The study of adaptive molecular evolution in natural populations has been severely limited by the difficulty of linking genetic
variation to phenotypic variation to fitness effects. Most studies connecting genotype, phenotype, and fitness have used reverse genetic
approaches to measure the functional effects of specific mutations in the laboratory because this relationship is difficult to measure in
natural populations, particularly for complex traits because of the "many-to-one" mapping of genotype to phenotype. Many of the
fundamental features of evolving systems, such as evolvability, epistasis, and pleiotropy, however, may be stronger determinants of
evolutionary outcomes in natural populations than in the laboratory because artificial selection and breeding schemes are generally more
simplistic relative to selection and demographic effects in natural settings. Snake venoms have emerged as a system for the study of the
genetics of adaptation in complex, polygenic traits because of their genetic tractability and role in feeding, digestion, and defense, all
of which are directly relevant to fitness. Because venom gene expression is tissue-specific (i.e., no pleiotropic constraints) and toxin
protein abundance directly influences venom efficacy, venoms are not inherently biased toward a particular mutational pathway, enabling a
systematic comparison of the molecular mechanisms underlying adaptive evolution. Venom phenotypes are manifest only upon injection into
another animal, and venom functions are directly measurable through various assays, allowing direct tests of adaptive hypotheses in
natural prey populations. In this work, we sought to create a genotype-phenotype-fitness map for the venom system of the eastern
diamondback rattlesnake (Crotalus adamanteus) and, for the first time, identify the genetic basis of adaptation for a complex, polygenic
trait in natural populations. Crotalus adamanteus is the largest species of rattlesnake and exclusively consumes endotherms. Crotalus
adamanteus is historically native to seven states in the southeastern United States but has recently been extirpated from Louisiana, is
endangered in North Carolina, and is currently under consideration for listing as threatened under the Endangered Species Act. In Chapter
1, we sequenced the venom-gland transcriptome and integrated mass spectrometry data to construct a transcriptome-proteome map for the
venom system. We then used this map to identify significant toxin-gene expression differentiation across the range of C. adamanteus,
providing candidate-genes for which to test the functional and evolutionary significance of the identified variation. In Chapter 2, we
used a similar approach and identified significant ontogenetic differentiation in toxin gene expression; further analyses determined that
ontogenetic effects explained more variation in toxin expression than geographic effects, although both juvenile and adult expression
patterns varied geographically, and time-series experiments in lab-raised individuals demonstrated that geographic and ontogenetic
expression differentiation were not environmentally induced but rather under genetic control. In Chapter 3, we used in vitro functional
assays to verify that the expression differences found in the previous two chapters corresponded to differences in venom function. We
found that, overall, the statistical differences in toxin expression outlined in the first two chapters equated to functional differences
in toxic activities in a predictable, tractable manner, suggesting that the differences identified in the first two chapters were, in
fact, biologically relevant. In Chapter 4, we used a target-enrichment approach to sequence the exons of all identified toxins in the
venom-gland transcriptome as well as several thousand neutral loci to ascertain the relative roles of expression versus coding-sequence
changes in a trait not inherently biased towards either mutational pathway. We found evidence for adaptive changes at both the expression
and sequence levels across the entire range, although expression differentiation did appear to be the more frequent molecular mechanism.
But, without functional characterizations of the identified sequence and expression evolution, it was difficult to characterize the
relative roles demography, selection, and drift played in generating the identified sequence and expression divergence. Although Chapter 3
did link expression variation to functional variation, these assays were not conducted in the actual target of venoms, natural prey. To
address these issues, we examined toxin sequence and expression evolution and estimated venom toxicity (i.e., fitness) in sympatric and
allopatric natural prey across an island-mainland population pair in Chapter 5 to, for the first time, construct a
genotype-phenotype-fitness map for a complex trait in natural populations. We found that expression differentiation was predominantly, or
exclusively, the genetic basis of polygenic adaptation, suggesting that over ecological timescales complex traits may preferentially
evolve through mutations affecting expression because more molecular mechanisms exist for altering the amount of protein produced than for
altering their functions through their primary sequences. In Chapter 1, we found significant expression differentiation in both high- and
low-abundance proteins across the range and over 1 million years of divergence, and in Chapter 4, we found both sequence and expression
differentiation across the same temporal and spatial scales. In Chapter 5, however, we only identified expression differentiation, and
found that this expression differentiation was restricted to low-expression proteins because of physiological and selective constraints on
high-expression proteins. These differences in the molecular mechanism underlying adaptive evolution were most likely the result of
temporal constraints on generating beneficial variation; because more molecular mechanisms exist for altering protein amounts than protein
function, the probability of generating a beneficial expression variant is greater than the probability of generating a beneficial point
mutation in the coding-region of a specific protein, and these differences in probability would be most pronounced over extremely short
timescales. Given enough time, however, both mutational pathways and proteins expressed at all levels can generate beneficial variation,
and these results provide qualitative predictions regarding the process of adaptation for a complex trait. / A Dissertation submitted to the Department of Biological Science in partial fulfillment of the
requirements for the degree of Doctor of Philosophy. / Fall Semester 2016. / November 8, 2016. / adaptation, gene flow, protein expression / Includes bibliographical references. / Darin Rokyta, Professor Directing Dissertation; Peter Beerli, University Representative; Greg
Erickson, Committee Member; Joseph Travis, Committee Member; Alice Winn, Committee Member.
| Identifer | oai:union.ndltd.org:fsu.edu/oai:fsu.digital.flvc.org:fsu_405605 |
| Contributors | Margres, Mark J. (authoraut), Rokyta, Darin R (professor directing dissertation), Beerli, Peter (university representative), Erickson, Gregory (committee member), Travis, Joseph, 1953- (committee member), Winn, Alice A. (committee member), Florida State University (degree granting institution), College of Arts and Sciences (degree granting college), Department of Biological Science (degree granting departmentdgg) |
| Publisher | Florida State University, Florida State University |
| Source Sets | Florida State University |
| Language | English, English |
| Detected Language | English |
| Type | Text, text |
| Format | 1 online resource (192 pages), computer, application/pdf |
| Rights | This Item is protected by copyright and/or related rights. You are free to use this Item in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you need to obtain permission from the rights-holder(s). The copyright in theses and dissertations completed at Florida State University is held by the students who author them. |
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