I. Project Summary

II. Project Description

A. Introduction

B. Specific Objectives

C. Significance

D. Relation to Current Knowledge and Long-Term Goals

E. Methods and Procedures

1. Rational

2. Selection of Species

3. Choice of Study Population

4. General Methods

5. Specific Methods and Analysis

F. Research Schedule

G. Impact on Education and Human Resources

IV. Literature Cited

V. Biographical Sketch

VI. Budget

VII. Budget Justification

VIII. Current and Pending Support

The Galápagos marine iguana (Amblyrhynchus cristatus) has recently been described as the first "lek mating" reptile. One typically invoked explanation of lek development and evolutionary stability is that of "female choice," where females expend energy actively searching for a suitable mate. One means of determining the value of this model involves following the offspring of one breeding season through their growth and developmental stages. Analyses of offspring survival and physical characteristics are then used to determine the benefits each female receives through "choice" of a particular mate. Additional analyses of male characteristics (including activity, morphological features, and reproductive success) can be used to determine male quality and traits females might base selection on. This project includes the collection of initial individual (male, female, and hatchling) characteristics (both measurements and behavior) for analysis as well as the production and characterization of the molecular markers necessary for parental assignment to offspring. This integrated use of behavior and molecular techniques will lead to both a greater understanding of the dynamics of the mating system as well as assist others interested in using molecular markers for paternity analysis in this species and others that are closely related.

A. Introduction

Much has been written about the general life history and mating behavior of the marine iguana (Amblyrhynchus cristatus) of the Galápagos (Carpenter 1966, Trillmich 1983, Laurie 1990, Laurie & Brown 1990a, b). This primarily herbivorous lizard is only found living along rocky Galápagos island shores (Archipiélago de Colón, Ecuador). During the greater part of the year groups of male and female marine iguanas coexist peacefully. However, as the breeding season (December-February) approaches adult males become increasingly territorial. There is often also a marked movement of adult males from their original locations (outside of the breeding season) to more widely spread sites. Males on established territories battle to remove intruding males. Receptive females generally mate once each breeding season. The majority of copulations occur within established territories and insemination is most often linked to the territory's owner.

The mating system of the marine iguana has recently been categorized as "lek" (Wikelski 1996, Wikelski et al 1996). This categorization follows many years of research and observation that produced conflicting opinions of the system's characteristics (Carpenter 1966, Trillmich 1983, Trillmich & Trillmich 1984, Wikelski et al 1996). Multiple hypotheses have been proposed to explain original development and stability of lek mating systems (female preference - Bradbury 1981, hotspots - Bradbury & Gibson 1983, hotshots - Beehler & Foster 1988, black hole - Stillman et al 1996). One consistent feature of all hypotheses is the need for "female choice." Female choice is defined as any energetic activity (either knowingly or unknowingly [alternatively expressed as - through learned vs. genetically predetermined behavior]) engaged in by a female for the purposes of "selecting" which male or type of male to mate with.

The "female preference" model identified by Bradbury (1981) uses the following simple rule to explain female choice of mate preference: Females prefer to mate with males that are territorial and clustered. This simple rule would drive males to group together and fight among themselves for territories. Males that could not compete with their peers would be excluded and not reproduce in high numbers.

Bradbury and Gibson's (1983) "hotspot" model is an extension of the female preference model and explains the location of male territories. The clustering of male territories is said to be determined by the sequential settling of males onto sites that are preferred by females. In this model active female choice is not as apparent and male- male interactions can influence reproductive success significantly.

"Hotshots," in the model proposed by Beehler and Foster (1988), group together as in the female preference model, because females prefer to mate with males in groups. However, there is a further refinement of the rule that states females prefer to mate with dominant males in the groups. This places increased importance on the competition between males and requires a mechanism to determine dominance hierarchy by females.

Stillman et al (1996) propose in their "black hole" model additional factors that may lead to females preferring grouped males. Mobile females are retained on (or decide to stay within) clustered male territories because there is some increased value or benefit over free-ranging movement. Some suggested benefits to remaining within a cluster of territories are: 1) easier sampling of mating partners, 2) avoidance of harassment, 3) decreased predation, or some other unidentified factor.

These descriptions of the principal components of the models point out the fact that each essentially differs from the others only in the reason why females are making the choice. The work of this study is part of a larger project examining the reproductive advantage that might be obtained by females mating with specific males. Wagner (1998) points out the difference between female "choice" and female "preference" and suggests that multiple test methods are necessary in order to determine the extent and importance of the two for a population. However, his tests are based on behavioral observations and manipulations. The current work will enable examination of the population at both a behavioral and genetic level in order to infer the significance of female choice in the mating system.
 

B. Specific Objectives

This study will examine the physical and behavioral traits associated with territorial reproductive behavior in the marine iguana (Amblyrhynchus cristatus). Additionally, it will provide the molecular marker tools necessary for complete paternity analysis of the species. Field work will require three visits to the study site.

Objective 1: Male Territoriality and Physical Traits. During the first visit to the study site male territorial location and behavior will be recorded. However, prior to behavioral observations, permanent marking and measurement of physical characteristics will be made, and a blood sample will be taken from each territorial male. This information will help to determine territory structure and aid in identification of potential morphological characteristics of value to females in their determination of male quality.

Objective 2: Female Reproductive Activity. While mating is taking place during the first visit, females will be identified, marked, and measured following each observed copulation. Later as the nests are dug and eggs laid, hitherto unmarked females will be captured, measured, and a blood sample taken. This will provide maternal data necessary for later classification as well as a measure of egg-deposition timing and potential.

Objective 3: Hatchling Identification. During the second visit, hatchlings of the previously monitored mating season will be captured as they emerge from the nest. Physical measurements and a blood sample will be taken. This will be followed by permanent marking for later identification. Data collected will enable paternity analysis and later survival data to be collected.

Objective 4: Survival Rates. During the third visit (one year after the initial mating season) the survival rate and growth of marked hatchlings will be assessed. Additionally, during all subsequent visits (second and third) marked male and female survivors will be noted. Such data may be used to analyze individual fitness as well as to help identify characteristics that lead to a higher probability of survival.

Objective 5: Development of Markers for Paternity Analysis: In order to determine reproductive success and the interrelationship of male and female traits on offspring viability, accurate matching of parents with offspring is necessary. To accomplish this, microsatellite molecular markers will be developed for DNA fingerprint analysis.
 

C. Significance

Many previous studies have examined the mating system of the marine iguana (Amblyrhynchus cristatus). However, these have all dealt exclusively with behavioral observations (Carpenter 1966, Dellinger 1996, Laurie 1990, Laurie & Brown 1990a, b, Trillmich 1983, Wikelski & Baurle 1996, Wikelski et al 1996, Wikelski & Trillmich 1997). This study will initiate the integrated use of both behavioral observations and molecular markers in determining individual reproductive success in this species. Previous studies have used biochemical assays (Higgins & Rand 1974, Higgins et al1974, Higgins & Rand 1975, Higgins 1977, Higgins 1978, Higgins 1980) or molecular markers (Rassmann 1997, Rassmann et al 1997a, b) in analysis of marine iguana populations, but none have developed and used techniques precise enough to determine paternity with certainty. A combined use of behavioral and molecular analyses will provide a more accurate picture of the mating system (female selection criteria and reproductive success, territorial male physical and behavioral characteristics and their effects on reproductive success, offspring developmental characteristics and their dependance upon parentage, and breeding male and female genetic characteristics/relatedness) than either of the methods can provide separately.

D. Relation to Current Knowledge and Long-Term Goals

This work will increase the current knowledge of marine iguana reproductive behavior and success in several ways. First, additional information concerning male territorial behavior (site fidelity, activity while on site, copulatory success rate) during the mating season will be gathered. This information may be combined with that of previous studies for comparison of behavior at different times (climatologically) and locations (geographically). Second, further information concerning female nesting activity (time, location, and guard behavior) as well as female physical condition at nesting will be gathered for analysis. Comparisons with previous studies during multiple years and at different sites will be of value to park managers. Third, hatchling physical and behavioral characteristics following emergence from the nest will be compared to the data of other studies. Fourth, permanent marking of males, females, and hatchlings will facilitate later work monitoring survival rate. The recapturing conducted for this study is only the beginning of the valuable data that might be obtained by other researchers for long-term analysis of the population. Fifth, development of the molecular tools necessary for paternity analysis for this species will be of great value to those who plan to conduct further research with this or a closely related species (such as the Galápagos land iguana, Conolophus). Additionally, this combination of work (field observations and laboratory analyses) provides the most valuable, persuasive, accurate, and complete picture of what is happening in the world and is a model I intend to follow in future work.
 

E. Methods and Procedures

At this point it is important to note that the marking, blood sampling, and measurement methods described for the animals have been successfully used in the past, and it has been determined that a minimal amount of pain and suffering is inflicted (Dellinger & von Hegel 1990, Laurie 1990, Laurie & Brown 1990a, b, Wikelski - thesis). Continued adherence to these known methods will utilize both humane treatments and provide comparable results.

1. Rationale

This project will comprise two distinct phases. First to be completed will be the original marking, sampling, and observations of all the individuals to be used in the population. The sampled population comprises the entire breeding assemblage on a small islet during one year. A one year follow up re-sampling of all survivors will be conducted. Following this collection of field data lab work will continue in the form of a search for appropriate molecular markers necessary for paternity tests. The markers of choice for paternity testing are microsatellite loci (Queller et al 1993, Pena & Chakrabority 1994, Ellegren et al 1995, O'Reilly & Wright 1995, Cooper et al 1997, Double et al 1997, Gullberg et al 1997, Hughes & Deloach 1997). These allow "fingerprint" profiles to be established for individuals followed by statistical comparison. In the selection of appropriate loci with the characteristics necessary for paternity testing additional loci will inevitably be discovered that provide useful information at a population or higher lever, but will not be appropriate for paternity analysis. These loci will be reported to other researchers and for use in additional work.

2. Selection of Species

The marine iguana is the first described reptilian lek mating species and its study has several advantages over other lek species (in addition to simple study of their unique way of life) as a drawing factor toward further study. In an analysis of a species' reproductive behavior (and for research in general) it is useful to have as large and complete a data set as possible. First, the animals are easily located and limited in their distribution. They are only found on the Galápagos archipelago of Ecuador. Secondly, they are easy to capture and work with. Darwin's (1909) oft quoted assessment is quite accurate: "The rocks on the coast are abounded with great black lizards between three and four feet long; . . . It is a hideous-looking creature, of dirty black colour, stupid and sluggish in its movements." While the specific descriptors used may not be agreed to by all, their implications provide an accurate portrait of their behavioral characteristics and general appearance within the habitat. Third, according to current knowledge the territorial males provide no resources (other than sperm) to attract females. This provides a good study subject when examining the potential value and implications of female choice of a mate. There are a reduced number of variables to work with since there is no input of male energy before or during hatchling development, while female energetic input following selection of nesting site and egg-laying is limited to a short period of nest guarding. Fourth, the majority of the iguanas' time is spent on the rocky beaches either sunning themselves or foraging within the intertidal zone. Male territories are within this region so behavioral observations are easily made. These characteristics make the marine iguana a species easily captured, measured, sampled, and observed. Because of the ease of data collection a large sample size may be used, providing more accurate results and dependable conclusions.

3. Choice of Study Population

Site selection for this work has been based upon examination of previously completed work on the islet (Trillmich 1983) and discussion with Martin Wikelski (pers. comm.) The site has the advantages of being small and separated (app. 1 km) from a larger island, Isla Santa Cruz. The small study area ensures that a complete breeding assemblage may be sampled and observed systematically. Additionally the islet is near the Charles Darwin Research Station (Estación Científica Charles Darwin) within Bahía Academy on Isla Santa Cruz. Such close proximity to the research station allows ample logistic support when necessary.

4. General Methods

Collection of Physical Measurements, Behavioral Observations, and Blood Samples: Data will be gathered on the small islet of Caamaño. The islet's small size enables collection of data from all territorial males, egg-laying females, and offspring during one reproductive season. The following physical measurement data will be collected:

Sex - Sex to be determined by visual observation or by probing with a sex probe (Dellinger & von Hegel 1990).

Snout-vent-length - Measured along the belly from the tip of the snout to the opening of the cloaca (to the nearest mm).

Tail length - Measured along the underside of the tail from the opening of the cloaca to the tip (to the nearest mm).

Hind leg length - Measured from the center of the body to the tissue between the first and second toes while the leg is held at 90 to body axis (to the nearest mm)

Head width - Measured widest point of the head behind the eyes (to the nearest 0.1 mm)

Weight - Weight to nearest 50 g for males and females and nearest 1 g for hatchlings.

Longest neck spine - Measured length of the longest spine along the neck, for males only (to the nearest 0.1 mm).

Longest back spine - Measured length of the longest spine along the back, for adult males and females only (to the nearest 0.1 mm).

Number of neck spines - Counted number of neck spines/nubs along the neck, for hatchlings only.

Cloaca depth - Measured depth of sex probe penetration, for hatchlings only (to the nearest 0.1 mm).

Ticks - Number of ticks found over the entire body.

Mites - Relative number of mites found around the neck of the animal. The scale used ranges from 0-3, with 0 representing very few or none and 3 indicating highest level with large patches of skin covered completely by the parasite.
 

The following data related to behavior (along with time and date of observation) will be collected:

Section - Division of islet shoreline or land region where the individual and behavior are observed. These will be arranged in approximately 30 m sections.

Zone - Location based on intertidal proximity. Distinctions range in four increments from very low (accessible only at low tides) to high within the bushes above the reaches of high tide.

Females in 1 m - Number of female sized iguanas located within 1 m radius of an adult male.

Number in 1 m - Total number of iguanas located within 1 m radius of the observed iguana.

Number marked - Total number of iguanas identified as marked within a 1 m radius of the observed iguana.

ID of nearest male - Identification number of the nearest marked male (to be recorded for males nearby - within 10 m).

Behavior - Observed behavior of individual. The most commonly observed behaviors have been identified and given short codes. Additional comments on location or behavior will be taken as necessary (such as "headbob" counts for territorial males).
 

Marking of individuals for observation and survival analysis will entail two procedures. First each animal will be permanently marked by branding with a hot wire. This practice has been used successfully in the past following determination of its humanity (Laurie 1990, Laurie & Brown 1990a, b, Snell pers. comm., Wikelski pers. comm.) and provides permanent means of identification. Individuals will be marked in three places (either side near the rear leg and on the belly) with a given number. Hatchlings will receive an additional mark (a line lengthwise) on the underside of the tail as an additional means of identifying them as cohorts for long-term observation. Secondly, each will have the identification number painted on its sides and a small bit of paint will be place along the neck spines and the outer edges of the crown of the head. Painting individual iguanas to facilitate re-identification and observation has been determined safe and humane (Trillmich 1983, Wikelski thesis).

Additionally, a blood sample will be collected from each individual. Blood will be collected from a caudal vessel at the time of measurement and permanent marking. A 1 ml sample will be taken from adult males and females while a 0.25 ml sample will be taken from hatchlings. Blood will be preserved in: 0.1 M EDTA, 0.1 M Trizma- base, and 2% SDS.
 

Systematic Behavioral Observations: Behavioral observations will be taken while moving around the islet in a randomized fashion. Selection of starting point and direction of movement (for observations made around the perimeter if the islet) will be determined randomly. Start point of observations made within the islet will be randomized as well. Both sets of observations (within and around the perimeter of the islet) will follow a predefined route. The route ensures that all locations are observed with equal frequency and at similar times. Reduced disturbance of animal activity is also believed to be facilitated by efficiently patterned movement. (The extra observer motion necessary for absolutely random observations would disturb many animals, and more often.) A regular route ensures that as much data will be collected as possible, and in as timely and efficient a manner as possible.

During observations every effort will be made to ensure as little disturbance as possible is incurred by the observer. When necessary binoculars will be used for identification. Observations will take place at all times during the day in an effort to obtain representative data.
 

Molecular Marker Development: Because of their greater specificity (single location within the genome) microsatellite markers are found to be easier to work with once developed. Paternity analysis has been performed using RAPD (random amplified polymorphic DNA, Tegelström and Höggren 1994) and VNTR (variable number tandem repeat, O'Reilly and Wright 1995) methods, however, such analyses are often performed on populations where one parent is known and/or sample size is small. The multiple sites amplified and visualized by these methods make allele frequency hard to determine. Additionally, due to the large number of bands involved accurate scoring of individual fingerprints is difficult. Microsatellite molecular markers will be searched for and developed following the basic guidelines outlined by Glenn (1998). This protocol has been selected because of its wide use and well tested application toward microsatellite discovery in many eukaryotic organisms. Once identified, markers will be tested on a sample (50) of the territorial males and breeding females (10) in order to determine the markers' variability and informative qualities. Markers with highest variability (largest number of alleles) within the population will be selected and reported as useful in paternity analysis. Any markers with sub-optimal variability will be reported as useful in broad comparisons between separate populations or within related species (Rassmann 1997, Rassmann et al 1997a).
 

5. Specific Methods and Analysis⁽¹⁾

Data collected from measurements, behavioral observations, and molecular marker development will be used for the following purposes:

Survivorship: Kaplan-Meier survival curves will be computed for the specific segments of the population (i.e. territorial males, females, and hatchlings). These survival data will add to and may be compared with those of previous projects. Additionally, a comparison of animals within the population segments with similar physical characteristics may be performed utilizing the Kruskal-Wallis test. These measures will provide information relating to the implied fitness (survival) value of the characteristics. An additional test analyzing data from all segments of the population may utilize Cox proportional hazards regression.

Physical Attributes vs. Behavior Comparisons: Physical measurements recorded at the time of capture will be used to analyze how they might correlate with behavior. Depending upon the number of groups to be analyzed either the Mann-Whitney (for 2 groups) or the Kruskal-Wallace (for 3+ groups) tests will be employed. Additionally, correlation or relationship between specific physical attributes may be determined (as well as between common behaviors).

Plots and Diagrammatic Representations: In addition to the statistical tests mentioned above plots will be made indicating site fidelity and movement patterns for individuals or groups/categories of individuals.

Molecular Marker Development and Analysis: Microsatellite molecular marker development will follow the procedure outlined in the Microsatellite Manual^© by Glenn (1998). Briefly, the major steps in the process are:

1) Extract DNA from blood sample

2) Prepare or obtain competent cells (bacterial cells that DNA may easily be inserted into)

3) Prepare vector for DNA insertion - cut and dephosphorylate

4) Prepare insert DNA for attachment to vector - cut and size-select

5) Ligate size-selected insert DNA into vector

6) Transform competent cells with ligation products (vector with selected DNA inserted)

7) Check insert size and transformation efficiency, re-transform and plate bacterial colonies if necessary

8) Lift/transfer colonies onto filters

9) Prepare probe (that will bind to specific sites within the inserted DNA)

10) Hybridize probe to colonies on filters

11) Expose filters to X-ray film

12) Select positive colonies and check for inserts

13) Isolate plasmid and single stranded DNA for sequencing

14) Sequence inserts

15) Design primers for repeat region of DNA insert

16) Optimize PCR conditions for primer use

17) Screen for allelic variation (60 individuals)
 

Arlequin ver. 1.1 (Schneider et al 1997) and Tools for Population Genetic Analyses - TFPGA (Miller 1997) will be used to analyze the allelic data. Allelic frequency, paired loci linkage disequilibrium, Nei's unbiased heterozygosity, neutrality of alleles tests, test of Hardy-Weinberg equilibrium, inbreeding coefficients (Wright's F_is, F_it, and F_st), and population genetic analyses based on an inferred analysis of variance (AMOVA) will all be calculated.

In their paper entitled "A unified approach to study hypervariable polymorphisms: Statistical considerations of determining relatedness and population distances," Chakraborty and Jin (1993) discuss the theoretical genetic specificity (certainty of identification) necessary to determine relationships at a given reliability within a population. They present a table (see Table 1) indicating the number of loci, with a variety of heterozygosity (H) values, necessary to distinguish a parent-offspring relationship.
 

Table 1. The number of loci (L) needed to determine parent-offspring relationship. Copied from Chakraborty and Jin (1993).
 

Heterozygosity (H)	Number of Loci (L)	Probability of Type I error	Probability of Type II error
0.90	6	0.027	0.000
0.80	11	0.040	0.000
0.70	18	0.031	0.042
0.60	26	0.041	0.048
0.50	48	0.028	0.045

(H₀=no genetic difference, so related and H₁=genetically different, so not related).

Type I error refers to the probability that the parent-offspring relationship will be rejected when in fact the relationship exists (rejecting H₀ when it ought to be accepted), while type II error refers to the probability that the parent-offspring relationship will be accepted when in fact the relationship does not exist (rejecting H₁ when it ought to be accepted).  It is notoriously difficult to distinguish between the parent-offspring and full sibling relationships.  However, in this project the parents and offspring are will defined groups (based on their physical attributes) so this will not create a problem.

Using this theoretical information the actual number of microsatellite loci necessary to meet the paternity analysis needs of the project will depend upon the composite heterozygosity of the loci identified.  In order for the future analysis of all individuals in the population to be most efficient it is imperative that the loci selected for use be as informative (heterozygous) as possible.  Avise (1994) defines heterozygosity as simply the percentage of loci that contain heterologous alleles within any one individual.  Thus, an individual's heterozygosity (h_i), locus heterozygosity (h_j), or population heterozygosity (H) may be calculated when the genetic profiles of a sample group utilizing multiple loci are known.  For this work a sample of 60 individuals will be used to characterized identified microsatellite loci.

1. Unless otherwise noted all statistical tests will be performed using SPSS for Windows 95 release 8.0.0
 

F. Research Schedule

Assisted by the flexible schedule afforded by the Natural Sciences Department of the Graduate School of Loma Linda University, research may be conducted both during the academic year and during summers.  With a starting date of January, 1997, the project should be completed within a 3½-year period as follows:

Winter 1997 - Initial marking, measuring, blood sampling, and observation of  territorial males and nesting females.

Spring 1997 - Second visit to capture, mark, measure, take blood sample, and observe hatchlings; identify and observe remaining marked males and females as well.

Summer 1997 - Begin initial work with molecular techniques and acquire experience using them.

Fall 1997 - Continue work with general molecular techniques and enter behavioral observation data collected to date.

Winter 1998 - Third visit to recapture marked hatchlings, identify and sample previously missed hatchlings; identify and observe remaining marked males and females as well.

Spring 1998 - Continue work with molecular techniques and continue entering data collected to date.

Summer 1998 - Begin initial work on identification of appropriate microsatellites for paternity analysis.

Fall 1998 - Identify and select potential microsatellites for characterization; continue entering data collected to date.

Winter 1999 - Select sample of 60 individuals to test identified microsatellites with for characterization and begin analysis; Continue entering data collected to date.

Spring 1999 - Continue analysis of microsatellites and identify additional ones if necessary.

Summer 1999 - Begin statistical analysis of physical measurements and behaviors; continue microsatellite identification and analysis.

Fall 1999 - Finish microsatellite identification and analysis; integrate analyses of genetics, physical measurements, and behavior.

Winter 2000 - Prepare initial thesis for submission and review.

Spring 2000 - Finalize thesis and submit to committee members.
 

G. Impact on Education and Human Resources

This research should have an enormous impact on the education provided graduate students at this institution.  In general people (especially biologists) are fascinated with research based in the Galápagos and become excited and most interested whenever nearly any aspect of work in the region is discussed.  Faculty (Drs. Carter and Hayes) and student (myself) alike have committed themselves to the time, energy, and departmental resources necessary for the successful completion of the project.  All concerned have determined that the work to be completed is directly related to research and academic goals that have been set.  The information obtained about both work needs and requirements at the site may be provided to those who will work under similar conditions in the future.  A wide range of research experiences will be encountered, from field work to molecular lab work and sophisticated computer analyses.  As far as possible information related to work in all of these areas will be placed within an Internet web site (http://members.spree.com/SIP/jsonnentag/iguana/iguana.htm) in order to assist others with similar interests and needs.  Additionally, information, techniques, and general assistance may be given to those that wish it.  In short, it is the purpose of this research not only to obtain information and disperse it as effectively and widely as possible, but to help those who request it to understand the knowledge that is gained more completely.

 The following equipment and facilities have been approved for use:

University Equipment

 Gel rigs (several of each)
  agarose
  PAGE
  sequencing
 Incubators -- several
 Refrigerator/Freezer -- several (down to -70  C)
 Spectrophotometer -- several
 pH mete -- several
 Centrifuges -- several (all sizes, from bench to highspeed refrigerated)
 Gel imaging -- several dedicated cameras/computers
 Balances -- several (all sizes)
 Autoclave
 PCR machines -- several
 Water baths -- several, including shaker model
 Water purification system
 Computer -- several w/ appropriate software
 8 mm video camera
 35 mm camera
 Film and developing for radioactive imaging
 Pipettors -- all sizes
 Hybridizing chamber
 UV cross linker
 Electroporation equipment
 Hot plate -- several

Logistical support provided by Charles Darwin Research Station (Estación Científica Charles Darwin):

 Permit from Parque Nacional Galápagos - research authorization
 CITES permit issued by Ecuadorian government (INEFAN)
 Travel aid to and from study site
 Food and supply acquisition and delivery
 Living quarters while not at study site
 Arrangement for TAME flights at reduced rate

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Beehler, B. M. and M. S. Foster.  1988.  Hotshots, hotspots, and female preference in the organization of lek mating systems.  Amer. Nat. 131(2): 203-219.

Bradbury, J. W.  1981.  The evolution of leks, p. 138-169 in R. D. Alexander and D. W. Tinkle, eds. Natural selection and social behavior: research and new theory. Chiron Press, New York.

Bradbury, J. W. and R. Gibson.  1983.  Leks and mate choice, p. 109-139 in P. Bateson, ed. Mate choice.  Cambridge University Press, Cambridge.

Carpenter, C.  1966.  The marine iguana of the Galapagos Islands, its behavior and ecology.  Proc. Calif. Acad. Sci. 34(6): 329-376.

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Gullberg, A., H. Tegelström, and M. Olsson.  1997.  Microsatellites in the sand lizard (Lacerta agilis): description, variation, inheritance, and applicability. Biochem. Gen. 35(7/8): 281-295.

Higgins, P. J. and C. S. Rand.  1974.  A comparative immunochemical study of the serum proteins of several Galapagos iguanids.  Comp. Biochem. Physiol. 49A: 347-355

Higgins, P. J., C. S. Rand, and J.-L. Haynes.  1974.  Galapagos iguanas: Amblyrhynchus and Conolophus serum protein relationships. J. Exp. Zool. 189: 255-258.

Higgins, P. J. and C. S. Rand.  1975.  Comparative immunology of Galapagos iguana hemoglobins.  J. Exp. Zool. 193: 319-397.

Higgins, P. J.  1977.  Immunodiffusion comparisons of the serum albumins of marine and land iguanas from different islands in the Galapagos Archipelago.  Can. J. Zool. 55: 1389-1392.

Higgins, P. J.  1978.  Immunochemical identity of the high and low molecular weight forms of Galapagos marine iguana hemoglobin. Comp. Biochem. Physiol. 59B: 129-132.

Higgins, P. J.  1980.  Physico-chemical properties of South American iguanid albumins. Experientia 36: 1388-1389.

Hughes, C. R. and D. Moralez Deloach.  1997.  Developing microsatellites when they are rare: trinucleotide repeat loci in the northern mockingbird Mimus polyglottos.  Mol. Ecol. 6: 1099-1102.

Laurie, W. A.  1990.  Population biology of marine iguanas (Amblyrhynchus cristatus).  I. Changes in fecundity related to a population crash.  J. Anim. Ecol. 59: 515-528.

Laurie, W. A. and D. Brown.  1990a.  Population biology of marine iguanas (Amblyrhynchus cristatus).  II. Changes in annual survival rates and the effects of size, sex, age and fecundity in a population crash.  J. Anim. Ecol. 59: 529-544.

Laurie, W. A. and D. Brown.  1990b.  Population biology of marine iguanas (Amblyrhynchus cristatus).  III. Factors affecting survival.  J. Anim. Ecol. 59: 545-568.

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Academic Credentials:

B. S. Biology, Walla Walla College, 1994
Washington State secondary teaching certification: biology, chemistry, history
Computer Science Certificate, Arizona Western College, 1991
 

Contact Information:

Jeffrey A. Sonnentag                                Natural Sciences Dept.
6262 Del Rosa Ave., #204                      Loma Linda University
San Bernardino, CA 92404                      Loma Linda, CA 92350
(909) 886-1047                                      (909) 824-4300 ext.48915

E-mail:
jsonnentag01g@ns.llu.edu                       sonnjef@sc.llu.edu
jsonnentag@juno.com                             jsonnentag@usa.net
 

Supporting Involvement in the Project:

The following individuals are associated with, have been involved in the design, and will be part of the implementation of the project:

Ronald L. Carter, Ph.D., Loma Linda University
Martin Wikelski, Ph.D., University of Illinois
William K. Hayes, Ph.D., Loma Linda University

A.   Personnel/Funding Duration:                                    Jeffrey Sonnentag/3.5 yrs.

B.   Salary and Wages:                                                  $ 0

C.   Fringe Benefits:                                                       $ 0

D.   Equipment:                                                              $ 2,000*

E.   Travel:                                                                     $ 3,500*

F.   Participant Support Costs (research assistants [4]):
     1.   Travel                                                                 $ 1,000*
     2.   Subsistence                                                         $ 200

G.   Other Direct Costs:
     1.   Materials and Supplies                                        $ 9,000*
     2.   Pub. Costs/Documentation/Dissemination            $ 500*
     3.   Consultant Services                                             $ 0
     4.   Computer Services                                              $ 0
     5.   Subawards                                                          $ 0
     6.   Other                                                                  $ 0

H.   Total Direct Costs:                                                   $ 16,200

I.   Indirect Costs (Graduate RA Stipend [over 4 yrs.]):   $ 24,100*

J.   Total Direct and Indirect Costs:                                 $ 40,300

* Included and explained in budget justification.

A breakdown and explanation of projected expenses marked by an asterisk (*) in the budget are given below.

Equipment:  $ 2,000:

Covers costs of food and supplies necessary for the research while at the study site.  This includes camping and other equipment necessary for capture and measurement of the iguanas.  Also included are the costs of transportation involved in delivery of the supplies to the study site.

Travel:  $ 3,500:

This covers personal travel for three round trips from Los Angeles, CA to the Galápagos via Quito, Ecuador (including all taxi, ferry, and lodging charges).

Participant Support Costs (research assistants [4]):
Travel:  $ 1,000:

Flight costs of four Ecuadorian research assistants from the continent are covered.  Additional assistants may be requested and arranged for at the Charles Darwin Research Station (Estación Científica Charles Darwin) or from the Galápagos National Park (Parque Nacional Galápagos).

Materials and Supplies:  $ 9,000:

Laboratory materials are included here.  Supplies (syringes, needles, and storage vials) for blood sample collection will cost around $200.  Approximately 200 inserts indicating microsatellite repeat content are expected to be sequenced at $20 per sequence ($4000).  Of the 200 sequences it is expected that around 80 will be promising enough to synthesize and test on the sample of 50.  At $16 per synthesized sequence a total of $1280 will be necessary.  Gels (agarose, polyacrylamide, and sequencing) will require materials costing $1000.  Film for visualization of radioactively labeled markers will cost approximately $500.  Reagents (such as Taq, dNTPs, and 32P) are expected to cost $1500, while miscellaneous supplies (pipette tips, petri dishes, growth media, gloves . . .) will require $500.

Pub. Costs/Documentation/Dissemination:  $ 500:

This sum includes the cost of copying and distributing original data collected to all members of the research project as well as journal publication and reprint duplicating and  distribution.

Indirect Costs (Graduate RA Stipend [over 4 yrs.]):  $ 24,100

This expense is typically covered by the Natural Sciences Department of Loma Linda University and not through individual laboratory budgets.  The stipend allotment would be made according to the following:

1st  year  $ 3100
2nd year  $ 5000
3rd  year  $ 8000
4th year  $ 8000

Support for this research (with the exception of the indirect cost of graduate research assistantship) has been limited to the laboratory research funds of Ronald L. Carter, Ph.D., of Loma Linda University.  Funds for the graduate research assistantship are distributed by the Natural Sciences Department of the Graduate School through Loma Linda University accounting.
 
 

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