The Molecular Systematics of the Side-Blotched Lizards (IGUANIA: PHRYNOSOMATIDAE: UTA)

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The Molecular Systematics of the Side-blotched Lizards (IGUANIA: PHRYNOSOMATIDAE: UTA)

Bradford Damion Hollingsworth

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E TH MOLECULAR SYSTEMATICS OF THESIDE-BLOTCHED LIZARDS

(IGUANIA: PHRYNOSOMATIDAE: UTA)

by BradfordDamion Hollingsworth

A Dissertation in Partial Fulfillment of the Requirements forthe Degree Doctor of

Philosophy in Biology

June 1999 0 1999

Bradford Hollingsworth

II Each person whose signature appears below certifies that this dissertation in their opinion is adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy.

Ronald L. Carter, Professor of Biology

Co-Chairperson L. Lee Grismer, Professor of Biology

H. Pau uchheim, Professor of Geology

William K. Hayes, Associate Profs r of Biology

e. Garry P. Lars 1, Assistant Research Scientist, Beckman Research Institute of the City of Hope

lii ACKNOWLEDGMENTS

I would like to thank the numerous individuals who assisted me during the course of this analysis. Most notable are those who contributed to my growth as a biologist and a person. My committee chairs Ron Carter and Lee Grismer patiently supported me and provided valuable advice both in the lab and field. In addition, I would like to thank Garry

Larson, Bill Hayes, and Paul Buchheim for their assistance as committee members and the remaining members of the faculty and student body in the Department of Natural Sciences at Loma Linda University for offering an educational atmosphere full of inspiration.

I am grateful to the Center of Molecular Biology and Gene Therapy for the use of their automated sequencer and the DNA Sequencing Facility at California State University,

Northridge and their technician Janine Wengert for generating numerous sequences. Many thanks to John J. Wiens at the Carnegie Museum of Natural History for his assistance on conducting the computer analyses and his insights into phylogenetic methods and evolutionary processes.

• I would like to thank the many individuals who helped me during the extensive field work done for this project, including Lee Grismer, Ron Carter, Mike Cryder, Erik Gergus,

Jesse Grismer, Eric Link, Gene Kim, Chris Mattison, Erik Mellink, Roland Sosa,

Humberto Wong, Nelson Wong, and the many Mexican ranchers and fisherman who served as guides and naturalists, in addition to providing me with food and shelter.

TABLE OF CONTENTS

LIST OF FIGURES vi

LIST OF TABLES viii

ABSTRACT 1

INTRODUCTION 3

MATERIAL AND METHODS 9

Sampling 9 Operational Taxonomic Units 9 Ingroup Monophyly 14 DNA Sequence Data 14 Tree Reconstruction 17 Selection of Outgroups 22 Confidence, Support, and Signal 26 Comparison With Previous Analyses 27

RESULTS 31 Confidence, Support, and Signal 31 Combined DNA Sequences 31 Maximum Parsimony Analyses 31 Maximum Likelihood Analyses 42 Comparison With Upton and Murphy (1997) S 45

DISCUSSION 61 Phylogenetic Analyses 61 Comparison with the Analysis of Upton and Murphy (1997) 64 Ancestral Nature of Insular Populations 65 Geographical Concordance 73 Cryptic Species Boundaries 79 Exclusivity and Non-exclusivity 83 Taxonomy 85

LITERATURE CITED 91

APPENDIX 101

Appendix 1: Results of the computer program PAUP 101 LIST OF FIGURES

FIGURES PAGE

1. Locality map showing the location of the eight insular endemic species of Uta. All continental landmasses and unmarked islands are inhabited by Uta stansburiana

2. Locality map showing the region of study and the 98 samples used in this analysis 10

3. Outgroup topologies of phrynosomatid genera: a) traditional topology of Etheridge and de Queiroz (1988) and Wiens (1993); b) combined tree of Reeder and Wiens (1996) and Wiens and Reeder (1997); and c) combined data tree of Schulte et al. (1998) 24

4. Strict consensus tree of the unweighted parsimony analysis using 98 ingroup samples and 11 outgroup taxa 33

5. Strict consensus tree of the weighted parsimony analysis (Tv:Ti = 5) using 98 ingroup samples and 11 outgroup taxa 40

6. Maximum likelihood tree using the same data set as in the parsimony analysis pruned to a total of 56 samples (see Table 6) 43

7. Reanalysis of Upton and Murphy (1997): a) unweighted parsimony analysis; and b) weighted parsimony analysis (Tv:Ti =5) 47

8. Reanalysis of data set from this analysis pruned to 22 taxa to match those of Upton and Murphy (1997; see Table 9): a) unweighted analysis using two outgroup species; b) tree from unweighted analysis (see Fig. 4) pruned to 22 taxa; c) unweighted analysis using 11 outgroup species unconstrained; and d) unweighted analysis using 11 outgroup species constrained to Reeder and Wiens (1996) topology 51

9. Analysis of the combined data set from DNA sequence generated in this analysis and Upton and Murphy (1997): a) unweighted parsimony analysis; and b) weighted parsimony analysis (Tv:Ti = 5) 55

10. Maximum likelihood tree using the combined data set from DNA sequence generated in this analysis and Upton and Murphy (1997): a) unweighted parsimony analysis; and b) weighted parsimony analysis (Tv:Ti = 5) 58

11. Phylogenetic tree from weighted analysis depicting the placement of ancestral insular populations (indicated with heavy bars) 66

FIGURES PAGE

12. Model of evolution for insular taxa derived from mainland ancestors from Grismer (1999c). A, B, C, and a4 are contemporary species depicting the chronological order of island colonization; A being first to colonize and C being the third to colonize. al = first continental ancestor; a, = second continental ancestor; a3 = third continental ancestor; and a4 = the last continental ancestor to evolve. The solid bars indicate the acquisition of derived character states in the continental ancestors 69 13. Map depicting the geographic position of the strongly supported groups within haploclade A (see Fig. 5 for designations) 75 14. Map depicting the geographic position of the strongly supported groups within haploclade B (see Fig. 5 for designations) 76 15. Map depicting the geographic position of haploclades A and B. Contact zone occurs between Santa Agueda (M-22) and San Bruno (M-21.) 80 16. Phylogenetic tee depicting the placement of currently named species and likely evolutionary species 87

vii

LIST OF TABLES

TABLE PAGE

1. Taxonomy of Uta based on Ballinger and Tinkle (1972) and Grismer (1994a)... 4

2. Scalar levels of the hierarchy of replicators, their mode of inheritance and replication, and their expected historical pattern 7

3. Specimen localities with their corresponding codes and species designation 11

4. Sequences of primers used in the PCR and sequencing reactions 15

5. PCR conditions used to amplify cytochrome b and cytochrome oxidase III gene fragments 16

6. Specimens selected for phylogenetic analysis using maximum likelihood 19

7. Summary of parameters in nucleotide substitution models 20

8. List of outgroup taxa used in the maximum parsimony and likelihood analyses 23

9. Samples selected from this analysis to combine with samples in the data set of Upton and Murphy (1997) 28

10. Comparison of likelihood scores from a single tree chosen from the parsimony analysis using the 56 taxa and the DNA sequence from cytochrome b and cytochrome oxidase 46

11. Likelihood ratio tests comparing statistical differences between increasingly complex models of DNA sequence evolution using 56 taxa and the DNA sequence from cytochrome b and cytochrome oxidase 46

12. Sequential Bonferroni correction for statistical tables for the comparison of models using the likelihood ratio test (see Table 9) 46

13. Results of the Wilcoxon Sign-Ranks Tests for the comparison between reduced taxa data sets and their most-parsimonious trees for the unweighted analyses 50

14. Results of the Wilcoxon Sign-Ranks Tests for the comparison between reduced taxa data sets and their most-parsimonious trees for the weighted analyses 53

15. Comparison of likelihood scores from a single tree chosen from the parsimony analysis using the 22 taxa data sets combined from this analysis and Upton and Murphy (1997) 60

viii TABLE PAGE

16. Likelihood ratio tests comparing statistical differences between increasingly complex models of DNA sequence evolution using the 22 taxa data sets combined from this analysis and Upton and Murphy (1997) 60

17. Sequential Bonferroni correction for statistical tables for the comparison of models using the likelihood ratio test (see Table 16) 60

18. Categories of strongly supported and geographically concordant groups of two or more populations 74

ix ABSTRACT

THE MOLECULAR SYSTEMATICS OF THE SIDE-BLOTCHED LIZARDS

(IGUANIA: PHRYNOSOMATIDAE: UTA)

Bradford Damion Hollingsworth

The lizard genus Uta comprises nine currently recognized species with the center of diversity occurring on islands in the Gulf of California and Pacific Ocean. The molecular evolution of the side-blotched lizards is analyzed using 1132 base pairs of mitochondrial

DNA sequence from the cytochrome b and cytochrome oxidase III genes. Samples come from 98 'individuals from 50 islands in the Gulf of California and Pacific Ocean and 48 adjacent populations from Baja California Sur, Baja California, California, New Mexico, and Sonora. All currently recognized species are represented. Both maximum parsimony and maximum likelihood were used to reconstruct phylogenetic relationships. This study is intended to expand on the analysis of Upton and Murphy (1997) who used 21 individuals representing five of the nine species. The deep divergence discovered by Upton and

Murphy (1997) for the two geographically proximate haplotype clades in central Baja

California is supported in this analysis. In Upton and Murphy (1997), the two haplotype groups contained samples located approximately 70 km apart in central Baja California; this distribution gap is narrowed to 10 km. The narrowing of the geographical gap between these deeply diverged haplotype clades and their geographical concordance is strong evidence of a cryptic species boundary. This deep substructuring is compared with the many geographically concordant haploclades nested higher in the tree. In addition, a number of insular populations are placed in a basal position with respect to their potential mainland source populations. This is counter-intuitive to the general notion that island populations are derived from current mainland populations. Due to their more restricted area and smaller effective population size, insular populations are also expected to evolve faster. Instead, this analysis has found that there has been enough evolution and extinction within the ancestors of mainland populations that they are now more closely related to each other, while many of the insular populations have preserved enough of their ancestral genetic sequence to remain in basal positions within the genetic tree. Lastly, the taxonomy of Uta is reevaluated in light of its molecular evolution.

2 INTRODUCTION

As the use of molecular data in phylogenetic analysis grows, issues focusing on sampling, accuracy, tree reconstruction, and the use of phylogenetic techniques at or below the species level have become increasingly prominent (e.g., DeSalle and Volger, 1994;

Graybeal, 1998; Hillis, 1995, 1996, 1998; Hillis and Huelsenbeck, 1992; Hillis et al.

1994; Hudson, 1990). As a result, molecular systematics, starting with the use of mitochondrial DNA sequences to analyze intra- and interspecific relationships (Avise et al.,

1987), has grown past its infancy and is now maturing as the efficiency of generating and

analyzing large amounts of data increases (Hillis, 1996). Consequently, it is currently

possible to undertake complex phylogenetic studies, using thousands of base pairs of

nucleotide sequence data, with reasonable hope of recovering evolutionary relationships

from hundreds of taxa or samples with a high degree of accuracy (Hillis, 1996;

Huelsenbeck and Rannala, 1997; Rannala et al., 1998; Soltis et al., 1998).

Until recently, most complex phylogenetic studies have concentrated on higher level

relationships (e.g., Soltis et al., 1998). In this study, the intra- and interspecific

relationships of side-blotched lizards (Uta) are analyzed using 98 populations and 1132

nucleotide base pairs from two mitochondria' DNA genes. This study is intended to

expand on the analysis of Upton and Murphy (1997) who used 21 individuals

unrepresentative of the entire diversity seen within the genus. The last systematic revision

of Uta recognized six species (Ballinger and Tinkle, 1972), while an additional three have

been subsequently described (Grismer, 1994a). In the last decade, a number of taxonomic

synonymies and changes in rank have taken place (e.g. Collins, 1991; Grismer, 1999a,b;

Upton and Murphy, 1997); however, for the sake of stability the taxonomy of Ballinger

and Tinkle (1972) and Grismer (1994a) will be followed here (Table 1).

3 4

Table 1. Taxonomy of Uta based on Ballinger and Tinkle (1972) and Grismer (1994a). Species Uta anti gua (Ballinger and Tinkle, 1968) Uta encantadae (Grismer, 1994a) Uta lowei (Grismer, 1994a) Uta nolascensis (Van Denburgh and Slevin, 1921) Uta palmeri (Stejneger, 1890) Uta squamata (Dickerson, 1919) Uta stansburiana (Baird and Girard, 1852) Uta stellata (Van Denburgh, 1905) Ilia tumidarostra (Grismer, 1994a) 5 The side-blotched lizards are distributed across most of western North America with the center of highest species diversity occurring in the region of the Gulf of California and Baja California peninsula (Ballinger and Tinkle, 1972; Grismer, 1994b,c). Eight of the nine species are insular endemics, while the single continental species, Uta stansburiana, also occurs on over 55 islands (Fig. 1; Ballinger and Tinkle, 1972; Grismer,

1993, 1999a,b). This wide-ranging distribution provides ample complexity for a study of this nature to be warranted. In fact, the analysis of Upton and Murphy (1997) reveals significant substructuring of the continental species, U. stansburiana, along the Baja

California peninsula and the possibility of cryptic species boundaries.

Despite the advances in molecular systematics and the theoretical issues concerning the use of phylogenetic methods at or below the species level, a number of issues are still being considered. Hudson (1990) emphasizes the importance of distinguishing between a pedigree, where diploid individuals are traced historically and the number of ancestors grows as one proceeds back through time, and a gene tree, where the absence of recombination results in a divergent, branching history. The use of non-recombining replicators (= haplotypes) is analogous to looking at species lineages in a phylogeny.

Therefore, when constructing hypotheses of relationships from clonally inherited sequences of mitochondrial DNA (= gene trees), it is generally understood that individual organisms (or their haplotypes) are used as terminals. In comparison, species (= lineages) are used as terminals when reconstructing a phylogeny (Davis and Nixon, 1992; DeSalle and Vogler, 1994).

The use of gene trees for interpreting interspecific relationships and other species- level processes requires two levels of reductionism through the scalar hierarchy of replicators (Table 2; Frost and Kluge, 1994; Hull, 1980). While both species phylogenies and gene trees are displayed in the form of a diagrammatic tree, they may represent Uta lowei Uta tumidarostra Uta encantadae El 14

stellata

Figure 1. Locality map showing the location of the eight insular endemic species of Uta. All continental landmasses and unmarked islands are inhabited by Uta stansburiana. Table 2. Scalar levels of the hierarchy of replicators, their mode of inheritance and replication, and their expected historical attern. Hierarchical Mode of Replication Mode Historical Examples , Level Inheitance , , Pattern

Genic Clonal Clonal replication Divergent Uniparental inheritance of mtDNA, or concerted evolution of nuclear DNA

Recombinational Chromosomal recombination Reticulate Nuclear DNA

Organismal Clonal Asexual reproduction Divergent Parthogenetic organisms

Recombinational Sexual reproduction Reticulate Biparental organisms i . Species "Clonal" Cladogenesis Divergent Speciation by lineage splitting

Recombinational Hybridization Reticulate Speciation through hybridization _ incongruent histories due to lineage sorting of ancestral polymorphisms (Avise, 1989;

Avise and Ball, 1990; Hudson, 1992; Nei, 1987; Moore, 1995; Neigel and Avise, 1986;

Pamilo and Nei, 1988; Wu, 1991). A gene tree will track a species phylogeny only when exclusive coalescence has occurred at every intemodal period (Moore, 1995). Therefore, critical evaluation is needed when interpreting interspecific relationships or other species- level processes from a single gene tree.

The goals of this study are to recover the complex history of side-blotched lizards, determine interspecific relationships, reveal patterns of biogeographic and evolutionary significance, substantiate the presence or absence of cryptic species boundaries, and uncover potential incongruence between the gene tree and species tree. ERIAL AND METHODS

Sampling

The sampling design used in this study centers on maximizing species diversity and their geographical coverage and minimizing problems associated with phylogenetic analysis. For Uta, the highest species diversity centers on the numerous islands in the

Gulf of California and Pacific Ocean, and the adjacent peninsula of Baja California and

Sonoran mainland (Grismer, 1999a,b). A total of 98 individuals are used in this analysis, representing all currently recognized species (Table 3). Fifty-one come from insular populations, while the remaining 47 are from circum-Gulf localities in Baja California Sur

(BCS), Baja California (BC), California (CA), and Sonora (SON; Fig. 2).

A number of recent empirical and simulation studies demonstrate that the addition of more taxa is not only beneficial in recovering the correct phylogeny, but under many instances, the accuracy of the phylogenetic estimate improves even if the total number of characters stays the same (Graybeal, 1998; Hillis, 1998; Poe, 1998; and Soltis et al.,

1998). Therefore, an attempt was made to include as many individuals from geographically intermediate populations as possible in order to improve phylogenetic accuracy by breaking up potentially long branches (Hendy and Penny, 1989; Swofford et al., 1996). Long-branch attraction was found by Felsenstein (1978) to lead to positively misleading results in parsimony analyses. However, the effort to sample as thoroughly as possible was limited to some extent by the relatively high financial costs involved with collecting DNA sequence data.

Operational Taxonomic Units

The conflation of scalar and specification hierarchies (Frost and Kluge, 1994) in systematic studies is readily apparent in the use of operational taxonomic units (OTUs) as

9 10

I-16 1-15 / 1-14 0 & 1-12 10 •• 1-09

0 1-071-06 I-05 1-02

1-03 1-01

Figure 2. Locality map showing the region of study and the 98 samples used in this analysis. 11

Table 3. S secimen localities with their cones ondin_ codes and species designation. Code Locality Species I-01 Isla Espirltu Santo, BCS Uta stansburiana 1-02 Isla Partida Sur, BCS Uta stansburiana 1-03 Isla Gallo, BCS Uta stansburiana 1-04 Isla Ballena, BCS Uta stansburiana 1-05 Isla San Francisco, BCS Uta stansburiana 1-06 Isla Pardito, BCS Uta stansburiana 1-07 Isla San Jose, BCS Uta stansburiana 1-08 Isla Magdalena, BCS Uta stansburiana 1-09 Isla Santa Catalina, BCS Uta squamata 1-10 Isla Monserrate, BCS Uta stansburiana I-11 Isla Galeras Este, BCS Uta stansburiana 1-12 Isla Galeras Oeste, BCS Uta stansburiana 1-13 Isla Danzante, BCS Uta stansburiana 1-14 Isla Carmen, BCS Uta stansburiana 1-15 Isla Los Coronados, BCS Uta stansburiana 1-16 Isla San Idelfonso, BCS Uta stansburiana 1-17 Isla San Marcos, BCS Uta stansburiana 1-18 Isla Tortuga, BCS Uta stansburiana 1-19 Isla Asuncion, BCS Uta stansburiana 1-20 Isla San Roque, BCS Uta stansburiana 1-21 Isla Natividad, BCS Uta stansburiana 1-22 Isla de Cedros, BC Uta stansburiana 1-23 Isla San Benitos Oeste, BC Uta stellata 1-24 Isla San Benitos Medio, BC Uta stellata 1-25 Isla San Benitos Este, BC Uta stellata 1-26 Isla San Pedro Nolasco, SON Uta nolascensis 1-27 Isla San Pedro Martir, SON Uta palmeri 1-28 Isla San Esteban, SON Uta stansburiana 1-29 Isla Datil, SON Uta stansburiana 1-30 Isla Alcatraz, SON Uta stansburiana 1-31 Isla Tiburon, SON Uta stansburiana 1-32 Isla Patos, SON Uta stansburiana 1-33 Isla San Lorenzo Sur, BC Uta antigua 1-34 Isla San Lorenzo Norte, BC Uta antigua 1-35 Isla Salsipuedes, BC Uta antigua 1-36 Isla Roca Lobos, BC Uta antigua 1-37 Isla Lagartija, BC Uta antigua 1-38 Isla Rasa, BC Uta stansburiana 1-39 Isla Cardonosa Este, BC Uta stansburiana 1-40 Isla Cardonosa Norte, BC Uta stansburiana 1-41 Isla Angel de La Guarda, BC Uta stansburiana 12

Table 3 (con't). Specimen localities with their corresponding codes and species desi nation. Code Locality Species 1-42 Isla Pond, BC Uta stansburiana 1-43 Isla Cabeza de Caballo, BC Uta stansburiana 1-44 Isla La Ventana, BC Uta stansburiana I-45 Isla Piojo, BC Uta stansburiana 1-46 Isla Smith, BC Uta stansburiana 1-47 Isla Mejfa, BC Uta stansburiana 1-48 Isla Granito, BC Uta stansburiana 1-49 Isla Encantada, BC Uta encantadae I-50 Isla Coloradito, BC Uta tumidorostra 1-51 Isla El Muerto, BC Uta lowei M-01 El Chorro, Cape Region, BCS Uta stansburiana M-02 Ensenada Los Muertos, BCS Uta stansburiana M-03 Playa Tecolote, BCS Uta stansburiana M-04 La Paz, 36 km. N of, BCS Uta stansburiana M-05 La Paz, 55.5 km N of, BCS Uta stansburiana M-06 La Paz, 82 km N of, BCS Uta stansburiana M-07 San Evaristo, BCS Uta stansburiana M-08 La Presa, BCS Uta stansburiana M-09 Rancho San Juanito, La Presa region, BCS Uta stansburiana M-10 Santa Rita, 20 km S of, BCS Uta stansburiana M-11 San Carlos, BCS Uta stansburiana M-12 Ciudad Insurgentes, 45 km E of, BCS Uta stansburiana M-13 Loreto, 96 km S of, BCS Uta stansburiana M-14 Agua Verde, BCS Uta stansburiana M-15 Puerto Escondido, BCS Uta stansburiana M-16 San Nicolas, BCS Uta stansburiana M-17 La Purfssima, BCS Uta stansburiana M-18 Arroyo Los Burros, BCS •Uta stansburiana M-19 Bahla San Juanico, 10 km S of, BCS Uta stansburiana M-20 Mulege, BCS Uta stansburiana M-21 San Bruno, BCS Uta stansburiana M-22 Santa Agueda, turn-off from Hwy 1, BCS Uta stansburiana M-23 Santa Rosalia, 20 km N of, BCS Uta stansburiana M-24 Tres Wrgenes, Geothermal Road, BCS Uta stansburiana M-25 San Ignacio, 37 km W of, BCS Uta stansburiana M-26 San Francisco de La Sierra, BCS Um stansburiana M-27 Bahia Asuncion, BCS Uta stansburiana M-28 Puerto Nuevo, BCS Uta stansburiana M-29 Punta Eugenia, BCS Uta stansburiana M-30 Guerrero Negro, BCS Uta stansburiana M-31 Bahla de Los Angeles, BC Uta stansburiana M-32 El Parador, 35 km N of, BC Uta stansburiana M-33 Catavifia, BC Uta stansburiana

Table 3 (con't). Specimen localities with their corresponding codes and species desi.nation. Code Locality Species M-34 Bahfa San Luis Gonzaga, BC Uta stansburiana M-35 Campo Cinco Islas Uta stansburiana M-36 San Felipe, BC Uta stansburiana M-37 Arroyo Santo Domingo, BC Uta stansburiana M-38 Temecula, CA Uta stansburiana M-39 Moorpark, CA Ilia stansburiana M-40 BahIa Kino, SON Uta stansburiana M-41 Punta Chueca, 15 km N of, SON Uta stansburiana M-42 Punta Sargent°, SON Uta stansburiana M-43 Puerto de La Libertad, SON Uta stansburiana M-44 El Desemboque, SON Uta stansburiana M-45 San Emetario, SON Uta stansburiana M-46 Bahia Cholla, SON Uta stansburiana M-47 Dona Ana, N.M. U. S. stejnegeri 14 fully transitory between the different hierarchical levels. For example, typology is a

common practice in molecular systematics where species are substituted for OTUs that in

reality represent genetic haplotypes (Doyle, 1992; Moritz et al., 1992). This is a potentially

problematic practice because the OTUs which are based on genic level properties become

transformed into species level entities at least two scalar levels above their origin.

Therefore, in this analysis, haplotypes will be treated and referred to at the population level,

one scalar level above their genic level origin.

Ingroup Monophyly

The currently recognized monophyly of Uta is based on 20 unambiguous

synapomorphies (Reeder and Wiens, 1996). Sixteen of the characters come from

mitochondrial ribosomal DNA sequence data and four from morphology and color pattern.

The morphological characters include the polymorphic presence of five non-autotomous

caudal vertebrae, the first supralabial contacting second infralabial, an increase in frequency

of white spots on nape, and the polymorphic presence of ventrolateral spots or stripes

(Reeder and Wiens, 1996).

DNA Sequence Data

Mitochondrial DNA sequence was collected using techniques out-lined in Hillis et

al. (1996) and Palumbi (1996a). All primer sequences are listed in Table 4. A 478 base- pair DNA fragment from the cytochrome b gene was PCR amplified using the primers

GLUDG-L (= L14704; Palumbi, 1996b) and H15149 (Kocher et al., 1989) following the thermocycler conditions listed in Table 5. In addition, DNA fragments from cytochrome oxidase III gene were PCR amplified using the same light strand primer L9193 and three different heavy strand primers positioned at H9829, H9925, and H9977 (David Orange, 15

Table 4. Se uences of rimers used in the PCR and se uencing reactions. Name Gene Sequence Application GLUDGL Cytochrome b 5I-TGA CU GAA RAA CCA YCG TTG-3' PCR/Sequence (L14704)

H15149 Cytochrome b 5t-AAA CTG CAG CCC CTC AGA ATG ATA PCR/Sequence it I GTC CTC A-3'

L9193 Cytochrome 5'-CAT GAT AAC ACA TAA TGA CCC ACC PCR/Sequence oxidase Ill AA-3'

H9829 Cytochrome S-GGA AGG AAG ACC CGA TGA TGA C-3' PCR oxidase III

H9925 Cytochrome 5-ACT ACG TCT ACG AAA TGT CAG TAT PCR oxidase ifi CA-3'

H9977 Cytochrome 51-ACT AAG AGA GTA GGA TCC TCA TCA PCR oxidase III ATA-3'

L9513 Cytochrome 51-CCA CTC AAG CCT AGC ACC AAC C-3' Sequence oxidase III 16

Table 5. PCR conditions used to amplify cytochrome b and cytochrome oxidase III gene fra ments. Thermocycler Conditions Primer Combination GLUDGL and H15149 60s(94°C), 60s(52°C), 60s(72°C) for 35 cycles L9193 and H9829 120s(94°C); 60s(94°C), 60s(52°C), 90s(72°C) for 35 cycles; 600s(72°C) L9193 and H9925 120s(94°C); 60s(94°C), 60s(52°C), 90s(72°C) for 35 cycles; 600s(72°C) L9193 and H9977 120s(94°C); 60s(94°C), 60s(52°C), 90s(72°C) for 35 cycles; 600s(72°C) 17 pers. comm. 1999) for various samples following the thermocycler conditions listed in

Table 5. All sequences were generated with Applied Biosystems, Inc., Fluorescent

Automated Sequencers 373 and 377. For cytochrome b, the two PCR primers were used in the sequencing reaction. For cytochrome oxidase III, the light strand primer L9193 and an internal primer, L9513, were used to generate 654 base-pairs of DNA sequence corresponding to the shortest of the three DNA templates used (e.g., template generated with primers L9193 and H9829). The two genes combined totaled 1,029 nucleotide base pairs, excluding primer sequences.

The computer program Sequencher 3.0 (Gene Codes Corporation, 1995) was used to verify peak calls and align sequences. Alignment of both genes was relatively straightforward. Pairwise alignments were first assembled for all ingroup samples and then combined with the alignment of assembled outgroup taxa. A single two base pair gap was inserted in the outgroup taxon Sceloporus grandaevus and a one base pair gap at the same site in Uma notata and U. scoparia. Aligned sequences were exported from

Sequencher 3.0 as a NEXUS file, formatted for phylogenetic analysis using MacClade

3.01 (Maddison and Maddison, 1992), and executed in PAUP* (version 4.0b2a;

Swofford, 1998). Formatting in MacClade 3.01 included codon position assignment and transition versus transversion weighting using Sankoff stepmatrices (Sankoff and

Rousseau, 1975). In PAUP*, base-pair composition, pair-wise variation, codon position variation, and total variation were calculated.

Tree Reconstruction

All phylogenetic analyses were performed using PAUP* (version 4.0b la;

Swofford, 1999). The combined DNA sequence data set was initially analyzed using unweighted (= equally) parsimony analysis on only phylogenetically informative 18 characters. Hypotheses were formulated using cladistic methods, with the principle that only shared derived character states are useful in recovering phylogenetic history (Hennig,

1966; Wiley, 1981). Shortest trees were sought using heuristic searches with 50 random addition sequence replicates per search and TBR branch swapping. Subsequent weighted parsimony analyses were conducted using the transversion to transition weight of five.

This weight was selected by calculating the transition to transversion ratio using likelihood analysis which resulted in a ratio of 4.90 for the data set including both ingroup and outgroup taxa and 6.03 for only ingroup taxa. Five was selected as a whole number average between the two calculations. Homoplasy levels (e.g., character incongruence) were evaluated with the consistency index (Kluge and Farris, 1969) and retention index

(Farris, 1989,1990). Because the consistency index is affected by uninformative characters, these were excluded from the calculations.

Maximum likelihood was used to compare the relative likelihoods of trees from the parsimony analyses, to compare the goodness-of-fit of different models of sequence evolution to the observed data, and to search for optimal likelihood trees. Due to the large number of taxa and computer software limitations, the likelihood analysis was unable to run to completion within the time constraints of this degree program (e.g., based on preliminary attempts at analyzing the total taxa data set, the analysis would need four or five years to complete). Therefore, the total number of taxa was judiciously pruned of "non- essential" specimens as judged by the shortest trees from the parsimony analyses

(Graybeal, 1998; Hillis, 1998; Poe, 1998). A total of 45 specimens were kept in the analysis based on a number of variables including their basal position within strongly supported clades, species designation, and biogeographic interest (Table 6).

Shortest trees (n = 138) were generated with an unweighted parsimony analysis, one of which was used to analyze six nested models of increasing complexity (Table 7; loosely following Huelsenbeck and Crandall, 1997; Sullivan et al., 1997): (1) Jukes- 19

Table 6. S secimen selected for h lo_enetic anal sis usin maximum likelihood. Code Locality Species I-01 Isla Espirltu Santo, BCS Uta stansburiana 1-07 Isla San Jose, BCS Uta stansburiana 1-09 Isla Santa Catalina, BCS Uta squamata 1-10 Isla Monserrate, BCS Uta stansburiana 1-15 Isla Los Coronados, BCS Uta stansburiana 1-17 Isla San Marcos, BCS Ilia stansburiana 1-18 Isla Tortuga, BCS Uta stansburiana 1-22 Isla de Cedros, BC Uta stansburiana 1-25 Isla San Benitos Este, BC Uta stellata 1-26 Isla San Pedro Nolasco, SON Uta nolascensis 1-27 Isla San Pedro Martir, SON Uta palmeri 1-28 Isla San Esteban, SON Uta stansburiana 1-31 Isla Tiburon, SON Uta stansburiana 1-33 Isla San Lorenzo Sur, BC Uta antigua 1-38 Isla Rasa, BC Uta stansburiana 1-41 Isla Angel de La Guarda, BC Uta stansburiana 1-49 Isla Encantada, BC Uta encantadae I-50 Isla Coloradito, BC Uta tumidorostra 1-51 Isla El Muerto, BC Uta lowei M-01 El Chorro, Cape Region, BCS Ilia stansburiana M-03 Playa Tecolote, BCS Uta stansburiana M-07 San Evaristo, BCS Uta stansburiana M-09 Rancho San Juanito, La Presa region, BCS Uta stansburiana M-11 San Carlos, BCS Uta stansburiana M-14 Agua Verde, BCS Uta stansburiana M-16 San Nicolas, BCS Uta stansburiana M-21 San Bruno, BCS Uta stansburiana M-22 Santa Agueda, turn-off from Hwy 1, BCS Uta stansburiana M-24 Tres Virgenes, Geothermal Road, BCS Um stansburiana M-25 San Ignacio, 37 km W of, BCS Uta stansburiana M-27 Bahla Asuncion, BCS Uta stansburiana M-31 Bahla de Los Angeles, BC Uta stansburiana M-33 Catavifia, BC Uta stansburiana M-35 Campo Cinco Islas Uta stansburiana M-37 Arroyo Santo Domingo, BC Uta stansburiana M-38 Temecula, CA Uta stansburiana M-40 Bahia Kino, SON Uta stansburiana M-42 Punta Sargento, SON Uta stansburiana M-46 Bahla Cholla, SON Uta stansburiana M-47 Dona Ana, N.M. U. s. stejnegeri

Table 7. Summary of Parameters in Nucleotide Substitution Models. Substitution Nucleotide Base Pair Invariable Among-site Rate Model Substitution Fres uenc Sites Variation Jc All six equal All four equal none none K2P Ti/Tv unequal* All four equal none none HKY85 Tifrv unequal* All four unequal* none none HKY85 + I Ti/Tv unequal* All four unequal* yes equal HKY85 +I+ F Ti/Tv unequal* All four unequal* yes gamma GTR+I+1" All six unequal* All four unequal* yes gamma *Settings for unequal parameters are estimated from data 21 Cantor (JC; Jukes and Cantor, 1969; assuming equal rates of change for transitions and transversions and equal base frequencies), with no invariable sites, and no among-site rate variation; (2) Kimura two parameter (K2P; Kimura 1980; assuming different rates of change for transitions and transversions and equal base frequencies), with no invariable site or among-site variation; (3) Hasegawa-Kishino-Yano (HKY85; Hasegawa et al., 1985); different rates for transitions and transversions and unequal base frequencies) with no invariable sites or among-site rate variation; (4) HKY85 with some sites assumed to be invariable but equal rates of change assumed at variable sites (=HKY85 + I; Hasegawa et al., 1985); (5) HKY85 with some sites assumed to be invariable and variable sites assumed to follow a gamma distribution (.111(Y85 +I + r; Gu et al., 1995); and (6) general time reversible (GTR; Yang, 1994; assuming a different rate for all six classes of substitutions), with some sites assumed to be invariable, and variable sites assumed to follow a gamma distribution (= GTR + + Specific model parameters for likelihood analyses were estimated from the data using PAUP* (e.g., base frequencies, transition-transversion ratios, proportion of invariable sites, gamma distribution shape parameters). Using maximum likelihood, the goodness-of-fit of different models to the observed data can be evaluated by comparing likelihoods for different models for the same tree. Statistical significance of differences of the models were evaluated using the likelihood-ratio test statistic —21ogA (the difference between the negative log likelihoods for the two models, multiplied by two), which should approximate a chi-square distribution, with the degrees of freedom equal to the differences in the number of parameters between the two models

(Yang et al., 1995). A sequential Bonferroni correction was employed because multiple tests were being performed (Rice, 1989). The best-fitting model was then used in a heuristic search to find the overall best likelihood topology. 22 Selection of Outgroups

The outgroups used in this analysis were selected from the phylogenies of

Phrynosomatidae proposed by Etheridge and de Queiroz (1988), Reeder and Wiens

(1996), Schulte et al. (1998), and Wiens (1993). A total of 11 different species, representing the genera Petrosaurus, Sceloporus (+ Sator sensu Wiens and Reeder,

1997), Uma, and Urosaurus, were used as outgroups in the parsimony analyses (Table 8).

In the likelihood analysis these were pruned to five species, one species to represent each of the five outgroup genera (Table 8). To test for root sensitivity, all parsimony analyses

, were subjected to both unconstrained and constrained outgroup topologies. In the unconstrained analyses, all outgroup tam were chosen to be paraphyletic with respect to the monophyletic ingroup, thus allowing the arrangement of the outgroup taxa to be based on the optimization of each character using the outgroup method (Maddison et al., 1984;

Watrous and Wheeler, 1981). In the constrained analyses, the hypotheses of Etheridge and de Queiroz (1988) and Wiens (1993), Reeder and Wiens (1996) and Schulte et al. (1998) were tested by using the constraints option in PAUP*. The topology of Etheridge and de

Queiroz (1988) and Wiens (1993), considered the more traditional arrangement, places

Sceloporus + Urosaurus, Petrosaurus, and the sand lizards (which includes Uma) as successive first, second, and third outgroups of Uta (Fig. 3a). In contrast, the combined data topology of Reeder and Wiens (1996) places Uta at the base of a (Petrosaurus

(Sceloporus + Urosaurus)) clade with the sand lizards forming a more distant second outgroup (Fig. 3b). The combined data topology of Schulte et al. (1998) is similar to

Reeder and Wiens (1996) in the placement of Uta at the base of the sceloporine clade (Fig.

3c); however, the arrangement of the genera within this clade is markedly different from the relationships proposed by both Etheridge and de Queiroz (1988) and Reeder and Wiens 23

Table 8. List of outgrou taxa used in the maximum arsirnony and likelihood analyses. Maximum Parsimony Analysis Maximum Likelihood Analysis Petrosaurus mearnsi Petrosaurus repens Petrosaurus repens Sceloporus grandaevus Sceloporus grandaevus Sceloporus occidentalis Sceloporus hunsakeri Uma scoparia Sceloporus occidentalis Urosaurus graciosus Sceloporus orcutti Uma notata Uma scoparia Urosaurus graciosus Urosaurus lahtelai Urosaurus nigricaudus 24

Figure 3. Outgroup topologies of phrynosomatid genera: a) traditional topology of Etheridge and de Queiroz (1988) and Wiens (1993); b) combined tree of Reeder and Wiens (1996) and Wiens and Reeder (1997); and c) combined data tree of Schulte et al. (1998). g (,) cr CD Cla CD 4< " CD Holbrookia CD 0-1 Cr) Holbrookia Uma \C) \C) Callisaurus • Callisaurus Phrynosoma Uma Uma Uta Phrynosoma Phrynosoma Sator Petrosaurus Uta Petrosaurus Uta Petrosaurus Urosaurus Urosaurus Urosaurus Sceloporus Sator Sceloporus Sceloporus 26 (1996). The topology of Schulte et al. (1998) resurrects the genus Sator and proposes the relationship (Sator (Petrosaurus (Sceloporus + Urosaurus))).

Confidence, Support, and Signal

Nonparametric bootstrap analysis (Felsenstein, 1985a) was used to evaluate confidence for reconstructed trees from the parsimony analyses. For each analysis, 2000 bootstrap replicates were performed using the fast-heuristic search option in PAUP*. Only bootstrap values of 50% and over are reported and clades under 70% are considered weakly supported. A 70% bootstrap value or above was found by Hillis and Bull (1993) to correspond to a 95% or greater probability of a group being correctly resolved.

Only unambiguous character state changes (those found using both ACCTRAN and

DELTRAN optimization routines) are discussed as support for a given clade, although ambiguously placed character state changes may also be important in tree reconstruction

(Reeder and Wiens, 1996). To examine the stability of individual clades, trees slightly longer than the most parsimonious tree were examined to see if the recovered topology was among the near-shortest trees of one step longer (Bremer, 1988; Hillis and Dixon, 1989).

Clades not supported in slightly longer trees (one step longer) are considered to be weakly supported, while those present in the slightly longer trees are not necessarily strongly supported (Wiens and Reeder, 1997).

The combined data set was examined for phylogenetic signal using the g1 statistic

(Fitch, 1979, 1984; Hillis, 1991; Hillis and Huelsenbeck, 1992; Huelsenbeck, 1991) because it is possible to generate a single most parsimonious tree from a data set that has levels of homoplasy comparable to random data (Hillis and Huelsenbeck, 1992). The gl statistic measures the skewness of the distribution of random trees (10,000 random trees used in this analysis) to discern phylogenetic information relative to random noise. Critical 27 values for random data were derived by extrapolating values from Table 2 of Hillis and

Huelsenbeck (1992). It would be preferable to generate critical values from the randomization of the combined data set (Wiens and Reeder, 1997), however, it was too large to make such an analysis feasible within the time constraints of this study.

Comparison With Previous Analyses

The results of the recent analyses of Upton and Murphy (1997) are compared to the results of this analysis. Upton and Murphy (1997) analyzed 21 populations of Uta using a total of 890 base pairs of mitochondria' DNA sequences from the cytochrome b (555 bp) and ATPase 6 (335 bp) genes. A total of 340 base pairs within the cytochrome b sequence, between position 14843 and 15398, overlaps between this analysis and theirs. Two comparisons were made. First, separate analyses were conducted on their combined data set (hereafter referred to as the "U&M data set") and the combined data set of this analysis pruned of the populations not represented in their analysis (hereafter referred to as the

"BDH data set"). Secondly, a combined data set (hereafter referred to as the "total combined data set") was constructed from the two analyses limited to the populations used in their analysis. Both unweighted (= equally) and weighted analyses were performed.

For the latter, a transversion to transition weight of 5:1 was chosen based on the transition to transversion ratio for the combined data set of 5.32.

In the separate analyses, DNA sequence data from Upton and Murphy (1997) was obtained from GenBank (accession numbers UA46695—UA46743) for 20 of the 21 populations used in their analysis (Table 9; the population from El Arco was unavailable from GenBank). Upton and Murphy (1997) fail to list which outgroup species were used to root their tree, although sequences from Petrosaurus meamsi and Urosaurus nigricaudus are included within their GenBank submissions. It is assumed that these two Table 9. Samples selected from this analysis to combine with samples in the data set of Upton and Murphy (1997). Accession Accession ROM Taxon Upton and Murphy Locality Sample From This Analysis % Difference Numbers Numbers Specimen (as listed by them) Cyt b ATPase 6 46718 46696 123 Urosaurus nigricaudus — 0-3.3 Urosaurus nigricaudui N 46719 46695 4109, Petrosaurus mearnsi — , 0-4.0 Petrosaurus lnearnsi 46723 46700 1525 Uta antigua Isla San Lorenzo Sur 1-33 Isla San Lorenzo Sur 5631 Uta palmeri Isla San Pedro Martir , I-27 Isla San Pedro Martir 46720 4 46697 Uta squamata 46722 46699 1518 Isla Santa Catalina 1-09 Isla Santa Catalina , 13934 Uta stansburiana 46724 46701 - Penasco, SON M-46 Bahia Cholla, SON 2.65% 46725 46702 5623 Uta stansburiana Isla Cedros 1-22 Isla de Cedros 2.65% 46726 46703 1231 Uta stansburiana Bajia de Los Angeles 1-46 Isla Smith 3.24% 1583 Uta stansburiana 46727 46704 , Isla Monserrate I-10 Isla Monserrate 2.35% , 46728 46705 5651 Uta stansburiana Isla San Esteban 1-28 Isla San Esteban 2.35% 46729 46706 — 14471 Uta stansburiana San Lucas , M-21 San Bruno, BC5 2.65% 46730 46707 1527 Uta stansburiana Isla Angel de La Guarda I-42 Isla Pond 2.35% 46732 46708 5544 Uta stansburiana Isla Santa Cruz, CA M-38 Temecula, CA 5.00% 15/9 Uta stansburiana 46733 46709 Isla Mejia ,I-47 Isla Mejia_ 2.65% Uta stansburiana 46735 46710 1672 Isla Danzante 1-13 Isla Danzante _ _ 2.35% 46736 46711 94 Uta stansburiana Isla Rasa 1-38 Isla Rasa 2.65% 46737 46712 852 Uta stansburiana La Presa M-09 Rancho San Juanito 3.24% 46738 46713 14383 Uta stansburiana Isla San Marcos 1-17 Isla San Marcos 2.06% 46740 46715 1247 Uta stansburiana Arroyo de San Regis M-32 El Parador, BC 5.00% 46742 46717 4586 Uta stansburiana San Ignacio M-24 Tres Virgenes, BCS 3.82% 5398 U. stansburiana stejnegeri _ _ , 46743 46716 M-47 Dona Ana, NM 46721 46698 4618 Uta stellata Isla San Benitos 1-25 Isla San Benitos Este 29 species were used as outgroups in their analysis. The BDH data set was modified from the original by pruning 78 populations to match the populations used in their analysis (Table

9). Populations from this study were chosen by comparing geographically proximal samples to those in Upton and Murphy (1997) and selecting the most similar based on percent differences (Table 9). In the combined analysis, the total combined data set was constructed from the data sets used in the separate analyses, excluding the overlapping cytochrome b sequence. The overlapping sequence was alternatively deleted from either source to determine if there is any effect on the results. The total combined data set includes 1,682 base pairs of sequence from the cytochrome b (693 bp), ATPase 6 (335 bp), and cytochrome oxidase III (654 bp) genes. The three data sets (U&M, BDH, and total combined) from both the separate and combined analyses were analyzed using maximum parsimony according to the procedures and parameters listed above for the original analysis.

All data sets and results were compared against each other and against the results of the original analysis. Incongruence between the data sets was examined in three ways.

First, data sets were analyzed and the support for conflicting clades was evaluated using non-parametric bootstrapping (Felsenstein, 1985a). Although this is not a statistical test of incongruence in the strict sense, it is a test of whether or not there are clades that are in strongly supported disagreement between data sets, and unlike other incongruence tests, can identify which clades are in conflict. Second, the incongruence length difference test

(ILD) proposed by Farris et al. (1994) was used to test for overall significant conflict between the data sets. This test involves finding the incongruence index (Michevich and

Farris, 1981) for the data sets analyzed separately, and then randomly repartitioning the combined data to generate a null distribution of this statistic assuming no significant conflict. All parsimony-uninformative characters were removed from each data set prior to analysis (not merely the invariant characters as recommended by Cunningham, 1997), and 30 1,000 replicates were analyzed for each pair of data matrices, using the "partition homogeneity" test in PAUP* (Swofford, 1999). Finally, the Wilcoxon signed-ranks test

(WSR, Templeton, 1983) was used to test whether or not a given data set significantly rejected the best tree from another data set (as recommended by Larson, 1994), based on the number of changes in each character on each topology. A list of changes was obtained using the "compare two trees" option in MacClade 3.01 (Maddison and Maddison, 1994).

The critical values for a two-tailed test (table 30 of Rohlf and Sokal, 1981) were used to determine statistical significance (Felsenstein, 1985b; Larson, 1994). RESULTS

Phylo genetic Signal

The g1 analyses indicate that all data sets used in this study contain substantial phylogenetic signal (as opposed to random data) based on significantly left-skewed g1 values at P < 0.01 (Hillis and Huelsenbeck, 1992). The lengths of 10,000 randomly sampled trees resulted in a g1 value of -0.270 for these taxa. The critical g1 value for random data for 10 four-state characters (such as DNA sequences) from 25 or more taxa is

—0.16 (P = 0.05; Hillis and Huelsenbeck, 1992). Therefore, the g1 value indicates that the data set is significantly more structured than random data.

Combined DNA Sequences Data

The data consist of 1132 aligned nucleotide positions from the cytochrome b and cytochrome oxidase III genes. This aligned sequence includes 103 nucleotide positions of the four terminal PCR primers of the two DNA fragments (located at the aligned positions

1-26, 632-675, 1099-1132). The total aligned sequence contains 31.9% adenine (A) nucleotides, 25.9% cytosine (C), 14.7% guanine (G), and 27.5% thymine (T). The data set contains a total of 356 parsimony informative characters, of which a minimum of 260 are informative within the ingroup. Of the 356 parsimony informative characters, 58

(16.3%) are located at first codon positions, 17 (4.8%) at second codon positions, and 281

(78.9%) at third codon positions.

Maximum Parsimony Analyses

The unweighted parsimony analysis was stopped after 30,000 trees were found due

to time and computer memory limitations. All PAUP results are presented in Appendix 1.

The results of the unweighted data set were sensitive to the different rooting topologies.

31 32 The unconstrained and Reeder and Wiens (1996) outgroup topologies produced the most

parsimonious trees (1895 steps) with identical strict consensus trees (Fig. 4). These trees

are preferred over those produced using the traditional and Schulte et al. (1998) constrained

outgroup topologies, each having most parsimonious trees of 1898 and 1901 steps,

respectively. The unconstrained and Reeder and Wiens (1996) trees differ from these

longer trees primarily in the arrangement of the most basal internal nodes. Of the studies

focusing on phrynosomatid relationships, the most comprehensive to date is that of Reeder

and Wiens (1996) who used all available morphological data and 779 bp of DNA sequence.

Because Reeder and Wiens (1996) is the most thorough analysis of phrynosomatid

relationships to date and both the unconstrained and Reeder and Wiens (1996) outgroup

arrangement produced the shortest trees, the most parsimonious trees from these two

analyses are favored over the longer trees produced with the other outgroup arrangements.

The most parsimonious trees each have a consistency index of 0.31 (excluding

uninformative characters) and a retention index of 0.76. Total character state

transformations for both ACCTRAN and DELTRAN optimizations are presented in

Appendix 1. Hereafter, only unambiguous character state transformations will be reported

as support for individual samples or haplociades. The results of the analysis retaining trees

up to one step longer than the shortest trees and bootstrap values of above 50% are reported

within each figure showing an evolutionary tree.

The strict consensus tree (Fig. 4) results in a gene tree with a basal polytomy of the

haplotype from the Dono Ana, New Mexico population (=Uta stansburiana stejnegeri)

and two haplociades, A and B (see Fig. 4 for haplociade designations). The Dono Ana

population is geographically the most discontinuous of all the samples included in this

study and has diverged from the ancestral condition (as determined by the outgroup

method) by 20 phylogenetically informative nucleotide positions in addition to five uninformative ones. Haploclade A (node 1) includes both peninsular and insular samples 33

Figure 4. Strict consensus tree of the unweighted parsimony analysis using 98 ingroup samples and 11 outgroup taxa.

34 Ono scoparia ,Utna notate Petrosaurus repress =Petrosaurus nrearnsi Urosaurus graciosus Urosaurus lahtelai Urosaurus nigricaudus Sceloporus grenulaevus Sceloporus occidortalis Sceloporus orcurti Sceloporus hunsakeri M-47 Dona Ana. N.M. 1-09 Isla Santa Catalina. BCS 72% I-I5 Isla Los Coronados 64% 1-13 Isla Danzante 4 I-14 Isla Carmen 63% 1-01 Isla Espiritu Santo 1-02 Isla Partida Sur 1-03 Isla Gallo 1-04 Isla Ballena M-03 Playa Tecolote 80% M-04 La Paz, 36km N of 2 74% M-01 El Chorro M-02Ensenada Los Muertos 2 84% r .,4.0_058 Lau N of 78% M-07 San Evaristo 78% r---- M-06 La Paz- 82km N of 10P M-10 Santa Rita 78% M-09 Rancho San bunko 12 87%i3 1-07 Isla San Jose 53% 85% 1-05 Isla San Francisco 14 1-06 Isla Panlito 66% M-I4 Agua Verde 80% M-12 Ciudad 1nsurgentes 5 16 62% M-13 Loreto, 96km S of 17 M-15 Puerto Escondido 69% 1-08 Magdalena 6 20 M-11 San Carlos 19 86% I-17 lsla San Marcos 21in M-20 Mulege 7 M-2I San Bruno 18 100% 1-12 Isla Galleras Genie I-10 Isla Monserrate 8 24 1-11 Isla Galleras Este 22 r 1-16 Isla Idelfonso 9 26 I M-I6 San Nicolas 25 76%76% M-19 Bahia San Juanico 271 6918r—...--. M-17 La Purissima 10 M-18 Army° Los Burros 1-38 Isla Rasa 1-39 Isla Cardonosa Este 100% I-40 Isla Cardonosa None 1-41 Isla Angel de La Guarda 30 1-42 Isla Pond 1-47 Isla. Mejfa 1-48 Isla Granito 1-36 Isla Roca Lobos 100% 58% 1-33 Isla San Lorenzo Sur 3- 34 1-34 Isla San Lorenzo None 2 33 67% 1-35 Isla Salsipuedes 35 1-37 Isla Lagartija 1-27 Isla San Pedro Martir M.24 Tres Virgenes 100% 31 9 52% E M-22 Santa Agueda 3 M-23 Santa Rosalia 40 M-26 San Francisco 38 1-19 Isla Asuncion 99% 20 Isla San Roque 411 M-27 Bahia Asuncion 81% M 28 Puerto Nuevo 42 M-29 Punta Eugenia 1-45 Isla Piojo M-3I Bahia de Los Angeles 77% 1-43 Isla Cohort de Caballo 84% 46 1-46 Isla Smith 37 74% 1-44 Isla La Ventana M-32 El Parador 44 1-50 Isla Coloradito M-34 Gonzaga 48 73% 52% 1-49 Isla Encantada M-33 Catavina 4 50 96% 51 Isla El Muerto 51 M-35 Campo Cinco Islas 100% M-25 San Ignacio 4 54 M-30 Guerrero Negro 1-22 Isla Cedros 1-24 Isla San Bonitos Medio 00%1 56 8 96% 1-21 Isla Natividad 571_4 62% — 1-23 Isla San Bonitos °este 58 1-25 Isla San Bonitos Este 1-18 Isla Tortuga 52 16:228 sanSan PedroEsteb:olasco 601= 1 lila 9 97% M-36 San Felipe 10 1= M-37 Santo Domingo 96% I-- M-38 Temecula 5 11 62.— M-39 Moorpark M-43 Porno de La Libenad M-44 El Desemboque *M-45 San Ernetario 84% M-46 Cholla Bay 63 75% 1-32 Isla Patos 12 M-42 Punta Sargent° Unweighted 1-30 Isla Alcatraz 1-31 Isla Tiburon M-41 Punta Chueca Parsimony Analysis 58% r— 1-29 Isla Datil 67/ M-40 Bahia Kino 35 from San Bruno and Isla San Marcos, BCS southward to El Chorro, BCS in the Cape

Region and is supported by nine nucleotide changes. This haploclade corresponds to the

"Southern" group of Upton and Murphy (1997). Haploclade A (node 1) has a 63% bootstrap value. Haploclade B (node 29) includes all populations north of haploclade A and is supported by six nucleotide changes. This haploclade is not supported by bootstrap values of above 50%. In both haploclades, there are numerous well-supported groups, all of which contain geographically proximal populations.

Within haploclade A (node 1), the population from Isla Santa Catalina (= Uta squamata) forms the most basal branch and has diverged from its ancestral node by 20 informative nucleotide positions in addition to four uninformative ones. The remaining populations form a haploclade (node 2) supported by 17 nucleotide changes and a 80% bootstrap value. Within this group, the first haploclade (Al) contains three insular populations from Islas Los Coronados, Danzante, and Carmen and is supported by 10 nucleotide changes with a 75% bootstrap value. The remaining samples form a haploclade

(node 5) that is weakly supported (<50%) in the bootstrap analysis, but contain a number of geographically concordant haploclades (A2—A10) and six unresolved haplotypes in the

strict consensus tree. Haploclade A2 (node 6) is formed by two of the southernmost populations from the Cape Region and in 93% of the most parsimonious trees forms a clade with five of the six unresolved populations: Islas Espiritu Santo, Partida Sur, Gallo,

Ballena, and Playa Tecolote. The placement of the population from 36 km N. of La Paz

appears to be the source of conflict for the unresolved nature of these populations in the

strict consensus tree.

Of the remaining groups, haploclade A3 (node 7) consists only of peninsular populations in the La Presa region in the southern Sierra Giganta and is supported by five nucleotide changes and a 74% bootstrap value. Haploclade A4 (node 12) consists of the populations from Islas San Francisco, Pardito, and San Jose and an associated peninsular 36 population from Rancho San Juanito, BCS. This group is supported by two nucleotide changes and a 78% bootstrap value; within this haploclade, Islas San Francisco, Pardito, and San Jose are adjacent islands and form a strongly supported haploclade (node 13).

Haploclade A5 (node 15) consists of populations from the eastern portion of the central

Sierra Giganta near Loreto and is supported by two nucleotide changes and a 66% bootstrap value. In the strict consensus tree, haploclade A4 and AS are a sister group wealdy supported by three nucleotide changes and a 53% bootstrap value. Haploclades A6

(node 20), A7 (node 21), and A9 (node 26) each consist of a insular population and its most adjacent peninsular counterpart. Haploclade A8 (node 23) consists of three insular populations from the northern coast of Isla Monserrate and two of its satellites, Islas

Galeras Este and Galeras Oeste, lying 5 km to the north. Haploclade Al0 (node 27) consists of three geographically proximal populations from the western portion of the central Sierra Giganta near La Purissima to the west coast of the peninsula at Bahia San

Juanico and is supported by four nucleotide changes and a 76% bootstrap value.

Within haploclade B (node 29), many of the internal nodes (31, 36, 38, 43, 52, 53,

59, and 64).are weakly supported by unambiguous characters and bootstrap values of less than 50%. In the strict consensus tree, these nodes form the majority of the structure within this haploclade. The most basal branch is formed by haploclade B1 (node 30) and consists of populations from Islas Rasa, Cardonosa Norte (=Pardito Norte), Cardonosa

Este, Angel de La Guarda and its three satellite islands, Granito, Mejfa, and Pond. This haploclade is strongly supported by 21 nucleotide changes and a 100% bootstrap value.

Haploclade B2 (node 32) consists of populations recognized as Uta antigua which includes Islas San Lorenzo Sur, San Lorenzo Norte, Salsipuedes and its two satellite islands, Roca Lobos and Lagartija. It is supported by 28 nucleotide changes and a 100% bootstrap value. The third branch within haploclade B (at node 36) is the population from

Isla San Pedro Martir (= Uta palmeri) which has diverged from its ancestral node by 19 37 informative nucleotide positions in addition to four uninformative ones. The remaining samples form a strongly supported haploclade (node 37; 84% bootstrap value), corresponding to the "Northern" group of Upton and Murphy (1997).

Haploclade B3 and B4 (node 38) form a weakly supported sister group relationship; both containing the first peninsular populations within haploclade B to branch off the tree. Haploclade B3 is from central Baja California immediately north of the northernmost population in haploclade A and is supported by 13 nucleotide changes and a

100% bootstrap value. The population from the turn-off to Santa Agueda along Mexican

Highway 1. is located 17 km from the nearest haploclade A population from San Bruno,

Haploclade B4 consists of populations from the Vizcaino peninsula and two of its near- shore islands, Islas San Roque and Asuncion and is supported by 14 nucleotide changes and a 99% bootstrap value.

Haploclades B5 and B6 (node 44) form a strongly supported sister group relationship supported by a 74% bootstrap value. Haploclade B5 consists of populations from Bahia de Los Angeles and El Paradox and the four more prominent islands within

Bahia de Los Angeles. Haploclade Bb consists of populations further to the north near

Bahia San Luis Gonzaga and its adjacent islands Islas Coloradito, Encantada, and El

Muerto. The population from Isla Coloradito (= Uta tumidarostra) is positioned most basal within haploclade B6 and has diverged from its ancestral node by four informative nucleotide positions in addition to one uninformative ones. The population from Isla

Encantada (= Uta encantadae) forms a weakly supported relationship with the population from Catavifia and has diverged from its ancestral node (node 50) by three informative nucleotide positions in addition to one uninformative ones. The population from Isla El

Muerto (= Uta lowei) forms a strongly supported relationship with the Campo Cinco Islas population, located immediately across from of island, and is supported by five nucleotide 38 changes and a 95% bootstrap value. Isla El Muerto has diverged from its ancestral node

(node 51) by two informative nucleotide positions in addition to one uninformative ones.

Haploclades B7 and B8 (node 53) form a weakly supported relationship between the populations from San Ignacio and Guerrero Negro in the Vizcalno desert and the islands extending off the tip of the Vizcaino peninsula. Haploclade B8 (node 55) is strongly supported by 12 nucleotide changes and a 100% bootstrap value. It consists of populations from Islas Cedros, Natividad, and San Benitos Este, Medio, and Oeste.

Interestingly, the three populations from Islas San Benitos (= Uta stellata) are not each others closest relatives. Islas San Benitos Oeste and Este (node 58) are more closely related to the population from Isla Natividad (node 57; 96% bootstrap value), than to Isla

San Benitos Medio. Instead, the latter population shares a weakly supported relationship

(53% bootstrap value) with Isla Cedros.

The remaining populations within the strict consensus tree (node 59 and above) are from northern Baja California, California, and Sonora, with the exception of Isla Tortuga.

This seemingly geographically disjunct population is located 30 km off the coast of Santa

Rosalia, BCS in the Gulf of California. The nearest population within this haploclade

(node 59) is found 150 km across the Gulf of California at Bahfa Kino, Sonora. The nearest peninsular population within the haploclade is located 470 km to the northwest at

San Felipe, BC. Also within the haploclade (node 59), the populations from Islas San

Esteban and San Pedro Nolasco form a weakly supported sister group relationship (node

60; 60% bootstrap value). The population from Isla San Pedro Nolasco Uta nolascensis) has diverged from its ancestral node by 15 informative nucleotide positions in addition to four uninformative ones. This relationship is interesting because Isla San Pedro

Nolasco is separated from Isla San Esteban by 140 km of uninterrupted water in the eastern portion of the Gulf of California. 39 Haploclades B10 and B11 each consist of strongly supported sister group relationships among geographically most proximal populations. Haploclade B10 (node 61) consists of the populations from San Felipe and Arroyo Santo Domingo in northern Baja

California and is supported by 10 nucleotide changes and a 97% bootstrap value.

Haploclade B11 (node 62) consists of populations from Temecula and Moorpark in southern California and is supported by nine nucleotide changes and a 96% bootstrap value.

Haploclade B12 (node 63) consists of all the populations from Sonora and its adjacent islands that include Islas Alcatraz, Tiburon, Datfl, and Patos. This haploclade is supported by two nucleotide changes and a 84% bootstrap value. Within this haplociade,

Isla Patos, located north of Isla Tiburon, forms a strongly supported sister group relationship (node 65; 75% bootstrap value) with the adjacent mainland population of Punta

Sargento, over the equally close Isla Tiburon. The sample from Isla Tiburon used in this analysis came from the opposite side of the island along the southwest coast in Arroyo

Sauzal. This population forms a strongly supported haploclade with the populations from

Punta Chueca, Bahia Kino, and Islas Datfi and Alcatraz (node 66; 92% bootstrap value).

In the parsimony analyses that weighted transversions greater than transitions by five steps, the strict consensus tree resulted in the same topology for all rooting topology options (Fig. 5). The weighted analysis (Tv:Ti = 5) resulted in 10,502 most parsimonious trees 2,406 steps in length, each with a consistency index of 0.32 and a retention index of

0.76. The relationships are identical to the unweighted analysis in 56 of the 67 nodes in the unweighted analysis (see Fig. 4). The majority of the differences between the weighted and unweighted trees occurs at the position of the haplotype from the Dono Ana, New

Mexico population (= Uta stansburiana stejnegeri) and many of the internal nodes within haploclade B. The Dono Ana, New Mexico population is positioned as the most basal branch within the ingroup. The basal position resolves the polytomy found in the 40

Figure 5. Strict consensus tree of the weighted parsimony analysis (Tv:Ti = 5) using 98 ingroup samples and 11 outgroup taxa.

41 Lima scoparia Ulna notata Petrosattrus repent Petrosaurus meamsi Urosaurus eraciosus Urosauntslahtelai Urosaurus nigricaudus Sceloporus grandaevits Sceloponts occidentalis r— seceeroPp°„rrl'iss. '11ttreigikerii M-47 Dona Ana. N.M. 1-09 Isla Santa Catalina. BCS 72% 1-15 Isla Los Comnados 3 64 1-13 Isla Danzante 1-14 Isla Cannen 77% 1-01 Isla Espiritu Santo 1-02 Isla Partida Sur 1-03 Isla Gallo I-04 Isla Ballena M-03 Playa Tecolote 89% M-04 La Paz. 361m N of 77% r--- M-01 El Chorro 2 6 ' M-02 Ensenada Los Muenos 73% i M-05 La Paz. 551m Not 74% 1 8 M-08 La Presa M-07 San Evaristo M-06 La Paz. 821m N of 9 66% 10 M- I 0 Santa Rita 61% M-09 Rancho San Juanito 1-07 Isla San Jose 2; 73% 4 3 89% 1-05 Isla San Francisco I 06 Isla Pardito 69% M-14 Agua Verde M-12 Ciudad Insurgentes 5 ri 75% 16 M-13 Loreto. 96km S of 17 M-15 Puerto Escondido 67% i -08 Magdalena 6 20 Ml! San Carlos 9 79% I 1- 17 Isla San Marcos 04-20 Mulege 7 21 M-2I San Bruno 1-12 Isla Galleras Oeste 99% 1-10 Isla Monserrate 8 23 24 1-11 Isla Galleras Este

2 1-16 Isla Ide!fonso 9 26 M-16 San Nicolas 72% M-I9 Bahia San Juanico I 10 27 58% M-17 La Purissima 28 M-18 Arroyo Los Burros 1-39 Isla Cardonosa Este I-40 Isla Cardonosa None 00% 1-41 Isla Angel de to Guarda 30 1-42 Isla Pond 1-48 Isla Granito 1-38 Isla Rasa 1-47 Isla Mejfa 1-27 Isla San Pedro Monk -9 1-36 Isla Roca Lobos 100% 57% 1-33 Isla San Lorenzo Sur 32 34 1-34 Isla San Lorenzo None 2 3 70% 1-35 Isla Salsipuedes 31 35 1-37 Isla Laganija 28 Isla San Esteban 1-26 Isla San Pedro Nolasco 9 97% M-36 San Felipe 10 6IP M-37 Santo Domingo 98% M-38 Temecula 11 62 01-39 Moorpark 1-18 Isla Tonuga 86% 57% M46 Cholla Bay 37 C 81% 04-45 San Emetario 63 f---- M43 Puerto de La Libertad 04-14 El Desemboque 68% 1-32 Isla Patos 12 65 54-42 Punta Sargento 1-30 Isla Alcatraz 1-31 Isla Tiburon 04.41 Punta Chueca 88%67i 1-29 Isla Datil N140 Bahia Kino 99% 04-25 San Ignacio 7 54 1 M-30 Guerrero Negro 84-22 Santa Agueda 100% M-23 Santa Rosalia 3 39 M.24 Tres Virgenes M-26 San Francisco 57% 1-19 Isla Asunci6n 100% 20 Isla Sat Roque Il 78% 04-27 Bahia Asuncion M-28 Puerto Nuevo 42 04-29 Punta Eugenia 59% I-22 Isla Cedros 100% 561 1-24 Isla San Bonitos Medio 8 55 90% 1-21 Isla Natividad 1-23 Isla San Benitos Oeste 58 1-25 Isla San Benitos Este Weighted Parsimony 145 Isla Piojo 51-31 Bahia de Los Angeles 75% 7% 1-43 Isla Caheza de Cahallo 5 46 146 Isla Smith Tv/Ti = :44 Isla La Ventana 81% 04-321! Pamdor 44 47 1-50 Isla Coloradito 73% M-34 Gonzaga 149 Isla Encantada 48 69% 6 • 49 2% Catavina 1-51 Isla El Motto 51 M-35 Campo Cinco Islas 42 unweighted analysis between the Dona Ana population and haploclades A and B; the latter two being sister groups (node A) within the weighted analysis, although this relationship is only weakly supported by six nucleotide changes and a bootstrap value of less than 50%.

Within haploclade B, the haploclades (B1—B12) remained strongly supported between the weighted and unweighted analyses, with the exception of haploclade B9.

However, there are a number of differences within the weakly supported internal nodes.

The position of haploclade B2 (node 32; = Uta antigua) and the population from Isla San

Pedro Martir (= U. palmeri) are reversed. Within the remaining haploclades (B3—B12; node 37 and above), there are six nodes unique to the weighted analysis (B—G), and the loss of seven nodes (38, 40, 43, 52, 53, 59, and 60) present in the unweighted analysis.

As a result, the structure seen within the unweighted analysis is reversed. In the weighted topology, the more nested haploclades B9—B12 (node 59; Fig. 4) are placed more basal to haploclades B3—B8. The loss of node 60 (haploclade B9) separates the populations from

Islas San Esteban and San Pedro Nolasco (= U. nolascensis) with the former positioned as the most basal branch among haploclades B3—B12. An unresolved polytomy occurs between the haplotype of Isla San Pedro Nolasco (= U. nolascensis), haploclades B10,

B11, B12, and the remaining haploclades B3—B7 occurs at node B (Fig. 5). Within haploclade B12, a strong north to south arrangement is developed with the addition of nodes C, D, and E. Haploclades B3—B7 form an unresolved polytomy at node F (Fig. 5).

Maximum Likelihood Analysis

The parsimony analysis of the reduced taxa data set (56 specimens) resulted in 138 shortest trees (Fig. 6). A single tree from these was used to test the various nucleotide substitution models. The FIKY85 I + F model (the second most parameter rich) of nucleotide sequence evolution has the best goodness-to-fit over the four other models tested 43

Figure 6. Maximum likelihood tree using the same data set as in the parsimony analysis pruned to a total of 56 samples (see Table 6). 44

Ulna scoparia Petrosaurus repens Sceloporus occidentalis Urosaurus graciosus Sceloporus grandaevus 1-27 Isla San Pedro Martir 1-38 Isla Rasa 1-41 Isla Angel de La Guarda M-47 Dona Ana, NM 1-09 Isla Santa Catalina M-16 San Nicolas 1-10_1sla_Monserrate M-11_San_Carlos r 1-17 Isla San Marcos I M-21 San Bruno M-14 Agua Verde 1-07 Isla San Jose flM-09 Rancho San Juanito 1-15 Isla Los Coronados M-07 San Evaristo M-03 Playa Tecolote 1-01 Isla Espiritu Santo M-01 El Chorro 1-33 Isla San Lorenzo Sur M-22 Santa Agueda M-24 Tres Virgenes 1-22 Isla de Cedros 1-25 Isla San Benitos Este M-25 San Ignacio M-27 Bahia Asuncion M-31 Bahia de Los Angeles 1-50 Isla Coloradito 1-49 Isla Encantada M-33 Catavina 1-51 Isla El Muerto M-35 Campo Cinco Islas M-38 Temecula M-37 Santo Domingo 1-26 Isla San Pedro Nolasco 1-28 Isla San Esteban I-18 Isla Tortuga Maximum Likelihood Tree M-46 Bahia Cholla -In likelihood = -7101.83 M-42 Punta Sargento (n = 45) 1-31 Isla Tiburon M-40 Bahia Kino 45 with a negative log likelihood score of -7602 (Table 10). Likelihood ratio tests show that this model significantly improve the likelihood score over the next less complex model

(HKY + I) and the next more complex model (GTR + I + F; Tables 11 and 12). The

HKY85 + I + F model was used to estimate phylogenies for the reduced taxa data set using maximum likelihood.

A single likelihood tree was found and is shown as a cladogram in Figure 6. The negative log likelihood score was —7101.83. The likelihood tree supports many of the relationships found in the weighted and unweighted parsimony analyses, except for the more basal nodes. In the likelihood tree (Fig. 6), the basal positions contain the populations from Isla San Pedro Martir (= Uta paimeri) and those associated with Isla

Angel de La Guarda, respectively. The population from Dona Ana, N.M. (= U. stansburiana stejnegeri) is found to be the sister haplotype to haploclade A, while the population from Isla San Lorenzo Sur is found to be the sister haplotype to haplociade B.

Comparison With Upton and Murphy (1997)

The reanalysis of Upton and Murphy (1997) produced two most parsimonious trees for the unweighted analysis with the strict consensus tree being compatible with their results (Fig. 7a). The shortest trees from the U&M data set were 583 steps with a consistency index of 0.45 and a retention index of 0.55. These trees are one step shorter than the shortest trees of Upton and Murphy (1997) with the difference being accounted for by the absence of the population from El Arco. In the reanalysis, the strict consensus tree relationships between populations from La Presa, Isla Monserrate, Isla San Marcos, and

San Lucas are unresolved (Fig. 7a). In the weighted analysis, two most parsimonious trees were found, each 1,189 steps with a consistency index of 0.51 and a retention index

Table 10. Comparison of likelihood scores from a single tree chosen from the parsimony analysis using the 56 taxa and the DNA sequence from cytochrome b and cytochrome oxidase III. Nucleotide Substitution Model -in Likelihood Value Jc 8974 K2P 8567 HKY85 8476 HKY85 + I 7669 HKY85 + I + F 7602* GTR+I-Fr 8090 *Best model.

Table 11. Likelihood ratio tests comparing statistical differences between increasingly complex models of DNA sequence evolution using 45 taxa and the DNA sequence from cytochrome b and cytochrome oxidase DI. The degrees of freedom is the difference in the number of parameters between the models (see Table 7). All tests are significant using a sequential Bonferroni correction (Rice, 1989) Model Comparison -21ogA degrees of P value freedom JC vs. K2P 814 1 <.005 K2P vs. HKY85 182 4 <.005 HKY85 vs. HKY85 + 1 1614 1 <.005 HKY85 + I vs. HKY85 + I + r 134 1 <.005 IIKY85 +I+Fvs.GTR+I-Fr 488 4 <.005

Table 12. Sequential Bonferroni correction for statistical tables for the comparison of models using the likelihood ratio test (see Table 11). P 1 P5 uncorrected P alk P < alk ? Accept chi-square value table values? P 1 .005 a& (.05 / 5) = .01 Yes Yes .005 P2 - 1 (.05 /4) = .025 Yes Yes P3 .005 cd1+ k (.05 / 3) = .067 Yes Yes .005 P4 a/1+ k (.05 /2) = .025 Yes Yes P5 .005 a/1+ k -i (.05 / i)=.05 Yes Yes 47

Figure 7. Reanalysis of Upton and Murphy (1997): a) unweighted parsimony analysis; and b) weighted parsimony analysis (Tv:Ti = 5). 48 Petrosaurus meanzsi Urosaurus nigricaudus Uta stansburiana stejnegeri

55% Uta palmeri

100% Isla Mejla Isla Rasa Isla Angel de La Guarda Uta antigua Uta squamata 72% Isla Danzante -La Presa Monserrate Isla San Marcos -San Lucas Isla San Esteban 84% Penasco, SON

Unweighted Parsimony 62% Isla Santa Cruz, CA Upton and Murphy (1997) Two Outgroup Species 85% Arroyo de San Regis Strict Consensus Tree Bahia de Los Angeles 2 Trees San Ignacio 583 steps CI = .45 100 Isla Cedros RI = .55 Uta stellata

Petrosaurus mearnsi Urosaurus iligricaudus Isla San Marcos 71% r-Uta squamata Isla Danzante La Presa San Lucas Isla Monserrate Uta palmeri 66% Uta stansburiana stejnegeri Isla Mejla 100% Rasa Isla Angel de La Guarda Uta antigua Isla San Esteban Weighted Parsimony San Ignacio (Tv:Ti = 5:1) 93% Upton and Murphy (1997) Penasco, SON 56% Two Outgroup Species t00% r: Isla Cedros Strict Consensus Tree 2 Trees Uta stellata

1189 steps 55% Isla Santa Cruz, CA CI= .51 .1221/-Arroyo de San Regis RI= .57 Bahla de Los Angeles 49 of 0.57. The weighted topology places the populations from southern Baja California at • the base of the tree, while Uta pahneri, Uta stansburiana stejnegeri, the populations associated with Isla Angel de La Guarda, and those from more northern localities are nested more sequentially in the tree, respectively (Fig. 7b).

In unweighted analyses of the BDH data set (pruned of 78 ingroup samples to match the samples used in Upton and Murphy), the results varied in the degree of resolution found in the strict consensus trees depending on the number of outgroups used.

When only Urosaurus nigricaudus and Petrosaurus meamsi are included, a single most- parsimonious trees was found, 578 steps in length, with a consistency index of 0.47 and a retention index of 0.60. This tree was significantly different (Table 13; P .01) from those produced with the U&M data set; however, after the application of a Bonferroni correction, the two topologies are most likely not significantly different. The unweighted tree of the BDH data set places the haploclade of Islas Angel de La Guarda, Melia, and

Rasa in the most basal position within the gene tree and Uta antigua (= Isla San Lorenzo

Sur) as sister group to the populations from the southern portion of Baja California (Fig.

8a). This tree is not significantly different (Table 13) from the shortest trees from the original analysis of 98 samples pruned down to the Upton and Murphy (1997) samples

(Fig. 8b). When 11 outgroup samples are used, either unconstrained or constrained to the

Reeder and Wiens (1996) topology, the strict consensus trees become less resolved with most of the internal nodes collapsing (Fig. 8c,d).

The incongruence length difference (ILD) found that there was not significant conflict between the U&M and BDH data sets (P = 0.82). Wilcoxon signed-ranks tests

(Tables 13 and 14) found that the U&M data set and its two unweighted and two weighted trees differed significantly from the single tree from the BDH data set in either the unweighted or weighted analyses. Likewise, the BDH data set and its unweighted and Table 13. Results of the Wilcoxon Sign-Ranks Tests* for the comparison between reduced taxa data sets and their, most-parsimonious trees for the unweighted analyses (= equal weights). Data Set and BDH BDH Tree U Sz. M U & M Combined Combined Combined Shortest Tree Pruned 1 Tree 1 Tree 2 Tree 1 Tree 2 Tree 3 Tree BDH Data Set n = 51 n=44 n=50 n = 43 n = 43 n= 43 Tree 1 = 662 T =357 Ts = 357 7= 36O 7= 362 Ts = 340 ns P .01t P .0051- ns ns ns M Data Set n = 38 n = 49 — n=10 n = 4 n = 1 Tree 1 7= 351 Ts = 240 Ts = 20 Ts = 2 T, = 0 ns P .005t ns ns ns & M Data Set n = 48 n = 54 — n = 22 n = 18 n = 15 Tree 2 . 564 Ts = 360 Ts = 110 T= 72 Ts = 49 ns P .05t ns ns ns Combined Data Set n = 37 n = 74 n= 15 n= 29 Tree 1 T =255 Ts = 1056 Ts = 49 T=168 ns ns ns ns Combined Data Set n = 35 n=75 n=7 n = 23 Tree 2 T =210 Ts = Imo T=9 Ts . 99 P .05* ns ns ns Combined Data Set n = 37 n=77 n=1 n = 17 Tree 3 Ts = 340 ,T=1151 T=O T=48 P .05* ns ns ns *The null hypothesis is that the data set and its shortest tree are not in conflict with the shortest tree from another data set. tAfter a Bonferroni correction, these findings are not significantly different. 51

Figure 8. Reanalysis of data set from this analysis pruned to 22 taxa to match those of Upton and Murphy (1997; see Table 9): a) unweighted analysis using two outgroup species; b) tree from unweighted analysis (see Fig. 4) pruned to 22 taxa; c) unweighted analysis using 11 outgroup species unconstrained; and d) unweighted analysis using 11 outgroup species constrained to Reeder and Wiens (1996) topology. 52

Petrosaurus mearnsi Petrosaurus mearnsi Urosaurus nigricaudus Urosairrus nigricaudus Isla Angel de La Guarda (1-42) Isla Rasa (1-38) Urn stansburiana stejnegeri (M-47) Urn squamata (1-12) Isla Mejfa (1-47) LE Isla Danzante (I-13) (Ira stansburiana stejnegeri (M-47) La Press (M-09) (Ira palmeri (I-27) Isla Monserrate (1-10) Uta antigua (1-33) Isla San Marcos (1-17) (Ira squamata(I-09) San Lucas (M-21) La Presa (M-09) Isla Rasa (1-38) Isla Danzante (1-13) Isla Angel de La Guarda (1-42) Isla Monserrate (1-10) [Isla Mejfa (1-47) Isla San Marcos (1-17) (Ira antigua (1-33) _LE San Lucas (M-21) Uta pahneri (1-27) Isla San Esteban (1-28) San Ignacio (M-24) San Ignacio (M-24) Bahia de Los Angeles (1-46) Isla Santa Cruz, CA(M-38) EArroyo de San Regis (M-32) Penasco, SON (M-46) Isla San Esteban (1-28) Arroyo de San Regis (M-32) Isla Santa Cruz Bahia de Los Angeles (1-46) EPenasco, SON (M-46) Isla Cedros (1-22) Isla Cedros (1-22) Uta stellata (1-25) Um stellata (1-25) BDH Data Set Total Data Set This Study (data set pruned) This Study (trees pruned) Two Outgroup Species 11 Outgroup Species Strict Consensus Tree (pruned to 2 for comparison) 1 Tree Unconstrained 578 steps Strict Consensus Tree CI = .47 30,000 Trees RI = .60 587 steps CI = .47 RI = .59

Petrosaurus mearnsi Petrosaurus iseams: Urosaurus nigricaudus Urosairrits nigricaudus Use stansburiana stejnegeri (M-47) Ufa stansburiana stejnegeri (M-47) (Ira antigua (1-33) antigua (1-33) Utapalmed (1-27) Lira pa/uteri (1-27) _EIsla Rasa (1-38) -Isla Rasa (1-38) Isla Angel de La Guarda (I-42) IsIa Angel de La Guarda (1-42) Isla Mejfa (1-47) *-1sla Mejfa (1-47) -Uta squamata (I-12) Urn .s-quatnata (1-12) f---La Presa (M-09) r—La Prcsa (M-09) AH..... I---Isla Danzante (1-13) Isla Danzante (1-13) Isla Monserrate (I-10) Isla .Monserrate (1-10) Isla San Marcos (1-17) Isla San Marcos (1-17) San Lucas (M-21) San Lucas (M-21) Isla San Esteban (I-28) Isla San Esteban (1-28) San Ignacio (M-24) San Ignacio (M-24) Isla Santa Cruz (M-38) Isla Santa Cruz (M-38) __/ Penasco, SON (M-46) -Penasco. SON (M-46) Arroyo de San Regis (M-32) Arroyo de San Regis (M-32) Bahia de Los Angeles (1-46) -Bahia dc Los A ngeles(I-46) Isla Cedros (1-22) Isla Cedros (1-22) (Ira stellata (I-25) Urn stellata (1-25) BDH Data Set BDH Data Set This Study (data set pruned) This Study (data set pruned) 11 Outgroup Species 11 Outgroup Species (pruned to 2 species for comparison) (pruned to 2 species for comparison) Unconstrained Reeder and Wiens Constraint Strict Consensus Tree Strict Consensus Tree 19 Trees 19 Trees 578 steps 580 steps CI = .47 CI = .47 RI = .60 RI = .60

Table 14. Results of the Wilcoxon Sign-Ranks Tests* for the comparison between reduced taxa data sets and their most-parsimonious trees for the weighted analyses (Tv:Ti = 5). Data Set and BDH BDH Tree U & M U & M Combined Combined Shortest Tree Pruned 1 Tree 1 Tree 2 Tree I Tree 2 Tree BDH Data Set n= 46 n = 70 n= 68 n = 43 n . 51 Tree 1 = 491 = 605 Ts = 540 Ts = 424 T's . 602 ns P .05t P .05f ns ns U & M Data Set n = 58 n=65 n . 67 n = 62 Tree 1 T5 = 728 Ts . 584 = 1013 Ts . 864 ns P .05t ns ns U & M Data Set n= 64 n = 65 n= 65 n= 58 Tree 2 T =934 Ts = 615 Ts . 961 Ts = 784 ns P .05t ns ns Combined Data Set n = 48 n=74 n= 109 n= 105 Tree 1 T( = 534 Ts = 1070 Ts = 2119 T=1959 ns ns ns ns Combined Data Set n =52 n= 84 n=104 n.97 Tree 2 = 621 Ts = 1401 Ts = 1963 T . 1574 ns ns ns ns *The null hypothesis is that the data set and its shortest tree are not in conflict with the shortest tree from another data set. tAfter a Bonferroni correction, these findings are not significantly different. 54 weighted trees differed significantly from the U&M data set trees. However, after the application of a Bonferroni correction, these topologies are most likely not significantly different. Neither of these data sets and their shortest trees could reject the pruned tree from the original analysis or the three trees from the unweighted and two trees from the weighted total combined data set. Similarly, the total combined data set trees could not reject the either the U&M or BDH trees. On the other hand, two of the three total combined data set trees (trees 1 and 2) can reject the pruned tree from the original analysis.

The analysis of the combined data set from Upton and Murphy (1997) and the one from this study (= total combined data set) produced a data set totaling 1,657 nucleotide sequences. No differences were found between shortest trees from the total combined data sets alternatively biased with the overlapping cytochrome b sequence from either this study and that of Upton and Murphy (1997). When rooted with only Urosaurus nigricaudus and

Petrosaurus meamsi, 330 phylogenetically informative characters produced three shortest trees 965 steps in length with a consistency index of 0.46 and a retention index of 0.56 in the unweighted analysis (Fig. 9a). The three trees were not significantly different from the two trees found in the Upton and Murphy (1997) reanalysis (Table 13), although there was less resolution in the placement of Uta antigua and the populations from Santa Cruz

Island, CA (= Temecula, CA) and San Ignacio, BCS (= Tres Virgenes, BCS) became sister haplotypes (see Fig. 8a). When compared to the trees from the BDH data set and the original analysis pruned of taxa (as described above), no significant differences were found

(Table 13). In the weighted analysis of the total combined data set, only slightly greater resolution was obtained in the strict consensus tree (Fig. 9b). Within the strongly supported haploclade containing Islas Angel de La Guarda, Mejla, and Rasa, the latter two form sister haplotypes. Further resolution over the unweighted analysis was also obtained with the placement of the haplociades from northern and southern localities as sister 55

Figure 9. Analysis of the combined data set from DNA sequence generated in this analysis and Upton and Murphy (1997): a) unweighted parsimony analysis; and b) weighted parsimony analysis (Tv:Ti = 5).

Petrosaurus rnearnsi Urosaurus nigricaudus Uta stansburiana stejnegeri (M-47)

100% Uta palmeri (I-27) - Isla Rasa (I-38) 100% 71% Isla Mejla (I-47) - Isla Angel de La Guarda (I-42) Uta antigua (1-33) Uta squamata (I-12) 80% Isla Danzante (I-13) 82% La Presa (M-09) 59% Isla Monserrate (I-10) Isla San Marcos (1-17) San Lucas (M-21) Isla San Esteban (1-28) Unweighted Parsimony 94% Penasco, SON (M-46) Total Combined Data Set Upton and Murphy + This Study San Ignacio (M-24) (taxa pruned) I Isla Santa Cruz (M-38) Two Outgroup Species Strict Consensus Tree 98% Arroyo de San Regis (M-32) 3 Trees 965 steps Bahla de Los Angeles(I-46) CI = .46 Isla Cedros (1-22) RI = .56 Uta stellata (1-25)

Petrosaurus mearnsi Urosaurus nigricaudus Uta stansburiana stejnegeri (M-47)

100% Uta palmeri (1-27) Isla Angel de La Guarda (1-42) 100% 53% 92% Isla Rasa (I-38) Isla Mejla (1-47) Uta antigua (1-33) Uta squamata (1-12) 95% Isla Danzante (1-13) 73% La Presa (M-09) Isla Monserrate (I-10) Isla San Marcos (1-17) San Lucas (M-21) San Ignacio (M-24) Weighted Parsimony (Tv:Ti = 5:1) Isla Santa Cruz (M-38) Total Combined Data Set Upton and Murphy + This Study Penasco, SON (M-46) (taxa pruned) 92% 99% Arroyo de San Regis (M-32) Two Outgroup Species Strict Consensus Tree Bahla de Los Angeles(I-46) 2 Trees Isla San Esteban (1-28) 1856 steps CI = .54 100 Isla Cedros (I-22) RI = .59 Uta stellata (1-25) 57 groups; however, less resolution was recovered among the more northern populations. In the tests that included the ii outgroup species, the total combined data set had a greater number of phylogenetically informative characters due to the larger data matrix.

The total combined data set biased with the overlapping sequence from this study had 442 phylogenetic informative characters compared to the 415 informative characters when the Upton and Murphy (1997) sequence is used. Despite this, both produced identical shortest trees congment with those produced when rooting with only U. nigricaudus and P. meamsi. The only difference between them is the greater resolution for the position Uta antigua, placing it similarly to that found in the U&M data set tree from the unweighted analysis (see Fig. 6a).

The maximum likelihood analysis of the total combined data set, using 22 taxa, produced a single tree with a likelihood tree score of 7460 (Fig. 10). One of the three parsimonious trees from the unweighted total combined data analysis was used to test the various nucleotide substitution models. The GTR + I + F model (the most parameter rich) of nucleotide sequence evolution has the best goodness-to-fit over the five other models tested with a negative log likelihood score of 7525 (Table 15). Likelihood ratio tests show that this model significantly improved the likelihood score over the next less complex model (HKY + I + F; Tables 16 and 17). The GTR + I + F model was used to estimate phylogenies for the total combined data set using maximum likelihood. The likelihood tree is nearly identical to the strict consensus tree from the unweighted parsimony analysis for the total combined data set (see Fig. 8a) with the exception that Uta antigua is placed as the sister haplotype to the populations from southern Baja California (Fig. 10). 58

Figure 10. Maximum likelihood tree using the combined data set from DNA sequence generated in this analysis and Upton and Murphy (1997): a) unweighted parsimony analysis; and b) weighted parsimony analysis (Tv:Ti = 5). 59

Petrosaurus mearnsi

Urosaurus nigricaudus

Uta stansburiana stejnegeri (M-47)

—Uta palmeri (1-27)

— Isla Angel de La Guarda (1-42) r Isla Rasa (I-38) L Isla Mejla (1-47)

Uta antigua (1-33)

Uta squamata (1-12) Isla Danzante (I-13)

— La Presa (M-09)

Isla Monserrate (I-10)

Isla San Marcos (I-17) San Lucas (M-21)

Isla San Esteban (1-28) Penasco, SON (M-46)

San Ignacio (M-24) Isla Cedros (1-22)

Uta stellata (1-25)

Isla Santa Cruz (M-38) — Arroyo de San Regis (M-32)

—Bahia de Los Angeles(I-46)

Maximum Likelihood Tree Combined Analysis Upton & Murphy (1997) and this analysis Two Outgroup Taxa 60 Table 15. Comparison of likelihood scores from a single tree chosen from the parsimony analysis using the 22 taxa data sets combined from this analysis and Upton and Murphy (1997). Nucleotide Substitution Model -in Likelihood Value JC 8671 K2P 8244 HKY85 8123 HKY85 + I 7579 HKY85 + I+ F 7931 GTR+I+F 7525* *Best model.

Table 16. Likelihood ratio tests comparing statistical differences between increasingly complex models of DNA sequence evolution using the 22 taxa data sets combined from this analysis and Upton and Murphy (1997). The degrees of freedom is the difference in the number of parameters between the models (see Table 5). All tests are significant using a sequential Bonferroni correction (Rice, 1989) Model Comparison -21ogA degrees of P value freedom JC vs. K2P 854 1 < .005 K2P vs. HKY85 242 < .005 HKY85 vs. HKY85 + I 1088 1 < .005 HKY85 + I vs. HKY85 + I + F 96 1 < .005 HKY85 +I+Fvs.GTR+I+F 12 4 < .025

Table 17. Sequential Bonferroni correction for statistical tables for the comparison of models using the likelihood ratio test (see Table 16). P f-P 5 uncorrected P a/k P

Phylo genetic Analyses

In this analysis, a number of concerns regarding experimental design and phylogenetic analysis were taken into consideration from the outset. Samples were included to ensure adequate coverage of side-blotched lizards from the region of interest with the intention of purposefully breaking up long branches as recommended by Graybeal

(1998), Hillis (1998), Hendy and Penny (1989), Poe (1998), Soltis et al. (1998), and

Swofford et al. (1996). Outgroup taxa representing both the first and second outgroup nodes were selected as recommended by Maddison et al.. (1984) and Watrous and Wheeler

(1981). Outgroup taxa were constrained to reflect a number of phylogenetic hypotheses for phrynosomatid relationships (Etheridge and de Queiroz,1988; Reeder and Wiens, 1996;

Schulte et al., 1998; Wiens, 1993) to test for root sensitivity (Messenger and McGuire,

1998). Phylogenetically informative characters were obtained from mitochondrial genes with the appropriate levels of rate variation to guard against site saturation (Graybeal, 1993;

Hillis, 1996; Yang, 1999) and, although the total amount of sequence used in this study is less than the amount shown by simulation studies to ensure a high accuracy of phylogenetic reconstruction (Hillis,1996; Hillis et. al.,1994; Rannala et al., 1998), the data set is considered large by current standards in molecular systematics. Both equal and differential weighting were performed to test for differing topological outcomes, with differential weights based on the intrinsic properties of the data as recommended by Knight and

Mindell (1993) and Reeder (1995). And finally, both maximum parsimony and maximum likelihood analyses were performed to guard against methodological shortcomings of one tree reconstruction method over the other (Huelsenbeck and Rannala, 1997).

Despite these considerations, a number of topological arrangements resulted from these variables. The primary cause of the different topologies is weak support over many

61 62 of the basal and internal nodes. As a result, many of the topological differences found when varying either the outgroups, number of taxa, weighting, or tree reconstruction method can be, in part, attributed to the undersampling of characters. However, this poor resolution may not be strictly a function of the phylogenetic content of cytochrome b or cytochrome oxidase III, because both of these genes have strong phylogenetic structure based on the g index and each has been shown to resolve branches that are older than, or

approximately equal to (e.g., Petren and Case, 1996; Schulte et al., 1989; Sites et al.,

1995), those in this study (also see Voelker and Edwards, 1998). The pattern of weak

support for some parts of the tree, while other parts are strongly supported, has been associated with the process of rapid speciation followed by relative stability (de Queiroz,

1987; Hollingsworth, 1998; Kraus and Miyamoto, 1991; Lanyon, 1988; Moore, 1995;

Rassmann, 1997; Reeder and Wiens, 1996; Sites et al., 1996; Wiens and Reeder, 1997;

Voelker and Edwards, 1998). Therefore, the weakly supported basal and internal nodes

within the side-blotched lizard trees may be difficult to recover even with the addition of large amounts of data compared to those used in this study.

Still, the best estimate of relationships should be those which approach the methodological recommendations determined by theoretical considerations and simulation

studies. The relatively large number of geographic samples used in this analysis effectively broke up potentially long branches; however, by including a large number of samples, the number of characters per terminal branch was lowered and the levels of homoplasy increased (Archie, 1989; Sanderson and Donoghue, 1989). Graybeal (1998) found that it is still preferable to add taxa, rather than characters, because accuracy improves more dramatically with the addition of the former (although Graybeal and others recommend adding both when given the opportunity). Therefore, the trees which include the total sample should be theoretically more accurate than those containing fewer samples, despite the weak support for basal and internal nodes. 63 Another factor affecting tree reconstruction is the selection of outgroups and their topological arrangement. Messenger and McGuire (1998) found that the selection of outgroups is extremely important in molecular analyses. In this study, four different outgroup arrangements were tested using 11 representative taxa from four closely related genera. Of the four arrangements, the unconstrained and Reeder and Wiens (1996) constrained topology resulted in the shortest-trees with an identical strict consensus tree.

The analysis of Reeder and Wiens (1996) represents the most thorough phylogenetic study of phrynosomatid relationships to date. Based on the shortest trees found in this analysis and the most robust analysis of phrynosomatid relationships, the trees rooted with the

Reeder and Wiens (1996) outgroup arrangement should be the best estimate of side- blotched lizard relationships.

Finally, trees were affected by the weighting scheme used. In phylogenetic studies, the most frequently employed weighting scheme is to use equal weights (i.e., unweighted) at all sites (Voelker and Edwards, 1998). The assumption of equal weights is itself a strong assumption (Swofford et al., 1996) that can be more arbitrary than weights based on empirical and theoretical considerations (Simon et al., 1994). However, Reeder (1995) found lower transition to transversion ratios in the mitochondrial 12S and 16S ribosomal

DNA genes for phrynosomatid lizards with respect to other studies of the same gene sequences. Reeder (1995) also found equivalent phylogenetic signal within the partitioned data set for transitions and transversions. Based on these results, he argued that there was no justification for down-weighting transitions relative to transversions. The transition to transversion ratio of Uta in this study, for cytochrome b and cytochrome oxidase III, was higher than that found for the 12S and 16S ribosomal genes. Knight and Mindell (1993) and Wheeler (1990) advocated differential weighting of transversions over transitions based on the intrinsic nature of the data. Therefore, there is ample justification for differentially weighting transversions over transitions for cytochrome b and cytochrome 64 oxidase III. Based on these considerations, the preferred trees in this analysis are those which are rooted with the most robust outgroup topology, weighted, and include the greatest number of samples. These represent the weighted trees from the total data set and rooted with the phrynosomatid relationships of Reeder and Wiens (1996). The structure of the weighted strict consensus tree (see Fig. 5) is similar in many respects to the unweighted analysis, with a number of exceptions at the basal-most node and within haploclade B. In the weighted analysis, the population from Dono Ana, New Mexico Uta stansburiana stejnegeri) is resolved at the base of the tree, with haploclades A and B forming sister groups. Within haploclade B, the populations associated with Isla Angel de La Guarda

(node 30; Fig. 5), Uta palmeri, and Uta antigua (node 32; Fig. 5) are the first three consecutive branches followed by a strongly supported clade of populations from both mainland and insular localities (node 37; Fig. 5). Of the 68 resolved nodes within the weighted tree, 37 are strongly supported with bootstrap values over 70%.

Comparison with the Analysis of Upton and Murphy (1997)

The results of the phylogenetic analysis of the data sets from this study, reduced to an equivalent sample as that used by Upton and Murphy (1997) and reanalyzed, are not significantly different to those generated by their analysis after a Bonfen-oni correction (see

Tables 13 and 14). When the shortest trees from either the unweighted or weighted analysis are pruned to the smaller sample of Upton and Murphy (1997), or the two data sets combined, neither results can significantly reject one another. Despite these results, incongruence is seen between the two studies, but is primarily confined to the basal branches within the tree where there is weak support for the internal nodes.

The smaller number of samples used by Upton and Murphy (1997) results in only a rough estimate of side-blotched lizard evolution. Because these lizards are distributed 65 across such a wide area and inhabit numerous islands, their tree represents only a small

fraction of the important details within the group and the recovered relationships reveal only

the more strongly supported clades. In comparison, the more thorough sampling and

outgroup representation of this study imparts a greater reliability in the positions of even

weakly supported nodes and reveals many of the details regarding repetitive patterns,

dispersal events, and substructuring.

Ancestral Nature of Insular Populations

Based on the weighted phylogenetic hypothesis (and nearly all other trees), a

distinct pattern emerges across the tree where insular populations are placed in ancestral

positions relative to their mainland counterparts (Fig. 11). Within haploclade A, of Uta

squamata from Isla Santa Catalina is the most basal branch, followed by a haploclade (Al)

containing the populations from Islas Carmen, Danzante, and Los Coronados (Fig. W.

Within haploclade B, the first four sequential branches consist of insular populations: Isla

Angel de La Guarda and its associated islands (BO, U. palmeri from Isla San Pedro

Mardi., U. antigua from the islands associated with Islas San Lorenzos (B2), and Isla San

Esteban, respectively (see Geographical Concordance below for a caveat concerning the

population from Isla San Esteban). Nested further within haploclade B, this pattern again emerges in haploclades B4, B6, and B12 (Fig. 11).

The ancestral nature of insular populations is counterintuitive for island biogeography. It is generally believed that insular populations are derived from a current mainland source, either by colonization, or were present at the time the island formed.

Once restricted, a insular population is expected to evolve relatively quickly due to their reduced effective population size (Ne ) and isolation. This would be especially true for islands colonized by the dispersal of only a limited number of individuals (e.g., founder 66

Figure 11. Phylogenetic tree from weighted analysis depicting the placement of ancestral insular populations (indicated with heavy bars). 67

Uma scoparia limo imam PetrosaunIS repens Porosaunts meamsi Urosaurus graciosus Urosairrus lahrelai Urosaluirs nigricaudus keloporus grandaevus Sceloporiis occidemalis Sceloporits orcuni - Sceloporus hunsakeri M-47 Dona Ana. N.M. 1-0916 Santa Catalina. BCS 72% 1-15 Isla Los COMMadOS 64% 1-13 Isla Danzante 4 1-14 Isla Cannes 1-01 Isla Espiritu Santo 1-02 Isla Panida Sur 1-03 Isla Gallo 1-04 Isla Baena M-03 Playa Tecolote 81-04 La Paz. 36km N 77% NI-01 El Chorro M-02 Ensenada Los Muertos 2 73% 6 M-05 La Paz. 55km N of 74% 8 18-011 La Press M-07 San Evaristo 3 66% 9 M-06 La Paz. 82km N of 10 M-10 Santa Rita 61% 11-09 Rancho San Juanito 12 73% 1 1-07 Isla San Jose 131 89% 1-05 Isla San Francisco 1-06 lsla Pardito 1 14 69% 61-I4 Agua Verde 15 75% 1 M-12 Ciudad Insurgentes 161 1 M-I3 Loreto. 96km S of M-15 Puerto Escondido 67%17 1-08 Magdalena

20 11-11 San Carlos 16 79% 1-17 Isla San Marcos M-20 Mulege 21 I 7 M-21 San Bruno 1-12. Isla Galleras Oeste 99% 1 1-10 Isla Monserrate 8 23 24 1-11 Isla Galleras Este 81% 1-16109 Idelfonso 9 26 M-16 San Nicolas 2. 72% M-19 Bahia San Juanico 10 27 58% M-17 1.3 Purissima 28 M-18 Arroyo Los BurrcYs 1-39 Isla Cardonosa Este 1-40104 Cardonosa Norte 1-41 Isla Angel .de La Guarda 1-42 Isla Pond 1-48 Isla Granito 1-38 Isla Rasa 1-47 Isla Mejfa 1-2709 San Pedro Manfr 1 36 Isla Roca Lobos 57% 1-33109 San Lorenzo Sur 34 1-34 Isla San Lorenzo None 2 70% 1-35 Isla Salsipuedes 35 1-37 Isla Lagartija 1-28 Isla San Esteban 1-26 Isla San Pedro Nolasco 97% M-36 San Felipe 61 M-37 Santo Domingo 98% M-38 Temecula • 62 M-39 Moorpark 1-18 Isla Tonuga 81-46 Cholla Bay M-45 San Emetario 6I-43 Puerto de La Libertad M-44E1 Desemboque 68% 1-32 Isla Pains 65 M-42 Punta Sargent° 12 I-30 Isla Alcatraz I-31 Isla Tibur6n 11-41 Punta Chucca 88% 1-29 Isla Datil 67 M40.Bahia Kino 99% NI-25 San Ignacio 541 M-30 Guerrero Negro 01-22 Sams Agucda 100% M-23 Santa Rosalia 3 01-24 is Virgenes 11-26 San Francisco 1-19 Isla Asunciln 100% 1-20 Isla San Roque 78% 11-27 Bahia Asuncion M-28 Puerto Nuevo 4, 01-29 Punta Eugenia 59% 1-22 Isla Cedros 100% 56 1-24 Isla San Denims hledio 90% 1-21 Isla Natividad 8 57 1-2309 San .Benitos Geste 58 1-25 .Isla San Benitos Este 1-45 Isla Piojo 1I-31 Bahia de Los Angeles 75% 77% 1-43 Isla Cabeza de Caballo 4 46 1-4604 Smith 5 81% 1-44 Isla La Vcntana 44 47 01-32 El Parador 1-50 Isla Coloradito 73% M-34 Gonzaga 48 69% 1-49 Isla Encantada 6 49 50 M-33 Catavi a 2% 8 1-51 Isla Muerto — M-35 Campo Cinco Islas 68 effect). However, this process appears to be equally effective for mainland populations as well. The continued evolution and extinction of mainland population haplotypes is essentially erasing the genetic history for the portion of the tree when these insular populations were formed. It remains a possibility that further sampling of localities will have little effect on resolving the weakly supported basal and internal nodes because the pattern of ancestral insular and evolved mainland populations preclude further sampling of intermediate localities.

Grismer (1999c) describes a model explaining the pattern of ancestral insular and evolved mainland populations (Fig. 12). Ancestral mainland populations represent the source locality for islands, yet themselves experience anagenic change and extinction, thus giving rise to more evolved mainland populations. For mitochondrial DNA haplotype evolution, the increased rate of evolution and extinction is predicted. First, when compared to nuclear-autosomal genes, the rate of evolution for mitochondrial sequences is much greater (Moore, 1995). Second, the probability of coalescence is greater for mitochondrial genes than it is for nuclear genes because coalescence is directly related to effective population size (Moore, 1995). For two nuclear genes, the expected time to coalescence is

twice the effective population size or 2Ne, where Ne = Nfe N., (Kreitman, 1991).

Because mitochondrial genes are haploid and only females are considered in the effective

population size, the expected time to coalescence is one-fourth that of nuclear genes or Ng =

Nfe = Ne/2 (Moore, 1995). The increased rate of nucleotide substitution and time to coalescence predicts that it is more probable that mitochondrial genes should obliterate their history. Therefore, Grismer's (1999c) model of ancestral insular and derived mainland evolution corresponds to the expected rate of haplotype evolution and coalescence in side- blotched lizards. 69

1st to 2nd to 3rd to continental colonize colonize colonize taxon

A a,

Figure 12. Model of evolution for insular taxa derived from mainland ancestors from Grismer (1999c). A, B, C, and a4 are contemporary species depicting the chronological order of island colonization; A being first to colonize and C being the third to colonize. al = first continental ancestor; a2 = second continental ancestor; a3 = third continental ancestor; and a4 = the last continental ancestor to evolve. The solid bars indicate the acquisition of derived character states in the continental ancestors. 70 This pattern is not restricted to side-blotched lizard evolution in the Gulf of

California. It is also found in the lizard groups Sauromalus (Hollingsworth, 1998),

Cnemidophorus hyperythrus complex (Grismer, 1998; Radtkey et al., 1997), C. tigris complex (Radtkey et al., 1997), and Sceloporus angustus and S. grandaevus (Wiens and

Reeder, 1997). In all of these examples, the insular taxa maintain a basal position within the evolutionary tree of their respective groups, in addition to retaining ancestral morphological characteristics. For example, Hollingsworth (1998) found that the two most ancestral branches in the evolution of Sauromalus were insular endemics that retained an ancestrally large body size. It is interesting that this pattern is also evident in morphological systems which would be expected to evolve at a similar (if not slower) rate compared to nuclear DNA.

In Uta, the majority of islands included in this pattern are continental or oceanic in origin. Within haploclade A, the population of U. squamata occurs on Isla Santa Catalina, an old continental island associated with the southern portion of the Baja California peninsula. It is believed that Isla Santa Catalina is old, but the exact age is unknown

(Gastil et al., 1983). Of the eight reptile species on the island, seven are endemic

(Grismer, 1999a,b). However, haploclade Ai, contains three populations from landbridge islands that are believed to be more recent in age, possibly as young as 10,000 years old

(Gastil et al., 1983). Within haploclade B, the four most basal lineages all occur on old continental or oceanic islands. Isla Angel de La Guarda and its satellite islands, along with the islands of the San Lorenzo block (Islas San Lorenzo Sur, San Lorenzo Norte,

Salsipuedes, Roca Lobos, and Lagartija) are continental in origin and Pleistocene in age

(Gastil et al., 1983). The San Lorenzo island group was isolated from Baja California through dip-slip faulting and is believed to have never been connected to Isla Angel de La

Guarda (Gastil and Krummenacher, 1977; Phillips, 1964; Rossetter, 1973). However, 71 Avise et al. (1974) noted that the islands are separated by water no more than 100 fathoms deep, while they are separated from Baja California by water as deep as 850 fathoms. This suggests that during low sea levels Isla Angel de La Guarda and the San Lorenzo island group may had shared a connection (Milliman and Emery, 1968) provided that the relatively shallow sea floor is not the result of recent fluvial deposition. Uta from Isla San

Esteban are also basal. This island is continental in origin and composed entirely of

Miocene age volcanics. This island probably formed as an erosional remnant of a larger structural block that was isolated from the mainland by sea-floor spreading in the

Pleistocene and not by Miocene eruptions (Gastil et al., 1983; Philips, 1964). The same pattern is found in U. palmeri from Isla San Pedro Martir is a oceanic island situated in the middle of Gulf of California and is believed to be Pliocene in age (Gastil et al., 1983).

With the exception of haploclade Al, the deeper insular haplotypes and clades correspond to older islands that have had either a continental or oceanic origin. The latter suggests that over-water colonization must have occurred very early on in the history of the haploclade.

Yet, the ancestral nature of insular populations is not restricted to the deeper lineages in the tree. This pattern is also seen in the relatively more recent haploclades B4,

B6, and B12. Within haploclade B4, two small, landbridge islands off the west coast of the Vizcaino peninsula, Islas Asuncion and San Roque, form a sister group to the strongly supported and more derived populations from the Sierra Vizcalno (node 42; Fig. 5). The sister group relationship between Islas Asuncion and San Roque is only weakly supported and most likely is the result of random convergence. In the unweighted analysis these two islands form a polytomy with the strongly supported Sierra Vizcaino haploclade (node 42;

Fig. 4). The Sierra Vizcalno extends 115 km from Bahfa Asuncion in the southeast tapering to Punta Eugenia in the northwest and reaches 900 m in elevation (Grismer et al.,

1994). The formation of the Sierra Vizcalno is the result of Miocene volcanic deposition and the range has been aerial since the beginning of the formation of the Gulf of California 72 (Gastil and Jensky, 1973). Until at least the Pleistocene, 10,000 years ago, the Sierra

Vizcalno, and the closely associated Sierra Santa Clara to the south and Isla Natividad and

Isla de Cedros to the north, existed as a large island or closely grouped archipelago of islands off the coast of western Mexico (Gastil and Jensky, 1973; Gastil et al., 1975).

During the late Pleistocene, a broad, land-positive connection formed with the rest of the

Baja California (Minch et al., 1976) because of lowered sea levels resulting from glaciation

(Auffenberg and Milstead, 1965). The populations from Islas Asuncion and San Roque have maintained their ancestral positions in the tree with respect to the populations from the

Sierra Vizcaino. In contrast, the haplotypes from the Sierra Vizcalno coalesced sometime before the land-positive connection united them with Baja California, but after serving as a source population to their two landbridge islands.

Within haploclade B6, the population from Isla Coronadito (= Uta tumidarostra) from the Encantada Archipelago occurs in the basal most position, while populations along the adjacent peninsula and Isla El Muerto (= U. lowei) and Encantada (= U. encantadae) are more derived. Grismer (1994a) hypothesized that the three species endemic to the

Encantada Archipelago formed a monophyletic group; however, their haplotype evolution indicates that each has had a separate origin with respect to the mainland. Uta lowei forms a strongly supported haploclade with the population lying immediately across from the island at Campo Cinco Islas, BC. Uta encantadae forms a weakly supported relationship with Catavifia. In contrast to these two species, the haplotype of U. tuniidarostra is relatively older and lacks a mainland relationship.

The pattern seen for haplociade B12 is more complicated. Haploclade B12 consists of 11 strongly supported populations from Sonora, and in the weighted analysis, forms a sister group relationship with Isla Tortuga; an island lying 30 km off the coast of Santa

Rosalia, BCS and 150 km across the Gulf of California to the nearest Sonoran population. 73 Isla Tortuga is a oceanic island composed of the remnants of a volcanic cinder-cone and is

believed to be late Pleistocene in age (Gastil, et al., 1983). This relationship is not only a

case of the ancestral maintenance of Isla Tortuga haplotype, but also a trans-gulfian

dispersal event. The over-water dispersal to Isla Tortuga is even more plausible because

the island contains a Sonoran rattlesnake, Crotalus tortugensis, proposed to have had an

over-water colonization (Grismer, 1999b). Crotalus tortugensis is endemic to Isla Tortuga

(Grismer, 1999a,b), yet is derived from C. atrox (Grismer, 1994d and references therein);

the latter does not occur in Baja California, but has its closest geographic locality in

Sonora. The strongly supported relationships among the Sonoran populations indicate that

at one point in time, the mitochondrial genome had coalesced after serving as the source

population to Isla Tortuga. Interestingly, the phylogeographic structure within haploclade

B12 indicates that dispersal occurred from north to south, starting with the Sonoran

populations at the top of the gulf and ending with the population from Bahia Kino, Sonora.

Geographical Concordance

Throughout the phylogenetic tree, there is strongly supported substructuring and

geographic concordance among populations. Three categories of patterns are evident: 1)

mainland-only groups; 2) island-only groups; and 3) mainland-island groups (Table 18).

Within haploclade A, there are ten strongly supported groups of two or more populations

(Fig. 13), while in haploclade B there are 12 such groups (Fig. 14). Of the 21 identified

groups, nine are formed from island localities and their geographically proximal mainland populations (Table 18). These mainland-island associations are nearly always between

geographically proximal locations indicating that the mainland region has served as the

source location to the islands (the population from Isla Tortuga is one exception, as discussed above, and possibly the populations from Islas San Esteban and San Pedro

Table 18. Categories of strongly supported and geographically concordant groups of two or more populations. Category Haploclade Mainland-only A3 AS A10 3 B3 4 B7 2 B10 2 Bil 2

Island-only Al 3 A4 (in part) A8 3 Bi 7 B2 5 B8 5

Mainland-Island A2 6 A4 4 A6 2 A7 3 A9 B4 5 B.5 6 B6 6 B12 12 75

Figure 13. Map depicting the geographic position of the strongly supported groups within haploclade A (see Fig. 5 for designations). 76

Figure 14. Map depicting the geographic position of the strongly supported groups within haploclade B (see Fig. 5 for designations). 77 Nolasco, as presented below). This pattern in particularly evident in haploclade A4 where

there is strongly supported substructuring with an ancestral mainland population from

Rancho San Juanito, BCS and the more derived populations from Islas San Jose, Pardito,

and San Francisco (contrary to the pattern of the ancestral nature of insular populations

presented above). Therefore, although this haploclade is categorized as a mainland-only

group, it also contains a strongly supported island-only group (Table 18). Unfortunately,

strongly supported structure within the remaining mainland-island groups is poorly

resolved or contains only two populations, thus preventing the determination of the

ancestral or derived nature of the insular populations. Of the remaining geographically

concordant groups, seven contain mainland-only populations, while six are island-only

groups (Table 18).

The large amount of strongly supported substructuring within Uta indicates that

these lizards are not as panmictic as has been believed (Ballinger and Tinkle, 1972) and

have inhabited the region represented in this study for a stable period of time. Avise et al.

(1989) describe five categories of phylogeographic patterns based on empirical examples,

two of which appear to be applicable to the substructuring seen in Uta. Category III is

explained as the geographic localization of mtDNA haploclades, in the absence of

phylogenetic breaks, and involves historically limited gene flow between populations in

species not subdivided by firm long-term zoogeographic barriers to dispersal (Avise et al.,

1989). For Uta, this category is applicable to the strongly supported haploclades from

mainland-only and mainland-island groups. These haploclades are geographically

localized, yet probably have limited amounts of gene flow between them. Of these two aspects, the presence or absence of gene flow is more difficult to substantiate. One point of corroboration may be the lack of strongly supported structure between haploclades, thus representing the ephemeral nature of the separation between these groups. In some cases, the partitions occur among localities less than 20 km apart. For example, haploclade A3 78 and A4 from the La Presa region are separated by no more than 10 km and have a 1.5% sequence divergence, yet are positioned in haploclades that are not each others sister group.

It is conceivable that these relatively small geographically concordant groups will be subsumed within one another through extinction and dispersal. Localized boundaries would be expected to fluctuate as one group goes extinct and the other disperses into it (or vice versa).

For island-only haploclades, category III of Avise et al. (1989) may not be as applicable because gene flow is further restricted by over-water dispersal. Category I of

Avise et al. (1989) describes a pattern of divergent clades, due to haplotype extinction and the coalescence of gene history at the intemodal period, correlated with long-term zoogeographic barriers. However, the presence or absence of gene flow between the island-only haploclades is just as difficult to substantiate in this situation as it was for geographically adjacent haploclades on the mainland. One caveat that supports the presence of gene flow between island and mainland populations is the prevalence of insular populations of side-blotched lizards. Within the Gulf of California and Pacific Ocean, Uta occurs on more than 70 islands (Ballinger and Tinkle, Grismer, 1993, 1999a,b). Three of these are oceanic in origin, thereby restricting their colonization to over-water dispersal.

In the unweighted analysis, the lack of geographical concordance is seen between the populations from Islas San Esteban and San Pedro Nolasco. These two populations form a weakly supported sister group relationship, as opposed to the weighted analysis, where Isla San Esteban is positioned slightly basal to Isla San Pedro Nolasco. The association between these two islands appears to be an anomaly because they are separated by approximately 140 km of uninterrupted water in the eastern portion of the Gulf of

California and have never had a geological contact (Gastil et al., 1983; Philips, 1964).

However, it is plausible that this relationship represents a human-facilitated introduction of

Uta to one or the other islands by the Seri Indians. A similar relationship occurs in 79 Ctenosaura hemilopha (Cryder, pers. corn., 1999), a large species used as a food source of the Seri Indians. There are numerous proposed cases of lizard introductions between islands in the Gulf of California involving the sea-faring Seri Indians (Malkin, 1962; Case,

1982; Bahre, 1983; Felger and Moser, 1985; Grismer, 1994b). The most notable involve chuckwallas which were transported and released onto smaller, near-shore islands because small island size would facilitate their recapture (Grismer, 1994b; Hollingsworth et al.,

1997). Before side-blotched lizards can be added to the list of possible introductions, further resolution is needed between the populations from Islas San Esteban and San Pedro

Nolasco.

Cryptic Species Boundaries

The difficulty in substantiating the presence of gene flow between mitochondrial

DNA haploclades, in the absence of other intrinsic markers (e.g., nuclear DNA, allozymes, or morphology) or zoogeographic boundaries, makes the determination of genetic barriers difficult. Clearly, whether two haploclades are genetically independent of one another can not be concluded with the use of mitochondrial DNA markers alone (Moore, 1995; Moritz et al., 1992). The primary reasons for this limitation is that paternal gene exchange can be actively occurring, or that genetic barriers still have limited gene flow (Moore, 1995; Moritz et al., 1992). These problems are particularly evident when considering the deep coalescence of haploclades A and B, and whether this divergence represents the presence of a genetic barrier between the two in central Baja California (Fig. 15). Both haploclades are strongly supported and deeply divergent, although the strong support in haploclade B is nested in the tree at node 37 (see Fig. 5). This clade contains the more northern mainland populations, in addition to numerous islands. 80

Figure 15. Map depicting the geographic position of haploclades A and B. Contact zone occurs between Santa Agueda (M-22) and San Bruno (M-21). 81 The two deeply diverged haploclades, separated geographically in central Baja

California, were first identified by Upton and Murphy (1997). In their analysis, the two haplotype groups contained samples located approximately 70 km apart, between San

Ignacio and San Lucas, BCS. This study narrows the geographical gap to 10 km, between

Santa Agueda and San Bruno, BCS, along the central Gulf Coast desert. Upton and

Murphy (1997) believed that a mid-peninsular seaway in central Baja California was the causal factor for the formation of northern and southern. While this is a plausible scenario, the geological evidence for a mid-peninsular seaway needs to be further substantiated (or found) for this explanation to be supported. This explanation is further diminished by the lack of strongly supported, concordant examples from other terrestrial vertebrates for the same geographic region. Had such a geological event of that magnitude occurred, we would expect to find similar patterns of differentiation in transpeninsular taxa. Regardless of the specific causal mechanism for the origins of haploclades A and B, their formation most likely involved geographic isolation from each other for a long enough period to allow the coalescence of their respective haplotypes to occur.

Upton and Murphy (1997) believed that gene flow was occurring among the peninsular populations without interruption, thus haploclades A and B were thought to be in secondary contact. With the narrowing of the geographic separation to 10 km and no signs of interdigitation between populations, it appears these haploclades are maintaining their historical integrity. The most proximate populations across this contact zone have a

8.7-9.0% sequence divergence compared to a 0.5-1.0% divergence between the nearest populations within their own haploclades. Based on the maintenance of a narrow geographical contact zone and the deep coalescence of their haplotype ancestry, it is equally likely that this secondary contact zone is a cryptic species boundary. This scenario is similar to the strongly supported and geographical partitioned haploclades throughout the 82 mainland (as discussed above). However, in comparison, this contact zone has maintained itself throughout the time it took for both haploclades to coalesce at the base of the tree.

In order to support a secondary contact zone with no genetic interruption, haploclades A and B would have to have merged only recently due to the faster expected rate of coalescence of the mitochondrial DNA genome (Moore, 1995). Unfortunately, in the absence of geological evidence, there is no way to know how long these two haploclades have been reconnected. The geographic region between Santa Agueda and San

Bruno is along a coastal alluvial plain and contains no apparent zoogeographic barriers.

Therefore, it is more likely that this contact zone is being maintained by a yet to be determined reproductive isolating mechanism, either behavioral or cytological in origin.

Deeply coalesced insular haplotypes and clades are even better candidates for the presence of species boundaries (Upton and Murphy, 1997). Not only is the likelihood for gene flow restricted by over-water dispersal, but their deep position in the gene tree excludes the mainland populations. This indicates that they have lacked maternal recruitment for a period dating back to their coalescence. At the same time, this period was long enough for the mainland populations to evolve and erase their ancestral connections to these islands.

There are six deeply coalesced insular haplotypes and clades that make good candidates for the presence of species boundaries. Three of the six are currently recognized species here: Uta antigua, U. pabneri, and U. squamata. The remaining three are currently recognized as U. stansburiana, of which, the group of seven populations associated with Isla Angel de La Guarda (haploclade B1) is the most likely to lack a genetic interaction with other populations (Upton and Murphy, 1997). This haploclade originated at the base of haploclade B, having been present on at least one of the component islands before the origins of both U. antigua and U. palmeri. The most likely point of origin was on the larger Isla Angel de La Guarda, to which the other satellite islands are landbridged. 83 The populations of three of the satellites, Islas Granito, Mejla, and Pond, were either

present at the time of their formation or colonized over the short distance that separates

them from the larger Isla Angel de La Guarda. On the other hand, Islas Cardonosa Este,

Cardonosa Oeste, and Rasa are as far as 23 km to the south and most likely were colonized

by over-water dispersal (Upton and Murphy, 1997).

The next candidate for the presence of a cryptic species boundary is the haploclade

composed of Islas Carmen, Danzante, and Los Coronados (haploclade Al). This group of

islands is positioned deep within haploclade A, however, this relationship is not strongly

supported due to the lack of bootstrap values of over 50% for node 5 (see Fig. 5) which

includes the remaining mainland and insular populations from the southern portion of the

Baja California peninsula. Therefore, the deep position within haploclade A is considered

tentative. Even still, the lack of a mainland association within this strongly supported

island-only haploclade places it as a good candidate as a cryptic species. As a result, the

other island-only haploclades, regardless of their position in the tree, should be considered

in future studies as candidates that lack a genetic interaction to the mainland.

The remaining population of Uta stansburiana from Isla San Esteban also makes a

good candidate as a cryptic species. However, the deep position within haploclade B is not

strongly supported, and as discussed previously, this population is placed as the sister

haplotype to U. nolascensis from Isla San Pedro Nolasco in the unweighted analysis. If

further analysis substantiates its position in weighted analysis, then it also appears to lack a

genetic interaction with the remaining Uta stansburiana.

Exclusivity and Non-exclusivity

The species status of both Uta stansburiana and U. stellata are called into question based on the results of this study. For both species, populations recognized under the 84 name of each species appear to be more closely related to populations of other species than

they are to populations of their own species. For U. stellata, the population from Isla San

Benitos Medio appears to be more closely related to the population of U. stansburiana

from Isla de Cedros than it is to conspecifics from Islas San Benitos Este and Oeste.

Likewise, numerous populations of Uta stansburiana occur more closely related to other

species than to other populations of U. stansburiana. This pattern of relationships for U.

stansburiana was also evident in the phylogenies of Ballinger and Tinkle (1972) and

Upton and Murphy (1997). Upton and Murphy (1997) referred to U. stansburiana as a

paraphyletic species; however, Grismer (1999c) correctly surmised that such terminology

is not appropriately applied to species and instead U. stansburiana should be considered a

non-exclusive lineage (Baum and Shaw, 1995; Graybeal, 1995).

A group of organisms is exclusive if their genes coalesce more recently within the

group than between any organism outside the group (Baum and Shaw, 1995; Graybeal,

1995). This interpretation of exclusivity of common ancestry relationships is equated to the

monophyly of the group (see Baum and Shaw, 1995; Baum and Donoghue, 1995; de

Queiroz, 1998; de Queiroz and Donoghue, 1990). Non-exclusive groups occur when

members outside the group of interest are share a more recent genetic history to with those inside the group (Baum and Shaw, 1995; Graybeal, 1995).

The non-exclusive nature of Uta stansburiana may be, in part, a result of the misapplication of taxonomic names. For example, if the exclusive populations of U. stansburiana from haploclade A (southern Baja California) and haploclade B1 (Isla Angel de La Guarda) are separated from the remaining U. stansburiana populations by species boundaries, then much of the non-exclusive associations to other species is removed from the tree. Still, U. stansburiana within haploclade B would remain a non-exclusive species 85 based on the presence of a number of insular endemics evolving out of this portion of the tree.

Due to the non-exclusive nature of Uta stansburiana, the interpretation of interspecific relationships may be incongruent with the gene tree. In order for a gene tree to track a species tree, it is necessary for exclusive coalescence to have occurred at each intemodal period (Moore, 1995). The identification of a non-exclusive species calls into question the interpretation of the species relationships for this portion of the tree (Graybeal, 1995)

The non-exclusive nature of Uta stellata is more problematic because different populations within this species are genetically more closely related to populations of U. stansburiana which is also a non-exclusive species. Grismer (1999c) found that the original and subsequent diagnoses of U. stellata of Van Denburgh (1905) and Ballinger and Tinkle (1972) could not be substantiated and therefore synonymized U. stellata with

U. stansburiana. Based on this and the non-exclusive nature of U. stellata with respect to the non-exclusive nature of U. stansburiana, the synonymy of Grismer (1999c) seems warranted (although, see caveat below).

Taxonomy

A number of taxonomic recommendations have been made throughout this discussion and will be further elaborated on and summarized here. These recommendations are based on the general lineage concept (sensu de Queiroz, 1998) using the monophyly criterion (e.g. Bremer and Wanntorp, 1979; de Queiroz and Donoghue,

1990; Donoghue, 1985; Mishler, 1985,) and its secondary criterion of exclusive coalescence (Baum and Shaw, 1995). These recommendations will also consider an additional secondary criterion of the non-exclusiveness of a species (Graybeal, 1995). 86 Because this analysis included only a single sample from each population, species endemic to single islands could not be evaluated for exclusive coalescence with respect to themselves. Of the nine species recognized at the onset of this analysis, six are insular endemics from single islands (Fig. 16). Two of six, Uta palmeri and U. squamata, are deeply diverged, while the remaining four, U. encantadae, U. tumidarostra, U. lowei, and U. nolascensis, are more recently derived (Fig. 16).

Two insular endemics, Uta stellata and U. antigua, occur on multiple islands and are represented in this analysis by more than two samples each (therefore, their exclusive coalescence can be evaluated). Uta stellata appears to be non-exclusive with respect to

different populations of U. stansburiana from haploclade B8. Because haploclade B8 is an

island-only group, there are two alternatives to resolve the non-exclusive nature of U.

stellata. First, the synonymy of U. stellata with U. stansburiana by Grismer (1999c)

could be followed. Second, the distribution of U. stellata could be expanded to include all

populations within haploclade B8, thereby adding Isla Natividad and Isla de Cedros to their

distribution (Fig. 16). Of these, the first is recommended until further evidence can

substantiate the presence or absence of a species boundary for haploclade B8. For U.

antigua, the five populations used in this analysis have exclusive coalescence and are

positioned deep within the tree (Fig. 16). It is believed a species boundary exists for this

taxon. Therefore, despite the lack of a morphological diagnosis (Grismer, 1999c), the

recognition of this species seems warranted.

The remaining taxonomic decisions involve the populational relationships of Uta

stansburiana. Within haploclade A, the deep position of the island-only Al haploclade,

containing populations from Islas Carmen, Danzante, and Los Coronados, makes it a good

candidate for the absence of gene flow and therefore should be recognized as an

evolutionary species (Fig. 16). Because the remaining populations of U. stansburiana 87

Figure 16. Phylogenetic tree depicting the placement of currently named species and likely evolutionary species.

2 to

Uta stejnegeri Uta squamata IIsla Carmen (Unnamed) 1-01 Isla E.spfritu Santo ...I-02 Isla Panida Sur 1-03 Isla Gallo 1-04 Isla BaRena M.43 Playa Tecoloie M 04 La P4. 30413 N of M (11 El Chorro M-02 Ensenada 1111 Muenos M-05 1.1 1111. 55k13 N of El-OS La Presa M-07 San Evaristo

-^ M-06 La Paz. 82km N of

M-10 Santa Rita ies 1v1-(Y) Rancho San luanito 1-07 Isla San Jose ec 1-05 Isla San Francisco I 0654 Pardito - M-14 Agua Verde d Sp M-12 Ciudad Insurgentes M-13 Loreto. 96km S of me M-15 Puerto Escondido 1-08 Magdalena

M-11 San Carlos Unna

....•••••••••••• 11-19 Bahia San hank() 11-17 La Purissima 1,1 18 Arroyo Los Burros

Isla Angel de La Guarda (Unnamed) . Uta palmeri -IUta antigua Isla San Esteban (Unnamed) Uta nolascensts M-36 Nan Felipe 11-37 Santo Domingo M-38 Temecula 11-39 Moorpark

• 1-18 Isla Tortuga M-46 Cholla Bay ...... ,....

11...45 san Emoafio iana

11-43 P311711 411 La Libertad r M-44 El Desemtxxlue 1-32 Isla Pams bu M-42 Punta Sargento

1-30 Isla Alcatraz tans 1-31 Isla 'Tiburon 11-41 Punta Chueca s 1-29 Isla 1-311 M40 Bahia Kino Uta

M-25 San Ignacio

M-30 Guerrero Negro ive 11-22 Santa Agueda

M-23 Santa Rosalia lus

M-24 Tres Virgenes c ...... ---.. M-26 San Francisco 1--...—.... 1-19 Isla Asuncion ex

L' .,...,,Er, ,...., 1-20 Isla San Roque n- M-27 :Bahia Asuncion

lk.1-28 Puerto Nuevo No ---- - 01-29 Puma Eugenia 1-22 Isla Cedms Uta stellata 1-211111 Natividad Uta .stellata —.1-45 Isla Piojo 14-31 Bahfa de I,os Angeles 1-43 1511 Callen de Caballo 1 461113 Smilh I-44 Isla La Vcntana Tv1-32 El Parador Uta tumidarostra M-34 Gonnga Uta encantadae M-33 Catavi a Uta lowei M-35 Campo Cinco Islas 89 within haploclade A are deeply divergent and geographically separated from those of

haploclade B, it is likely a species boundary exists between these to groups at the point in

which the contact each other in central Baja California. As a result, the populations of U.

stansburiana above node 5 should be recognized as an evolutionary species (Fig. 16) and

not subdivided further until the presence of genetic barriers can be substantiated. Within

haploclade B, the deep position of the island-only B I haploclade, containing populations

associated with Isla Angel de La Guarda, makes it a good candidate for the absence of gene

flow and therefore should be recognized as an evolutionary species (Fig. 16). Likewise, the deep position of the population from Isla San Esteban is indicative of the absence of gene flow and therefore this population should be recognized as an evolutionary species (Fig. 16).

The remaining populations of Uta stansburiana within haploclade B, above node

37, pose a more difficult taxonomic problem due to the nested insular endemics: U. encantadae, U. tuinidarostra, U. lowei, and U. nolascensis. The separation of U. stansburiana into exclusive groups is also unwarranted due to the likelihood that gene flow is presently occurring between haploclades. Therefore, U. stansburiana from above node

37 should be recognized as a non-exclusive species.

Finally, the single sample of Uta stansburiana stejnegeri included in this analysis is positioned at the base of the weighted tree. This same relationship is supported by the analysis of Upton and Murphy (1997). Uta stansburiana stejnegeri was elevated to a full species by Collins (1991) and subsequently followed by Upton and Murphy (1997).

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APPENDIX 1

Results of the Computer Program PAUP* 4.0

A list of apomorphies is presented below. Refer to Figure 4 for node designations. Characters refer to sequence alignment position. Unambiguous nucleotide base changes are indicated with a double arrow; ambiguous changes with a single arrow.

Branch Character Steps CI Change Node 1 (Haploclade A 29 1 0.333 C --> T 135 1 0.250 C ==> T 245 1 0.250 T --> C 317 1 0.062 C --> T 329 1 0.375 353 1 1.000 A ==> C 431 1 0.300 A ==> T 479 1 0.143 G --> A 566 1 0.214 A ==> T 717 1 0.222 C ==> T 751 1 0.200 A --> G 801 1 0.222 C ==> T 804 1 0.273 C ==> A 843 1 0.375 A --> G 867 1 0.200 C ==> T 939 1 0.200 C ==> T 966 1 0.167 T --> C 1092 1 0.167 A --> C Uta squamata 74 1 0.250 C ==> T 131 1 0.667 C ==> T 149 1 0.273 C ==> T 154 1 0.250 T ==> C 212 1 0.200 C ==> T 254 1 0.143 T ==> C 287 1 0.200 C ==> T 377 1 0.111 T ==> C 425 1 0.400 A ==> G 437 1 0.154 T ==> C 458 1 0.200 T ==> C 476 1 0.600 A ==> G 512 1 0.125 T --> C 513 1 0333 T ==> C 578 1 0.375 A ==> G 795 1 0.333 T ==> C 810 1 0.429 A ==> G 813 1 0.333 T ==> G

101

102

Branch Character Steps CI Change 912 1 0.125 T ==> C 942 1 0.182 T ==> C 951 1 0.083 C ==> T 972 1 0.167 C ==> T 978 1 0.125 C ==> T 1008 1 0.167 C ==> T 1017 1 0.062 C ==> T Node 2 87 1 0.167 C --> T 134 1 0.500 C --> T 152 1 0.333 A ==> C 159 1 0.333 T ==> C 161 1 0.667 A ==> T 224 1 0.250 C --> T 257 1 0.400 A ==> G 308 1 0.250 G ==> A 398 1 0.214 C ==> T 410 1 0.250 C ==> T 434 1 0.158 A ==> G 467 1 0.167 A ==> G 473 1 0.143 C ==> T 503 1 0.286 T ==> C 557 1 0.167 T --> C 599 1 0.400 C ==> T 741 1 0.091 C --> T 765 1 0.300 C ==> G 840 1 0.400 A ==> C 894 1 0.250 T ==> C 957 1 0.167 T ==> C 1092 1 0.167 C --> T 1095 1 0.600 G ==> T Node 3 59 1 0.200 A --> G 695 1 0.500 T ==> C 751 1 0.200 G --> A 771 1 0.333 C ==> T 822 1 0.222 T ,=> C 945 1 0.200 T ==> C 984 1 0.333 A ==> G 990 1 0.500 A ==> G 1023 1 0.167 T ==> C 1054 1 0.143 C ==> T 1083 1 0.500 A ==> G 1089 1 0.083 C ==> T Node 4 885 1 0.091 C ==> T 912 '1 0.125 T ==> C Node 5 63 1 0.250 110 1 0.250 729 1 0.400 C ==> T 792 1 0.250 C ==> T 798 1 0.200 T ==> C

103

Branch Character Steps CI Change 843 1 0.375 G --> A 861 1 0.091 C --> T 987 1 0.111 T ==> C 1074 1 0.375 C ==> T 233 1 0.273 A ==> G Node 6 717 1 0.222 T ==> C 756 1 0.250 C ==> T 807 1 0.600 A ==> T 840 1 0.400 C ==> T 841 1 0.125 C ==> T Node 7 212 1 0.200 C ==> T 470 1 0.091 G ==> A 473 1 0.143 T ==> C 832 1 0.250 C ==> T 978 1 0.125 C ==> T Node 8 765 i 0.300 G ==> A 1002 1 0.375 T ==> C Node 9 398 1 0.214 T ==> C 401 1 0.231 T ==> C Node 10 885 1 0.091 C ==> T 1017 1 0.062 T ==> C Node 11 537 1 0.200 C ==> T 861 1 0.091 T ==> C 1062 1 0.167 A ==> G Node 12 65 1 0.250 A ==> G 620 1 0.250 C ==> T Node 13 50 1 0.667 A --> T 102 1 0.167 T ==> C 359 1 0.333 A ==> G Node 14 133 1 0.143 T ==> C 229 1 0.250 A ==> G 626 1 1.000 A ==> T Node 15 825 1 0.250 T ==> C 1074 1 0.375 T ==> G Node 16 245 1 0.250 C ==> T 807 1 0.600 A ==> G Node 17 194 1 0.143 T ==> C Node 18 134 1 0.500 T --> C 236 1 0.200 G ==> A 296 1 0.750 A ==> G 317 1 0.062 344 1 0.333 A ==> G 404 1 0.286 A --> G 563 1 1.000 C ==> T 837 1 0.333 1062 1 0.167 A ==> G 1086 1 0.333 A ==> G Node 19 84 1 0.333 C --> T 294 1 0.667 C --> T

104

Branch Character Steps CI Change 367 1 0.250 A --> G 368 1 0.500 C --> T 372 1 0.250 A --> G 377 1 0.111 T --> C 383 1 0.250 T --> C 434 1 0.158 G --> A 498 1 0.167 T --> C 584 1 0.200 614 1 0.500 A --> G 751 1 0.200 G --> A 801 1 0.222 T ==> C 1089 1 0.083 C ==> T Node 20 837 1 0.333 885 1 0.091 C ==> T 942 1 0.182 T ==> C 975 1 0.500 C ==> T 1017 1 0.062 C ==> T 1041 1 0.222 T ==> C Node 21 717 1 0.222 T ==> C 741 1 0.091 T ==> C 777 1 0.100 T ==> C Node 22 116 1 0.167 T ==> C 194 1 0.143 T --> C 398 1 0.214 T ==> C 765 1 0.300 G ==> A 861 1 0.091 T --> C 972 1 0.167 C ==> T Node 23 200 1 0.500 A ==> G 317 1 0.062 C --> T 341 1 0.231 A ==> G 375 1 0.286 T ==> A 405 1 0.667 C --> A 431 1 0.300 T ==> C 489 1 0.333 A ==> T 759 1 0.375 C==>A 780 1 0.500 A ==> G 927 1 0.500 A ==> G 1056 1 0.750 A ==> G Node 24 405 1 0.667 A --> T Node 25 110 1 0.250 T --> C 404 1 0.286 G --> A 443 1 0.250 C ==> T 473 1 0.143 T --> A 554 1 0.167 C ==> T 873 1 0.167 T --> C 939 1 0.200 T ==> C Node 26 63 1 0.250 T ==> C 194 1 0.143 C --> T 257 1 0.400 G ==> A

105

Branch Character Steps Cl Change 341 1 0.231 A --> C 437 1 0.154 T ==> C 491 1 0.400 C ==> T 801 1 0.222 T ==> C 837 1 0.333 G --> A 1014 1 0.429 A ==> T Node 27 56 1 0.750 A --> C 101 1 0.250 A --> T 359 1 0.333 A --> G 374 1 0.286 380 1 0.375 A --> G 434 1 0.158 G --> A 501 1 0.333 T --> C 527 1 0.250 C --> T 530 1 0.167 535 1 0.500 C --> T 537 1 0.200 C --> T 789 1 0.500 A ==> G 858 1 0.200 C ==> T 861 1 0.091 C --> T 1017 1 0.062 C ==> T 1092 1 0.167 T ==> C Node 28 921 1 0.167 C ==> T Node 29 (Hap1oclade B) 62 1 0.286 A ==> C 65 1 0.250 A --> G 122 1 0.167 C ==> T 176 1 0.111 T ==> C 260 1 0.333 A ==> G 362 1 0.400 C --> A 443 1 0.250 C ==> T 494 1 0.143 822 1 0.222 T ==> C 930 1 0.286 T --> A 996 1 0.333 A --> G Node 30 65 1 0.250 G --> T 155 1 0.667 A ==> G 197 1 0.500 T ==> C 248 1 0.500 T --> C 275 1 0.333 T ==> A 320 1 0.100 T --> C 389 1 0.333 A ==> G 401 1 0.231 T ==> C 404 1 0.286 A ==> G 434 1 0.158 A ==> T 473 1 0.143 C ==> A 611 1 0.667 A ==> G 741 1 0.091 744 1 0.400 C --> T 750 1 0.143 C ==> T 106

Branch Character Ste 's CI Chan 'e 798 1 0.200 T ==> C 813 1 0.333 T ==> C 924 1 0.375 C ==> T 928 1 0.333 C ==> T 933 1 0.500 C ==> T 981 1 0.143 C ==> T 987 1 0.111 T ==> C 1047 1 0.300 A ==> T 1053 -1 0.286 T ==> C 1074 1 0.375 C ==> T 1089 1 0.083 C ==> T Node 31 71 1 0.375 A --> G 152 1 0.333 A --> C 153 1 0.200 A ==> G 452 1 0.333 T --> C 581 1 0.143 C ==> T 861 1 0.091 C --> T 894 1 0.250 T ==> A 912 1 0.125 T ==> C 930 1 0.286 942 1 0.182 T ==> C 1062 1 0.167 A --> G Node 32 (Uta antigua) 53 1 0.333 C ==> T 59 1 0.200 A ==> G 98 1 0.300 A ==> G 149 1 0.273 C ==> T 191 1 0.200 T ==> C 230 1 0.250 C ==> T 254 1 0.143 T ==> C 317 1 0.062 C ==> T 374 1 0.286 T ==> C 437 1 0.154 T ==> C 467 1 0.167 A ==> G 479 1 0.143 G --> A 490 1 1.000 C ==> A 501 1 0.333 T ==> C 527 1 0.250 C ==> T 530 1 0.167 C ==> T 551 1 0.500 A ==> G 557 1 0.167 T ==> C 566 1 0.214 A ==> T 587 1 0.600 C ==> A 617 1 0.250 T ==> C 751 1 0.200 A ==> G 777 1 0.100 T ==> C 804 1 0.273 C ==> A 940 1 0.500 A ==> C 999 1 0.286 A ==> G

107

Branch Character Steps Cl Change 1038 1 0.200 C ==> T 1054 1 0.143 C ==> T 1093 1 0.250 C ==> T Node 33 188 1 0.333 A ==> G Node 34 98 1 0.300 G ==> T 443 1 0.250 T ==> C Node 35 473 1 0.143 C ==> A 1077 1 0.286 C ==> A 924 1 0.375 C ==> A Node 36 329 1 0.375 G --> A 516 1 0.167 T ==> C 771 1 0.333 C ==> T 801 1 0.222 C ==> T 996 1 0.333 G --> A Uta palmeri 35 1 0.500 C ==> T 50 1 0.667 A ==> C 65 1 0.250 G --> T 102 1 0.167 T ==> C 146 1 0.333 T ==> C 152 1 0.333 C --> A 188 1 0.333 A ==> G 341 1 0.231 A ==> C 401 1 0.231 T ==> A 476 1 0.600 A ==> G 498 1 0.167 T ==> C 599 1 0.400 C ==> T 694 1 0.667 C ==> A 759 1 0.375 C ==> A 810 1 0.429 A ==> G 813 1 0.333 T ==> C 837 1 0.333 A ==> G 894 1 0.250 A ==> C 941 1 0.333 T ==> C 957 1 0.167 T ==> C 1044 1 0.286 C ==> A 1047 1 0.300 A ==> G 1095 1 0.600 G ==> A 1098 1 0.667 A ==> G Node 37 71 1 0.375 G --> A 77 1 0.333 T ==> C 154 1 0.250 T ==> C 245 1 0.250 T ==> C 308 1 0.250 G ==> A 377 1 0.111 T ==> C 383 1 0.250 T ==> G 434 1 0.158 A --> G 443 1 0.250 T ==> C 458 1 0.200 T ==> C 473 1 0.143 C ==> T

108

Branch Character Steps CI Change 590 1 0.200 C ==> T 750 1 0.143 C ==> T 798 1 0.200 T ==> C 805 1 0.500 C ==> T 900 1 0.111 C ==> T 930 1 0.286 951 1 0.083 C ==> T 978 1 0.125 C ==> T 981 1 0.143 C ==> T 1050 1 0.286 C ==> T 1057 1 0.250 C ==> T 1062 1 0.167 G --> A 1074 1 0.375 C ==> T 1089 1 0.083 C ==> T 1092 1 0.167 A ==> C Node 38 350 1 0.222 A ==> G 533 1 0.250 T --> C 566 1 0.214 A ==> C 841 1 0.125 C ==> T Node 39 162 1 0.250 C ==> T 164 1 0.333 C ==> A 242 1 0.167 A --> G 383 1 0.250 G ==> A 398 1 0.214 C ==> T 402 1 0.667 C ==> T 434 1 0.158 G --> A 449 1 0.125 A ==> G 512 1 0.125 T ==> C 605 1 0.250 C ==> T 608 1 0.500 A ==> G 700 1 0.667 A ==> G 921 1 0.167 C ==> T 1008 1 0.167 C ==> T 1065 1 0.600 A ==> T Node 40 149 1 0.273 C ==> T 173 1 0.333 A --> G 437 1 0.154 T ==> C 533 1 0.250 C --> T 566 1 0.214 C ==> T 617 1 0.250 T ==> C Node 41 74 1 0.250 C ==> T 86 1 0.500 A ==> G 92 1 0.250 A ==> G 101 1 0.250 A --> G 264 1 0.167 T ==> C 302 1 0.250 C ==> T 372 1 0.250 A ==> G 376 1 0.667 A ==> T 380 1 0.375 A ==> G

109

Branch Character Steps CI Change 470 1 0.091 G ==> A 777 1 0.100 T ==> C 936 1 0.500 A ==> T 957 1 0.167 T ==> C 969 1 0.167 T ==> C 1060 1 0.200 ==> T Node 42 176 1 0.111 C ==> T 843 1 0.375 A ==> G 885 1 0.091 C ==> T 987 1 0.111 T ==> C Node 43 149 1 0.273 C ==> T 437 1 0.154 T ==> C 804 1 0.273 C ==> T Node 44 84 1 0.333 C ==> T 101 1 0.250 A ==> G 236 1 0.200 G ==> A 521 1 0.375 C ==> T 581 1 0.143 T ==> C 867 1 0.200 C ==> A 963 1 0.200 1054 1 0.143 C ==> T 1059 1 0.375 A ==> T Node 45 350 1 0.222 A ==> G 617 1 0.250 T ==> C 825 1 0.250 T ==> C 969 1 0.167 T ==> C 1041 1 0.222 T ==> C Node 46 383 1 0.250 G ==> A 942 1 0.182 C ==> T 951 1 0.083 T ==> C Node 47 79 1 0.500 C ==> Node 48 260 1 0.333 G ==> A 572 1 0.250 T ==> C 623 1 0.250 C ==> T 741 1 0.091 C ==> T 987 1 0.111 T ==> C Node 49 383 1 0.250 G ==> A 566 1 0.214 A ==> G 798 1 0.200 C ==> T Node 50 516 1 0.167 C ==> T Uta encantadae 264 1 0.167 T ==> C Node 51 65 1 0.250 G ==> A 158 1 0.250 T ==> C 470 1 0.091 G ==> A 518 1 0.286 A ==> G 912 1 0.125 C ==> T Uta lowei 386 1 0.200 C ==> T Uta tumidarostra 320 1 0.100 T ==> C 401 1 0.231 T ==> C

Branch Character Steps CI -----6,..-- Change 861 1 0.091 T ==> C 963 1 0.200 1077 1 0.286 C ==> T Node 52 449 1 0.125 A ==> G Node 53 176 1 0.111 C ==> T 841 1 0.125 C --> T 990 1 0.500 A ==> G 993 1 0.500 A ==> G Node 54 143 1 0.250 C ==> T 236 1 0.200 G ==> A 264 1 0.167 T ==> C 305 1 0.333 C ==> T 317 1 0.062 C ==> T 376 1 0.667 A ==> T 407 1 0.400 A ==> T 437 1 0.154 C ==> T 539 1 0.286 A ==> T 554 1 0.167 C ==> T 969 1 0.167 T ==> C 981 1 0.143 T ==> C Node 55 53 1 0.333 C ==> T 179 1 0.333 A --> G 320 1 0.100 T ==> C 377 1 0.111 C ==> T 617 1 0.250 T ==> C 777 1 0.100 T ==> C 822 1 0.222 C ==> T 873 1 0.167 T ==> C 903 1 0.286 C ==> T 939 1 0.200 C ==> T 963 1 0.200 A ==> G 1017 1 0.062 C ==> T Node 56 329 1 0.375 A ==> G 401 1 0.231 T ==> A Node 57 278 1 0.667 A ==> T 470 1 0.091 G==>A 924 1 0.375 C ==> A Node 58 560 1 0.500 A ==> G Uta stellata (Oeste) 153 1 0.200 G ==> A 774 1 0.333 C ==> T Uta stellata (Este) 338 1 0.667 C ==> T 867 1 0.200 C ==> T 951 1 0.083 T ==> C Uta stellata (medio) 398 1 0.214 C ==> T 885 1 0.091 C ==> T 1023 1 0.167 C --> T Node 59 101 1 0.250 A --> G 167 1 0.667 A --> T

1 1 1

Branch Character Steps CI Change 254 1 0.143 T --> C 284 1 0.500 A --> G 347 1 0.333 T --> C 434 1 0.158 G --> A 750 1 0.143 T ==> C 1047 1 0.300 A ==> G 1059 1 0.375 A --> C Node 60 254 1 0.143 T ==> C 350 1 0.222 A ==> G 383 1 0.250 A --> G 401 1 0.231 T ==> C 885 1 0.091 C ==> T Node 61 133 1 0.143 T ==> C 152 1 0.333 C ==> A 182 1 1.000 A ==> G 200 1 0.500 A ==> G 320 1 0.100 T ==> C 437 1 0.154 C ==> T 518 1 0.286 A ==> G 801 1 0.222 T ==> C 855 1 0.500 A ==> G 1065 1 0.600 A ==> G Node 62 233 1 0.273 A ==> C 341 1 0.231 A ==> T 374 1 0.286 T ==> C 513 1 0.333 T ==> C 569 1 0.250 C ==> T 744 1 0.400 C ==> T 858 1 0.200 C ==> T 1008 1 0.167 C ==> T 1022 1 1.000 T ==> A Node 63 813 1 0.333 T ==> C 1032 1 0.600 A ==> G 1059 1 0.375 C --> T Node 64 900 1 0.111 T ==> C Node 65 473 1 0.143 T ==> C 518 1 0.286 A ==> G Node 66 122 1 0.167 T ==> C 347 1 0.333 C ==> T 741 1 0.091 C ==> T 984 1 0.333 A ==> G 1089 1 0.083 T ==> C Node 67 817 1 0.167 T ==> C Uta nolascensis 83 1 0.667 A ==> G 108 1 0.200 G ==> A 287 1 0.200 C ==> T 290 1 0.333 C ==> T 375 1 0.286 11 ==> C 434 1 0.158 G ==> A 112

Branch Character SteEs CI Chanse 449 1 0.125 G==>A 458 1 0.200 C==>T 470 1 0.091 G==>A 473 1 0.143 T==>A 735 1 0.200 T ==;> C 750 1 0.143 T==>C 765 1 0.300 C==>A 861 0.091 T==>C 924 1 0.375 C==>A 978 1 0.125 T==>C 1053 1 0.286 C --> T Uta stansburiana stejnegeri 257 0.400 A==>T 284 1 0.500 A==>G 341 1 0.231 A==>T 347 0.333 T==>C 386 0.200 C==>T 398 1 0.214 C==>T 434 1 0.158 A==>G 464 0.200 C==>T 518 0.286 A==>G 530 1 0.167 C --> T 539 1 0.286 A==>G 566 1 0.214 A==>C 590 1 0.200 C --> T 825 1 0.250 T:==>G 841 1 0.125 C==>T 870 0.250 C==>T 903 0.286 C --> T 941 1 0.333 T==>C 942 0.182 T==>A 951 0.083 C==>T 978 0.125 C==>T 1011 1 0.400 A==>G 1044 1 0.286 C --> T 1047 1 0.300 A==>G 1059 0.375 A==>T