Molecular Systematics & Evolution of the CTENOSAURA HEMILOPHA

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9-1999

Molecular Systematics & Evolution of the CTENOSAURA HEMILOPHA Complex (SQUAMATA: IGUANIDAE)

Michael Ray Cryder

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Graduate School

MOLECULARSYSTEMATICS & EVOLUTION OF THECTENOSAURA

HEMJLOPHA COMPLEX (SQUAMATA: IGUANIDAE)

Michael Ray Cryder

A Thesis in PartialFulfillment

of the Requirements forthe Degree Master of

Science in Biology

September 1999 0 1999

Michael Ray Cryder

11 Each person whose signature appears below certifies that this thesis in their opinion is adequate, in scope and quality, as a thesis for the degree Master of Science.

,Co-Chairperson Ronald L. Carter, Professor of Biology

Arc 5 ,Co-Chairperson L. Lee Grismer, Professor of Biology and Herpetology

- -/(71— William Hayes, Pr fessor of Biology

111 ACKNOWLEDGMENTS

I would like to express my appreciation to the institution and individuals who helped me complete this study. I am grateful to the Department of Natural Sciences,

Lorna Linda University, for scholarship, funding and assistantship. I wish to thank

Ronald L. Carter and L. Lee thismer for their advice and the use of their facilities, and I wish to thank the other member of my guidance committee, William Hayes for his advice and comments. I would also like to thank the remaining members of the faculty and student body for constantly inspiring me and providing an educational environment full of thought and support.

I am grateful to Bradford Hollingsworth for providing technical assistance, friendship, and advice. His assistance on sequencing protocol, conducting the computer analyses and his understanding of phylogenetics was invaluable to my project. Many thanks to the Center of Molecular Biology and Gene Therapy at Loma Linda University

for generating the DNA sequences needed for this project.

Larry Buckley helped me in acquiring tissues and DNA sequences and I sincerely

appreciate his efforts, comments and advice concerning spiny-tailed iguanas. I would

like to thank the many individuals who helped me complete the field work done for this

project, including Lee Grismer, Brad Hollingsworth, Humberto Wong, Jesse Grismer,

Gene Kim, Nelson Wong, Victor Velasquez and the numerous Mexican ranchers,

fisherman and friends who provided me with food, shelter and knowledge of the

surrounding areas. I would also like to give a special thanks to my wife, Elissa, whose

tireless support and sacrifice allowed me to complete my graduate studies. To my kids,

Zachary and Miranda: never give up and always strive for the best.

iv TABLE OF CONTENTS

LIST OF FIGURES vi

LIST OF TABLES viii

ABSTRACT 1

INTRODUCTION ..... 3 Phylogenetic Analysis...... 13 Biogeographic Topics__ ...... 14

MA i ERIALS AND METHODS ...... 15 Cytochrome b and Cytochrome thddase III ..•••••••••••••.••.•••••.•.• 15 Sample Availability.••••••.•••••••••••••••.•.••••••••••••••••••• ...... •.••••.•• 15 Technical Protocols.••••.....•••••.••••••••••.•••••••••••.•••.•.•••••..•.••••••• 17 Alignments_ ...... •••••••...... ••••••••.•••••••••••.•••••••••• ••••••••• 21 Tree Reconstruction...... 22 Outgroup Selection...... 23 Confidence and Support...... •••••••••••••••••• 23

RESULTS • •• ••• • • • ••• ••• t•• ••• ••• ••• ••• ••• ••• •• • ••• • 25 Phylogenetic Signal... 25 Combined DNA Sequences... .. 25 Maximum Parsimony Analysis__ ...... 25

DISCUSSION 33 C. nolascensis + C. conspicuosa...... 33 Insular Populations__ ...... ...... 36 C. conspicuosa of Isla Cholludo...... ••••••••••••••••••••••• 38 C. nolascensis + C. macrolopha...... •••••••••••••.•••••••••• 38 C. hemilopha + C. h. insulana...... 39 (C. macrolopha I C. nolascensis )+(C. hemilopha I C. h. insulana)... 39

LITERATURE CITED 40

APPENDIX ...... 46 Appendix I: Results of the computer program 46 Appendix II: Habitat and Specimen Photos of the C. hemilopha complex . 49 LIST OF FIGURES

Figure 1. Proposed relationships of Ctenosaura: a) de Queiroz (1.987a,b) strict b) Hollingsworth (1998) strict consensus tree from a consensus tree; frequency bin analysis 8 Figure 2. Relationships based on congruence between de Queiroz (1987a,b) and Hollingsworth (1998) and the addition of three more recently described species

Figure 3. Distribution of the Ctenosaura hemilopha complex ... ..... 11 Figure 4. Mammalian mitochondrial DNA gene map ... ...... 16 Figure 5. Strict consensus tree of the unweighted parsimony analysis using 22 ingroup samples and 2 outgroup taxa ... 27 Figure 6. Map showing the relative distance between; Islas San Esteban and San Pedro Nolasco in the Gulf of California_ . 28 Figure 7. Map of relative geographic distances between all populations within the Ctenosaura hemilopha complex._ ..... 31 Figure 8. Phylogenetic tree from witweighted analysis depicting the placement of the anomalous associations within Ctenosaura nolascensis 34 Figure 9. Map showing the geographic intermediacy of Isla Tiburon to Isla San Esteban and Mainland Sonora 37 Figure 10. Ctenosaura nolascensis basking in the morning sun on. tor) of a cactus on Isla San Pedro Nolasco, Sonora, Mexico._ .... 49 Figure 11. Rocky habitat of Ctenosaura nolascensis on Isla San Pedro Nolasco, Sonora, Mexico (top); Large male C. nolascensis basking on a granite boulder at the top of Isla San Pedro Nolasco, Sonora, Mexico (bottom)...... 50 Figure 12. Large male Ctenosaura nolascensis sitting above nesting site on Isla San Pedro Nolasco, Sonora, Mexico (top); Green juvenile from Isla. San Pedro Nolasco, Sonora, Mexico (bottom)... ..... 51 Figure 13. Female Ctenosaura nolascensis basking on granite outcrop on Isla San Pedro Nolasco, Sonora, Mexico (top); Male C. nolascensis foraging in plants for food on Isla San Pedro Nolasco, Sonora, Mexico (bottom)... ... 52 Figure 14. Isla San Esteban, Sonora, Mexico, as seen from a southern shore of Isla Tiburon (top); Arroyo Limantur on Isla San Esteban, Sonora, Mexico 53 Figure 15. Large male Ctenosaura conspicuosa basking in the sun on top of a giant cactus in Arroyo Limantur on Isla San Esteban, Sonora, Mexico (top); Small male C. conspicuosa seeks refuge amongst the rocks and dense foliage on Isla San Esteban, Sonora, Mexico (bottom)... 54 Figure 16. Ctenosaura conspicuosa male seeking shelter in a rock crevice of Arroyo Limantur on Isla San Esteban, Sonora, Mexico (top); Juvenile C. conspicuosa from Isla San Esteban, Sonora, Mexico (bottom)... 55 Figure 17. Ctenosaura conspicuosa basking in the sun on a cactus within Arroyo Limantur on Isla Esteban, Sonora, Mexico... 56 Figure 18. Isla Cholludo, Sonora, Mexico (top), Northern shore of Isla Cholludo, Sonora, Mexico (bottom)... 57

vi Figure 19. Dense forest of cacti covering Isla Choiludo, Sonora, Mexico (top); Ctenosaura conspicuosa perched on a rock on Isla Cho'ludo, Sonora, Mexico ..•••.• 58 Figure 20. Arroyo above Rancho Santuaria in El Chorro, Baja California Stir, Mexico (top); Rocky mountainside above El Chorro, Baja California Sur, Mexico that contains crevices that serve as habitat for Ctenosaura hemilopha (bottom)... 59 Figure 21. Ctenosaura hemilopha habitat found on rocky mountainsides, south of San Bartolo, Baja California Sur, Mexico (top); Male C. hemilopha basking in the sun on a tree branch just south of San Bartolo, Baja California Sur, Mexico (bottom)... ..... 60 Figure 22. Green (unicolor) juvenile Ctenosaura hemilopha from Rancho Santuario in El Chorro, Baja California Sur, Mexico... 61 Figure 23. Northwest facing arroyo on Isla Cerralvo, Baja California, Mexico that provides habitat for Ctenosaura hemilopha (insulana) (top); Southwest shoreline habitat of C. hemilopha (insulana) on Isla Cerralvo, Baja California, Mexico (bottom)...... ...... 62 Figure 24. Arroyo on the northwest facing side of Isla Cerralvo, Baja California, Mexico, that provides habitat for Ctenosaura hemilopha (insulana) (top); Large male C. hemilopha (insulana) basking in the sun on Isla Cerralvo, Baja California, Mexico (bottom)... 63 Figure 25. Ctenosaura conspicuosa asleep on top of a cactus in Arroyo Limantur at sundown on Isla San Esteban, Sonora, Mexico... 64

vii

LIST OF TABLES

Table 1. Iguanid genera ...... , • . •• .. . • • ...... 4 Table 2. Species and subspecies of the genus Ctenosaura...... 5 Table 3. Specimen localities with their corresponding codes and subspecies/ species designations ...... 18 Table 4. Raw Percent Pairwise Sequence Divergences among C. hemilopha

taxa. ...... ••• ••• ••• ••• ••• f•• ••• ••• ••• ••• ••• ••• ••• ••• ••• ••• ••• ••• ••• ••• ••• ••• ••• 30

viii ABSTRACT

MOLECULAR SYSTEMATICS & EVOLUTION OF THE CTENOSAURA HEMILOPHA COMPLEX (SQUAMATA: IGUANIDAE) by Michael Ray Cryder

Molecular variation within Ctenosaura hemilopha and among other Ctenosaura

species are used to identify species boundaries, assess suitable systematic characters,

identify evolutionary patterns within C. hemilopha mitochondrial DNA sequences and reconstruct species and area relationships among the various taxa. The molecular evolution of the C. hemilopha complex is analyzed using 1109 base pairs of mitochondrial DNA sequence from the cytochrome b and cytochrome oxidase III genes.

Samples come from 22 individuals representing each of the five allopatric populations.

The results of a parsimony analysis showed a strongly supported, partially resolved set of relationships. The strict consenses tee formed in this analysis resulted in a gene tree that is comprised of two well-supported basal haploclades, A and B. The most basal haploclade (A) includes all individuals from Islas San Esteban and Cholludo

(C. conspicuosa) and three of the five individuals from Isla San Pedro Nolasco (C. nolascensis). Haploclade B includes all individuals from mainland Sonora (C. macrolopha), Baja California (C. hemilopha), Isla Cerralvo (C. hemilopha (insulana)), and the remaining two samples from Isla San Pedro Nolasco. The anomalous positions of the C. nolascensis samples as well as the positions of each of other populations within the complex are discussed for their informativeness and possible relationship to the most recent hypotheses developed concerning Iguanid relationships. 3

INTRODUCTION

Lizards in the family Iguanidae (sensu Frost and Etheridge, 1989) are the most geographically widespread group within the Iguania. They range throughout North,

Central and South America, the Galapagos Islands, the Greater and Lesser Antilles, and various islands in the Fiji and Tonga archipelagos. The family consists of eight extant genera and approximately 35 species (Buckley and Axtell, 1997; de Queiroz, 1987, 1995;

Etheridge, 1982; Hollingsworth, 1998; Kohler, 1995; Kohler and Streit, 1995) of moderate to large lizards (Table 1) measuring 127 — 745 mm snout-vent length

(Hollingsworth, 1998). The spiny-tailediguanas', Ctenosaura, collective range extends from north-western Mexico to Panama (Table 2). Their distribution is confined to arid and tropical lowlands (below 500 m) along both the Atlantic and Pacific coasts, with inland extensions as far as the lowland habitats persist (Bailey, 1928; Etheridge, 1982).

The genus Ctenosaura is a neotropical, monophyletic group of iguanids (de

Queiroz, 1987a,b; Frost and Etheridge, 1989; Hollingsworth, 1998). It includes thirteen species: C. acanthura, C. alfredschmidti, C. bakeri, C. clarki, C. defensor, C. flavidorsalis, C. hemilopha, C. melanosterna, C. oedirhina, C. pa/earls, C. pectinata, C. quinquecarinata, and C. siimi/is (Buckley and Axtell, 1997; de Queiroz, 1987a and b;

Etheridge, 1982; KOhler, 1995; KOhler and Klemmer, 1994; Table 2). The last major taxonomic revision of the genus was conducted by Bailey (1928). Since then, a number of studies have incorporated Ctenosaura into larger analyses of the Iguanidae de Queiroz,

1987a and references therein; Hollingsworth, 1998; Sites et al., 1996) or expanded species descriptions to include discussions or analyses of the genus as a whole (de

Queiroz, 19871); Buckley and Axtell, 1997). The taxonomy used here follows Etheridge Table 1. Iguanid genera (from Etheridge, 1982; de Queiroz, 1987a). Number of Distribution Genus/ Common Name Species Iguana Laurenti 1768 Mexico, Central and South Green Iguanas America, Lesser Antilles Cyclura Harlan 1824 Greater Antilles Rock or Ground Iguanas Amblyrynchus Bell 1825 Galapagos Archipelago, Galapagos Marine Iguanas Ecuador Ctenosaura Wiegmann 1828 13 Mexico and Central Spiny-tailed Iguanas America Brachylophus Wagler 1830 Fiji and Tonga Islands Banded and Crested Iguanas Conolophus Fitzinger 1843 2 Galapagos Archipelago, Galapagos Land Iguanas Ecuador Dipsosaurus Hallowell 1854 Southwest U.S.A. and Desert Iguanas Northwest Mexico Sauromalus Demeril 1856 Southwest U.S.A. and Chuckwallas Northwest Mexico Table 2. Species and subspecies of the genus Ctenosaura (Buckley, 1997). Species Subspecies Distribution C. acanthura none East coast of Mexico from Shaw 1802 Tamaulipas to Tobasco C. simihs C. s. simi/is Southern Mexico and the Gray 1831 Barbour and Schreve 1834 Yucatan Peninsula to Panama C. s. multipunctata Old Providence Island, Barbour and Schreve 1834 Columbia C. pectinata none West coast of Mexico from Wiegmann 1834 central Sinaloa to Chiapas C. hemilopha C. h. conspicuosa San Esteban and Cholludo Cope 1863 Dickerson 1919 Islands, northern Gulf of California, Mexico

C. h. insulana Cerralvo Island, southern Dickerson 1919 Gulf of California, Mexico

C. h. hemilopha Southern Peninsula of Smith 1972 Baja California, Mexico

C. h. macrolopha Southern Sonora and Smith 1972 Northern Sinaloa, Mexico

C. h. nolascensis San Pedro Nolasco Island, Smith 1972 northern Gulf of California, Sonora, Mexico Table 2 (continued) Species Subspecies Distribution C. qiunquecarinata none Disjunct arid forests from Gray 1842 southern Mexico to Costa Rica

C. defensor none Campeche and Yucatan, Cope 1866 Mexico

C. palearis none Middle Rio Montagua valley, Stejneger 1899 Guatemala

C. bakeri none Utila Island, Honduras Stejneger 1901

C. clarki none Rio Balsas valley, Bailey 1928 southwest Mexico

C. oedirhina none Roatan Island, Honduras de Queiroz 1987

C. flavidorsalis none La Paz, Honduras KOhler and Klemmer 1994

C. alfredschmidti none Pablo Garcia, Campeche, KOhler 1995 Mexico

C. melanosterna none Middle Rio Aguan valley Buckley and Axtell 1997 and the Cayos Cochinos, Honduras (1982) with subsequent species descriptions by de Queiroz (1987b), Buckley and Axtell

(1997), Kohler (1995) and Kohler and Klemmer (1994).

The first phylogenetic analysis of Ctenosaura was completed by de Queiroz

(1987a) and expanded in de Queiroz (1987b) to include Ctenosaura oedirhina (Fig. la).

Subsequent to de Queiroz (1987a,b), Hollingsworth (1998) analyzed the phylogenetic relationships again for the majority of species using an expanded data set (Fig. lb). Both de Queiroz (1987a,b) and Hollingsworth (1998) found strong support for the monophyly of the genus. In the analysis by Hollingsworth (1998), nine unambiguous synapomorphies were found. The interspecific relationships reported by de Queiroz

(1987a,b) and Hollingsworth (1998) were similar, with exception of the placement of

Ctenosaura hemilopha. Hollingsworth (1998) placed C. hemilopha at the base of the

Ctenosaura clade, while de Queiroz (1987a and b) placed it within Ctenosaura and as the sister taxon to the species formerly recognized as Enyaliosaurus.

Subsequent to de Queiroz's (1987a,b) phylogenetic analyses, three additional species of Ctenosaura have been described. Buckley and Axtell (1997) described C. melanosterna from Honduras and found it to be the sister taxon of C. palearis. KOhler

(1995) described C. alfredschmidti from southern Mexicoand found it to be the sister taxon to C. defensor. Kohler amd Klemmer (1994) described C. flavidorsalis and believed it to be allied with C. quinquecarinata. Therefore, the discovery of additional species only involves relationships within the Enyaliosaurus clade and should not affect either of the proposed positions of C. hemilopha within the genus (Fig. 2)

Within Ctenosaura hemilopha, five subspecies have been recognized (Smith, 1972; C similis

C pectinata r.1 C acanthura

C hemilopha C hemilopha C patearis

C acanthura C bakeri

C oedirhina C pectinata C qufriquecarinata '64 C inili C darki

C bakeri

C defignsor C palearis

C oedirhina

C quinquecarinata

C clarki

C defimsor

iti ic na r te ;N. hm

os 0 dsc lem lfre rne a

Figure 2. Relationships based on congruence between de Queiroz (1987a,b) and Hollingsworth (1998) and the addition of three more recently described species. 10 see Appendix 2), four of which are elevated to species level in a recent revision by

Grismer (1999a) who applies an evolutionary species concept to the entire Gulf of

California herpetofauna. Grismer (1999a) synonymizes C. h. insulana from Isla

Cerralvo, Baja California, with C. h. hemilopha from southern Baja California. The remaining three subspecies are each elevated to species. Ctenosaura macrolopha occurs in southern Sonora and northern Sinaloa (Smith, 1972), Ctenosaura nolascensis is

endemic to Isla San Pedro Nolasco, and Ctenosaura conspicuosa is endemic to Isla San

Esteban and also occurs on Isla Cholludo (Fig. 3). Grismer (1994a) suggested that, based

on distributional evidence, there appears to be a mainland Mexico lineage composed of

•C. macrolopha and its insular derivatives C. nolascensis and C. conspicuosa (referred to

as the "macrolopha group") and a Baja California lineage composed of C. hemilopha and

C. insulana (referred to as the "hemilopha group"). The distributional patterns, and their

recognition as evolutionary species, makes a systematic study of this group inviting.

The intent of this study is to analyze the evolutionary relationships between the

various species within the Ctenosaura hemilopha complex. An analysis of this type

provides the opportunity to study the evolutionary and biogeographic patterns between

continental and insular populations. The treatment of C. hemilopha by Smith (1972), and

his recognition of five subspecies, was based on an biological species concept (BSC;

Mayr, 1963). Grismer's (1999a) reevaluation using an evolutionary species concept is

highly congruent to the taxonomy of Smith (1972) except for the recognition of C.

insulana and the shift in taxonomic rank. The operational paradigms of the BSC often

placed similarly appearing organisms, such as the various populations of C. hemilopha,

as subspecies within the same species. The five allopatric subspecies which comprise the 11

Ctenosaura hemilopha

hemilopha conspicuosa insulana

. 3. - Distribution of the Ctenosaura hemilopha complex. 12 group could not be directly evaluated using the BSC because the most important criterion, reproductive compatibility, cannot be evaluated (Frost and Hillis, 1990) due to their allopatry. As a result, Smith's (1972) evaluation was based on the overall morphological similarity. Any attempt to assess reproductive compatibility on the basis of similarity would require that non-objective grouping or ranking, according to the interpreted observations or inferences of the evaluator. Furthermore, since reproductive compatibility among individuals is a shared primitive feature (see Frost and Hillis, 1990 and references therein), any attempt to equate overall similarity with evolutionary relatedness may result in inconsistent conclusions (Rosen, 1978).

Simpson (1959), Wiley (1978), Frost and Hillis (1990) and Frost and Kluge

(1994) developed strong theoretical arguments detailing why the Evolutionary Species

Concept (ESC) is a more objective choice than the BSC. A species, as viewed under the

ESC, is a "single lineage which maintains its identity from other lineages and has its own evolutionary and historical fate (Wiley, 1978)." More recent additions to the ESC include a species as a "sexual plexus through time (Frost and Hillis, 1990)" and "the largest integrating entities immediately below the level of non-integrating clade, which translates to the most inclusive taxa whose parts are on the same phylogenetic trajectory

(Frost and Kluge, 1994)." Owing to its lineage-based philosophy, the Ctenosaura hemilopha complex will be assessed here using the criteria of the ESC.

Since no formal phylogenetic analysis of the C. hemilopha complex has ever been conducted, this analysis will attempt to reconstruct the relationships between the four evolutionary species recognized by &ismer (1999a) by using mitochondrial DNA sequence data. This hypothesis of relationships will then be compared to the proposed 13 relationships from Grismer (1994a) to reassess the synonomy of Ctenosaura hemilopha

insulana with C. h. hemilopha.

Phylogenetic Analysis. The primary goal of this and most systematic studies is to provide insight into the historical structure of a group of organisms and the evolutionary processes that underlie its diversity. The tendency for a given amount of variation among populations to be substantially higher than that within populations allows mtDNA to be used to estimate historical patterns of individuals, and thus to explore specific patterns of historical biogeography (Birmingham and Avise, 1986; -Bowen et al., 1989; Moritz et al.,

1993; Joseph and Moritz, 1994). This phylogeographic mode of evaluation provides a qualitative evaluation of genetic population structure within species (Avise et al., 1987).

The amount and distribution of variation within versus among populations depends on the population sizes and rates of gene flow, both in the past and present. The most common pattern in terrestrial vertebrates is to have less variation within than among geographic populations (Avise et al., 1987). Variants detected within populations generally have low

levels (<1%) of sequence divergence. Much of the variation in animal mtDNA is due to base pair substitutions, with transitions greatly outnumbering transversions (Brown et al.,

1982). However, it is often possible to differentiate many mtDNA haplotypes within populations if sufficient nucleotides are used to sample the genome (Dowling and Brown,

1993).

The unique characteristics of mtDNA also result in some disadvantages.

mtDNA's lack of recombination makes it comparable to a single allozyme locus with

many alleles. As a result, any estimates of gene diversity obtained from mtDNA are

expected to show a larger valiance than comparable estimates made using a large number 14 of nuclear loci. Thus, the extremely fast evolution of some mitochondrial genes can result in convergence, confounding phylogenetic relationships even within some species

(e.g. Lansman et al., 1983; Dowling and Brown, 1989).

Biogeographic Topics. A phylogeny is a hypothesis that makes testable predictions about the historical relationships among groups of organisms and the biogeographic regions that they inhabit. It is difficult to draw conclusions about the biogeographic history of any taxon unless the phylogenetic relationships of the organisms are known. The difficulty in supporting the presence or absence of gene flow between mitochondrial DNA haploclades, in the absence of other intrinsic markers (e.g. nuclear

DNA, morphology, and allozymes), makes the determination of genetic barriers difficult

(Hollingsworth, 1998). It is clear that whether or not two haploclades are genetically independent of one another cannot be determined using only mtDNA markers (Moore,

1995; Moritz et al., 1992). Despite this, the phylogeographic history is useful in determining the historical patterns of dispersal and those geographic features that have influenced the movement of organisms. 15

MATERIALS AND METHODS

Choice of the Mitochondrial Cytochrome b and Cytochrome oxidase 111 Loci. To accomplish the objectives of this project, the data must contain enough variation for the resolution of evolutionary relationships. Smith (1972) analyzed the morphology of these lizards and found some variation, but not enough to construct a robust phylogenetic hypothesis. This analysis will use mtDNA sequence data which provides sufficient variation to construct hypothesis of relationship.

Vertebrate mtDNA is well-characterized and useful in molecular systematic studies (Moritz et al. 1987). It is a small, circular molecule ranging between 15-42 kb in length (Fig. 4), it lacks recombination, and is inherited clonally through maternal lineages. DNA sequences from the cytochrome b and cytochrome oxidase III genes have been analyzed for informative variation. These regions were chosen because they are known to evolve relatively rapidly compared to other genes and may therefore provide useful patterns of variation to estimate relationships among the closely related taxa

(Graybeal, 1993). This gene also lacks insertions and deletions, so aligning the sequences of various taxa is straightforward (de Queiroz and Lawson, 1994). Since there are no previous DNA sequence studies of Ctenosaura hemilopha, the choice of cytochrome b and cytochrome oxidase III was determined during the initial stages of this analysis. Enough intraspecific variation was found to warrant using these regions.

Sample Availability. Most DNA samples were prepared using standard extraction techniques (see below) from tissue samples collected in the field or acquired from others.

Necessary field work involved travel throughout Baja California, Sonora, and selected 16

Figure 4. Mammalian mitochondrial DNA gene order (White and Densmore, 1992). 17

Gulf of California islands to collect fresh tissue samples from each of the previously mentioned localities. Samples from 24 individuals were used in this analysis, representing each of the currently recognized taxa (Table 3). Fifteen come from insular populations, while the remaining 9 are from mainland localities in Baja California (B.C.) and Sonora (SON; see Fig. 3).

Technical Protocols. DNA was extracted from muscle/blood/liver using standard methods of cell lysis, organic extraction, and ethanol precipitation. Two procedures for

DNA isolation were used. Both use similar principles, but different techniques. The phenol-chloroform extraction method described in the Simple Fool's Guide (Palumbi et al., 1991) uses a lysis buffer composed of strong detergents (SDS and EDTA) to disrupt lipid membranes and strong organic solvents (phenol and chloroform) to dissolve proteins. DNA is then precipitated from solution using ethanol to separate it from any left over salts and aid in its recovery. This method is quick (2-3 h) and works well with all kinds of tissues, including those containing large amounts of connective tissue (e.g. muscle and liver). The salting out extraction method (Miller et al., 1988) also uses a lysis buffer containing SDS and EDTA to disrupt cell membranes, but replaces the strong solvents with a protease K solution that enzymatically digests proteins. Proteins are precipitated from solution using a concentrated salt solution, and then DNA is precipitated using ethanol. This method avoids hazardous organic solvents; however it involves a lengthy digest time (12 h) and works well only with tissues containing little connective tissue (e.g. blood). Both methods recovered genomic DNA containing both nuclear and mitochondria' DNA. 18

Table 3. Specimen localities with their corresponding codes and species designation. Code Locality Subspecies/Species C-01 Isla San Esteban, SON C. conspicuosa C-02 Isla San Esteban, SON C. cionspicuosa C-03 Isla San Esteban, SON C. conspicuosa C-04 Isla San Esteban, SON C. conspicuosa C-05 Isla Choiludo, SON C. conspicuosa C-06 Isla Choiludo, SON C. conspicuosa * N-01 Isla San Pedro Nolasco, SON C. nolascensis N-02 Isla San Pedro Nolasco, SON C. nolascensis N-03 Isla San Pedro Nolasco, SON C. nolascensis N-04 Isla San Pedro Nolasco, SON C. nolascensis •N-05 Isla San Pedro Nolasco, SON C. nolascensis * M-01 Bahia San Carlos, SON C. macrolopha M-02 Bahia San Carlos, SON C. macrolopha M-03 Bahia San Carlos, SON C. macrolopha M-04 Bahia San Carlos, SON C. macrolopha H-01 San Bartolo, BC C. h. hemilopha * H-02 El Choro, BC C. h. hemilopha H-03 El Choro, BC C. h. hemilopha 1-01 Isla Cerralvo, BC C. h. insulana 1-02 Isla Cerralvo, BC C. h. insulana 1-03 Isla Cerralvo, BC C. h. insulana 1-04 Isla Cerralvo, BC C. h. insulana * Mel-01 Honduras C. melanosterna Sim-01 _ Yucatan Peninsula C. simi/is

*Individuals used in the cytochrome b analysis 19

Once DNA isolates were obtained, DNA quantification was performed. The TKO

MiniFlourometer was used to quantify the concentration of total genomic DNA by detecting the amount of flourescent dye (Hoechst Dye) that binds to the DNA strands.

Concentrations were recorded in ng/g1 (nanograms per microliter) and subsequently diluted into stock and working solutions for use in PCR amplifications. Primers were selected from published reports or personal contact of researchers doing similar studies.

Cytochrome b primers were chosen from the Simple Fool's Guide (Palumbi et al., 1991) and Kocher et al. (1989). Cytochrome oxidase III primers were obtained from David

Orange from the University of Nevada, Las Vegas. Trial and error experiments of various primer pairs were conducted to determine which would work for C. hemilopha.

The following primers were optimized for each of the DNA samples used (Table 3):

Cytochrome b pair (450 bp)-GLUDG-L 5'-TGA CTT GAA RAA CCA YCG TTG-3' and

CTEN-8 H CTG TOG CGC CTC GGA AGO ATA TTT GGC CTC A-3';

Cytochrome oxidase III (705 bp)- 1,861.8-0O3 5'-CAT GAT AAC ACA TAA TGA CCC

ACC AA-3', H9323-0O3 5'-ACT ACG TCT ACG AAA TGT CAG TAT CA-3' and

(internally designed) L9513-0O3 5'-CCA CTC AAG CCT AGC ACC AAC C-3'.

When PCR conditions were optimized to assure the reaction avoided the amplification of artifact products and that the primers and nucleotides were completely exhausted, PCR amplification was performed. Balanced-piimer PCR reaction mixtures included 5.0 gl 10X Buffer, 5.0 gi of 2.0 mM MgCl2, 5.0 µ1 of 2.0 mM dNTP's, 0.50 Ill

Taq Polymerase and 2.0 gl (10 pmo1/121) template DNA for both 50 pi cytochrome b and cytochrome oxidase III reactions. However, upstream and downstream primer (25 pmol4t1) amounts for the two reactions varied. Cytochrome b required 0.40 1.11 of 20

GLUDG-L and CB3-H with 31.70 1.11 of ddF120. Cytochrome oxidase III required 2.0 iii of L8618-0O3 and H9323-0O3 with 28.50 of old1-120. Each reaction mixture was subjected to the following thermal cycler conditions: 35 cycles of denature (94° C, 60 sec), anneal (50° C, 60 sec), extend (72° C, 90 sec), and a terminal extension (72° C, 10 min). These conditions gave excellent results for both sets of primers. Often it was possible to increase the stringency of the reaction by annealing at 52-55°, and thereby improve the quality of the product (visualized by decreases in the number and prominence of non-specific bands). All PCR reactions included a negative control to check for contamination. The negative control contained all the reagents except for template DNA. When contamination occurred, as indicated by a positive result (band) in the negative control lane, the entire PCR set was discarded and the amplification was repeated until no contamination was detected. All contaminated reagents were discarded.

I found that this, in conjunction with careful laboratory organization and cleanliness, was adequate to prevent any significant contamination problems.

The Mini-PROTEAN II gel apparatus was then used to verify the size and quality of the PCR products. Products ranging between 100-705 bp were separated using a non- denaturing 3.5% polyacrylamide gel. The gel was subjected to a 90 v charge for 45 minutes. Following product migration, the gel was stained with ethidium bromide and visualized under UV light.

Once size and quality of the PCR products was affirmed, gel purification was performed. This separates the PCR products by size on a TAE agarose gel and allows the product of interest to be removed from the gel. A 1.5% TAE agarose gel was used instead of a TBE gel to maximize PCR fragment recovery when using the GeneClean 21

Gel Extraction kit. GeneClean was used primarily because it recovers high yields. It

contains a silica matrix called Glassmilk that binds single and double stranded DNA

without binding lower size fragments such as the primer oligonucleotides. Gel fragments

containing the DNA bands were excised and subjected to the procedures listed in the

GeneClean kit.

Gel purification products were again run (in small amounts) onto a 3.5%

polyacrylamide gel to visualize and determine the approximate concentration needed for

Automated Flourescent Sequencing. The Center of Molecular Biology and Gene

Therapy (CMBGT) at Loma Linda University provides a core facility service for the

automated flourescent DNA sequencing. The CMBGT uses an ABI 373 DNA Sequencer

at a relatively low cost per sample ($15.00 - $18.00 / sample).

The ABI 373 DNA Sequencer automatically analyzes DNA molecules labeled

with any of four different, codon specific, flourescent dyes. After samples were loaded

onto the system's vertical gel, they underwent electrophoresis, laser detection, and

computer analysis. Sequence data was returned within 24-48 hours. The sequence data

for both regions of study was then analyzed, both by hand and with the assistance of

various computer programs (see below).

Alignments. Once the sequence data was acquired, it was edited by hand to verify

electrochromatogram peak calls. Sequences were then aligned. Sequence alignment was

performed by Sequencher 3.0 (Gene Codes Corporation, 1995) using a pairwise

alignment procedure. Pairwise alignments were first assembled for all ingroup samples

and then combined with the alignment of assembled outgroup taxa. Alignments of both

genes was relatively straightforward due to the lack of insertions and deletions. Aligned, 22 sequences (approximately 404 bp for cytochrome b and 705 bp for cytochrome oxidase

III) were analyzed separately and then combined into a complete data set. The two genes totaled 1,109 nucleotide base pairs.

Aligned sequences were exported from Sequencher 3.0 as a NEXUS file, formatted for phylogenetic analysis using MacCiade 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 4.0b1a; Swofford, 1999). The combined DNA sequence data set was initially analyzed using unweighted (= equally) parsimony analysis on only phylogenetically informative characters. Initial trees were constructed using the nucleotide sequence data from cytochrome oxidase III. An analysis was run on the combined cytochrome b and cytochrome oxidase III data for all available representatives from the five subspecies.

Cytochrome b sequence was only available for the individuals N-01, H-02, 0-01, M-01, and Mel-01 (Table 3).

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. Homoplasy levels

(e.g. character incongruence) were evaluated with the consistency index (Kluge and 23

Farris, 1969) and retention index (Farris, 1989,1990). Because the consistency index is affected by uninformative characters, these were excluded from the calculations.

Outgroup Selection. Character states were polarized using the outgroup comparison method (Maddison et al., 1984). Outgroups were selected based on the relationships of Ctenosaura proposed by Hollingsworth (1998; Fig. 1b). Two representatives were chosen (based on availability) and sequenced for analysis in this study. Ctenosaura similis from southern Mexico forms a close relationship to C. hemilopha and was therefore chosen to comprise the first outgroup. The second functional outgroup was C. melanosterna from Honduras.

Confidence and Support. Nonparametric bootstrap analysis (Felsenstein, 1985) was used to evaluate confidence for reconstructed trees from the parsimony analyses. For each analysis, 500 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 optimizing 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 24 be weakly supported, while those present in the slightly longer trees are not necessarily strongly supported (Wiens and Reeder, 1997). Raw pairwise sequence divergences were calculated to obtain an estimate of the percentage differences between each of the C. hemilopha taxa. The percent divergences between each allopatric population were subsequently compared to the relative distances between the population sites.

Due to the nature of mitochondrial DNA, a number of concerns regarding experimental design and phylogenetic analysis were considered. First, samples were included to ensure adequate sampling of each of the populations within the Ctenosaura hemilopha complex throughout their respective geographic ranges (see Table 3; Fig. 3).

Outgroup ta.xa representing both the first and second outgroup nodes were selected as recommended by Maddison et al. (1984) and Watrous and Wheeler (1981). And finally, 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). 25

RESULTS

Phylogenetic Signal. The gi analyses indicate that all data sets used in this study contain substantial phylogenetic signal (as opposed to random data) based on significantly left-skewed gi values at P < 0.01 (Hillis and Huelsenbeck, 1992). The lengths of 10,000 randomly sampled trees resulted in a gi value of -0.220 for these taxa.

The critical gi 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 gi value indicates that the data set is significantly more structured than random data.

Combined DNA Sequence Data. The nucleotide sequence data consists of 1,109 aligned nucleotide positions from the cytochrome b and cytochrome oxidase III genes.

This aligned sequence includes 94 nucleotide positions of the four terminal PCR primers of the two DNA fragments (located at the aligned positions 1-21,631-674, 1079-1109).

The total aligned sequence contains 27.8% adenine (A), 31.7% cytosine (C), 14.9% guanine (G), and 25.6% thymine (T) nucleotides. The data set contains a total of 93 parsimony informative characters, of which a minimum of 67 are informative within the ingroup. Of the 93 parsimony informative characters, 14 (15.1%) are located at first codon positions, 6(6.4%) at second codon positions, and 73 (78.5%) at third codon positions.

Maximum Parsimony Analysis. The analysis resulted in a strongly supported, partially resolved set of relationships. The unweighted parsimony analysis was stopped after 10,000 trees were found due to time and computer memory limitations. The most parsimonious trees each have a consistency index (CI) of 0.81 (excluding uninformative 26 characters) and a retention index (RI) of 0.92. Total character state transformations for both ACCTRAN and DELMAN optimizations are presented in Appendix 1. Only unambiguous (i.e. informative) character state transformations will be discussed from this point on when describing the support for individual samples or haploclades. 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. 5) results in a gene tree that is comprised of two well-supported basal haplociades, A and B (see Figure 5 for haploclade designations).

Haploclade A (node 1) includes all individuals from Islas San Esteban and Cholludo (C. conspicuosa) and three of the five individuals from Isla San Pedro Nolasco (C. nolascensis). This haploclade is strongly supported by twenty-one nucleotide changes and a bootstrap value of 100%. Haploclade B (node 4) includes all individuals from mainland Sonora (C. macrolopha), Baja California (C. hemilopha), Isla Cerralvo (C. hemilopha/C. h. insularui), and the remaining two samples from Isla San Pedro Nolasco.

This haploclade is supported by nine nucleotide changes and a bootstrap value of 100%.

Within haploclade A (node 1), three individuals from Isla San Pedro Nolasco (C. nolascensis) form a haploclade (node 2; Al) supported by four informative nucleotide changes and a 100% bootstrap value. All of the samples from Islas San Esteban and

Cholludo (C. conspicuosa) also form a haploclade (node 3; A2) which is supported by eight nucleotide changes and a bootstrap value of 78%. This relationship is interesting because of the fact that Isla San Pedro Nolasco is 140 lan south of Isla San Esteban and there is nothing but water separating the two islands (Fig. 6).

Within haploclade B (node 4), the sister group relationship between the four 27

C. melanostema ; Mel-01 C similes ; Sim-01 C. nolascensis 1; N-01 100% C. nolascensis 4; N-04 C. nolascensis 5; N-05 100% C. conspicuosa 1; C-01 C. conspicuosa 2; C-02

78% C. conspicuosa 3; C-03 C. conspicuosa 4; C-04 C. conspicuosa 5; C-05 C. conspicuosa 100% 6; C-06 C. macrolopha 1; M-01

83% C. macrolopha 2; M-02 6 C. macrolopha 3; M-03 100% C. macrolopha 4; M-04 5 100% C. nolascensis 2; N-02 2 C. nolascensis 3; N-03

C. hemilopha 1; H-01

100% 100% C. hemilopha 2; H-02 C. hemilopha 3; H-03

86% C. insulana 1; 1-01 8 insulana 2;1-02 100% C. insulana 3; 1-03 10 C. insulana 4; 1-04

Unweighted Parsimony Analysis

Figure 5. Strict consensus tree of the unweighted parsimony analysis using 22 ingroup samples and 2 outgroup taxa. 28

117° 110° Mexicali I F 150 km ulZife'l Sta te 32° s 32° Ale Vico Paso ;2San atlas

0 Puertocitos El Rosar Catavina

San Esteban Bahia de Los Angel San Pedro Nolasco 280 28°

San Ignacio

05 Loreto 0

• La Presa region Paz

La Laguna Isla Cerralvo — 23° Cabo San Lucas

117° 110°

Figure 6. Map showing the relative geographic distance between Islas San Esteban and San Pedro Nolasco in the Gulf of California. 29 individuals from mainland Mexico and the two remaining individuals from Isla San

Pedro Nolasco (node 5) is supported by eight nucleotide changes and a 100% bootstrap value. All of the individuals from mainland Mexico (C. macrolopha) are grouped together (node 6; B1), supported by four nucleotide changes and a 83% bootstrap value.

The two individuals from Isla San Pedro Nolasco (node 7; B2) form a group supported by two nucleotide changes and a bootstrap value of 100%.

The remaining structure within haploclade B contains the individuals from Baja

California and Isla Cerralvo, located off the east coast of the Cape Region (see Fig. 6).

This haploclade (node 8) is supported by five nucleotide changes and a 86% bootstrap value. The three individuals from the Baja California peninsula group together (node 9;

B3), supported by two nucleotide changes and a 100% bootstrap value. The four individuals from Isla Ceffalvo form a well-supported haploclade (node 10; B4). This group is supported by four nucleotide changes and a 83% bootstrap value.

Each of the allopatric populations were compared to obtain an understanding of undirected sequence divergence. The results from pairwise comparisons are illustrated in

Table 4. All possible combinations of taxa are shown, including the two different associations of C. nolascensis. The average range of sequence divergence within haploclade A ranged between 0 — 0.01%. The average range of sequence divergence within haploclade B ranged between 0 — 0.02%. Comparisons between haploclades and B ranged between 0.03 — 0.06%. The geographic distances between all populations were calculated and plotted (Fig. 7). An attempt was made to plot geographic distance against percent divergences, but no significant correlation was found (Pearson correlation

= .359 with low significance). It appears that the separate groupings of Ctenosaura Table 4. 11!•aw Percent Pairwise Sequence Divergences throughout the Ctenos ura hemilopho,' complex.

Cconspiemosa Cnokacensis Comer° phi Cnollascensis Chendlopha C h. (1, 4, S) (2,3) insea na

. . Cconspicaeosa 0.01 % . 01.04% 0.03-0:04 • : 0.03-0.06 0 %..' .. %a,b: % a,b ,

Cnolascensis % 3 .5 (1, 4, 5) % a 0.04 % aj.) 0.04 % 0.04 % a'b 0.0 4

Cvnacrolopha % b •• 0 ?A b 0,01-0.02 0.01 % 0.0 % b

Cnolascensis (2, 3) 0 % 0.01-0.62, 0.01-0.02 % b %

Chesfrnol iipha . :0: • coot % b

C. h. insw.fina 0% b

Blue (a) = Haploclade A Red: (a,b) = Haploclade A ± B . . : .Green (I)) = Haploclade B. : • .

Isla San Esteban to Mainland Mexico (A) = 45 - 50 km Isla San Esteban to Isla Cho'ludo (B) = 15 - 20 km Isla San Esteban to Isla San Pedro Nolasco (C) = 130 -140 km Isla San Pedro Nolasco to Mainland Mexico (D) = 10 - 12 km Isla San Esteban to Cape Region (E) = 600 -700 km Isla San Pedro Nolasco to Cape Region (F) = 450 - 500 km Isla CerraIvo to Cape Rgion (G) = 11- 12 km Mainland Mexico to Cape Region (H) =300 - 400 km

Mainland Mexico

Sonora

Baja C

Isla CerraIvo

Figure 7. Map of relative geographic distances between all populations within the Ctenosaura hemilopha complex. 32 nolascensis (N-01, 04,05 and N-02, 03) and the nature of their origins skew the data considerably. If the separate populations found on Isla San Pedro Nolasco are not considered, then a correlation between distance and divergence appears for the complex.

The fact that Ctenosaura conspicuosa exists as a separate population on Isla San Pedro

Nolasco suggests that problems of time, dispersal mechanisms, vicariance and human- facilitated movements of organisms may be contributing to the lack of correlation seen here. 33

DISCUSSION

The parsimony analysis conducted on the miDNA sequences obtained in this study showed a number of interesting relationships throughout the Ctenosaura hemilopha complex.

C. nolascensis + C. conspicuosa. Based on the unweighted phylogenetic hypothesis produced in this study, two strongly supported, partially resolved haplociades were found (Fig. 5). The most problematic pattern is the position of the samples from

Isla San Pedro Nolasco (Ctenosara nolascensis; Fig. 9; nodes 3 and 7; A2 and B2).

Three of the individuals (N-01, 04,05) group in haploclade A, while the remaining two

(N-02, 03) group in haploclade B. All of the individuals from Islas San Esteban and

Cholludo and the three from Isla San Pedro Nolasco (haploclade A) form a strongly supported sister group relationship (node 1; A1,2). In haploclade B, the two remaining samples of C. nolascensis are each others closest relatives and form a strongly supported sister taxa relationship with the individuals of C. macrolopha from mainland Sonora

(node 5). This divergent phylogeographic pattern is unexpected for an insular endemic found on only a single island, Isla San Pedro Nolasco.

When comparing sequence divergence, the haploclade A C. nolascensis are

0.04% different then from haploclade B C. nolascensis (see Table 4). While not greatly different in the broad sense, this percent difference is relatively large within the C. hemilopha complex. In comparison, each group of C. nolascensis is only 0.01 — 0.02% different from their respective haploclade sister group (see Table 4). Lastly, the two associations of C. nolascensis within haploclades A and B, are structured in such a way 34

C meianosterna ; Mel-01 C similis ; Sim-01 C nolascensis 1; N-01 100% C. nolascensis 4; N-04 C. nolascensis 5; N-05 100% C. conspicuosa 1; C-01 C. conspicuosa 2; C-02 A 78% C. conspicuosa 3; C-03 2 C. conspicuosa 4; C-04 C. conspicuosa 5; C-05 C. conspicuosa 6; C-06 100% C. macrolopha 1; M-01

83% C. macrolopha 2; M-02 1 6 C. macrolopha 3; M-03

100% C. macrolopha 4; M-04

5 100% C. nolascensis 2; N-02 2 C. nolascensis 3; N-03

C. hemilopha 1; H-01 100% 100% C. hemilopha 2; H-02 3 C. hemilopha 3; H-03

86% C. insulana 1; 1-01 8 C. insulana 2; 1-02 100% 4 C. insulana 3; 1-03 10 C. insulana 4; 1-04

Figure 9. Phylogenetic tree from unweighted analysis depicting the placement of the anomalous associations within C. nolascensis. 35 that the individuals from each other's closest relatives, separate from the other samples within each clade.

The lack of geographic concordance of individuals from Isla San Pedro Nolasco suggests that a relatively recent dispersal event has occurred from either Ctenosaura macrolopha from mainland Sonora or C. conspicuosa from Islas San Esteban or

Cholludo. Because the Sonoran coastline is only about 15 km from Isla San Pedro

Nolasco, and both are geologically related, it is reasonable to interpret the relationship between C. macrolopha and the two individuals of C. nolascensis as natural. However, the association between Islas San Esteban and San Pedro Nolasco is more suspicious.

Because these two islands are separated by nearly 140 km of water and have never had a geologic contact (Gastil et al., 1983; Philips, 1964), it is possible that this relationship represents a human-facilitated introduction by the Seri Indians who once inhabited Isla

San Esteban (Grismer, 1994b).

There are other examples of introductions of lizards between islands throughout the Sea of Cortes that have involved the Seri (Malkin, 1962; Case, 1982; Bahre, 1983;

Felger and Moser, 1985; Grismer, 1999b, Hollingsworth, 1999). A similar relationship occurs within Sauromalus (chuckwallas) where large individuals were transported and released onto smaller, coastal islands in the Gulf of California (Grismer, 1994b;

Hollingsworth et al., 1997). These small island transplantations facilitated their recapture and provided a dependable food source for the Seri during long fishing expeditions

(Grismer, 1994b; Hollingsworth et al., 1997).

The relationship formed between C. conspicuosa and the three C. nolascensis samples (N-01, 04, 05) presents a number of questions that need further consideration. 36

Grismer (1994b) has suggested a number of factors that contradict the natural presence of

C. conspicuosa on Islas San Esteban and Choiludo suggesting that it too has been influenced by human-facilitated movements. First, the coastal distribution of C. macrolopha on the mainland only reaches Guaymas, which is approximately 120 km south of any coastal locality opposite Isla San Esteban (Smith, 1972; Grismer, 1994b).

Likewise, Ctenosaura does not inhabit Isla Tiburon, the largest island in the gulf, which lies directly between Isla San Esteban and the mainland (Fig. 10). If the presence of C. conspicuosa is a natural occurrence, then it would be expected to occur on Isla Tiburon or the adjacent mainland (Grismer, 1994a,b). Finally, Isla San Esteban is the only island within the Gulf of California where Sauromalus and Ctenosaura live in symaptry

(Grismer, 1994b). Despite these observations, the mtDNA sequence data suggests that a dispersal event carried individuals from Islas San Esteban or Cholludo to Isla San Pedro

Nolasco, and not the opposite direction.

Insular Populations. The most common belief concerning island populations is that they are generally derived from a mainland source, either by being present at the time the island was formed, or by colonization. Once isolated, these insular populations generally evolve relatively rapidly because of the small founding population size.

However, there are more and more cases where it appears that this process is just as likely for mainland populations (Grismer, 1999b; Hollingsworth, 1999). Mainland population haplotypes continue to evolve and experience extinction patterns that may result in their genetic history being erased for the portion of the tree when the insular populations were formed (Hollingsworth, 1999). The structure of the tree created by this analysis is organized in such a way that two insular groups branch off before the 37

Figure 10. Map showing the geographic intermediacy of Isla Tiburon to Isla San Esteban and the mainland. 38 mainland group. With respect to both Ctenosaura macrolopha from mainland Sonora and C. hemilopha from peninsular Baja California, each is sister taxa to a insular group, while all are the sister group to the entirely insular haploclade A. The basal nature of these insular samples follows the ancestral insular and derived mainland pattern recognized by thismer (1999b) and Hollingsworth (1998). Both recognize the basal position of insular lineages relative to more derived mainland counterparts.

C. conspicuosa of Isla Cholludo. The occurrence of Ctenosaura conspicuosa on

Isla Cholludo is also problematic. The two individuals (C-05, 06) from this island have the identical haplotype as all individuals from Isla San Esteban (C-01 to 04; Fig. 9).

Since these two islands do not share a close geologic history (Gastil et al., 1983), and C. conspicuosa is not found on the two nearest islands, Isla Turner or on Isla Tiburon, the most plausible explanation for the presence on Isla Cholludo is their dispersal from Isla

San Esteban by the Sefi Indians. As commented on earlier, the Seri are believed to be responsible for transporting large iguanas (such as Ctenosaura and Sauromalus) onto smaller, suitable islands throughout the Gulf of California (Grismer, 1994b;

Hollingsworth et al., 1997).

C. nolasensis + C. macrolopha. The association formed between the population from Isla San Pedro Nolasco (C. nolascensis) and C. macrolopha from mainland Sonora appears to be a natural event (Fig. 9; node 5; B1,2). A strongly supported sister group relationship is formed between all of the mainland Sonoran samples (C. macrolopha) and the remaining two samples from Isla San Pedro Nolasco (14-02, 03). The most likely scenario for this relationship is that Isla San Pedro Nolasco population originated from the mainland, either by dispersal or through vicariance at the time the island was formed. 39

The clade of individuals representing Ctenosaura macrolopha indicates that enough time has passed for the coalescence of the mitochondrial haplotype, suggesting that the ancestral mainland population has genetically gone extinct.

C. hemilopha + C. h. insulana. The relationship between the populations from the

Cape Region (C. hemilopha) and Isla Cerralvo (C. h. insulana) is a well-supported association (node 8; B3,4). Isla Cerralvo is a large, continental island that lies approximately 11 km off the coast of southern Baja California (Grismer, 1994b). Based on the rather short geographic distance between the peninsula and the island, it is probable that these populations form two closely related groups. Grismer (1999a) has proposed the recognition of a single taxon, synonymizing C. h. insulana and C. h. insulana. However, C. insulana forms a well-supported group separate from the peninsular C. hemilopha. Yet, in comparing sequence divergences, both are similar with only 0.001% divergence. Thus, I believe that Grismer's (1999a) taxonomy is supported by the data set produced here.

(C. macrolopha and C. nolascensis) + (C. hemilopha + C. h. insulana).

Haploclade B (node 4), contains a strongly supported, highly resolved sister relationship between the Sonoran (C. macrolopha and C. nolascensis) and Baja Californian (C. hemilopha and C. h. insulana) populations. This, in part, supports the relationships proposed by Grismer (1994a), in which, based on distributional evidence, a mainland

Mexico lineage (composed of C. macrolopha and C. nolascensis) and a Baja California lineage (composed of C. hemilopha and C. h. insulana) originated during the formation of the peninsula. However, the sequence divergence (0.01 — 0.02%) indicates a relatively more recent association. LITERATURE CIT ED

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APPENDIX 1

Results of the Computer Program PAUP* 4.0

Unweighted Analysis. A list of apomorphies is presented below. Refer to Figure

6 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) 73 1 0.500 C------>T 113 1 1.000 C---->T 133 1 0.500 T----->C 238 1 1.000 A----->G 265 1 1.000 C=--->T 272 1 1.000 C- -->T- 319 1 0.667 T--->C 352 1 0.500 C --> T 370 1 0.500 T --> C 379 1 1.000 C----->T 385 1 1.000 C----->T 397 1 1.000 T.---->C 412 1 0.500 439 1 1.000 C---->T 451 1 0.500 C----->T 532 1 1.000 C=-->T 605 1 1.000 C----->T 607 1 1.000 A==>G 616 1 0.500 C --> T 678 1 1.000 C --> G 679 1 0.500 T --> C 685 1 1.000 C---- ->T 722 1 1.000 A--->G 749 1 1.000 C----->T 754 1 1.000 A==>G 760 1 1.000 C---->T- 775 1 1.000 C --> T 844 1 0.500 C --> T 1000 1 1.000 C --> T 1045 1 1.000 C------>T

Branch Character Steps CI Change 1075 1 1.000 C --> T Node 2 118 1 1.000 A------>G 274 1 0.500 A==>G 339 1 1.000 C----->T 601 1 1.000 C==>T Node 3 103 1 0.500 T----->C 277 1 1.000 C----->A 319 1 0.667 C--->A 398 1 1.000 C--- - ->T 436 1 1.000 C----->T 544 1 1.000 A---->C 604 1 1.000 A--->G- 643 1 0.500 C------>T 829 1 0.333 967 1 0.500 T -> C Node 4 (Haploclade B) 22 1 1.000 A.- --->G 226 1 1.000 T---->C 322 1 1.000 A----->G 328 1 0.667 G---->A- 415 1 1.000 T---- ->C 502 1 1.000 T----->C 625 1 1.000 C---->T 658 1 1.000 C----->T 844 1 ' 0.500 C --> T 1030 1 1.000 C---->T Node 5 37 1 1.000 A---->G 73 1 0.500 C==>T 76 1 0.667 G----->A- 94 1 1.000 T----->C 241 1 1.000 C.--->T 328 1 0.667 G----->A 610 1 1.000 T----->C- 904 1 1.000 C==>A Node 6 118 1 1.000 A.------>G- 206 1 1.000 G---->A 301 1 1.000 C==>T 505 1 0.500 C--=>T Node 7 307 1 1.000 A---=>C 1087 1 1.000 C------>T 1108 1 0.500 T --> C Node 8 145 1 1.000 A------>C 209 1 1.000 C==>T 214 1 1.000 A----->C 775 1 1.000 C --> T 48

Branch Character Step CI Change 846 1 1.000 874 1 1.000 Node 9 265 1 1.000 T----=>C 512 1 1.000 331 1 0.500 829 1 0.333 Node 10 132 1 1.000 274 1 0.500 331 1 0.500 451 1 0.500 49

APPENDIX II

abitat and specimen photos, taken throughout this study, from each of the populations found within the Ctenosaura hem ilopha complex.

Figure ii. Ctenosaura nolascensis basking in the morning sun on top of a cactus on Isla San Pedro Nolasco, Sonora, Mexico. - F ,xure OCKV flj1fljL of' Ctenosaura noiasrensis on Isla San Pedro Nola _,71co 7 no ascensis o ath-P top n sia an P-efiro sco onora, Mexico (1,01:Loi T iire 13. Large (,tenosaura noiascemcis slain nesting site on S , _ Pedro 1 Mexico (top)• Gre, from Isio San earu - r=, 7., Nola-co, 52

Figure 14. Female Cienosaura nolascensis basking on granite outcrop on Isla San Pedro Nolasco, Sonora, Mexico (top); Male C. nolascensis foraging in plants for food on Isla San Pedro Nolase°, Sonora, Mexico (bottom). ure 15 \2n hZt an .Mexico as s-f---_re from the souther shore of

, vo L;rnintur on Isla SanT-7-stuDa S-tnora drc :b LO:11)

- -

16, Larg,---_ Ctenosau- consi, itmasa- bnskin - in the sun on ciant cartus in rro LT) r =on Isla f'cln Pst ban,Soncra, k xico Small mate co,-----Lspiruos- ammo-, rocKs densi- mnage on Isla 4in tste bail. Sonora Mexico bortom ) 55

Figure 17. Ctenosaura conspicuosa male seeking shelter in the rock crevices of Arroyo Limantur on Isla San Esteban, Sonora, Mexico (top); Juvenile C. conspicuosa from Isla San Esteban, Sonora, Mexico (bottom).

?nosaura cons picuost, hrqskii the sun on a cactus withii Liniaitur on Isla San -EsI I a II , ora, Mexico. 57

Figure 19. Isla Cho'ludo, Sonora, Mexico (top); Northern shore of Isla Cholludo, Sonora, Mexico (bottom). 58

Figure 20. Dense forest of cacti covering Isla Cho'ludo, Sonora, Mexico (top); Ctenosaura conspicuosa perched on a rock on Isla Choiludo, Sonora, Mexico (bottom). 59

Figure 21. Arroyo above Rancho Santuaria in El Chorro, Baja California Sur, Mexico (top); Rocky mountainside above El Chorro, Baja California Sur, Mexico that contains crevices that serve as habitat for Ctenosaura hernilopha (bottom). Figure 22. Ctenosaura hemilopha habitat found on rocky mountainsides, south of San Bartok), Baja California Sur, Mexico (top); Male C. hemilopha basking in the sun just south of San Bart°lo, Baja California Sur, Mexico (bottom). 61

Figure 23. Green (unicolor) juvenile Ctenosaura hemilopha from Rancho Santuario in El Chon-o, Baja California Sur, Mexico. 62

Figure 24. Northwest facing arroyo on Isla Cerralvo, Baja California, Mexico that provides habitat for Ctenosaura hemilopha (insulana) (top); Southwest shoreline habitat of C. hemilopha (insulana) on Isla Cerralvo, Baja California, Mexico (bottom).

- 1 Arrovoa on the northwest facing side of isia Cerralvo. Baja Calif that provides habitat for tenoaarc henzilopha ulana) male C. hem ilo ha (insulana) basking in the sun on Isla Cerralvo, Califou exico (bottom). hf'otzITYta Lir49 Li;!j-A,, CAU

Ctenosaura consuosa asleep on ton of a cactus imPri7.7 sundown on ISIci an. Af_.ra, ico,