Small Grant Proposal, May 2011

Genetic analysis of local climate adaptation in D. melanogaster

Felicia King
PROPOSAL SUMMARY. The study of adaptation is at the heart of evolutionary biology. Nevertheless, after
over a century of rigorous empirical and theoretical developments, we still lacks a comprehensive view of the
mode and tempo of adaptive evolution. The migration of the fruit fly species Drosophila melanogaster from
tropical Africa to more temperate climates is an excellent system for the study of adaptation. D. melanogaster
has only recently colonized temperate climates, yet displays a well characterized set of traits and behaviors that
promote survival under the stresses of winter.
The projects outlined here aim to study the adaptation of D. melanogaster to temperate climates by
investigating genetic loci that are known to vary latitudinally along seasonal and elevational transects in
Northern California. I hypothesize that “Northernly” winter-adaptive genetic variants (alleles) will vary linearly
with elevation and through the season, being most prevalent at high elevation and early in the growing season.
Investigating whether or not such patterns exist will significantly contribute to understanding the mode and
tempo of adaptation in D. melanogaster and will fill fundamental gaps in our knowledge of the ecology of the
species.
Here I propose to assay frequencies of known climate-adaptive alleles in sampled fly populations using
pyrosequencing, a DNA sequencing technology. The results from this analysis will provide genetic data that I
will use to construct the narrative of D. melanogaster's adaptation to the temperate climate of the West Coast.
The project discussed in this proposal will form a critical component of my Honors Thesis in Ecology
and Evolutionary Biology at Stanford University. The project outlined here complements my other ongoing
projects to evaluate seasonal and elevational variation in traits and behaviors associated with winter survival.

OBJECTIVES. I propose to investigate adaptation of D. melanogaster to temperate climates in Central

California by looking genetic variation (i) across elevation gradients, (ii) through the growing season, or (iii)
both. This proposal is aimed at my third objective—Objectives 1 and 2 are summarized to give context for the
genetics work.
Objective 1. Collect flies along elevation gradient through a complete growing season. An ongoing effort, we
began taking collections in February and will take monthly collections up through November.
Objective 2. Assay phenotypes associated with temperate adaptation. To test my hypothesis that winter-

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adaptive phenotypes will be more prevalent in high elevations and early season collections, I will measure
known climate-adaptive traits including diapause incidence, thorax length, development time, starvation
resistance, cold tolerance, and fat content.
Objective 3. Assay frequencies of known alleles underlying climate adaptation. I will perform pooled
sequencing of the collected D. melanogaster at loci linked with known winter-adaptive traits. Candidate loci
include: couch potato (cpo)1; timeless (tim)2; UDP-glucose pyrophosphorylase gene (UGPase)3 and Alcohol
dehydrogenase (Adh)3,4; and
Insulin-like Receptor gene (InR)5.
Variation in these genes have well
documented effects on winter
success and are known to vary
latitudinally. Using
pyrosequencing, I will assay for
allele frequency at these genes. I
will use these data to test two models of the origin and maintenance of clines.

BACKGROUND AND SIGNIFICANCE.

D. melanogaster is a tropical fruit fly that has colonized much of the world over the last 10,000 years 6.
As these flies expanded their range they experienced new environmental pressures that likely resulted in
adaptation. In particular, D. melanogaster have colonized more temperate climates and have evolved in
response to the principal manifestation of temperate climates, cold winters.
One strong line of evidence to support the claim that flies have adapted to harsh winters comes from the
study of clinal variation in allele frequencies. Populations of D. melanogaster along latitudinal gradients in
several continents show robust clinal changes in allele frequencies at a number of loci 4,7. Although these clines
could have been produced solely by stochastic, demographic processes associated with the migration of D.
melanogaster, a number of observations argue strongly against this purely demographic model and in favor of
climatic adaptation. Clines in allele frequency at many loci are repeatable across several countries 8,9,10. Single-
nucleotide polymorphisms (SNPs) close to clinally varying loci generally do not show concordant shifts as
would be expected from a purely demographic model, but instead show panmixia, implying an adaptive
model11,12. Lastly, some of the observed clines have shifted over the last 20 years in a pattern predicted by

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anthropogenic climate change13. Pulling from this evidence, it is very unlikely that demography alone has
produced all or even most of the clines in allele frequencies; consequently, natural selection has most likely
played a role in generating this clinal variation.
Two specific models describing how natural selection could generate clinal variation have been
proposed. The classic model by Haldane14, often referenced as the “migration-selection balance” (MiSB) model,
posits that clines are consequence of the joint effects of migration between neighboring demes and selection
coefficients that vary linearly along the cline or vary strongly at the ends of the cline. Alternatively, clines could
be explained as a byproduct of selection coefficients that vary temporally over the course of a season 15. In this
scenario, referred to as the “seasonal phase cline” (SPC), one allele is favored in the winter, but suffers a fitness
disadvantage in the summer. By this logic, the winter-favored allele will be present in relatively high
frequencies at the beginning of spring, but will decrease through the summer months in proportion to the length
of the growing season. It has been proposed that due to the short growing season at higher latitudes, the winter-
favored allele will not decrease in frequency as substantially in populations closer to the Equator 15. And, in a
complementary fashion, due to shorter, milder winters felt by the populations at lower latitudes, the winter-
favored allele will not rise to the same high frequency it does at higher latitudes. A similar scenario plays out
across elevational clines: the short growing season at high elevations and the shorter, milder winters at low
elevations predict analogous allelic frequency patterns.
The seasonal phase cline model predicts that estimates of allele frequency should vary through the
growing season in a predictable way, whereas the MiSB model predicts that allele frequencies will be relatively
constant through time once populations reach equilibrium. Thus, by assessing the dynamic change in allele
frequency through time, I will be able to test these alternative cline generating hypotheses. If alleles present at
higher frequency at high elevation are also at high frequency in the early part of the growing season and decrease
in frequency as the season progresses, then I will conclude that these clines are maintained by the SPC
mechanism.

METHODS.
I will use pyrosequencing to estimate allele frequency at 5 genes known to vary in a clinal fashion or
known to strongly affect the phenotypes I will measure in Objective 2. Pyrosequencing is a highly validated and
efficient way to assess allele frequency in pooled samples 16,17. Allele frequency estimation by pyrosequencing
works by measuring relative heights of nucleotide peaks in a sequencing run; these heights are used to determine

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the proportion of DNA molecules that contain each allele. To perform pyrosequencing, I will pool individuals
from each collection (collections will be made by myself and other members of the Petrov Lab) and extract
DNA. I will then use PCR to amplify a 100 bp region surrounding the adaptive SNPs in each gene. These
amplicon pools will be sent to Stanford's PAN facility for pyroseqencing. To accurately estimate allele
frequency from DNA pools, I need to calibrate pyrosequencing reactions with pools of DNA with known 50:50
allele frequencies. To this end, I will use the Drosophila Genetic Resource Panel, which is a population of
isofemale lines from Raleigh, NC each with individually resequenced genomes. I will identify strains that bear
each allele, cross them to make heterozygotes, PCR amplify the regions of interest and send them to the
pyrosequencer along with my samples. I will use linear models to test for changes in allele frequency through
time and space.

RESOURCES AND PREPARATION.

Previous experience in the lab has prepared me for undertaking this project. The summer before
entering Stanford I worked as a lab assistant at UCSC, gaining time-intensive experience with lab protocol, DNA
extraction, and qPCR. As a member of the Petrov Lab, I have familiarized myself with the layout and capacities
of the lab, performed DNA extractions, run PCR, and completed lab safety training. Professor Petrov and one of
his postdoctoral students, Alan Bergland, will mentor me in this project.
Fall quarter I took Bio41: Molecular Biology, which explored the mechanisms of molecular evolution,
focusing on historical experimental methodology, and discussed DNA sequencing technologies. I am currently
taking Bio131: Fundamentals of Molecular Evolution to become more familiar with key molecular evolutionary
processes.
This project will culminate in an Honors Thesis, required for my concentration in Ecology and
Evolutionary Biology within the Biology major. I plan to continue and expand on this project for the next two
years, ultimately producing a paper for publication. I will present the results of this work at the next Bay Area
Population Genomics IV Conference and at the 2012 Evolution Society Meeting.
I received UIRP fellowship from the Woods Institute at Stanford, which provides $5,600 to fund the
first two objectives and provide a summer stipend. These funds will not cover the cost of genotyping with
pyrosequencing.

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TIMELINE AND WORKFLOW CHART.

Tasks Week Week Week Week Week Week Week Week Week Week
1 2 3 4 6 7 8 9 10 11

Collections, identifying isofemale lines Temp Temp Temp

Sample Sample Sample
1 2 3

Extracting DNA from some progeny and set up new

generation
Pyrosequencing

Starting to assay for the F2 for phenotypes

Fig 5: (above) A magnified view of the first 11 weeks of the summer season field and lab work schedule that includes the
collection of 3 temporal samples and processing of 2. The five week cycle shown here repeats every month, with each new
field collection. Pyrosequencing will be an ongoing effort beginning this June and continuing into next winter.

FUNDING ALLOCATIONS. The market price for a single run with pyrosequencing is roughly 10 dollars.
The following are the expected costs of pyrosequencing 21 temporal samples (7 time points from 3 populations)
and 5 elevational samples around 5 genes (tim, Adh, UPGase, cpo, InR):

Anticipated Cost Estimated Cost

3 control runs per gene (two homozygotes, 1 heterozygote) $200
3 populations in central valley * 7 time points $1050
5 populations from central valley to sierra * 1 time point $250
Total: $1500

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1 Schmidt, P. S., C. T. Zhu, et al. (2008). “An amino acid polymorphism in the couch potato gene forms the basis for
climatic adaptation in Drosophila melanogaster.” Proc Natl Acad Sci U S A 105(42): 16207-16211.

2 Tauber, E, F. Sandrelli, et al. (2007). “Natural selection favours a newly derived allele of the circadian clock gene
timeless in European Drosophila melanogaster populations.” Science 316: 1895-1898.

3 Aquardo, C.F., S.F. Desse, et al. (1986). “Molecular population genetics of the alcohol dehydrogenase gene region
of Drosophila melanogaster.” Genetics 114: 1165-1190.

4 Sezgin, E., Duvernell, D.D., Matzkin, et al. (2004). “Single locus latitudinal clines and their relationship to
temperate adaptation in metabolic genes and derived alleles in Drosophila melanogaster.” Genetics 168: 923-031.

5 Paaby, A.B., M.J. Blacket, et al. (2010). “Identification of a candidate adaptive polymorphism for Drosophila life
history by parallel independent clines on two continents.” Molecular Ecology 19: 760-774.

6 David, J. R. and P. Capy. (1988). “Genetic variation of Drosophila melanogaster.” Trends in Genetics 4: 106-111.

7 Flowers, J. M., E. Sezgin, et al. (2007). “Adaptive evolution of metabolic pathways in Drosophila.” Mol Biol Evol
24(6): 1347-1354.

8 Gonzalez, J., T. L. Karasov, et al. (2010). “Genome-wide patterns of adaptation to temperate environments
associated with transposable elements in Drosophila.” PLoS Genet 6(4): e1000905.

9 Knibb, W. R. (1988). “Chromosome inversion polymorphisms in Drosophila melanogaster. II. Geographic clines
and climatic associations in Australasia, North America and Asia.” Genetica 58: 213-221.

10 Turner, T. L., M. T. Levine, et al. (2008). “Genomic analysis of adaptive differentiation in Drosophila
melanogaster.” Genetics 179(1): 455-473.

11 Berry, A. and M. Kreitman (1993). “Molecular analysis of an allozyme cline: alcohol dehydrogenase in Drosophila
melanogaster on the east coast of North America.” Genetics 134(3): 869-893.

12 Gockel, J., W. J. Kennington, et al. (2001). “Nonclinality of molecular variation implicates selection in maintaining
a morphological cline of Drosophila melanogaster.” Genetics 158(1): 319-323.

13 Umina, P. A. (2005). “A rapid shift in a classic clinal pattern in Drosophila reflecting climate change.” Nature 308:
691-693.

14 Haldane, J. B. S. (1948). “The theory of a cline.” Journal of Genetics 48: 277-284.

15 Rhomberg, L. R. and R. S. Singh (1989). “Evidence for a link between local and seasonal cycles in gene
frequencies and latitudinal clines in a cyclic parthenogen.” Genetica 78: 73-79.

16 Gruber, J. D., P. B. Colligan, et al (2002). “Estimation of single nucleotide polymorphism allele frequency in DNA
pools by using Pyrosequencing.” Human Genetics 110: 395-401.

17 Lavebratt, C. and S. Sengul (2006). “Single nucleotide polymorphism (SNP) allele frequency estimation in DNA
pools using Pyrosequencing.” Nature Protocols 1: 2573-2582.