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”).