenbeck, 1992). Given that the data have significant cladistic structure, the phylogenetic

analyses were performed.

One most parsimonious tree (MPT)was found fiom the COI gene data, with 3665 steps,

CI of 0.222 and RI of 0.297 (Fig. 2-2A). BSPs were calculated, and eight nodes received values over 0.70. The suboptimal trees were also examined. Eight nodes earned decay indices over three. They were exactly the same nodes, which have BSPs over 0.70. Five genera (groups) with multiple representatives, Gallotia, Timon, Lacerta (s. str.), Podarcis, Meroles, were confirmed to be monophyletic. However, the rnonophyly of Adolfis and Archaeolacerta were questioned.

The monophyly of the subfamily Gallotiinae which includes genera Gallotia and Psammodromus, as we11 as the subfamily Lacertinae which includes the remaining lacertids, was also well-supported. In the Lacertinae clade, Takydromus, the only Asian lacertid, was placed at the base of the clade. An Eurasian lacertid clade grouped most of the Eurasian lacertids together, including Eremias, which was grouped into Akican lacertids by Arnold (1989a) and Fu (1998).

The African lacertids did not group together, but rather they formed three clades, which were located at the base of the Lacertinae clade next to Takydromus.

One MPT was found from the cyt-b data, with 3319 steps, CI of 0.279 and RI of 0.275. The BSPs were calculated and one node obtained a value over 0.70. Six nodes earned decay indices over 4. The node with BSP over 0.70 received the highest DI (Fig. 2-2B). Again, the monophyly of the subfamily Gallotiinae was well-supported. The subfamily Lacertinae was also present on the tree, although one African genus (Acanthodacfylus) fell out at the base of the tree instead of being grouped with the other Afiican lacertids. It obtained a medium decay index (5).

Neither the African lacertids nor the Eurasian lacertids grouped together. Figure 2-2. Trees fiom independent analyses of the four major genes. The numbers above the lines are bootstrap proportions over 0.50. The numbers below the Iines are decay indices. Bold lines indicate well-supported nodes. Taxa name abbreviations: Adolfirs j. =Adol_Jsjacksoni;

Adolfis v. = Adoljiis vauereselli; Gallotia g. =Gallotia galloti; Gallotia s. =Gallotia stehlini; L.

(Archaeolacerta) b. =Lacerta bedriagae; L. (Archaeolacerta) m. =Lacerta monticola; L.

(Archaeolacerta) v. =Laceria valentini; Lacerta m. (sstr.) =Lacerta media; Lacerta s. (sstr.) dacerta schreiberi; Meroles c. =Meroles ctenodactylus; L. (Teira) a. =Lacerta andreanszkyi; L. (Teira)p. =Lacerta perspicillata; L. (Timon) I. =Lacerta lepida; L. (Timon)p. =Lacerta pater; L. (Zootoca) =L. vivipara; Meroles s. =Meroles suborbitalis; Podarcis h. =Podarcis hispanica; Podmcis m. =Podarcis muralis; Podarcis s. =Podarcis sicula. A. The most parsimonious tree from the COI gene data. Decay indices over three are

mapped on the tree. B. The most parsimonious tree from the cyt-b gene data. Decay indices over four are

mapped on the tree.

C. The strict consensus tree of the two most parsimonious trees fiom the 12s gene data.

Decay indices over four are mapped on the tree.

D. The strict consensus tree of the five most parsimonious trees from the 16s gene data.

Decay indices over two are mapped on the tree. Tropidosaura Pedioplanis Adolfus v. Meroles c. Meroles s. Adolfus j. Ophisops Nucras Mesalina Latastia Heliobolus Acanthodactylus L. (Teira) a. Eremias L. (Emon) p. L. (Timon) I. L. (Archaeolacerta) v. Algyroides Podarcis m. Podarcis h. Podarcis s. L. (Archaeolacerta) m. L. (Zootoca) L. (Archaeolacerta) 6. L. (Teira) p. Lacerta s. (s. str.) Lacerta m. (s. str.) Takydromus - Gallotia g. Gallotia s. Psammodromus Ameiva Cnemidophorus Nucras - Latastia Adolfus v. Tropidosaura Meroles s. Eremias Pedioplanis Adolfus j. Heliobolus L. (Teira) a.

Ophisops I

L. (Timon) p. I L. (Teira) p. Podarcis s. Lacerta m. L. (Archaeolacerta) b. L. (Archaeolacerta) v. L. (Archaeolacerta) m. Algyroides Takydromus

L. (Zootoca) I Psammodromus !? Gallotia g. In5 Acanfhodacfylus % Meroles s. lchnotropis Meroles c. Tropidosaura Pedioplanis Nucras Latastia Heliobolus Mesalina Adolfus j. A canthodactylus L. (Teira) a. Poromera Ophisops Adolfus v. Eremias Psammodromus Gallotia g. L. (Zootoca) Podarcis s. Podarcis m. Podarcis h. L. (Teira)p. L. (Archaeolacerta) m Algyroides L. (Archaeolacerta) v. Lacerta s. (s. str.) Lacefla m. (s. str.) L. (Timon) p. L. (Timon) I. L. (Archaeolacerta) b. Takydromus Ameiva Cnemidophorus Neusticurus Podarcis s. . Podarcis m. Podarcis h. 1. (Archaeolacerta) m. L. pa)p. 1. (Zootoca) L. (Archaeolacerfa) b. Lacetta s. (s.str.) Lacerta m. (s. str.) Algyroides L. (Archaeolacerta) v. Takydromus 1. (limon) p. L. (Timon )I. Psammodromus Gallotia g. Adolfus v. L. (Teira) a. Adolfus j. Eremias Mesalina Acanthodactylus Ophisops Tropidosaura Poromera Pedioplanis Meroles s. Meroles c. Ichnotropis Nucras Heliobolus La tastia Ameiva Cnemidophorus Although both data sets resulted in filly resolved trees, the small number of well- supported nodes indicated that the data were inconsistent. The low HERS, 0.073 and 0.046 respectively, showed the same trend.

The results from these two genes were Iargely compatible. Among the well-supported nodes, none is in conflict. Because both genes are from the mitochondria1 energy-generating pathway, they may be under simiIar evolutionary constraints. Thus, a combined data approach was appropriate (Doyle, 1992; Huelsenbeck et al., 1996). Because the monophyly of five genera

(groups) with multiple representatives were confirmed by COI data, only one representative fiom each of these groups was included in the combined protein encoding gene data.

Seven MPTs were found fiom the combined protein encoding gene data, with 6505 steps, CI of 0.264, and RI of 0.260. Four MPTs resolved the ingroup as paraphyletic. The other three trees maintained the monophyly of the ingroup, and among the three, the ambiguities were the relationships among the outgroups. When accepting the monophyly of the ingroup, the data produced only one topology for the ingroup (Fig. 2-3A). BSPs were calculated and two nodes obtained values over 0.70. Three nodes received decay indices over three, which included the two with BSP>0.70. Combining the data fiom the two genes did not improve the resolution. Only two nodes had BSPs over 0.70. The HER was 0.050, still very low.

A tree search using the first and second codon positional changes resulted in two MPTs. However, the two trees showed very little consistency - the strict consensus only resolved six terminal nodes including an association of Gallotia and Psarnrnodromus. No basal nodes were resolved.

Using amino-acid sequences as the characters resulted in 16 MPTs, with 1069 steps, CI of

0.683, RI of 0.4 13. The topology was much like the tree resulting from the 16s gene data (see below and Fig. 2-2D). The strict consensus tree grouped Psarnmodromus and Gallotia together, as well as the Eurasian lacertids. These two clades formed a sister group relationship. Most of the Afiican lacertids were placed at the base of the tree and formed a pectinate pattern. Figure 2-3. Trees fi-om combined protein encoding gene data and combined rRNA encoding gene

data, with primary outgroups. The numbers above the lines are bootstrap proportions over

0.50. The numbers below the lines are decay indices over three. Bold lines indicate well-

supported nodes. Taxa name abbreviations refer to Figure 2-2.

A. The strict consensus tree of the three most parsimonious trees from COI and cyt-b

combined data, when the ingroup was constrained as a monophyletic group (see

text). Both BSPs and DIs were calculated under topological constraints.

B. The strict consensus tree of the four most parsimonious trees from 12s and 16s

combined data. Mssalina Nucras Latastia Acanthodactylus Heliobolus L. (Teira) a. Tropidosaura Adolfirs v. Pedioplanis Adolfus j. Eremias . L. (Timon) p. L. (Archaeolacerta) v. L. (Zootoca) L. (Teira) p. Podarcis s. Lacerta m. (s. str.) L. (Archaeolacerta) m. L. (Archaeolacerta) 6. A lgyroides Meroles s. Ophisops Takydromus Gallotia g. Psammodromus Cnemidophorus Ameiva Neusticurus L. (Timon) L. (l7mon) I. Lacerta s. (s. str.) iacerta m.(s.str.) L. veira) L. (Archaeolacerta) L. (Archaeolacerta) Algyroides L. (Zootoca) L. (Archaeolacerta)

Podarcis s. Podarcis m. Podarcis h. 100 Psammodromus A >5 L--'1 Gallotia g. 1L. .(Teira) a. I 7Tropidosaura Poromera Pedioplanis Meroles s. Meroies c. lchnotropis L Latastia Nucras Heliobolus Acanthodactylus - Ophisops

1 Mesalina Ameiva Cnemidophorus Neusticums 12s and 16s

A 948 bp fragment of the 12s gene was sequenced and aligned for 32 species. For two species, Cnemidophorus tigris and Mesalina brevirostris, a shorter fragment was sequenced. A

1510 bp fragment of 16s was sequenced and aligned for 32 species. A 78 bp fragment of the adjacent ~RNA~~gene was also resolved; no separate analysis was conducted because of its small size and these data were only used in the six gene combined data set. Sequences of two other species, Poromera fordii and Ichnotropis squamulosa, were provided by D. J. Harris. A total of

183 bp fiom 12s and 395 bp from 16s were excluded fiom the analyses because of ambiguous alignment.

Uninformative characters were excluded fiom the phylogenetic analysis. The 12s gene yielded 290 potentially phylogenetically informative characters. The 16s gene yielded 446 potentially phylogenetically informative characters. The 12s gene and 16s gene data resulted in g, of -0.7513 and -0.9883 (10,000 replicates), respectively, and in a significant PTP of 0.001.

Both measurements indicated the presence of significant character covariance in the data sets

(Faith and Cranston, 1991; Hillis and Wuelsenbeck, 1992).

Two MPTs derived fiom the 12s gene data, with 1444 steps, CI of 0.343 and RI of 0.416

(Fig. 2-2C). The ambiguities were the relationships among the three species in Podarcis. Five nodes obtained BSPs over 0.70. Eight nodes earned decay indices over four; five of them had

BSPs over 0.70. Three groups with multiple representatives, Podarcis, Lacerta (s. str.) and

Lacerta (Timon),were resolved as monophyletic and received strong support from the data. Two other terminal nodes were also weI1-supported. However, differing from the COI and cyt-b data, the Gallotiinae clade was placed in the middle of the tree rather than at the base. All members of the Afiican clade, previously identified by Arnold (1989a) and Fu (1998), were grouped together.

The 12s gene data placed Takydromus at the base of the tree. The other Eurasian lacertids formed a pectinate pattern and were located at the base of the tree next to Takydromus.

Five MPTs were obtained fiom the 16s data, with 2338 steps, CI of 0.324 and RI of

0.426 (Fig. 2-2D). BSPs were calculated and eight nodes on the strict consensus tree received values over 0.70. Seven nodes earned decay indices over two and they were among the nodes

with BSPs over 0.70. Similar to the results of 12s gene, the monophyly of Podarcis, Lacerta (s. str.) and Lacerta (Timon)were well-supported. Again, the Gallotiinae was placed in the middle of the tree instead of at the base. In contrast to the 12s data, the Eurasian lacertids were grouped together at the top of the tree. The African lacertids formed a pectinate pattern and were located at the base of the tree.

The results from these two genes appeared markedly different. However, the differences were mainly due to tree rooting at different parts of the network. In spite of the differences, there is no conflict among well-supported nodes. A combined data analysis was therefore conducted.

Four MPTs were resolved from the combined data, with 3834 steps, CI of 0.327, and RI of 0.410 (Fig. 2-3B). BSPs were calculated and seven nodes obtained values over 0.70. Ten nodes earned decay indices over three. The Afiican lacertids were located at the base of the trees. Since the 12s gene suggested another possible rooting which put Eurasian lacertids at the base of the tree, a constrained search was conducted. Forcing the African lacertid to be monophyletic placed the Eurasian lacertids at the base of the tree. Two MPTs were found with

3838 steps, CI of 0.326, RI of 0.409.

Using transversional changes only resulted in three MPTs, with 1334 steps, CI of 0.296,

RI of 0.452. The African lacertids were located at the base of the trees. The same constrained search was applied as above, and resulted in nine MPTs with 1339 steps, CI of 0.295, lU of

0.449.

The Tree Root Where should the tree be rooted? The data from the four genes presented different tree roots (Fig. 2-4). However, only the COI solution is well-supported by the data, which is the root between the Gallotiinae and the Lacertinae. The combined protein data agreed with the COI data and strongly supported this solution. Forcing the 12s data with a COI-like root resulted in MPTs Figure 2-4. The tree root comparison - contradiction from the independent analyses of the four major genes. Gallotiinae clade Lacertinae clade Lacertinae clade Gallotiinae clade

@ Q 8 %=&4 .$ +' *Q $? 9 African lacertids 0 Eurasian lacertids African lacertids Eurasian lacertids with 1452 steps, 6 steps longer than the unconstrained topology. Similar constraints on the 16s

data resulted in MPTs with 2342 steps, an increase of 4 steps. The constrained and

unconstrained topologies are almost equally parsimonious. Evidently, the rRNA data misrooted

the tree. The characters supporting the erroneous root may have resulted from homoplastic

changes.

The choice of a COX-like root was hrther reinforced by the six-gene combined data. One

MPT resulted when all data were pooled together, with 10,378 steps, CI of 0.287, and FU of

0.275 (Fig. 2-5A). Not surprisingly, the tree root was located between the Gallotiinae and the

Lacertinae, and the monophyly of these two clades were well-supported by the data. Mayer and

Benyr (1994) also supported this solution.

Because the primary outgroup misrooted the 12s and 16s tree, it could possibly have an

adverse effect on the tree topology as well. To avoid this problem, a FIGIFOG analysis was

conducted.

FIG/FOG Analysis

The Gallotiinae formed a ideal functional outgroup for the Lacertinae. Subsequently, the reIationships among Lacertinae were further examined using FIGEOG analyses and excluding the

primary outgroups. The 16s gene data resulted in two MPTs, with 1961 steps, CI of 0.322, RI of 0.392. The

Lacertinae was separated into two clades. The African lacertid clade included Acanthodactylus,

Adolfus, Heliobolus, Ichnotropis, Lacerta (Teira) andreanszkyi, Latastia, Meroles, Mesalina,

Nucras, Pedioplanis, Poromera and Tropidosaura, which are mainly distributed in Africa, the

Arabian peninsula and the middle east, and two Eurasian genera, Eremias and Ophisops. The

Eurasian clade included all of the remaining European lacertid groups, Podarcis, Lacerta (Teira) perspicillata, Lacerta (Archaeolacerta), Algyroides, Lacerta (Timon), Lacerta (s. str.), Lacerta

(Zootoca) and the sole Asian lacertid genus, Takydromus. Figure 2-5. The most parsimonious trees from six-gene combined data. The numbers above the

lines are bootstrap proportions over 0.50. The numbers below the lines are decay indices

over five. Bold Iines indicate well-supported nodes. Taxa name abbreviations refer to

Figure 2-2. A. The tree with the primary outgroups. B. The tree without the primary outgroups. Tropidosaura Pedioplanis Meroles s. Meroles c. Adolfis j. Eremias Adolfus v. Ophisops L. (Teira) a. Nucras Latastia Heliobolus Mesalina Acanthodactylus L. (Archaeolacerta) v. - L. (Archaeolacerta) m. Algyroides L. (Timon) p. Lacerla m. (s.str.) L. (Archaeolacerta) b. L. (Zootoca) L. (Teira) p. Podarcis s. Takydromus . Psarnmodromus P Gallotia g. -. 1%3 Ameiva mCD Cnemidophorus Neusticurus Tropidosaura Pedioplanis Meroles s. Meroles c. Adolfus j. Eremias Adolfus v. Ophisops L. (Teira) a. Nucras Latastia Heliobolus

L. (Archaeolacerta) v. 73 L. (Archaeolacerta) m.

yr----L. (Archaeolacerfa) b. L. (Zootoca) Takydromus The 12s gene data resulted in three MPTs, with 1173 steps, CI of 0.334, and RI of

0.393. Except for Lacerta (Zootoca), the Lacertinae again split into two groups: the African clade and the Eurasian clade. Lacerta (Zootoca) was located at the base, being sister-group to all the other members of Lacertinae.

The combined 12s and 16s gene data resulted in one MPT, with 3179 steps, CI of 0.322, and FU of 0.380 (Fig. 2-6A). BSPs were calculated and seven nodes obtained value over 0.70. Six nodes earned decay indices over four, and they are among the seven nodes with BSP over 0.70.

The two-clade pattern of the Lacertinae was exactly the same as the result of the analysis of the

16s gene alone. Using only transversion data resulted in 17 MPTs. Again, the two groups of the

Lacertinae were maintained on the strict consensus tree.

The COI gene data resulted in two MPTs, with 3386 steps, CI of 0.228, and RI of 0.292

(No tree is shown). Takydromus was placed at the base. Eurasian lacertids always grouped together. African lacertids grouped together on one MPT, but, on the other, they formed three groups and were located at the base of the tree next to Takydromus. Interestingly, on one tree,

Eremias was grouped in the Eurasian clade, but on the other it grouped with the African lacertids.

The cyt-b gene data resulted in seven MPTs, with 3 108 steps, CI of 0.282, and RI of

0.273. Eurasian lacertids formed a monophyletic group. The relationships among African lacertids were largely uncertain and most of them remained at the base and formed a "bush."

The combined COi and cyt-b data resulted in four MlPTs, with 6005 steps, CI of 0.270, and RI of 0.256 (Fig. 2-dB). Except Eremias, all other Lacertinae clade members were grouped into an Eurasian clade and an African clade, in the same way as for the 16s gene. Eremias bounced between these two clades.

Combining all six genes resulted in one MPT (Fig. 2-5B). Both the African and Eurasian clades were resolved, and with significant support in terms of BSP.

In summary, the Lacertinae clearly formed two groups: the Eurasian group and the African group. Both rRNA genes unanimously grouped Iacertids into two clades. The combined rRNA data showed significant support for both clades as indicated by bootstrap and decay Figure 2-6.Trees fiom combined protein encoding genes data and combined rRNA encoding gene

data, without primary outgroups. The numbers above the lines are bootstrap proportions

over 0.50. The numbers below the lines are decay indices. Bold lines indicate well-supported

nodes. Taxa name abbreviations refer to Figure 2-2.

A. The most parsimonious trees fiorn 12s and 16s genes combined data. Decay indices

over four are mapped on the tree.

8. The Adams consensus tree of four most parsimonious trees fiom COI and cyt-b genes

data. Decay indices over two are mapped on the tree. No nodes obtained BSP over

0.50. Tropidosaura Poromera Pedioplanis

Meroles c. lchnotropis y+F5;iyNucras Acanthodactylus Ophisops Mesalina Adolfus v.

Podarck s. 100 I I Podarcis m. I >6 Podarcis h. L. reira) p. L. (Archaeolacerta) b. L. (Archaeolacerta) m, A Igyroides I00 L. (Timon) p. I 53 26' L. rimon) I. 100 Lacerta s. (sstr.)

I 26 Lacerta m. (s.str.) L. (Archaeolacerta) v. L. (Zootoca)

Takydromus I Gallotia g. Psammodromus Tropidosaura Adoltis v. Pedioplanis Adolfus j.

Ophisops Nucras Mesalina Latastia Heliobolus Acanthodactylus L. (Teira) a. 9

IAlgyroides - L. (fimon) p. L. (Archaeolacerta) v. L. (Teira) p. Lacerta m. (s-str.) 1. (Archaeolacerta) m. L. (Archaeolacerta) b. L. (Zootoca) Podarcis s. Takydromus p-- Gallotia g. analyses (Fig. 2-6A). COI and Cyt-b generally agreed with the RNA data although neither clade obtained significant bootstrap support (Fig. 2-6B). The results were compiled in Table 2-3.

Relationships within the Eurasian and African Groups

Although analyses of each gene and combined data sets achieved a small number of MPTs (Table 2-3), the relationships among members within the two clades have little in common. The

BSPs of most nodes were below 0.50. Because of the large number of potentially

phylogenetically informative characters among the two clade members (Table 2-4), the

inconsistency and low levels of nodal support were not likely caused by insufficient data, but

rather a lack of structure in the data. To evaluate the character covariance, a PTP analysis was applied.

The combined protein encoding gene data set and the rRNA encoding data set were

partitioned into two data sets. One was consisting of only African clade members, and the other of Eurasian clade members (for taxa components refer to Fig. 2-6).

Very little structure was observed in the data among Eurasian clade members (Table 2-4).

The combined protein data do not have significant structure (PTP=0.170); the pattern appearing

in the data set does not differ significantly from a pattern of randomly generated data.

Therefore, the relationships resulting fiom the data should not be regarded as representing the

genealogy. The rRNA combined data have significant structure (PTP=0.001, HER=0.226). The

data strongly supported the monophyly of Lacerta (Timon), Lacerta (s. str.) and Podarcis as indicated by BSPs. Keeping only one member of each of the three well-supported groups, little

structure was resolved (PTP=0.034, HER=0.023). Although statistically the PTP is significant

(10.05), the character covariance is minimal. Thirtythree of the 1000 randomized data sets

produced trees shorter than the MPTs. Considering the little consistency in terms of tree topology between the 12S, 16s data and the combined data, and the low BSPs, no conclusion

about the relationships of the Eurasian clade members can be confidently drawn, except the well-

supported monophyly of Lacerta (Timon), Lacerta (s. str.) and Podarcis.

The protein encoding gene data of the African clade members showed very little

structure. Although the PTP is statistically significant (PTP=O.OIS), the low HER (0.015) indicated that the structure in the data is minimal. This is also reflected by the low BSPs, among

which the highest resulting from the FIGIFOG analysis is 0.47. In contrast, the rRNA data have a

substantial amount of structure (PTP=0.001, HER=0.080). The MPTs are 59 steps shorter than the shortest tree fiom the randomized data. However, a single well-supported node could make

an otherwise random data set yield in a significant PTP (Fu and Murphy, 1999). Two well-

supported nodes on the tree resulted fiom the 12s and 16s combined data (Fig. 2-6A). These

nodes may carry a large portion of the structure in the sequence data. Subsequently, two of the

three taxa from the clade with the highest BSP (0.97), Zchnotropis and Meroles ctenodactylus,

were deIeted fiom the data set. Consequently, the character covariance at this node was

removed. The remaining data set was subjected to PTP test again and a significant PTP was

maintained (PTP=0.001, HER=0.036). However, the MPT is only 16 steps shorter than the

random tree. When two of the three taxa fiom the cIade with the second highest BSP (0.72), Latastia and Heliobolus, were deleted fiom the daa a marginally significant PTP emerged

(PTP=0.025, HER=0.019), with a low HER. The MPT is actually 7 steps longer than the random tree. Consequentially, a conclusion was reached that two nodes were supported by significantly covaried characters, which associated Heliobolus, Latastia, Nucras, and Ichnotropis, Meroles ctenodacrylus, Meroles suborbitalis together, respectively. A PTP analysis of the six- gene combined data set reached the same conclusion (Table 2-4).

DISCUSSION

The Preferred Phylogeny The preferred tree was constructed based on emphasizing the well-supported elements

(Fig. 2-7), a similar idea to Lanyon's (1993) phylogenetic framework concept. The subfamiIy

Gallotiinae, which includes Gallutia and Psammodromus, was well-supported by all data. The subfamily Lacertinae, which includes all remaining lacertids, is supported by the protein encoding Figure 2-7. The preferred tree, with consideration of well-supported elements. Taxa name abbreviations refer to Figure 2-2. L Tropidosaura Poromera Pedioplanis Meroles s. Meroles c. lchnotropis I Nucras r Laiastia Heliobolus Adolfus v. Adoltirs j. Mesalina Acan thodactylus Ophisops Eremias L. (Teira) a. -Podarcis s. Podarcis m. Podarcis h. L. vein)p. L. (Archaeolacerta) b. L. (Archaeolacerta) m. Algyroides L. (Timon) p. L. (Timon) I. ~acertas. (s.str.) Lacerta m. (s.str.) L. (Archaeolacerta) v. L. (Zootoca) Takydromus Gallotia g. Gallotia s. Psammodromus gene data, especially COI. The grouping of the Eurasian clade and African clades is mainly

supported by rRNA data, although the protein data also provide some support. Relationships

among the Eurasian clade members were left unresolved except for the monophyly of Podarcis,

Tirnon, and Lacerta (s. str.) which are well-supported by COI, 12s and 16s gene data.

Relationships among African clade members mainly represent the rRNA data. The genera Meroles and Ichnotropis are closely related, likewise Nucras, Latastia and Heliobolus. A11 other relationships could not be resolved.

Hypothetical Recent Explosive Speciation

Why cannot 4,708 bp of the mtDNA sequence resohe a well-supported tree? It is not because the genes are overly conservative. The six-gene combined data yielded 1664 potentially phylogenetically informative characters for 29 taxa (Table 2-3). The numbers of informative characters for the relationships among the Eurasian lacertids and African lacertids are also large.

For instance, the two protein coding genes yielded 644 potentially informative characters for 10

Eurasian lacertids. It is not because the genes are overly variable either. If so, the data would have resolved the terminal nodes and not the basal nodes. The mtDNA sequence data collected in this study successfully recovered the deep divergence of lacertids with confidence, but failed to decipher more recent divergence. This failure is due to inconsistency of the data, and this inconsistency likeIy has arisen from recent explosive speciation events.

Lutz et al. (1 986) suggested that the ancestor of western European lacertids may have undergone rapid multiple divergence since the early Miocene. This is probably true for lacertids on both Eurasia and Africa. After the division of the ancestor of African lacertids from the ancestor of Eurasian lacertids, the subsequent speciation rates suddenly accelerated. This

acceleration in Africa may have been correlated to the change of climate. Since the late , Miocene, northern Africa has become progressively more arid (Duellman and Trueb, 1994). The divergence of Afiican lacertids was possibly associated with this change in climate, which led to rapid multiple speciation events. This is evidenced by the fact that the greatest divergence of

Afiican lacertids is affiliated with adaptations to arid habitats.

The explosive speciation hypothesis can also account for the lack of resolution of the

morphological study by Arnold (1989a). In the explosive speciation scenario, the speciation events were so close to each other in time that none or only a few characters were fixed on the

internodes, which makes the phylogenetic reconstruction difficult. This hypothesis can be tested

by collecting more data. If the tree becomes better resolved by increasing the size of the data set,

then the explosive hypotheses would be falsified.

Comparison of Hypotheses The three previous phylogenetic hypotheses of lacertids were made by Arnold (1989a;

hereafter referred to as ENA), Mayer and Benyr (1994; hereafter referred to as MB) and Fu

(1998). The data used by Fu (1998) were incorporated into this study, and therefore are regarded

as part of this study. ENA and MB's hypotheses differ fiom my study in several aspects (Fig. 2-

8)- The monophyly of the Gallotiinae, which includes genera Gallotia and Psarnmodromus,

was recognized by all three studies. However, the monophyly of the Lacertinae, which includes all remaining Iacertids, was only recognized by MB and my study. Although placement of the

Gatlotiinae and their relatives was unresolved, ENA tentatively associated the Gallotiinae and its relatives with [Algyroides, Archaeolacerta, Lacerta saxicola complex, Podarcis and Podarcis

(Teira) dugesii], which made the clade Lacertinae paraphyletic. This relationship was not supported by the DNA data. The monophyly of the African lacertid group was recognized by all three studies, but the composition of the group slightly differs. ENA and my study included Eremias, Ophisops and Mesalina in the Afiican clade while ME3 included them in the Eurasian clade. My study also included Lacerta (Teira) andreanszkyi in the African clade, which neither ENA nor MI3 did. Figure 2-8. Comparison of hypotheses.

A. The simplified trees presented by Arnold (1989a).

B. The simplified trees presented by Mayer and Benyr (1994).

C. The simplified trees resulted from this study. African clade African lacertids A Ophisops, Mesalina, Eremias Eurasian lacertids Zootoca, Lacerta (s.str.) Gallotia Psarnmodromus IEurasian lacertids -L. andreanszkyi African clade B African lacertids L. andreanszkyi Eurasian clade Eurasian lacertids Lacertinae

v Ophisops, Mesalina, I Eremias Lacerta clade Lacerta (s. str.) 4Zootoca Gallotiinae Gallotia Psarnmodromus

L. andreanszkyi African lacertids Lacertinae I I

I -Zootoca, Lacerta (sstr.) Gallotia Psammodromus Monophyly of the Eurasian lacertid group was recognized by MB and my study, but not by ENA. Notwithstanding, the compositions are different. My study included Eurasian lacertids

into this clade, except for Eremias and Ophisops. MB included fiemias, Ophisops and Mesalina

into this group, and excluded Lacerta s. str. and Zootoca, which together formed the sister group

of (African clade + Eurasian clade). ENA placed the Eurasian lacertids at the base of his tree making them paraphyletic.

ENA, MB and my study recognized the difficulty in reconstructing the phylogeny among Eurasian lacertids, This uncertainty is clearly revealed by the largely unresolved tree from

morphology and poorly supported nodes fiom the DNA sequence data.

Taxonomic Inferences

Mayer and Benyr (1994) proposed a two-subfamily classification with the genera Gallotia and Psammodrornus forming the subfmiIy Gallotiinae, and the rest forming the subfamily

Lacertinae. The DNA sequence phylogeny supports this classification and the monophyly of the

subfamily Lacertinae. Considering the number of genera in the current subfamily Lacertinae, an

alternative three-subfamily classification is also valid which includes Gallotiinae and two other

subfamilies corresponding to Eurasian and Afiican Iacertid clades.

The genus Lacerta is not monophyletic (Arnold, 1973; Bohme and Corti, 1993); a taxonomic revision is required. Most of the current subgenera should be recognized as genera, i.e.

Lacerta s. str., Timon, Zootoca. The subgenus Teira is not monophyletic. The two species

grouped into different clades, the Eurasian and Atiican Iacertid cfades. The subgenus Archaeolacerta is not monophyletic either. The three representatives used in this study were

only grouped together on the cyt-b tree, bu.t not the others.

Historical Biogeography Presently, 17 of 24 lacertid genera occur in Afiica, six genera are found in Europe and

adjacent central and western Asia (including the paraphyletic genus Lacerta), and one genus, Tabdrornus, is distributed in East Asia. Although fossil records are comparatively rare, most of

them are fiom the Cenozoic of Europe and a single questionable record is known fiom the Miocene of Morocco (Estes, 1983b).

Estes (1983a) postulated that lacertids originated from one of the large continental

islands in the European region during the Late Jurassic as a result of a vicariance event at the

separation of North America and Eurasia. The DNA sequence phylogeny supports the European- origin hypothesis. The basal lacertid genus, Gallotia, occurs only on the volcanic Canary Islands,

and the genus Psammodromus is found on the Iberian Peninsula, which is one of the oldest

former islands of Europe. Accepting that lacertids have a European origin, the distribution of

lacertids in East Asia and Africa can be best explained by dispersal events.

The dispersal event of the African clade was likely a Miocene event, not, as Estes

(1983a) suggested, a Cretaceous event. Since the middle Miocene (about 19-20 MYA), Africa

had a restricted link with Eurasia via Arabia. The common ancestor of African lacertids probably

dispersed to Afiica after the connection. Moreover, the climate has also changed dramatically

since the Miocene. In particular, southwestern Asia and northern Africa have become

progressively more arid. The common ancestor of African lacertids, and Erernias and Ophisops

may have adapted to a xeric habitat during that time, penetrating the arid region of Africa

southward and westward. The Eurasian remnants evolved into Eremias and Ophisops and dispersed further northward to central Asia. The AfXcan invader radiated to form the present

African clade. Some groups adapted secondarily to mesic habitat. The only Indian lacertids, genus Cabrita (included in Ophisops by Bischoff, 1991c), probably also reached the Indian subcontinent through south-western Asia. Because the relationship between Asian lacertid,

Takydrornus, and other Eurasian lacertids are unresolved, the time and pattern of dispersal cannot be established at this time. Future Direction of Research on This Topic

If the explosive speciation hypothesis is true, collecting more data would be unlikely to improve the resolution of the phylogeny. Phylogenetic reconstruction at the family level may prove very diff~cult,if not impossible. Although the large picture is difficult, the close associations of a few genera (or other natural groups) are possible (e.g., the association of Ichnotropis and Meroles based on this study). Future phylogenetic studies should focus on lower levels, such as genera or subgenera. Most of the currently recognized genera probably are monophyletic. When working on the phylogeny of a genus (or other assumed monophyletic groups), a large number of possibly closely related outgroup taxa should be used. This would test the monophyly of the ingroup while searching for the phylogeny of the ingroup members. For DNA sequence data, international databases, such as GenBank, will supply the data for choice of outgroups. My experience on Caucasian rock lizards showed that phylogenetic construction is more fiuiffil at a lower taxonomic level. Chapter 3. From a firm phylogenetic foundation to the constraints on the evolution of parthenogenesis in Caucasion rock lizards

Abstract. - A phylogeny of 15 bisexual species of Caucasian rock lizards was constructed using DNA sequences data from cytochrome-by ATPase 6 and 16s genes. The well-supported elements were extracted and formed a firm foundation for phylogenetic inferences. Three major groups were identified. The mdis group included Lacerta rudis, L. valentini, L. portschinskii and L. parvula; the smicola group included L. saxicola, L. bra n uneri, L. lindholmi, L. alpina, and L. praticola; the caucasica group included L. caucasica, L. dughestmica, L. derjugni, L. rnixta, L. clarkorum, and L. raddei (nairensis). The latter two groups are sister groups. The parentage of the seven known parthenogenetic lacertas was mapped on the phylogeny, and patterns were revealed. The hybridization leading to parthenogenetic species only involved inter-clade hybridization between two major clades, the caucasica group and the rudis group. The hybridization is directional. The caucasica group only contributed to maternal parents and the rudis group to paternal parents. These patterns can be best explained by the phylogenetic constraints on the origin of parthenogenesis. The analysis did not support the "one-allele hypothesis" for control of phylogenetic constraints. Rather, the phylogenetic constraints are likely through complex interactions of many factors. The natural scarcity of unisexual species is the consequence of constraints from many aspects; no single explanation accounts for all of the limitations. INTRODUCTION Unisexuality in vertebrates is rare and bizarre. At present, approximately 50 known

species of unisexual vertebrates occur among 22 genera of fishes, amphibians and reptiles @awley, 1989). They are of interspecific hybrid origin. The formation of unisexuality is under

complicated constraints. Many have been studied, e.g., developmental constraints in fishes, genetics in lizards, and ecology (reviewed by Veijenhoek, 1989). However, examination of the

phylogenetic constraints on the origin of unisexuality has only just begun. Darevsky et al. (1985) first noticed that some lineage dependent factor(s) might have limited the formation of

unisexuality. Murphy et al. (in review) further explored this aspect in Caucasian rock lizards. The Caucasian rock lizards (genus Lacerta: the saxicola complex) have been extensively

studied. Indeed, unisexuality in higher vertebrates first became known fiom this group. True

parthenogenesis occurs only in Caucasian lizards, as well as some other squarnate reptiles. Seven

unisexual species of Caucasian Lacerta have been described up to date. Their parentage and

clonal variation have been thoroughly investigated via morphology, allozyme electrophoresis,

and rntDNA analyses. In addition, the species boundaries among bisexual species have also been

firmly established. ConsequentIy, the Caucasian rock lizards offer an exceptional case to

examine the phylogenetic constraints on the origin of unisexuality. The phylogeny of the Caucasian rock lizards has been investigated previously. Darevsky

(1967) proposed the first "tree" based on morphological characters. However, the lack of

rigorous phylogenetic methods Iefi its nodes as educated guesses. Moritz et al. (1992a) using

mtDNA restriction fragment analysis examined the relationships among four parthenogenetic species and three of their maternal parental species. Murphy et al. (1996) and Fu et al. (1997),

using allozyme data and mtDNA sequence data, respectively, presented the most comprehensive

analyses to date. Despite all these attempts, contradictions and ambiguities remain (see Fu et al.,

1997). Recent molecular surveys have resuited in the discovery of more cryptic species, and fbrther clarified the species boundaries (MacCulloch et al., in press). The identities of several species used in the previous studies have been changed and questioned. Therefore, a re-evaluation

of the phylogeny is necessary.

A main objective of this study is to resolve a solid phylogeny of the Caucasian rock

lizards and to discriminate its well-supported components. To this end, new data are collected,

and the previously published data are re-evaluated. A total of 285 1 base pairs of mtDNA

sequences are compiled and analysed. Lanyon's (1993) "phylogenetic framework" concept is

employed to reconstruct a firm phylogenetic foundation. Finally, the phylogenetic constraints

on the origin of parthenogenesis in the Caucasian rock lizards are examined.

MATERIALS AND METHODS

Specimens Examined Fifteen bisexual species of the Caucasian rock lizards were examined for the phylogenetic

reconstruction. Choice of appropriate outgroups was obscured by a largely unresolved phylogeny

of family Lacertidae (Arnold, 1989; Fu, 1998; Chapter 2). Because the Caucasian rock lizards

were unambiguously grouped into the Eurasian lacertid group, multiple Eurasian lacertids were used as the outgroup, including Algyroides jitzingeri, Lacerta media, L. vivipara and L. monticola

(Maddison et al., 1984).

Fieldwork was conducted during 1992-1996. Live specimens were euthanized by an overdose of sodium pentobarbital before processing, following accepted animal welfare protocols.

Blood, heart, liver and tail muscle tissues were collected and fiozen in liquid nitrogen for use in multiple molecular investigations. Voucher specimens and locality data are listed in Appendix I.

Due to commonplace polymorphism in this group, two cases of suspicious species identities confounded the results of Murphy et al. (1996) and Fu et al. (1997) (see "Discussion: taxonomic remarks" for details). Subsequently, two precautions were taken. First, all specimens were identified by both morphology and allozyme properties. Murphy et al. (1996) reported all currently recognized bisexual species have at least two "fixed" alleles. The unique alleles were used for species identification. Second, multiple specimens were used for most species. In all cases except Lacerta clarkorum, the fmt half of cytochrome-b (cyt-b) gene was sequenced for all

specimens. For L. clarkorum, ATPase 6 gene was sequenced for all specimens. One specimen of Lacerta alpina used in a previous study (Fu et al. 1998) was originally identified by morphology. However, the recent allozyme identification indicated that it differed fiom other specimens of L. alpina in possessing several unique alleles. It was included in this

study and labelled as ''L. alpina." Another similar case, specimens of L. saxicola used by Murphy et al. (1996) and Fu et al. (1997) differed significantly from more recently acquired specimens. They were also included in this study and labelled as '2. saxicola."

Genes Selected Two protein encoding genes, cyt-b and ATPase 6, fiom the mitochondrial genome were

used to reconstruct the phylogeny. In most vertebrates, they are moderately variable and have

been used extensively for phylogenetic reconstruction, especially cyt-b (Irwin et al., 1991;

Kornegay et al., 1993).

One ribosomal RNA gene, 16S, was also selected. The 16s gene, as well as other mtDNA

rRNA and tRNA genes, provide the structural RNAs for mitochondrial protein synthesis. Ribosomal RNAs are functionally important, which makes them relatively resistant to

evolutionary change (Mindeil and Honeycutt, 1990). This trait may help overcome the problems of polymorphism common to many morphological attributes in Caucasian rock lizards.

Amplification and Sequencing Protocols Standard phenol-chloroform methods were used to extract DNA from tail muscle or liver tissues. Laboratory protocols follow Chapter 2. In one case, L. raddei, a procedure to puriQ

mitochondrial DNA was employed due to the presence of a presumptive pseudogene of cyt-b in the nuclear genome (see Chapter 4). Polymerase Chain Reaction (PCR)was used for amplifying the DNA sample and double stranded DNA was sequenced directly using 33~labelled terminator cycle sequencing protocol. Laboratory protocols follow Chapter 2. Primers used for PCR and sequencing are listed in Table 3-1. Both heavy and light strands were sequenced for most regions.

DNA Alignment The alignment of 16s gene was accomplished by computer program ClustalW with the following parameters: gap opening= 10.00; gap extension= 0.05 (version 1.6, Thompson et a]., 1994).

Minor modifications of the computer output alignments were made by eye using ESEE (version

3,0, Cabot and Beckenbach, 1989). Sites with ambiguous alignment were excluded from the phylogenetic analysis, because the homoIogy cannot be assumed confidently (Hillis and Dixon,

1991).

Phylogenetic Analysis

The computer programs PAUP (version 3.1.1; Swofford, 1993), PAUP* (version 4.0bl;

Swofford, 1998), and MacClade (version 3.04; Maddison and Maddison, 1992) were used for the analysis.

Tree length distribution skewness (gl statistics; Huelsenbeck, 1991 ; Hillis and

Huelsenbeck, 1992), permutation tail probability (PTP;Archie, l989a; Faith and Cranston,

1991) and homolasy excess ratio (HER; Archie, 1989b; Fu and Murphy, 1999) were used for assessing character covariance (sometime interpreted as cladistic structure; Faith and Cranston,

1991) in the data sets. All gl values were calculated with 10,000 randomly chosen trees; all PTPs and HERS were calculated with 1000 randomization replicates, without randomizing the outgroups. The maximum parsimony criterion was used for inferring phylogeny. Each base site was treated as a non-additive (=unordered) character. Alignment gaps were treated as "missing data." Both unweighted (=equally weighted) and weighted analyses were applied. In the weighted scenario, the transition substitutions of the 16s gene data and the third codon positional substitutions of the cyt-b and ATPase 6 genes were down-weighted to zero. Because transversion are less likely to occur and less frequently observed than transitions, applying a greater weight to Table 3-1. Primers used for DNA amplification and sequencing in Chapter 3.

Human position' Gene Sequence Reference

16s 5' CCC GAA ACC AAA CGA GCA A 3' This study

16s 5' CCG ACT GTT TAC CAA AAA CAT 3' This study

16s 5' CTA CCT IITG CAC GGT TAG GAT ACC GCG GC 3' This study

16s 5' CCG GAT CCC CGG CCG GTC TGA ACT CAG ATC ACG 3' Polumbi (1996)

ATPase 6 5' ATG AAC CTA AGC 'ITC TTC GAC CAA TT 3' Haddrith, pers. comm.

ATPase 6 5' ATA AAA AGG CTA AT' GTT TCG AT 3' Haddrith, pers. comm.

ATPase 6 5' ACG AAT ACG TAG GCT TGG ATT A 3' This study

cyt-b 5' CCA TCC AAC ATC TCA GCA TGA TGA AA 3' Kocher et al 1989

cyt- b 5' GCC CCT CAG AAT GAT ATT TGT CCT CA 3' Kocher et a1 1989

cyt-b 5' TGA GGA CAA ATA TCC TTC TGA GG 3' This study

cyt- b 5' 'ITG CTG GGG TGA AGT TTT CTG GGT C 3' Haddrath, pers. comm.

cyt- b 5' CAT GAA ACT GGA TCA AAC AAC CC 3' This study

~RNA~I~ 5' GTC 'ITC AGT TTT TGG TTT ACA AGA C 3' Haddrath, pers. comm.

such changes may more accurately reflect the genealogical relationships, especially the deep

divergences (Hillis et al., 1994). The same is the 1st and 2nd codon positional change. To

search for the optimal tree, a heuristic search with 50 random addition sequence replicates was

used in either PAW or PAUP*.

The three genes were initially analysed independently, because different genes may have

experienced different evolutionary pathways. Further, the corroboration fiom independent data

sets provides strong evidence for the reliability of phylogenetic trees (Hillis, 1987; Lanyon,

1993; Miyamoto and Fitch, 1995). Lanyon's (1993) phylogenetic framework concept was used to form the consensus of the independent analyses. Only the relationships which were strongly supported by at least one independent data set, but not contradicted by an alternative strongly supported topology, were present in the framework. Conflicting alternative relationships which were both strongly supported by data set(s) were not present on the framework, a procedure termed "Lanyon's consensus". When no strongly supported topologies conflicted, a combined data analysis was conducted.

Two methods were applied to evaluate the nodal support. Bootstrap (Felsenstein, 1985) has been the most commonly used method for inferring confidence. All bootstrap proportions

(BSPs) were calculated fiom 1000 replicates, and for each bootstrapping replicate, 10 random addition sequence replicates were conducted in PAUP. In this study, the cut-off value of 0.70 suggested by Hillis and Bull (1993) was applied. A node with BSP greater than 0.70 is regarded as well-supported. However, one shouId be aware of its always-violated IID (identical independent distribution) assumption, and other statistic-related problems. These problems may compromise its strength, and thus the BSPs should not be taken as an absolute measurement of confidence estimation (Kluge and Wolf, 1993; Swofford, 1993; but see Sanderson, 1995). Another method, decay analysis (Bremer, 1988), was also conducted. The decay index (DI) was used to rank the recovered nodes, which is defined as the number of additional tree steps required to collapse nodes on the strict consensus tree when all trees equal to or less than the additional length were kept. Genetic Distance AnaIysis

The genetic divergence has been regarded as the major constraints in the origin of

parthenogenesis in lizards (the balance hypothesis; Moritz et aI., 1989). Therefore, the pairwise

distance between species was calculated in PAUP*. The Kamura 2-parameter model was empirically chosen based on properties of the data.

RESULTS

Cyt-b Gene

A 1045 base pair (bp) fragment was sequenced and aligned for 35 specimens representing

20 species (Appendix 11). Sequence of an outgroup member, Lacerta vivipara, was obtained from

GenBank (accession number U69834). Several sequences were used in other studies (Fu et al.,

1997; Chapter 2,4 and 5). No insertions/deletions were found. A 37 bp fragment of adjacent

~RNA~'was also resulted for all species. No separated analysis of the ~RNA~~gene was conducted because of its small size. These data were only used in the combined data analysis.

Multiple specimens were sequenced for most ingroup members. When using multiple specimens, except L. mixta and L. raddei, only one specimen from each species was sequenced for the complete fragment and only the first half of the fiagment was sequenced for the rest. All specimens of L. miHa and L. raddei were sequenced for the complete fragment. Five species, L. daghestanica, L. derjugini, L. alpina, L. parvula, and L. &is, revealed identical sequences between the two specimens used. The pairwise differences ranged from 15.6% to 24.5% between outgroup and ingroup species, 1.9% 16.4% among ingroup species.

A total of 392 potentially phylogenetically informative characters were found for parsimony analysis. A g, of -0.6184 and a PTP of 0.001 indicated that there is significant character covariance in the data set (Faith and Cranston, 1991; Hillis and Huelsenbeck, 1992).

Consequentially, the phylogenetic analysis was performed. Equally weighting all characters resulted in one tree with 1398 steps, CI of 0.408, RI of

0.632 (Fig. 3-1A). BSPs were calculated, and 16 nodes received values greater than 0.70. Fifteen of the 16 nodes received the highest DIs (24).

All but two species with multiple specimens were proven to be monophyletic. The questionable "L. smicolayy(Dambay) was grouped with L. nairensis. The suspicious "L. alpind' was placed between the two L. rnixta samples. Lacerta raddei and L. brauneri appeared to be paraphyletic.

All species except L. parvula grouped into three well-supported major clades. The rudis group included L. rudis, L. valentini and L. portschinskii; the saxicola group inchded L. saxicola,

L. brauneri, L. lindholmi, L. alpina, and L. praticola; the caucasica group included L. caucasica,

L. dughestanicu, L. derjugini, L. rnixta, L. clarkorum, and L. raddei-nairensis complex. Lacerta parvula joined the saxicola group, but the association is not well-supported. The rudis group has been recognized by previous studies (Murphy et al. 1996; Fu et al. 1997), which included L. parvula as well. Several sister group relationships among species were also realized and well- supported, e.g., L. rnixta with L. clarkorum; L. caucasica with L. daghestunica; L. rudis with L. valentini. The tree also clustered the saxicola group with the rudis group, but this sister group relationship was not well-supported.

In attempt to test the relationships among the deep divergence, the 3rd positional changes were excluded from the analysis. The 102 remaining informative characters produced 24

MPTs, with 313 steps, CI of 0.428 and RT of 0.659. Again, the three major groups were resolved, but the saxicola and caucasica groups formed a sister group relationship. The rudis group located at the base; L. parvula was grouped with the clade of (the saxicolu group + the caucasica group). The ambiguities were mainly among the members of the saxicola group.

Results are summarized in Table 3-2. Figure 3-1. The most parsimonious trees from the independent analyses of the individual genes.

The numbers above the iines are bootstrap proportions greater than 0.50. The numbers below the lines are decay indices. The numbers after the taxon names refer the specimen number in

Appendix 11, when multiple specimens were used for one species. Bold lines indicate well- supported nodes.

A. The most parsimonious tree from cyt-b gene. B. The most parsimonious trees from ATPase 6 gene. Dashed lines show the ambiguities.

C. The most parsimonious tree from 16s gene. L. mixta-1 "L. alpina" L. mixta-2 L. clarkorum-1 L. daghestanica-1812 L. caucasica L. derjugini-l&2 L. raddei - 1 L. raddei -2 L. nairensis '1.saxl'cola" L. raddei -3 L. brauneri-1 L. brauneri-2 L. brauneri-3 L. saxicola L. lindholmi L. alpina-1 &2 L. praticola-I L. pra ticola-2 L. pama-1 &2 L. valentini-1 L. valen tini-2 L. rudis-l&2 L. portschinskii-1 L. portschinskii-2 L. media L. monticola Algyroides L. vivjpara L. mixta-1 Y. alpina" L. mixta-2 L. clarkorum-1,2,4,5 L. clarkorum-3 L. derjugini-I L. daghestanlca-I L. caucasica L. raddei-I rn 1: I L. brauneri-1 I I I 100 L. lindholmi 8 I 1 i >6 L. saxicola I a L. pama-1 1 a L. portschinskii-1

L. praticola-I I L. media loo L. saxico~a 68 >9 I L. alpina-l

L. mixta-1 "L, alpina" 96 L. daghestanica-1 1 99 7; L. caucasica 1 L. derjugini-I 79 'g L. clarkorum- 1 1r 4

I Atgyroides L. media L. monticola L. vivipara Table 3-2. Summary of the results of Chapter 3. bp= base pair, PIC= phylogenetically informative character, MPT= most parsimonious tree, TL= tree length, CI= consistency index, RI= retention index, TS= transition, TV= transversion, PTP= permutation tail probability,

HER= homoplasy excess ratio. All g, are calculated with 10,000 random trees. All PTPs and HERS are calculated with 1,000 randomization replicates and without randomizing the outgroups.

Gene bp aligned bp included number number number TL CI RI TSITV g, PTP HER in analyses of PICs of taxa of MPTs ratio

cyt-b 4.31 -0.6184 0.489

1st&2nd 4.29 n/a n/a

ATPase 6 3.48 -0.7832 0.477 1st&2nd 4.61 nla n/a

16s 2.57 -0.7148 0.419

TV nla n/a nia

tRNA nla n/a nia

Combined 3.66 -0.7908 0.344

TV+l st&2nd n/a n/a nla ATPase 6 Gene

A 596 base pair fiagment was sequenced and aligned for 19 specimens representing 13

species. A shorter fragment (403 bp) was sequenced fiom L. portschinskii. Lacerta clarkorum had two haplotypes with one bp difference. Attempts to sequence three ingroup species (L.

valentini, L. &is, and L. alpina) and three outgroup members (Algyroides Btzingeri, L. vivipara

and L. monticola) failed. No deletions/insertions were found. Pairwise differences ranged from

20.0% to 23.5% between the outgroup and ingroup species, and fiom 5.2% to 19.6% among ingroup species.

A total of 157 potentially phylogenetically informative characters were resolved. A g, of

-0.7832 and a PTP of 0.001 indicated that there is significant character covariance in the data

set (Faith and Cranston, 1991; Hillis and Huelsenbeck, 1992). Equally weighting all characters

resulted in two MPTs, with 393 steps, CI of 0.545, RI of 0.615 (Fig. 3-IB). BSPs were caIculated

and eight nodes received values greater than 0.70. Five of the eight nodes received the highest DIs (24). The two haplotypes of L. clarkorum grouped together. The suspicious "L.alpina" was

placed in between the two haplotypes of L. mikta, as with cyt-b. All caucasica group members

identified by cyt-b clustered together, and were well-supported by the data. Members of the

saxicola group were grouped together with one exception; L. praticola was placed at the very

base of the tree rather than in the saxicola group. The position of L. parvula was ambiguous. It was either grouped with the rudis group (L. portschinskii only, in this case) or with the clade of (the caucasica group + the saxicola group). The placements of L. praticola, L. parvula, and the sister group relationship of the caucasica group with the saxicola group were not well-supported.

The 1st and 2nd positional substitutions yielded 56 potentially informative characters,

and resulted in 16 MPTs. Lacertaparvula was resolved as the sister group of L. portschinskii. Other relationships agreed with results from using all changes, except the highly unresolved

I relationships within the saxicola and the caucasica groups. Results are summarized in Table 3-2. 16s Gene

An 1173 base pair fragment was sequenced and aligned for 20 specimens representing all 19 species. Due to ambiguous alignment, a 27 base pair fragment was excluded fiom the analysis.

Painvise comparison treating gap as "missing data" ranged fram 11.0% to 16.2% between

ingroup and outgroup species, and from 0.6% to 10.4% among ingroup species.

A total of 239 potentially phylogenetically informative characters were used for

parsimony analysis. A g, of -0.7148 and a PTP of 0.001 indicated that there is significant character covariance in the data set (Faith and Cranston, 1991; Hillis and Huelsenbeck, 1992).

Equally weighting all characters resulted in one MPT, with 712 steps, CI of 0.504 and FU of

0.604 (Fig. 3-1C). Eight nodes received BSP values greater than 0.70, and these are among the

nine nodes which received the highest DIs (24). All species were assembled into three groups; the

memberships of the groups were identical to the results of cyt-b, except the placement of L. parvula, which joined the rudis group with strong support (BSP=71; DI4). The rudis group and

the caucasica group formed a well-supported sister group relationship.

Transversion substitution alone produced 204 potentially phylogenetically informative characters, and resulted in three MPTs, with 201 steps, CI of 0.524, and RI of 0.712. The three major clades were maintained, and the caucasica group and the saxicola group formed a sister group relationship. Ambiguities occurred in the position of L. parvula. It was placed either at the very base of the tree, with the rudis group, or with the clade of (the saxicola group + the caucasica group). Results are summarized in Table 3-2.

Combined Data Examining the resulting trees from the three independent analyses, none of the well- supported nodes (i.e., BSP>0.70) was in conflict. Therefore, a combined data approach was appropriate (Doyle, 1992; Huelsenbeck et al., 1996). The mysterious "L. alpha" and '2. saricola" were excluded. Only one specimen from each of the 19 species was used. Missing data were used to represent species without ATPase 6 sequences. A total of 769 potentially phylogenetically informative characters were produced. Equally weighting all characters resulted in one MPT, with 2393 steps, CI of 0.463, and RI of 0.522 (Fig. 3-2A). Ten nodes received

BSPs greater than 0.70, and they were exactly the same ten nodes which received the highest DIs. All species assorted into three groups; memberships were exactly the same as resolved in the

16s gene alone. The monophyly of each of the three groups was well-supported. Lacerta

parvula grouped into the rudis group, but the association was not well-supported. The saxicola

group and the caucasica group formed a well-supported sister group relationship.

Use of transversions only from the RNA genes, and the 1st and 2nd codon positional substitutions of the protein genes yielded 397 potentially phylogenetically informative

characters. Analysis resulted in four MPTs with 656 steps, CI of 0.465, and RI of 0.538 (Fig. 3-

2B). Eight nodes received BSPs greater than 0.70, and they were the same nodes which received

the highest DIs. The three clades grouped exactly the same as using all changes, and the

monophyly of the three groups and the sister group relationship of the saxicola group with the caucasica group were well-supported. However, the relationships within the saxicola and

caucasica groups were highly unresolved. All results are summarized in Table 3-2.

The Genetic Divergence among the Three Major Clades

To evaluate the genetic divergence among the species and three the major clades, a

pairwise distance comparison was conducted. Because a transversion bias was observed (Table 3-

2), the Kimura 2-parameter distance was used. The results were presented in Table 3-3. Not surprisingly, the intra-clade divergences are smaller than inter-clade. Although phylogenetically the caucasica group and the saxicola group are closer to each other than either is to the rudis

group, the genetic divergence showed no significant difference among the three (p10.05).

Figure 3-2. The most parsimonious tree from the four gene combined data. The numbers above the lines are bootstrap proportions greater than 0.50. The numbers below the lines are decay indices. The numbers after the taxon names refer the specimen number in Appendix I, when multiple specimens were used for a species. Bold lines indicate well-supported nodes.

A. The most parsimonious tree from the unweighted analysis.

B. The strict consensus tree of the four most parsimonious trees from the weighted analysis. 100 L. mixtal 59 18 L. clarkorum-1 2 1 00 ' L. derjugini-1 L 20 99 L. daghestanica-1 99 1 15 13 L. caucasica

_I L. raddei-1

82 82 L. brauneri-1 I I 8 60 5 L. lindholmi 3 100 L. saxicola 97 >22 : L. alpina-1 15 L. praticola-1

A lgyriodes L. media L. vivipara L. mixta-1 L. clarkorum- I 68 L. derjugini-1 1 100 81 L. daghestanica-1 9 4 L. caucasica L. raddei-I 82 L. brauneri-1 3 100 L. lindholmi 7, L. saxicola 89 5 L. alpina-1

j L. praticola-t

Algyriodes L. media L. vivipara DISCUSSION The Lanyon's Consensus and the Preferred Phylogeny

Well-supported elements were first isolated from the resulting trees of independent

analyses (Fig. 3-I A, lB, 1C). No conflict occurred among the well-supported elements. The

preferred tree was constructed using these elements (Fig. 3-3). The individual genes did not

resolve well-supported relationships among the three major clades, but the four genes combined did. Both weighted and unweighted analyses strongly associated the caucasica group with the

saxicola group. Therefore, this relationship is also depicted on the preferred tree. The well-

supported elements from the combined data are almost filly congruent with the independent

analyses. They only differ in that the former united the saxicola and caucasica groups, and the latter included L. parvula in the rudis group. The preferred tree grouped all species into three

major clades: the rudis group including L. rudis, L. valentini, L. portschinskii and L. parvula; the

saxicola group including L. saxicola, L. brauneri, L. lindholmi, L. alpina, and L. praticola; and

the caucasica group including L. caucasica, L. daghestanica, L. derjugini, L. mrjcta, L.

clarkorum, and L. raddei (nairensis). Among the three clades, the saxicola group and the

caucasica group form sister group relationship. The relationships among the rudis group

members are fully resolved and well-supported. Nevertheless, some relationships within the other two groups remain uncertain. The position of L. derjugini in the caucasica clade is ambiguous.

It has been associated with L. clarkorum (Fig. 3-l B), with the clade (L. daghestanica + L. caucasica) (Fig. 3- lC), the clade (L. mixta + L. clarkorum) (Fig. 3-2A), and placed as the sister taxon of the above four species (Fig. 3-IA). No placement is well-supported by the data. The relationships among L. alpina, L. saxicola, and the clade of (L. brauneri + L. lindholmi) are unresolved, reflecting conflict among the characters.

Methods for testing confidence limit, as well as the general usage of statistic methods in phylogenetics, have been long subjected to debate (e-g., Siddall and Kluge, 1998). Traditional ways of nodal evaluation (e.g., number of synapomorphies of each internode) can only rank the recovered nodes, but cannot provide an answer to questions such as "is this node good enough to Figure 3-3. The preferred tree, which represent the firm foundation of the phylogeny. Letter a, b, c, F designate the well-supported elements fiom Fig. 3-1A (cyt-b), 3-1B (ATPase 6), 3-1C

(1 6s) and Fig. 3-2A (four gene combined), respectively. L. mixta L. clarkorum bc A L. derjugini F abc L. daghestanica F L. caucasica L. raddei a L. brauneri F F L. Iindholmi abc F L. saxicola a - L. alpina F -L. praticola L. valentini L. rudis L. portschinskii L. parvula warrant an explanation of other biological observations?" Statistical methods provide options at

this aspect. As for the two methods used in this study, decay analysis is a derived form of

traditional ways and bootstrapping represents statistic methods. In all cases of this study, they showed the same or similar ranking of the recovered nodes, and they are consistent fiom

independent analyses to the combined.

Taxonomic Remarks

Monophyly of the Caucasian rock lizards is well confirmed. The two ccground"lizards, L. praticola and L. derjugini, indisputably belong to the group. Their preference of grassland

habitat is likely a parallel secondary adaptation. Lacerta nairensis is not a valid species. Accepting as a species makes L. ruddei

paraphyletic (Fig. 3-1A). Previous phylogenetic studies reached the same conclusion (Moritz et

al., 1992a; also see Chapter 4). Allozyme electrophoresis showed no fixed allele between L. raddei and L. nairensis populations, which is evidence for the presence of gene flow among their

populations (Bobyn et a]., 1996).

The DNA data do not support the species status of Lacerta dryada (Darevsky and

Tuniyev, 1997). Three of my five specimens of L. clarkorum (3,4,5) are fiom the geographic range of L. dryada, and morphologically they also resemble the diagnostic characters of L. dryada. However, two of the three L. dryada shared identical ATPase 6 sequence with two specimens of L. clarkorum. The other specimen is one bp different. Comparing to the substantial interspecific variation among other Caucasian rock lizard species (min. 5.2%), the species status of L. dryada is considered questionable. A recent allozyme study did not detect any fixed allele between the two proposed species (MacCulloch, pers. comm.).

Lacerta brauneri appeared to be paraphyletic on the cyt-b tree (Fig. 3-1A).

Phylogenetically, L. brauneri-3 (Anapa) is closer to L. saxicola than to other two populations of

L. brauneri. However, the allozyme study showed that there was no fixed allele among the three populations of L, brauneri, but there were more than three fixed alleles between populations of L. brauneri and L. saxicola (MacCulloch et al., in press). For the species to represent their

genealogical history (evolutionary species concept; Wiley, 1981; also see Frost and Hillis, 1990),

the population of L. brauneri fiom Anapa should be included into L. saxicula. However, more

specimens should be examined before making a taxonomic revision.

Lacerta saxicola fiom Darnbay (labelled as "L. suxicola" on the tree) strongly grouped

with L. nairensis (BSP=100; Fig. 3- 1 A). Morphologically it also resembles L. nairensis.

However, the allozyme data showed three fmed alleles between the L. saxicola (Dambay; n=3)

and populations of L. nairensis (Murphy et al., 1996). At the present time, the taxonomic status

of the Dambay L. saxicola has not been determined. Because the Dambay population was used as

the representative of L. saxicola in both Murphy et al. (1996) and Fu et al. (1997), the

relationships oft. saxicola to others depicted on their phylogenies are questionable.

The mysterious "L. alpina" was placed between the two haplotypes of L. rnixta (Fig. 3-

1A, lB, 1C). Clearly, its mtDNA resembles L. mixta. Examination of another sample of four L.

mixta showed little variation among individuals of L. rnixta (see Chapter 5). The other two

specimens of L. alpina were identical and grouped into the suxicola group, distantly reIated to L.

mixta. The best explanation for this observation is that the '2. alpina" specimen is a hybrid

between L. rnixta and L. alpina. The rntDNA of "L. alpina" represent its maternal lineage, L. mixta. Currently, L. alpina and L. rnixta are not sympatric; therefore the hybridization must be

an historical event. Because their mtDNA are extremely similar, the event must have been

recent. Unfortunately, Fu et al. (1997) used this hybrid specimen to represent L. alpha, and also

proposed a historical hybridization event hypothesis. Their hypothesis assumed the hybrid

specimen represented the L. alpina, which is clearly incorrect. They mistakenly concluded that

L. mixta acquired the L. alpina mtDNA. Comparison of Hypotheses Phylogenetic studies of Caucasian rock lizards include Darevsky (1967), Murphy et al.

(1996) and Fu et al. (1997). The data of Fu et aI. (1997) are included here; therefore no comparison is made. Darevsky (1967) presented the first phylogeny of the Caucasian rock lizards based on

morphology (Fig. 3-4A). Several associations recognized by Darevsky are also supported by the

DNA data, i.e., L. rudis was united with L. valentini; L. portschinskii with L. parvula; L. caucasica with L daghestanica; L. saxicola, L. brauneri with L. lindholmi. He also associated the latter two clades with I;. raddei. The most distinctive differences between his hypothesis and the DNA phylogeny are the placement of L. alpha and L. mixta. Darevsky did not include the two ground lizards, L. detjugini and L. praticola in his tree. Murphy et al. (1996) presented a phylogeny derived from allozyme data (Fig. 3-4B).

Using "mutation coding" (Murphy, 1993), 35 presumptive loci yielded 20 potentially phylogenetically informative characters. Their phylogeny grouped the species into four clades. The arrangement of the rudis group is identical to this study, but the memberships of the saxicola group and the caucasica group are largely different. Whereas the placements of L. praticola and L. derjugini on their phylogeny were regarded as tentative, the DNA data resolved their positions. A clear difference between the allozyme phylogeny and the current DNA phyIogeny is the placement of L. alpina. Allozymes associated it with the clade of (L. caucasica + L. daghestanica), but the DNA data strongly grouped it with L. scuricola, L. lindholmi and L. brauneri. These three species were previously under the name of L. saxicola, and they were not included in the allozyme study.

Constraints on the Origin of Parthenogenesis The interspecific hybrid origin of parthenogenetic species in Caucasian rock lizards has been well established (Darevsky et al., 1985; Darevsky, 1992) and the parentage of the seven known parthenogenetic species has been confirmed by a series of recent molecular studies (Moritz et al., 1992a; MacCulloch et al., 1995a, 1997a; Murphy et al., 1997; Fu et a]., 1998; Figure 3-4. Comparison of hypotheses.

A. Darevsky's (1967) tree. The lines at the right side depict the grouping of the DNA data.

B. Murphy et al.'s (1996) tree. The number above the lines are bootstrap proportions great than 0.50. The Iines at the right side depict the grouping of the DNA data. I- I- L. alpina L. caucasica L. daghestanica L. raddei Lo brauneri L. lindholmi L, saxicola L. mixta Lo pawula L. portschinskii 1 L. valentini L. rudis -,I

L. alpina I L. daghestanica L. caucasica 7 L. raddei L. mixta

L. valentini 1 56 ,69 wdis 1 L. portschinskii -L. parvula I Chapter 4). Lacerta raddei (including L. nairensis) has been identified as the maternal parents of

L. rostombekowi, L. unisexualis, L. zrzzelli, L. sapphirina, and L. bendimahiensis. Lacerta rnixta has been identified as the maternal parents of L. armeniaca and L. dahli. Lacerta valentini has been identified as the paternal parents of L. armeniaca, L. unisexdis, L. uzzelli, L. sapphirina, and L. bendimahiensis. Lacerta portschinskii has been identified as the paternal parents of L. dahli and L. rostornbekowi.

To examine the phylogenefic constraints, the parentage of the seven parthenogens was mapped on the phylogeny (Fig. 3-5). Patterns were revealed. The hybridization events that led to the formation of parthenogenesis only involved two of the three major clades, the caucasica group and the rudis group, but not the saxicola group. The hybridization only occurred between the major clades, as observed by Murphy et al. (in review), and not within the clades.

Furthermore, the hybridization is "directional:" the maternal parents came fiom the caucasica group and the paternal parents fi-om the rudis group. These patterns are best explained by the existence of phylogenetic constraints, i.e., lineage-dependent factors at least partially control the formation of parthenogenesis. These factors restricted the involvement of the saxicola group and the hybridization direction.

Many hypotheses have proposed that parthenogenesis resulted fiom a simple genetic mutation, usually at a single locus (see Moritz et al., 1992b). Although it may be true for some organisms (e.g., maize, Golubovskaya, 1979), it is not the case in squamate reptiles. Darevsky et al. (1985) argued that a lineage dependent genetic factor or factor(s) determines clonal reproduction in hybrids. Moritz et al. (1992b) refuted the idea, and stated that it is the amount of genetic divergence across many genes that is critical, and not the presence of a specific allele at one locus [= Darevsky et al.'s (1985) "genetic factor"]. The DNA sequence data support

Darevsky et al.'s (1995) explanation that the formation of parthenogenetic species is lineage dependent. However, this does not imply that the presence of a particular allele is the key to achieve parthenogenesis, and it does not rule out the role of genetic divergence. If a single allele is responsible, given the absence of the saxicola group in the formation of parthenogenesis, as Figure 3-5. Phylogenetic relationships among the bisexual species of Caucasian rock lizards, and the parentage of unisexual species. Abbreviations for the species are as follows: arme = L. armeniaca; bend = L. bendimahiensis; duhl = L. dahli; rost = L. rostombekowi; sapp = L. sapphirina; unis = L. unisexualis; and uzze = L. uzzelli. bend SaPP uzze dahl arme unis rost

I I The cakasica The saxicola The rudis group I well as other species in the caucasica group and the rudis group, then the allele has to be independently gained andfor lost several times. For example, the allele could be independently gained four times, gained once and lost at least five times, or gained twice and lost a minimum of three times. Considering the natural rarity of parthenogenesis, it is unlikely that such an allele could independently evolve multiple times. It is also unlikely that so many species lost the allele.

What are the causative agents of phylogenetic constraints and how do they operate?

Murphy et al. (in review) probed several aspects from heteromorphic sex chromosomes, heterosis, and paleogeography, but could not find a single satisfactory explanation. The directional hybridization suggests that sex chromosomes may play an important role, but at present time there is not enough convincing evidence to reach a conclusion. Phylogeny influences and leaves traces of all biological aspects of the organisms, e.g., genetics, physiology, development, biogeography, ecology etc. In my opinion, Darevsky et a1.k (1985) lineage- dependent factors are most likely to be a multifunctional complex. It operates through co- effects of many factors at multiple levels. In a more obscure scenario, the phylogenetic constraints are possibly purely intrinsic, and may not be explicitly caused by one or a few detectable factors. In other words, although the patterns are observable, the processes themselves may not be. This hypothesis is falsifiable. It predicts that other undescribed (if there are any) parthenogens in Caucasian rock lizards would originate from hybridization between bisexual species of the caucasica group and the rudis group. If new-found parthenogens were not fkom these two groups, then this hypothesis would be falsified. Moritz et al. (1992b) examined the constraints on the formation of parthenogenetic Cnemidophorus, and concluded that it is genetic divergence rather than phyiogenetic relationships. However, their phylogeny only included the parental species, not all bisexual species. This approach may hide the potential patterns. For deducing a convincing conclusion, a rigorous phylogeny including all bisexual species of Cnemidophorus is required. Future research in this direction is highly desirable. The constraints on the formation of parthenogenesis are multi-faceted. And different aspects of the constraints are interactive. Interspecific hybridization in Caucasian rock lizards is commonplace and well-documented (Table 4-4; Darevsky, 1967; Murphy et a]., in review).

However, there are only six documented cases in which parthe"ogens formed. Lacerta

armeniaca, L. dahli, L. rustombekowi, L. unisexualis and L. uzzelli have a single common origin

(reviewed in Murphy et a]., in review; Chapter 4). Lacerta sapphirina and L. bendimahiensis likely share a single origin (Chapter 4). Thus, hybridization leading to parthenogenesis is

extremely rare. A single aspect of the constraints (e.g., genetic or phylogenetic) could not expIain this rarity.

Moritz et al. (1989) put forward the "balance hypothesis," which states that there is a

narrow range of (genetic) divergence within which unisexual lineages have a reasonable

probability of becoming established. A certain degree of divergence between the parents is

required to produce unreduced gametes in hybrids; too much divergence would lower the viability

and/or fecundity of hybrids. The amount of divergence is critical. It is reasonable and evidenced

that the genetic constraints played a critical role in the formation of Caucasian lacerta

parthenogens. My data showed that the divergence between the two pairs of parental species is

moderate and narrow in range (0.12212-0.13730, Table 3-3). However, divergence alone cannot

explain why the saxicola group was never involved in the formation of parthenogens. If the

divergence of cyt-b, ATPase 6 and 16s genes represent the divergence of the organisms (and the

divergence of the genes which control the oogenesis, initiation of development, etc.), then the

genetic distances are suitable (Table 3-3). Many pairwise distances between the saxicola group

members and species from other two groups approximate the pairwise distance between the

parental species of the parthenogens, but no parthenogen has been formed. Furthermore, the

balance hypothesis cannot explain the "directional" aspect of the hybridization. The documented interspecific hybridization events were also considered (Table 3-4). In

general, interspecific hybridization is common among sympatric species, both intra- and inter- clades, although not all sympatric species hybridize. Interestingly, there are no documented

hybrids between the rudis and saxicda groups although there are cases of sympatry (L.rudis with L. brauneri; L. paticola with L. panula). The average genetic distance between these two Table 3-4. The documented inter- and intra-clade hybridization events among Caucasian rock lizards.

Hybrid cross references

intra-

L. caucasica x daghestanica Darevsky, 1967; Fu et al., 1995 L. parvula x L. rudis Darevsky, 1967

L. rnixta x L. derjugini Darevsky, I 967

L. alpina x L. brauneri Darevsky, 1967 ,

L. saxicola x 1;. brauneri Darevsky, 1967 inter-

L. alpina x caucasica Darevsky, 1967; Fu et a]., 1995

L. raddei x portschinskii Darevsky, 1967

L. caucasica x L. saxicola Darevsky, 1967

L. rnixta x alpina Fu et al., 1997; this study

L. rudis x clarkorurn Darevsky and Tuniyev, 1997

L. derjugini x L. parvula Darevsky, 1967 groups is slightly higher than others, but the ranges largely overlap. The unknown factor(s)

which inhibit the hybridization may also be responsible for the exclusion of the saxicola group in

the formation of parthenogens.

Ecological aspects of the constraints in Caucasian rock lizards are also very interesting. Wright and Lowe (1968) found that parthenogenetic Cnemidophorus prefer disturbed habitats,

acting as biological "weed species." This may be the consequence of the competition with their

parental bisexual species. Parthenogenetic lacertas occur in more diverse habitats. Sometimes,

they occupy extreme habitats, especially with respect to aridity. At many localities two or more

parthenogenetic species occur sympatrically without any bisexual species (Darevsky, 1992).

Evidence also indicates that parthenogens sometimes are more tolerant to environmental change

than their bisexual counterparts. In Kareli (Georgia), L. rudis and L. dahli co-existed in a

completely isolated small spring site during the 1950's through the 1970's. However, due to

human use, the water supply has been reduced recently. My 1995 survey found that L. rudis had vanished, but L. dahli survived. Danielyan (197 1) reported that the eggs of parthenogenetic

species are more resistant to drying compared with those of their parental species.

Notwithstanding, in many cases the parthenogens tend to have a more restricted habitat compared to their bisexual parents. Where the parthenogenetic species co-exist with their parents, they often occupy only a portion of the available habitat. For example, L. raddei and L. bendimahiensis co-occur at Muradiye Waterfall (Turkey), and the parthenogenetic species has a far more restricted distribution than its matema1 parental species. This aspect deserves more attention and is a direction of the future research in Caucasian rock Iizards. Other constraints, such as paleobiogeography, heterosis, also contribute to the scarcity of parthenogenesis (see Murphy et al., in review). At last, chance alone may also play an important role in the formation of a new parthenogen. Chapter 4. Divergence of the cytochrome b gene in the Lacerta raddei complex and its

parthenogenetic daughter species: evidence for recent multiple origins

Abstract. - Questions concerning the origin of parthenogenesis in Caucasian rock lizards, and genetic divergence among bisexual Iizards of the Lacerta raddei complex, was examined using sequences fiom the mitochondria1 cytochrorne-b gene. The maternal parent of parthenogenetic

L. uzzelZi, L. sapphirina, and L. bendimahiensis was confirmed to be L. raddei. Whereas, substantial variation was revealed among bisexual populations of L. raddei and L. nairensis populations, very low or no variation was found among the parthenogenetic species. A phylogenetic tree including eleven populations of L. raddei and L. nairensis, as well as ten populations of its five daughter parthenogens was constructed. Owing to paraphyletic relationships, L. nairensis was considered conspecific with L. raddei. Evaluation of the parthenogenetic species suggested that separate hybridization events between L. raddei and L. valentini might have occurred at least twice. One resulted in L. sapphirina and L. bendimahiensis, and the other one or more resulted in L. unisexualis and L. uzzelli. The females involved were distantly related. Lacerta unisexualis and L. uzzelli likely had a separate origin, but the femaIes involved were closely related. Lacerta sapphirina and L. bendimahiensis likely share a single origin and are considered conspecific. INTRODUCTION

Parthenogenesis in Caucasian rock lizards (genus Lacerta) has intrigued biologists since it

was discovered nearIy a half century ago (Darevsky, 1957, 1958). It originates from interspecific hybridization between bisexual species, and the eggs, produced without meiosis,

develop into genetically identical offspring (Uuell and Darevsky, 1975; Darevsky et al., 1985).

The Lacerta raddei complex, including L. r. ruddei, L. r. vanensis, and L. nairensis, has been

identified as the maternal parents of five (out of seven) parthenogenetic species: L.

rostornbekow i, L. unisexualis, L. uzzelli, L. sapphirina, and L. bendimahiensis (Darevsky, 1967,

1992; Darevsky and Danielyan, 1977; Schmidtler et al., 1994; MacCulloch et al., 1997a; Fu et

al., 1998). Figure 4-1 depicts the hypothetical parentage of the seven parthenogenetic species of Caucasian rock lizards.

A recent series of molecular studies have examined population variation in Caucasian rock lizards. Moritz et al. (1992a) using mtDNA restriction fragment analysis, and MacCulloch et al. (1995% 1997a), Murphy et al. (1997), and Fu et al. (1998) employing allozyrne electrophoresis, examined divergence of the parthenogenetic species. Little variation was found.

For example, L. rostombekowi expressed no variation in and among the four populations studied (MacCulloch et al., 1997a). Bobyn et al. (1996) found little intra-population genetic variation in

L. raddei and L. nairensis, two proposed parental forms, although both species displayed relatively high levels of substructuring among populations. Similar low levels of variation were found in other species (Fu et al., 1995; MacCulloch et al., 1995a, 1997a). In addition, all but one originally proposed bisexual parents were confirmed; Moritz et al. (1992a) proposed that L. valentini was the maternal parent of L. uzzelli, whereas Darevsky and Danielyan (1977) proposed that it was L. nairensis.

The objectives of this chapter are to examine the diversity of cytochrome-b (cyt-b) gene sequences in the L. raddei complex, and its parthenogenetic daughter species. Further, the divergence between the parthenogens and their parent species are compared, and questions pertinent to the origin of parthenogenesis in Caucasian rock lizards are addressed. Figure 4-1. Hypothetical parentage of parthenogenetic Caucasian rock lizards. Species in shadowed area are the parthenogens. "?" indicates uncertainty.

MATERIALS AND METHODS Five populations currently assigned to Lacerta raddei and six currently assigned to L.

nairensis were examined along with four populations of L. unisexualis, three of L. rostornbekowi,

and one each of L. uzzelli, L. sapphirina, and L. bendimahiensis. Moritz et al. (1992a) found

that mtDNA nucleotide diversity within populations was significantly lower than among

populations in Caucasian rock lizards (0.003 vs. 0.018 in L. nairensis and 0.000 vs. 0.03 I in L.

raddei). Bobyn et al. (1996) also suggested that populations of L. raddei and L. nairensis are

disjunct, with reduced gene flow among them. Therefore, only one specimen fiom each

population was sequenced. Two other species of rock lizards, L. derjugiini and L. pamla, were

used for outgroup comparison (Fu et a]., 1997). Lacerta valentini was also sequenced as a

potential alternative maternal parent of L. uzzelli. Voucher specimens and locality data are listed

in Appendix 3 and shown in Fig. 4-2. Fieldwork was conducted during 1992-1996. Live specimens were euthanized by an

overdose of sodium pentobarbital before processing, following accepted animal welfare protocols.

Blood, heart, liver and tail muscle tissues were collected and frozen in liquid nitrogen for use in

multiple molecular investigations. All voucher specimens were deposited in the herpetological

. collections of the Royal Ontario Museum. Standard phenol-chloroform methods were used to extract DNA fiom tail muscle or liver

tissues. Laboratory protocols followed Chapter 2. After the initial sequencing, a pseudogene was

found, which presumably was a nuclear copy of mtDNA cyt-b gene. Subsequently, both whole

DNA and purified mtlDNA extractions were conducted. Purification of mitochondria was

accomplished by repeatedly centrifuging at 4000 rpm and 14,000 rpm on a Canlab Biofige A

machine to separate mitochondria fiom nuclei after manually homogenizing liver tissues using a conventional glass homogenizer (modified fiom Palumbi, 1996). This method successfully

limited the nuclear DNA to an undetectable level on the sequencing gels.

Polymerase Chain Reaction (PCR) was used for amplifying the DNA samples, and P~~labeled

terminator cycle sequencing kits (Amersharn) were used for DNA sequencing. Protocols Figure 4-2. Map of Transcaucasus with the distribution of localities from which sexual and parthenogenetic species were collected. The numbers at each locality correspond to those in

Appendix 111.

followed Chapter 2. Six primers were used for amplifying and sequencing the target cyt-b gene

segments (Table 4-1). All sequences were completed on both directions with 80-90% overlap.

DNA sequences were edited in ESEE (version 3, Cabot and Beckenbach, 1989).

Nucleotide statistics, including base composition, substitution patterns, and amino-acid

codon usage, were used to evaluate the dynamics of sequence evolution. These were computed

using MEGA (version 1.01, Kumar et al., 1993). The divergence of populations was measured by

p-distance in MEGA, which is defined as the proportion (p) of nucleotide sites at which the two

sequences compared are different (Kumar et al., 1993).

The phylogenetic analysis using maximum parsimony was conducted with PAUP (version

3.1.1, Swofford, 1993). Bootstrap proportions (Felsenstein, 1985) were calcuIated with 1000 replicates. All eleven populations of L. raddei and L. nairensis, as well as all ten parthenogenetic populations, were included in the tree construction. The well-established mother-daughter

relationships (except L. uzzelli) and strict maternal inheritance of mtDNA warranted the

monophyly of the ingroup. Three other lacertas, L. parvula, L. derjugini, and L. valentini, were

also included as outgroups.

Phylogenetic tree was used to test hypotheses related to the origin of parthenogenesis.

By testing the monophyly of the parthenogenetic species, it can be determined whether they were from a single origin or multiple origins. Phylogenetic affiliation between the parthenogenetic species and one local maternal population will indicate a constrained, localized origin. The association of parthenogens with several divergent maternal populations will indicate multiple origins. The extent of divergence between maternal populations and parthenogenetic species will shed light on the age of the parthenogens.

RESULTS

A total of 1044 base pair (bp) sequences were resolved for all 24 specimens; these correspond to positions 14843-15886 of the human mtDNA sequence (Anderson et al. 1981).

All sequences can be translated into amino-acid residues with vertebrate mtDNA genetic code. I Table 4-1. Primers used for amplifLing and sequencing cyt-b segments in Chapter 4.

Human position' Gene Sequence References

L14841 cyt-b 5' CCA TCC AAC ATC TCA GCA TGA TGA AA 3' Kocher et al., 1989

H15 149 cyt- b 5' GCC CCT CAG AAT GAT ATT TGT CCT CA 3' Kocher et al., 1989

L15153 cyt-b 5' TGA GGA CAA ATA TCC TTC TGA GG 3' This study

HI5488 cyt-b 5' TTG CTG GGG TGA AGT l'"TT CTG GGT C 3' 0.Haddrath (pers. comm.)

L15369 cyt- b 5' CAT GAA ACT GGA TCA AAC AAC CC 3' This study

H15915 ~RNA~'" 5' GTC TTC AGT TIT TGG TTT ACA AGA C 3' 0.Haddrath (pers. comm.)

' Letters L and H refer to light and heavy strands, and the numbers refer to the position of the 3' ends of the primers in the complete human rntDNA sequence (Anderson et al., 1981). assumed that the mtDNA cyt-b sequences are neither a nuclear copy nor a pseudogene. The

fragment consisted of approximately 91% of the cyt-b gene. No insertions and/or deletions were

found.

Nucleotide Composition The nucleotide composition of Lacertu (Table 4-2) was similar to previous analyses of

the entire cyt-b gene in birds and mammals (Irwin et al., 1991; Kornegay et al., 1993). The third

codon position was strongly biased against guanine, a characteristic of vertebrate mtDNA (Kocher et al., 1989). A bias against thymine, which has been reported for avian and mammalian

cyt-b (Kocher et al., 1989; Irwin et al., 1991; Kornegay et al., 1993), was not found. The

second codon position was thymine-rich, and the four bases in the first codon position were relatively equally distributed - observations also made for birds (Kornegay et al., 1993) and mammals (Irwin et al., 1991). Very little difference in base composition occurred between the

bisexual L. raddei complex and its parthenogenetic daughter species.

Substitution Patterns

Substantial divergence was observed among L. raddei-nairensis populations. A total of

122 sites (out of 1044) was variable among the 1 1 populations examined. Thirty-two sites varied at first codon position, 5 at the second, and 85 at the third, with a ratio of approximately

6: 1 :17. Distribution of the variable sites was uneven (Fig. 4-3), implying the existence of fbnctional constraints. Pairwise comparisons were used to examine the types of mismatches.

Tabulation of specific mismatch types showed that T-C mismatches were dominant, followed by

A43 mismatches (Table 4-3). These two types made up almost 90% of all mismatches.

Transitions comprised 50.0-94.9% of total mismatches, with an average of 90.2%. No variation was detected among either the four populations of L. unisexualis or the three populations of L. rostombekowi. Lacerta uzzelli was identical to L. unisexualis (Table 4-3), Table 4-2. Nucleotide composition of cyt-b gene at different codon positions for Lucerta ruddei-nuirensis and its parthenogenetic daughter species (%).

FIRST SECOND THIRD

Species A T C G A T C G A T C G

L. ruddei-nairensis 27.5 24.6 27.4 20.5 20.4 41.6 25.3 12.6 33.8 27.2 35.9 3.1

L unisexualis 27.9 24.1 27.9 20.1 20.4 41.4 25.6 12.6 34.2 27.0 35.9 2.9

L. uzzelZi 27.9 24.1 27.9 20.1 20.4 41.4 25.6 12.6 34.2 27.0 35.9 2.9

L. rostombekowi 28.4 25.0 27.0 19.5 20.4 41.7 25.3 12.6 34.8 28.2 35.1 2.0

L. bendimahiensis 28.4 25.0 27.0 19.5 20.4 41.7 25.3 12.6 34.5 28.2 35.1 2.3

L. supphirinu 28.4 25.0 27.0 19.5 20.4 41.7 25.3 12.6 34.5 27.9 35.3 2.3 Figure 4-3. Distribution of variable sites across cyt-b in non-overlapping windows among L. ruddei-nuirensis populations. Top bars are protein sequence, size of windows=lO sites, total variable site~22;bottom bars are DNA sequence, size of window~30sites, total variable sites= 1 22. I Protein

DNA

5 10 15 20 25 30 35 Non-overlapping windows Table 4-3. Frequencies of mismatch types of cyt-b gene among populations of Lacerta raddei- nairensis and the parthenogenetic species (%).

L. raddei-nairensis 29.63 59.98 5.10 3.04 1.43 0.81

L. unisexualis+L. uzzelli 0 0 0 0 0 0

L. bendimahiensis+l. sapphirina 0 100 0 0 0 0

L. rostombekowi 0 0 0 0 0 0 but differed considerably from L. valentini by a 14.4% mismatch. Lacerta sapphirina was most

similar to L. bendimahiensis with 1 bp mismatch (T-C, third codon position).

Protein-level Variation

The codon usage patterns of L. raddei-nairensis are listed in Table 4-4. They revealed a

strong bias against guanine. A bias favoring adenine and cytosine was evidenced by seven out of

20 codon families having adenine and/or cytosine with frequencies twice as high as thymine.

This pattern correlated with the base composition which favors adenine and cytosine, and is

against guanine at the third codon position.

Cyt-b is the only protein in complex 111 of the mitochondria1 oxidative phosphorylation

system that is encoded by the mtDNA (Hatefi, 1985). Because of its functional importance and

structural limitations, we might expect variability in cyt-b to be relatively low. A total of 24

sites (out of 348) was variable among L. raddei-nairensis populations. Distribution of the

variable sites is shown in Fig. 4-3. The variable sites showed certain correlation to the functional

domains (Howell, 1989). Howell (1989) suggested that four segments of cyt-b were very

conservative, which comprise the putative Qoreaction center. In these Lacerta data, none of the

24 variable sites was located on these segments. Fifteen of the 24 variable sites were found on

the transmembrane hydrophobic segments, which are regarded as relatively more variable. Certain domains, for example transmembrane domain VI, were hypovariable. Among the 26 amino-acid residues, four were variable in the lacerta data. No protein-level variation was found

among parthenogenetic species.

Phylogenetic Analyses

A total of 184 potentially phylogenetically informative characters were resolved. A parsimony analysis treating all characters as non-additive (=unordered) and equally weighted resulted in two trees with 336 steps, CI of 0.622, and FU of 0.779. The number of MPTs was reduced to one by altering outgroups. Deleting outgroup L. valentini or L. parvula resulted in one Table 4-4. Average codon usage frequencies of cyt-b gene in Lacerta raddei-nairensis. Numbers in parentheses are the relative synonymous codon usage frequencies. MPT, which was one of the above two MPTs. This tree was chosen as the preferred tree (Fig. 4-

4). Bootstrap proportions over 0.50 were mapped on the tree. The parthenogenetic species did not cluster together, but rather formed three groups on

the tree. Lacerta unisexualis and L. uzzelli grouped together, and they were most closely related to populations of L. nairensis from Apnaguch, Adis I1 and Aragatz. Lacerta sapphirina and L. bendimahiensis grouped together and they clustered most closely with L. raddei fiom Muradiye.

The three populations of L. rostombekowi clustered most closely with L. raddei fiom

Egegnadzor, which agrees with Moritz et al. (1992a).

Neither all populations of L. raddei nor of L. nairensis clustered together. The

population of L. raddei from Gosh was most closely associated with the population of L.

nairensis fiom Tumanyan, and not other populations of L. raddei. Populations of L. nairensis

fiom Yerevan and Adis I were most closely clustered with populations of L. raddei fiom Chosrov

and Geghart, not other populations of L. nairensis.

Variability within Species Groups and between Parthenogens and Their Closest

Maternal Populations The simplest distance, p-distance was used to measure the variability. The p-distances

among the populations within the L. raddei-nairensis complex are listed in Table 4-5. Not

surprisingly, the greatest distance (0.0747-0.776) occurred between the northern populations

(Gosh and Tumanyan) and southern populations (Muradiye and Egegnadzor). The smallest p-distance occurred between populations from Chosrov and Geghart (0.0019).

The p-distances within parthenogenetic species are summarized in Table 4-6, along with average p-distances between the parthenogens and their closest "sister taxa" (Table 4-6). In general, the distances between parthenogens and their closest maternal populations are much less than the average distance among populations in the L. raddei-nairensis complex. At the protein level, there was one amino-acid residue difference between populations of L. raddei fiom Adis 11,

Apnaguch, and Aragatz, and the parthenogenetic L. unisexualis and L. uzzelli. It derived fiom a Figure 4-4. One of the most parsimonious trees from PAUP. Species in shadowed area are parthenogens. L. nairensis Apnaguch (D L. nairensis Adis II 1: L. nairensis Aragatz J- ,nn J- L. raddei Egegnadzor .I

I

-L. raddei Muradiye I 7L. nairensis Yerevan bJ QK 13 * VJ L. nairensis Adis I 100 L. raddei Geghart L. raddei Chosrov L. raddei Gosh L. nairensis Turnanyan 1$ L. derjugini 5 L. parvula wo L. valenfini E" Table 4-5. The p-distance among populations of Lacerta raddei-nairensis.

Populations

1 L. raddei Chosrov

2 L. raddei Geghart

3 L. raddei Egegnadzor

4 L. raddei Muradiye 5 L. raddei Gosh

6 L. nairensis Adis I1

7 L. nairensis Adis I

8 L. nairensis Yerevan

9 L. nairensis Apnaguch

10 L. nairensis Aragatz

11 L. nairensis Turnanyan Tabie 4-6. The average within species p-distance among populations of Lacerta raddei-nairensis

and the parthenogens, and the average p-distance between the parthenogens and their closest mother populations.

--

Average Range

L. raddei-nairensis 0.0384f0.0204 0.00 19-0.0776 L. unisexualis-L. uzzelli 0.0000f0.0000 - L. sapphirina-L. bendimahiensis 0.00 10 - L. rostombekowi 0.0000f0.0000 - L. unisexualis-l. uzzelli

vs. L. nairensis Apnaguch, Adis 11, Aragatz 0.0109f0.00 15 0.0096-0.0I25 L. sapphirina-L. bendimahiensis

vs. L. raddei Muradiye 0.0053f 0.0006 0.0048-0.0057 L. rostombekowi

vs. L. raddei Egegnadzor 0.0 1 05f0.0000 0.0 105-0.0105 second codon position substitution. Two amino-acid differences were detected between the pair

of parthenogenetic L. sapphirina and L. bendimahiensis, and the bisexual L. raddei fiom Van. No differences separated L. rostombekowi and L. raddei from Egegnadzor.

DISCUSSION

Dynamics of Sequence Evolution Cyt-b has been used in a large number of systematic and population studies. Compared to

other taxa, the L. ruddei-nairensis complex showed slightly higher levels of divergence @-

distance,, =0.0776). For example, sand darter fish, Etheostoma vitreum, had 3 variable sites in

402 bp (p-distance =0.0075) among its populations (Wiley and Hagen, 1997); rainbow fish,

Melurzotaenia splendida splendida, had less than 4% divergence among populations (Zhu et al.,

1994); Moritz et aI. (1989) reported that the greatest intraspecific mtDNA divergence among

Cnemidophorus was 6.7% (p-distance =0.067). However, lizards fiom populations in close

geographic proximity often had less than 1% divergence of the mtDNA, which is typical for terrestrial vertebrates.

Departures from random substitution patterns in cyt-b sequences have been well documented (Meyer, 1994). In the case of the L. raddei-nairensis complex, composition biases could explain much of the non-uniformity in substitution types. Third position substitutions dominated variations among populations (70%), as did transitions (90%). The strong bias against guanine at the third position made the G-A transition substitution rare. ConsequentIy, transition substitution T-C dominated the mismatch types. Composition bias also explained the uneven codon usage. Functional constraints on the evolution of cyt-b sequences best explained the distribution of variable sites across the gene.

The Taxonomic Validity of L. nairensis Populations assigned to L. raddei and L. nairensis did not cluster together on the phylogenetic tree. This finding is concordant with the allozyme study of Bobyn et al. (1996). An examination of 36 allozyme loci did not find any fixed allelic differences between populations

of L. raddei and L. nairensis. The most striking difference between these two "species" is the copulatory position. In I;. nairensis, the males hold the female by the thigh only, while in L.

raddei the males hold the female by either the thigh or the flank (Darevsky, 1967). However, this character may be habitat-related. Darevsky (1967) found that among species using both

positions, thigh holding was more fiequent at either high elevations or low population densities. Notwithstanding, if the taxonomy is to reflect genealogical relationships, then both species

cannot be recognized. The name Lacerta raddei Boettger, 1892 has clear priority over L.

nairensis Darevsky, 1967.

Parentage of the Parthenogens

Lacerta raddei is the maternal parent of L. unisexualis and L. rostombekowi, with L.

valentini and L. portschinskii being the paternal parents, respectively (Darevsky, 1992; Moritz et

al., 1992a; MacCulloch et al., 1997a; Fu et al., 1998). This study identified L. raddei as the

maternal parent of L. uzzelli, and unpublished allozyme data showed that L. valentini was the

paternal species, as Darevsky and Danielyan (1977) originally proposed. In contrast, Moritz et

al. (1992a) concluded that L. valentini was the maternal parent because L. uzzelli and L. valentini

showed an identical restriction fiagment map. However, the specimens used by Moritz et al. and

I are from different populations. Assuming both findings to be correct, this discrepancy indicates

fascinating multiple origins for L. uzzelli, one in which L. valentini was the maternal parent and L. raddei the paternal species, and the other vice versa. Sampling more L. uzzelli populations is highly desirable. The cyt-b data also confirmed that L. raddei is the maternal parent of L. sapphirina and L. bendimahiensis as Schmidtler et aI. (1994) predicted. A recent allozyme study confirmed that L. valentini is also the paternal parent of L. sapphirina, and L. bendimahiensis

(unpublished data). The Phylogenetic Tree

Although the use of phylogenetic methods at the population level has been questioned

(Nixon and Wheeler, 1990), they are suitable for this study. Mitochondria1 DNA inheritance is predominantly maternal in animals, and no recombination has been detected (Zouros et a].,

1994). Therefore, the resulting tree represents the genealogical relationships of the female hapIotypes, and no reticulate evolution is involved. Moreover, the sampled populations are well isolated from one another, i.e., there is an absence of, or very limited of continuous gene flow.

This is evidenced by the low intra- and high inter-population variation, indicated by both mtDNA and allozyrne data (Moritz et al., 1992a; Bobyn et al., 1996). Consequently, males and females likely experienced identical evolutionary pathways. Thus, the evaluation of cyt-b represents the genealogical history of the populations. The phylogenetic tree grouped the Gosh and Tumanyan populations together, and placed this clade at the base of the tree. These two populations occur on the northern slope of the

Transcaucasus, and are the only populations located in the Kura river drainage. Caucasian rock lizards typicaIly require a habitat with high humidity (Darevsky, 1967). This trait has often constrained their distribution patterns to river valley systems. The absence of a direct valley connection appears to effectively isolate the Gosh and Tumanyan populations from the other populations.

The tree also clusters the Egegnadzor and Muradiye populations together; they have the southem-most distribution. Muradiye occurs in the Lake Van drainage system, and Egegnadzor occurs in the Aras River system. Geographically, Egegnadzor is closer to the central populations than to the Muradiye population. The association of the populations fiom Egegnadzor and

Muradiye indicates a relatively old historical connection as revealed in a medium level distance

@-distance = 0.0335). All the remaining seven populations are distributed in central Armenia, in the Aras River system. They formed two groups on the tree: populations fiom Apnaguch, Adis II and Aragatz Mt., and populations fiom Geghart, Chosrov, Adis I and Yerevan. Origin of Parthenogenesis

The successful hybridization events between the same parental species have occurred more than once, which led to the formation of multiple clones (or species) of parthenogens.

The cyt-b tree divided the parthenogens into three groups (Fig. 4-4): a L. rostombekowi clade, a

L. misexualis-L. uzzelfi clade, and a L. sapphirina-l. bendimahiensis clade. The latter two clades shared same parental species, L. raddei and L. valentini, and they were closely associated with distantly related maternal populations. If the parental identifications of L. unisexualis and L. uzzeNi, as well as L. sapphirina and L. bendimahiensis are correct, then hybridization events between L. raddei and L. valentini must have happened at least twice. Furthermore, because of the distant relationship of the maternal ancestors, evidently different populations were involved in the hybridization events, i.e., the origins of the two parthenogenetic groups occurred at different regions andlor during different time periods. It is more likely that the hybridization events occurred at different geographical populations, because of the close association of the parthenogens with different local populations (Table 4-6, and Fig. 4-4). Considering the genetic divergence between the parthenogens and their closest maternal populations, the hybridization event(s) leading to L. unisexualis and L. uzzelli probably happened much earlier than the one leading to L. sapphirina and L. bendimahiensis. This is the first documented case in which different species (clones) of parthenogenetic vertebrates arose from multiple hybridization events involving distantly related females (and males), which has been reported in some gynogenetic and hybridogenetic vertebrates (Moritz et al., 1989). Multiple clones resulting from multiple origins have also been observed in parthenogenetic Cnenzidophorus and Heteronotia, in which only closely related females were involved (Moritz et al., 1989). A different geographic population of L. raddei involved in the hybridization event with L. portschinskii, which led to the formation of L. rostombekowi (Fig. 4-4). Extremely low variation in both mtDNA and nuclear genes (MacCulloch et al., 1997a) suggested that L. rostombekowi likely originated from a single

F, hybrid, i.e., single origin. Although L. unisexualis and L. uzzeZZi grouped together, indicating that their mtDNA

shared a common ancestor, it is unlikely that they shared a single origin. Lacerta unisexualis and

L. uzzeZZi are identical in terms of their mitochondrial DNA sequences obtained in this study.

Anatomically, L. unisexualis is distinguished on the basis of two nasal scales which are in contact

whereas they are separated by the rostra1 and frontonasal scales in L. uzzeZZi (Darevsky and

Danielyan, 1977). Both scale patterns also occur in their parental species. The absence of

variation within the parthenogens suggests that a genetic mechanism underlies the scale variation, as opposed to the differences resulting fiom epigenetic expression. Consequently,

these two species likely had different ancestors, i.e. each of them had a unique origin, despite

their genetic identity. The simplest explanation is that the two ancestors were siblings of F,

hybrids from same parents. The two siblings had identical mtDNA, but different nuclear DNA,

which resulted fiom heterozygous nuclear DNA of their parents. Alternatively, the two

ancestors were from two hybridization events. In this case, the females which involved in the hybridization must be closely related, and they likely belonged to the same geographically

restricted population. The hybridization events probably occurred in the same time period,

which resulted in the extremely similar mitochondrial genomes.

Not all currently recognized parthenogenetic species appeared to have a unique origin.

Lacerta sapphirina and L. bendimahiensis were also grouped together on the tree (Fig. 4-4),

indicating that their mtDNA shared a common ancestor as well. Cyt-b sequences of these two

species showed only one bp difference. Anatomically they are indistinguishable. The species is

distinguished by coloration (Schmidtler et al., 1994). However, recent field observations have

revealed that this character varies considerably. The preliminary allozyme examination of their

nuclear genes also failed to distinguish the two parthenogens (unpublished data). Given the lack of diagnostic anatomical characteristics, and near genetic identity, they likely originated fiom a

single F, hybrid; a single origin is the best explanation for the history of the species. If true, then the one-bp difference between the two parthenogens likely formed within the species as the result of a point mutation. Sampling more populations and sequencing more individuals of these two

parthenogens would provide more evidence. Murphy et al. (in review) hypothesized that a single origin may be the rule for each

species of parthenogenetic Caucasian rock lizard upon examining four parthenogens, L. unisexualis, L. rostornbekowi, L. dahli and L. armeniaca. A1 lozyme studies revealed that

parthenogens with multiple clones were composed of one major clone with additional

geographically restricted clones, the latter often involving only a few individuals. It is more likely that the restricted ctones were formed in the parthenogens as mutants, rather than representing unique origins (Murphy et al., 1997; Fu et al., 1998; Murphy et al., in review). The

cyt-b data supported a single origin of L. unisexualis and L. rostombekowi. However, the data

also revealed that multiple origin with both closely and distantly related females also occurred in Caucasian rock lizards, which resulted in multiple species fiom same parents.

Low intraspecific variation and high similarity to their parental forms have been a

general role in parthenogenetic lizards (Moritz et al., 1989). These have been explained by

young age (Darevsky et al., 1985, Moritz et al., 1989) and severely constrained origin, i.e.,

genetic constraints (Moritz et al., 1989), ecological constraints (Vrijenhoek, l989), and

phylogenetic constraints (Murphy et al., in review; Chapter 3), as well as others. The cyt-b data

confirmed this role. While the L. sapphirina-l. bendirnahiensis clade only has one variable site, the L. unisexualis-L. uzzelli group, and L. rostombekowi do not vary at all. This contrasts strongly with the substantial variation observed among L. raddei populations. The average of

only 1% difference between the parthenogens and their closest maternal population is much

lower than the average divergence among extant populations of L. raddei (3.8%). This lower

level of divergence is also concordant with the aliozyme data, which revealed very little clonal variation. Only one done has been detected in L. rostombekowi (MacCulloch et al., 1997a);

three in L. armeniaca (MacCulloch et al., 1995a), three in L. unisexualis (Fu et al., 1998); and five in 1;. dahli (Murphy et al., 1997). Although all parthenogens are recent origin in general, the different distances between the parthenogens and their closest maternal populations suggested different ages of the parthenogens. For example, L. sapphirina and L. bendimahiensis are probably much younger than L. unisexualis, L. uzzelli and L. rostombekowi.

The validity of the molecular clock hypothesis has been severely questioned (e.g., Hillis et al., 1996b). Clearly, a universal, radioactive decay style dock does not exist. However, a few studies do show that there is roughly a linear relationship between time and nucleotide divergence

(e.g., Irwin et al., 1989). By using the fossil record or geological events to calibrate the clock, an approximate estimation of time is possible. This is an appealing way to estimate the age of the parthenogens, which has always been among the most commonly asked questions. Unfortunately, a reliable event, which can be used to caIibrate the cyt-b clock in Caucasian

Lacerta, has not been found.

Species Concept in Unisexual Species There are at least four approaches to this issue (see review in Dawley, 1989). Frost and

Wright's (1988) view was favored in my study, in which they argued that each unisexual lineage of independent hybrid origin constitutes a unique historical entity and thus a separate species

(evolutionary species concept). Frost and Hillis (1990) further elaborated on this concept. Due to the clonal reproductive mode, the biological and the phylogenetic species concepts are obviously not applicable to unisexual species. The evolutionary species concept is advantageous in that it is compatible with both unisexual and bisexual species, and it reflects the evolutionary history of the species. However, it has practical problems as well. For example, not all currently existing clones have a unique hybrid origin. There are clones originating from within the parthenogens, and often it is difficult to distinguish these two types of clones. A solution to this problem is to keep all clones under one name until the unique origin of a clone is firmly established. Chapter 5. Limited genetic variation in Lacerta mixia and its parthenogenetic

daughter species: evidence from cytochrome b and ATPase 6 genes

Parthenogenesis in Caucasian rock lizards (genus Lacerfa) originated fiom interspecific

hybridization between bisexual species (Darevsky et al., 1985; Darevsky, 1992). Seven

parthenogenetic species have been described up to date. Lacerta mixta has been identified as the

maternal parents of two parthenogenetic species: L. armeniaca, and L. dahli (Darevsky, 1992;

Moritz et al., 1992a; MacColluch et al., I995a; Murphy et al., 1997).

Examining the mtDNA variation in the maternal parental species and comparing that to their parthenogenetic daughter species will better our understanding about the age, and modes of origin of parthenogenesis. Chapter 3 analysed cytochrome-b (cyt-b) variation in L. raddei and its parthenogenetic daughter species. This chapter examines the other maternal parental species,

L. mixta, and its daughter species using cyt-b and ATPase 6 gene sequences as molecular makers.

Two populations of L. mixta, six of L. dahli, and seven of L. armeniaca were examined in this study. Three specimens fiom each population of L. mixta and one from each population of L. dahli and L. armeniaca were sequenced. Specimens voucher and locality data are listed in

Appendix IV. Laboratory protocols followed Chapter 2. A I044 base pair (bp) fragment of cytochrome-b was amplified and sequenced using the same primers presented in Chapter 4. A ' 596 bp fragment of ATPase 6 was amplified and sequenced using three primers: L8552 5' ATG

AAC CTA AGC TTC TTC GAC CAA TT 3'; H8956 5' ATA AAA AGG CTA ATT GTT TCG

AT 3 ' (0. Haddrath, pers. comm.); H9 148 5' ACG AAT ACG TAG GCT TGG ATT A 3 ' (this study). The letters L and H refer to light and heavy strands, and the numbers refer to the positions of the 3' ends of the primers in the complete human mtDNA sequence (Anderson et al., 1981).

Limited divergence was observed among the six samples of L. mixta (Table 5-1).

Variation among populations was lower than within populations. The largest pairwise difference among the six samples was 0.67%. A total of eight sites varied in cyt-b, and three in ATPase 6. Table 5-1. Cyt-6 and ATPase 6 gene divergence within and among popuIations of Lacerta rnixta and its parthenogenetic daughter species.

------

Species Number Number Number Within Among individuals populations types populations populations

L. rnixta 6 2 3 0.609/0.670% 0.06 1 %

L. dahli 6 6 1 - 0%

L. armeniaca 7 7 1 - 0% These variable sites sorted the six samples of L. mixta into three types (Appendix V). All

variations occurred on the third codon position, and all were transitional substitutions. In cyt-b, T-Cmismatches and A-G mismatches each consisted of half of the total eight mismatches. In

ATPase 6, all six mismatches were A-G type. No protein level variation was observed. No

insertions/deletions were found.

No variation was found among populations of parthenogenetic L. arrneniaca and L. dahli

(Table 5-1). The 13 populations of L, armeniaca and L. dahli were identical for both cyt-b and

ATPase 6. Both species shared the exactly same sequences with type A of L. mixta for both

genes (Appendix V). The cyt-b nucleotide composition of the three species was similar to the L. raddei

complex reported in Chapter 4 (Table 5-2). The third codon position was strongly biased against

guanine; the second codon position was thymine-rich; and the first codon position were relatively

equally distributed of the four bases. The composition of the ATPase 6 gene was largely the

same as cyt-b, with favored adenine at the first and third codon positions (Table 5-2). Divergence in L. mixta is low compared to that of L. raddei. For example, the largest

pairwise difference of cyt-b among L. mixra is 0.77%, while it is 7.76% in L. raddei (Chapter 4). The seemingly limited divergence in L. mixta may partially result from the small population

sample size (n=2), and the short geographic distance between the two populations (= 30 krn). The distribution of E. rnixta was constrained in a small area of western Kura river valley in Georgia Republic. Political instability in Georgia limited the scope of the field collecting.

Although more samples are desirable, a significant increase in observed divergence is unlikely,

considering its restricted distribution.

Neither of the two parthenogenetic species showed any intraspecific variation (Table 5-

1). The samples used in this study covers the distribution of the two parthenogens, and should represent the divergence of the species. The extremely low divergence of the two parthenogens

indicated that each of them is likely established from one F1 hybrid female which successfidly formed one parthenogenetic lineage (single origin). This is concordant to previous studies and generally true in many parthenogenetic lizards (e.g., Moritz et al., 1989, 1992a). However, this is in contrast to the finding among L. raddei and its daughter species, in which not only closely- related but also distantly-related L. raddei were involved in the hybridization and multiple hybridization events occurred between the same parental species resulting in rnultipIe clones.

No interspecific variation was observed in the two parthenogens Lacerta armeniaca and

L. dahli even though they have different paternal parents, L. valentini and L. portschinskii, respectively. The females involved in the hybridization had the same or extremely similar mitochondria1 genomes. Again, this contrasts with the observations of great intraspecific variation in L. raddei and its daughter species. Although L. unisexualis and L. uzzelli, which were from the same parental species, are identical (Chapter 4), the other parthenogenetic species with different paternal parents displayed substantial rnfDNA variation (e.g. 2.97% difference between

L. rostombekowi and L. sapphirina; 3.35% between L. rostombekowi and L unisexualis; Chapter

4). The low variation in the maternal parent L. rnixta may also account for the lack of variation between L. dahli and L. arrneniaca. The parthenogenetic species showed no divergence from the maternal parents. This suggests a recent origin for the parthenogenetic Caucasian rock lizards. In comparison, L. raddei and its daughter species showed more variations (e.g., L. rostombekowi vs. L. raddei Egagnadzor,

1.05%; 1;. sapphirina vs. L. raddei Muradiye, 0.5396, Chapter 4). The origin of L. armeniaca and L. dahli are likely to be much more recent than the other five parthenogenetic Caucasian rock lizards. SUMMARY

A phylogeny of the family Lacertidae grouped the lacertids into two clades, which correspond to the current subfamilies Gallotiinae and Lacertinae. The former included genera

Gallotia and Psamrnodromus and the latter include the remaining lacertids. The Lacertinae was firther split into two groups: the African group included all Afiican and Arabian lacertids as well as two Eurasian genera, Eremias and Ophisops; the remaining Eurasian lacertids were included in the Eurasian group. The relationships within the African and Eurasian groups were largely unresolved, although a large number of informative character were present. A recent explosive speciation hypothesis was invoked to expIain the lack of resolution in the analysis. The common ancestor of the Eurasian group, as well as the ancestor of the African group, experienced simultaneous or almost simultaneous multiple speciations. These explosive speciation events left few or no characters fixed on the internodes, which made the phylogenetic reconstruction difficult. The formation of unisexuality in lacertids is phylogenetically constrained. A phylogeny was constructed for Caucasian rock lizards, among which the seven known unisexuals originated from interspecific hybridization. Three major groups were identified. The rudis group included

Lacerta rudis, L. valentini, L. portschinskii and L. parvula; the saxicola group included L. saxicola, L. brauneri, L. lindholmi, 1;. alpha, and L. praticola; the caucasica group included L. caucasica, L. daghestanica, L. derjugni, L. mixta, L. clarkorurn, and L. ruddei (nairensis). The latter two groups formed a sister group relationship. The formation of unisexuality involved inter-clade hybridization, and only between the caucasica group and the rudis group. Furthermore, the hybridization was directional, in that the caucasica group only contributed maternal forms while the rudis group only paternal forms. These patterns can be best explained by the phylogenetic constraints on the origin of unisexuality. The phylogenetic constraints are likely intrinsic and their operation is through co-effect of many factors at multiple levels, although sex chromosomes may play an important role. The divergence of cytochrome-b gene in Lacerta raddei, a maternal form, was substantial, but low divergence was found within its parthenogenetic daughter species. A phylogeny of the mtDNA haplotypes suggested that parthenogenetic species have different types of origin. All populations of parthenogenetic L. rostombekowi shared a common ancestor and may have a single origin; L. sapphirina and L. bendimuhiensis shared a common ancestor and may share a single origin as well; Lacerta unisexualis and L. uzzelli likely originated from separate hybrids, but the hybridization events involved closely related females, because they shared a maternal common ancestor. However, the latter four unisexuals, which are from the same parents, did not cluster together on the phylogeny. The maternal parents of L. sapphirim-L. bendimahiensis and of L. unisexualis-l. uzzelli are distantly related. The hybridization events between I;. raddei and L. valentini which successfully established unisexuals might have occurred at least two times. The divergence of cytochrome-b and ATPase 6 genes was very low in the maternal form L, mi- and zero for both its parthenogenetic daughter species, L. dahli and L. armeniaca. Each of the two unisexuals may have a single origin and their maternal parents are cIosely related. Une phyloghie de la farnille des LacertidJs a mis les espPces dans deux groupes, qui correspondent aux sous-familIes courantes, GallotiinJs et Lacertids. Le premier comprend les genres Gallotia et Psammodromus et le dernier comprend les LacertidJs restant. Les LacertinJs ont 6th fendus davantage dans deux groupes: le groupe africain comprend toutes espPces africaines et arabiennes et deux genres eurasiennes, Eremias et Ophisops; les LacertidJs eurasiens restant ont CtCs inclus dam le groupe eurasien. Les rapports dam le groupe afiicain et le groupe eurasien Ctaient en grande partie non rhsolus, bien qu'un grand nombre des caractPres instructifs ktait pr6sent. Une hypothhse d'un JvJnement de spJciation recent et expIosif a Ctd invoquCe pour expliquer le manque de rdsolution dans I'analyse. L'ancgtre commun du groupe eurasien, ainsi que 11anc6tredu groupe africain, ont subis des spJciations multiple simultandes ou presque simultandes. Ces CvCnements du spJciation explosifs ont laissC peu ou pas de caractPres arrangks sur les internodes, qui rend dificile la reconstruction phylogJnJtique.

La formation de I'unisexuaIitJ chez les LacertidJs est phy1ogJnJtiquement gBnde. Une phylogJnie a Me construite pour les l6zards des rochers caucasiens, parmi qui le sept espPces unisexuelles connues sont provenues d'hybridation interspJcifique. Trois groupes majeurs ont 6tC identifihs. Le groupe du rudis compris Lacerta rudis, L. valentini, L. portschinskii et L. parvula; le groupe du saxicola compris L. saxicola, L. brauneri, L. lindholmi, L. alpha, et L. praticola; le groupe du caucasica compris L. caucasica, L. dughestanica, L. derjugini, L. mixta, L. clarkorum, et L. raddei (nairensis). Les derniers deux groupes ont form6 un groupe. La formation de

I'unisexualitJ est la rJsulte d'hybridation inter-groupe, et seul entre le groupe du caucasica et le groupe du rudis. En outre, l'hybridation &tit directionnelle, dans laquelle le groupe du caucasica seul a contribuk les formes maternelles et le groupe du rudis seul formes paternelles. Ces rnodeles peuvent titre bien expliquds par les contraintes phylogJnJtiques sur l'origine de I'unisexualitJ. Les contraintes phylogJnJtiques sont possiblement intrins&queset leur opkration est a travers co-effet de beaucoup d'agents A niveaux multiples, bien que les chromosomes du sexe peuvent jouer un r6le important. La divergence du g&nedu cytochrome-b dam Lacerta raddei, une forme maternelle, &it substantielle, mais divergence basse se trouvait dam son descendant parthenogJnJtique. Une phylogJnie des haplotypes de I'ADN a sugg6rB que les espkces parthenogJnJtique sont d'origines diffkrentes. Toutes populations de I'espkce parthenogJnJtique L. rostombekowi sont descendues d'un anc&e commun et peuvent avoir une origine simple; L. sapphirina et L. bendimahiensis sont descendues d'un ancetre commun et peuvent aussi avoir une origine simple; Lacerta unisexualis et L. uzzelli ont possiblement provenus d'hybridations uniques, mais les 6venements de

Ithybridation ont impliqubs des femelles Jtroitement apparentkes, parce que ces-derniPres sont descendues d'un ancdtre maternel commun. Cependant, les quatre derniPres especes unisexuelles, qui sont descendues des m&mesespbces paternelles et maternelles, ne foment pas un groupe dam la phylogJnie. Les parents matemels de L. sapphirina-L. bendimahiensis et de L. unisexualis-L. uzzelli ne sont pas Jtroitement apparent&. Les kvenements de l'hybridation entre L. raddei et L. valentini qui ont dtablis des especes unisexuelles ont pu arrivJs au moins dew fois. La divergence des genes cytochrome-b et ATPase 6 Btaient trhs bas dans I'espbce maternelle L. mixta et nu1 pour les deux descendants parthenogJnJtiques, L. dahli et L. armeniaca. Ces deux derniPres peuvent avoir une origine seule des parents maternels parentJs attentivement. Literature Cited

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Appendix I. Specimens examined in Chapter 2. Abbreviations: CAS California Academy of

Sciences, San Francisco; LSUMZ Museum of Natural Science, Louisiana State University; MNCN Museo Nacional de Ciencias Naturales, Madrid; MVZ Museum of Vertebrate Zoology, University of California at Berkeley; ROM Royal Ontario Museum, Toronto.

Acanthodactylus erythrurus, Spain: Chdiz: Punta Palorna, MNCN 1193 1; Adolfics vauereselli,

CAS201617, Uganda: Kabale Dist.: Bwindi Impenetrable National Park; Adolfus jacksoni,

Uganda: Kabale Dist. : Bwindi Impenetrable National Park, CAS20 1605; Algyroides fitzingeri, Italy: Sardinia, ROM 24642; Ameiva ameiva, Guyana: Tukeit, ROM 20530; Cnemidophorus

tigris maximus, Mexico: Baja California, ROM RWM647; Eremias velox, Russia: Daghestan,

ROM 23498; Gallotia galloti, Spain: Canary Is., no voucher number available; Gallotia stehlini,

Spain: Canary Is., no voucher number available; Weliobolus spekii, Kenya: Rift Valley Prov.:

Kajiado Dist., CAS 198923; Lacerta (Archaeolacerta) bedriagae, Italy: Sardinia, ROM 24640;

Lacerta (Archaeolacerta) monticola, Spain: Avila: Sierra de Gredos, MNCN 1383 1; Lacerta

(Teira) andreanszkyi, Morocco: Marrakech: OukbimedBn, MVZ 1782 13; Lacerta (Teira) perspicillata, Morocco: Rabat-Salt: Rabat, MVZ186202; Lacerta (Timon) lepida, Spain: Cadiz:

Benalup de Sidonia, MVZ186068; Lacerta (Timon) pater, Morocco: kin-~euh,MVZ178286; Lacerta media (sstr.), Armenia: Abovyan, ROM 24267; Lacerta schreiberi, Spain: Avila: Cuevas

del Valle, MNCN13904; Lacerta (Archaeolacerta) valentini, Armenia: Sevan, ROM 23861;

Lacerta (Zootoca) vivipara, Russia: St. Petersburg, ROM 24750; Latastia longicaudata, Kenya:

Rift Valley Prov.: Kajiado Dist., CAS 198982; Meroles ctenodactylus, South Afiica: Cape Prov.: 37.1 km S Alexander Bay, LSUMZ H-13 110; Meroles suborbitalis, Namibia: East Spitzkoppe, no voucher number available; Mesalina brevirostris, no location data and voucher number available;

Neusticurus sp., Guyana, ROM 22892; Nucras tessellata, South Afiica: Cape Prov.: Richtersveld

National Forest, LSUMZ H-13 111; Ophisops elegans, Armenia: Chosrov, ROM 23506; Pedioplanis namaquensis, South Africa: Cape Prov.: Richtersveld National Park, LSUMZ H-

13 109; Podarcis sicula, Italy: Tuscany, ROM 2463 7; Podarcis muralis, Spain: Huesca: BaiIos de

Benasque, MNCN23640; Podarcis hispanica, Morocco: T6touan: Asilah, MVZ186232; Psamrnodrornus algirus, Morocco: Tanger: Cap Spartel, MVZ178376; Takydrornus sexlineatus,

Vietnam: Sapa, ROM 26345; Tropidosaura gularis, South Africa: East Cape: Montague Pass, no voucher number available. Appendix IT. Specimens examined in Chapter 3. The numbers in the parentheses are used to

distinguish specimens when multiple specimens were used for a species, which correspond to the numbers behind the taxon names on the trees (Figure 3-1).

Algyroides Jitzingeri, ROM24642, Italy: Sardinia; Lacerta alpina, ROM24375 (I), ROM24372

(2), Russia: Krasnodar, Aisho Mountains, 4S002'N, 039°00'E; "L. alpina", ROM24373, Russia: Krasnodar, Aisho Mountains, 4S0021\J, 039°00'E; L. b. brauneri, ROM28215 (I), Russia:

Krasnodar Dist., vicinity of Sochi, 43"35'N, 03g046'E; L. b. darevskii, ROM Field 10812 (2), Russia: Krasnodar District, Dagomys, 44°40'N, 0390501E; L. b. szczerbaki, ROM.24335 (3),

Russia: Anapa, 44'54'N, 0370201E; L. caucasica, ROM24357, Russia: Daghestan, Khvarshi,

42"2 1'N, 046O06'E; L. clarkorum, ROM26524 (I), ROM26523 (2), Turkey: Mahden, 4 1°12W,

041°42'E, ROM24885 (3), ROM24887 (4), ROM24888 (5), Georgia: Adzgarua, TskaIi Gorge; L.

daghestanica, ROMZ603 ( 1), Russia: Daghestan, Kuli, 42OO 1' 18'W, 047°14'42"E; ROM23 537

(2), Russia: Daghestan, Jengutai, 42°40'36"N, 047OI4'7"E; L. derjugini, ROM26585 ( l), Georgia:

Bakuriani, 41°40N, 0430301E; ROM24377 (2), Georgia: Achaldaba, 41°54'24"N, 043°30'05"E; L.

lindholmi, ROM2493 1, Ukraine, Sebastopol, Feolent Point; L. media, ROM24267, Armenia:

Abovyan, Arailer Mt., 40"24N, 044029'E; L. mixta, ROM24369 (I), ROM24367 (2), Georgia:

Achatdaba, 4 1"54'24"N, 043°30'05"E; L. rnonticola, MNCNl383 1, Spain: Avila: Sierra de Gredos; L. nairensis, ROM23805, Armenia: Adis Mt., 40"23W, 044O42'E; L. parvula, ROM24384 (I), ROM26644 (2), Georgia: Achaldaba, 4 1O54'24''N, O43O3 0'05 "E; L. portschinskii, ROW3953 (I), Armenia: Stepenavan, 4 1'0 1'1 S"N, 044O22'54"E; ROM24862 (2), Armenia: Gosh, 40°44'51"N, 045°01'26"E; L. praticola, ROW4346 (I), Russia: vicinity of Sochi, 43O35'N, 03g046'E; ROM 24904 (2), Russia: Krasnodar, Tulskaya, 44O31'NY040°14'E; L.

raddei, ROW3629 (I), Armenia: Geghart, 40°08'15"N, 044°49'06"E; ROM23736 (2), Armenia: Gosh, 40'44'5 1"N, O45OO l'26"E; ROM2824 1 (3), Turkey: Muradiye, 39°00'N, 043'44'E; L.

rudis, ROM.4313 (1 ), ROM243 14 (2), Georgia: Achaldaba, 4 1"54'24"N, 043'3 O'OS'E; L. saxicola, ROM26971, Russia: Kislovodsk, 43O56'N, 042O44'E; "L. saicola", ROM24388, Russia: Dambay, 43'1 5'N, 041°45'E; L. valentini, ROM23861 (I), Armenia: Sevan, 40°30'58"N,

044°56'26"E;ROM23862 (2), Armenia, Adis Mt., 40"23'N, 044O42'E; L. vkvipura, ROM24750,

Russia: St. Petersburg. Appendix ID. Specimens examined in Chapter 4. The numbers in the parentheses correspond to

those in the distribution map (Fig. 4-2).

Lacerta raddei (n=5) - ROM236 19, Armenia: Egegnadzor, 3g045'N, 04S008'E (1 5);

ROM23681, Armenia: Chosrov National Park, 40°00'54"N, 044O54'56"E (14); ROM23629,

Armenia: Geghart, 40°08'1 S'W, 044°49'06"E (13); ROM.23736, Armenia: Gosh, 40°44'5 1'W,

045"01726"E (3); ROM2824 1, Turkey: Muradiye, 39°00'N7 043O44'E (1 6).

Lacerta nairensis (n=6) - ROM23805, Armenia: Adis Mt. 11, 40°23'N7 044O42'E (12); ROM24780, Armenia: Turnanyan, 41 000'00"N7 044O4OY12"E (1); ROM24843, Armenia:

Apnaguch, 40°27'N, 044O22'E (7); ROM23801, Armenia: Adis Mt. I, southern slope of Gehaim

ridge (1 1); ROMZ 8 19, Armenia: Aragatz Mt., 40'2 1'54"N, 044' 15' 12"E (8); ROM26609,

Armenia: Yerevan, 40' 1 1 '5OY'N, 044O29'48"E (1 0).

Lacerta unisexualis (n=4) - ROM24242, Armenia: Ankavan, 40°38'1 5"N, 044O32'54"E

(5); ROM24985, Armenia: Kutchak, 40" 18' N, 043 O4O' E (9); ROM26800, Armenia:

Nozaduz, 40°30' N, 044O20' E (6); ROM283 18, Turkey: Horason, 3g050N, 042O2OYE

(18). Lacerta rostombekowi (n=3) - ROM23985, Armenia: Papanino, 40°44'N, 044O49'E (4);

ROM24983, Armenia: Spitak, 40'5 1'N, 044" 19'E (2); ROM Field 12134 (no voucher specimens available), Armenia: Gosh, 40'44'5 1'W, 04S001 '26"E (3).

Lacerta uzzelli (n=l) - ROM 28293, Turkey: Horason, 3g050N, 042O2OYE(1 8).

Lacerta bendimahiensis (n=l ) - ROM28249, Turkey: Muradiye, 39"00'N, 043O44'E (16).

Lacerta sapphirina (n=l ) - ROM28276, Turkey: Patnos, 39" 14'N, 042'52'E (17).

Lacerta portschipkii (n=I ) - ROM 23953, Armenia: Stepenavan, 4 1'01 ' 15"N, 044O22'54"E.

Lacerta derjugini (n=l) - ROM 26585, Georgia: Bakuriani, 4 1"4OYN, 043'3 O'E.

Lacerta valentini (n= 1) - ROM 23 861, Armenia: Sevan, 40°30'59''N, 044'56' 16"E. Appendix IV. Specimen examined in Chapter 5.

L. rnixta (n=6) - ROM24369, ROM24366, ROM24367, Georgia: AchaIdaba, 41°S4'24"N, 043O3 lY05"E; ROM26604, ROM26605, ROM26606, Georgia, Bakuriani, 41°40'N, 043O3OYE

L. dahli (n=6) - ROM24078, Armenia: Papanino, 40°44'N, 044'49'E; ROMUO3 1, Armenia: Stepanavan 41 "01 '1 S'N, 044"22'54"E; ROM24939, Armenia: Tumanyan, 4 1"00'00"N,

044'40' 12"E; ROM26529, Georgia: Kodjovi, 4 1"38'32"N, 044'4 1'02"E; ROM26547, Georgia: Kareli, 42'0 1'N, 043'52'E; ROM26562, Georgia: Manglisi, 4 1*43'N, 044"25"E;

L. armeniaca (n=7) - ROM24133, Armenia: Sevan, 4O030'59"N, O44O56' 16"E; ROM24 1 18,

Armenia: Papanino, 40°44'N, 044O49'E; ROMM 152, Armenia: Ankavan, 40'3 8'1 5 "N,

044O32'54"E; ROM24 192, Armenia: Steponavan, 4 1°0 1' 15"N, 044O22'54"E; ROM24753,

Armenia: Sevan Pass 40a41' 12"N, 044'5 1'20"E; ROM24979, Armenia: Kutchak Kutchak,

40" 18' N, 043O40' E; ROM24997, Armenia: Tumanyan, 4 1OOO'OO"N, 0440407l2"E;

ROM26514, Georgia: Bakuriani, 41°40'N, 043O3OYE. Appendix V. The cyt-b and ATPase 6 genes sequences of Lacerta mixta and its parthenogenetic daughter species. Type A = L. mixta: ROM24369, ROM24366, ROM26604; All L. armeniaca;

A11 L. dahli; Type B = L. mixta: ROM24367, ROM26606; Type C = L. mixta: ROM26605. ATPase 6

Type A TGT ATC CCA AGC CTC CTA GGA GTA CCT TTA ATT ATA CTA GCT TTA TTT TTC CCA CTA ATA ATC TGA TTC ACA ACT AAC CGC CTC ATC CAA Type B ...... Type C ......

Type A AAT CGA TAC TCA ACT ATT CAA TCC TCA CTT CTT ACT TAT ATT ACA AAA CAA ATA ATA TTA CCA ATT AAT ATT TCA GGC CAC AAA TGA GCA Type B ...... Type C ...... G ...

Type A AGT ACC TTC ATC ACA CTA ATA CTA ATA CTC ATA CTA CTT AAC ACC CTG GGC CTT CTA CCA TAT ACT TTT ACC CCC ACC ACC CAA CTC TCA Type B ...... Type C ......

Type A . ATA AAT ATA GCT CTT GCC ATG CCA GCT TGA TTA ATA ACA GTT TTA ACT GGG CTA CGA AAT CAA CCC ACA ACC TCA TTA GGC CAC CTC CTA Type I3 ...... A ...... Type C ...... A ......

Type A CCA GAG GGC ACA CCC ATC TTA TTA ATT CCT ATG TTA GTT TTA ATC GAA ACA GCT AGC TTA CTC ATC CGC CCA ATT GCT TTA GGC GTA CGA Type B ...... A...... Type C ...... A,......

Type A CTA ACA GCC AAC CTA ACA GCC GGA CAC CTA TTA ATT CAA CTT ACC TCA ACA GCA GTA CTT GCT CTA ATA AAT ACC ATA ACC ACT ACC GCA Type B ...... Type C ...... Type A ATA ATT ACC CTA TTG ATA CTC ATT TTA TTA TCC TGT CTG GAA GTG GCC GTT GCC Type B ...... Type C ...... 8:: ma- 4.- s:: ma* m.. U-• m-• 8:: U** me. 5:: me* me* 4.. g:: Us. 2:: U.. 2 : : 3:: . 0.. w.. 2:: k 9:: 2:: 2:: B.. 4.- P.. 5:: m** woo g:: 0.. 0.. Wo* g:: Us- mq* U*. 3;; m-. E.. m.. ma* m-. 8:: U*. m-• 4.. 5:: 2:: g:: 2:: U*- U** ; 8:: 3 m.. wE* -. 2:: U U-• 0.. 4.- B..E:: E::13" 8:: 0.. 2:: U.. g:: g:: 2: g:: wU . B*. p.. U.. $1: Us* U 4.. U p.. u.. 4.. t+.* E.. 8:: E.. p.. U.. U g::. . U-• U 4.. 2:: 0. Usw 2:: m-• k.. "-0 g:: 2:: u A,. r).. 0.. k.. 3:: u.. U ::2:: U.. U.. U** 2 : : g:: U-• 4 U-. 0.- Us*g:: gii a** 4.- U.. 8:: Bag U.- B.. 4.. 4.. g:: k.. 2:: 2:: 8:: U.. Us* y? 3:: U** U 8 : : 4.. 0.. 2:: k*. 2:: 2:: U.. k3 . 0.. 0.- 0.. 2:: E:: g:: g:: U-• p.. m.. 3:: 3:: m*. 4.- H-• me* He* t+ 2::

emu C, WaJW WWQI WWW aJwaJ saw WaJaJ WWaJ aww RRR EC RRR RRR ERR RRR ERR gRR $;$ U E*l3k mmH kk@ Hmt3 HHB mmm wmm BEE