Proposed Research Jonathan Warner Atwell Timothy James Grieves Dawn Michelle O’Neal

Physiological and genetic mechanisms underlying population divergence across

an altitudinal gradient

Proposed Research

Jonathan Warner Atwell

Timothy James Grieves

Dawn Michelle O’Neal

Introduction

• Understanding mechanisms that underlie adaptive divergence is a central goal of evolutionary biology and ecology

• Investment in reproduction vs. survival is a ubiquitous life-history trade-off, and optimal strategies should vary with environment

• Few studies have robustly characterized physiological and genetic substrates of divergent life-history strategies

Local adaptation to seasonal environments

From Bronson 1990

•Breeding season length varies with latitude

• Life-history traits also vary with breeding season & latitude, including testosterone and immune function

•Similar differences are seen across an altitudinal gradient

• How has selection shaped the regulatory physiology of divergent mouse populations across an altitudinal gradient?

• Breeding phenology• Seasonal testosterone & immune function

• What are the genetic mechanisms underlying physiological variation?

• Divergent candidate genes

General Questions

Rocky Mountain Biological Laboratory

Alpine tundra

prairie grasslands

Subalpine forest

(Storz et al. 2004)

Field Capture methods

•Capture in baited sherman traps•Sample February-September

•Morphological measures•Assessment of reproductive condition (fondling)•Retro-orbital blood sample•Ear-tag and release

Breeding season

0

5

10

15

20

25

30

35

jan

jan fe

b

march

march

april

may

may

june ju

ly juy

aug

sept

spet oc

tnov

nov

dec

0

5

10

15

20

25

30

35

jan

jan

feb

march

march

april

may

may

june ju

ly juy

augsept

spet oc

tnovnov

dec

0

5

10

15

20

25

30

35

jan

jan

feb

march

march

april

may

may

june ju

ly juy

augsept

spet oc

tnov

nov

dec

Alpine tundra

Subalpine forest

Prairie grasslands

Measuring seasonal T and Innate Immune Function profiles…

-Capture free-living individuals each month before, during, & after breeding in both populations

-Collect sub-orbital blood samples

-Centrifuge blood, freeze plasma

-Conduct EIA assays to measure [ T ]

-Bacterial Killing Assays (Petri Dishes)

-Heamolytic Complement (EIA Plate)

= anti-testosteroneantibody

Y

Y Y

YY

Y

Y

Y

Y

Y

YY Y Y YYY Y YY YY

Y Y YY YY

= hormone in serum sample

= labeled hormoneof known amount

Y Y YY

ColorColor

Y Y

How do you measure hormones?

• Hypothesis: seasonal variation in T expression

Mountain MicePrairie Mice

T TTT

Immune Function

• Innate– Nonspecific antigen defense mechanisms – Bactericidal assay

• Measure of susceptibility

– Hemolytic complement• Measure of ability to respond

• Acquired/adaptive– Developed in response to specific antigens

• KLH– Antibody production

Alpine tundra

Subalpineforest

Prairie grassland

Hemolytic Complement Activity by Altitude

CH50

Alti

tud

e

0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6

Prairie Grasslands

Subalpine Forest

Alpine Tundra

anti

-KL

H I

gG

(%

pla

te p

osi

tive

) Alpine Tundra

Prairie Grasslands

0

20

40

60

80

Acquired Immune Response (KLH)

Common garden study…

-Differences in behavior, hormones, immunity could be genetic or plastic

-Collect 20 males and females from each population from early in life (or pregnant females).

-Establish in common captive rearing conditions

-Collect same measures across season, as in free-living studies

-We expect to document genetic change underlying phenotypic divergence.

Microarray study to identify important loci...

-Construct cDNA library

-Sacrifice individuals during peak breeding from each free-living population

-Compare gene expression profiles

-Bayesian statistics to identify genes that differ

-Use molecular database to evaluate roles of genes that differ between populations

Conclusions• Utilizing a wide array of methods and techniques, we will

be able to assess the mechanisms underlying divergence in breeding phenology, hormonal, and immunological traits.

Broader Impacts:

-Train a diverse group of undergraduates, high-schoolers, and senior “citizen scientists,” in field and lab methods.

-Identify crucial physiological and genetic mechanisms that influence the ability of populations to persist in differing environments (e.g. global climate change, habitat destruction, disease epidemiological patterns)

-Dissemination of our results will include public outreach programs (e.g. “A Moment of Science,” Discovery Channel for Kids, National Geographic, Fox News’s “Nutty Professors”).