1. Physiological and genetic mechanisms underlying population divergence across an altitudinal gradient Proposed Research Jonathan Warner Atwell Timothy James Grieves Dawn Michelle O’Neal

2. 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

3. 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

4. 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

5. Rocky Mountain Biological Laboratory

6. Alpine tundra prairie grasslands Subalpine forest (Storz et al. 2004)

7. 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

8. Breeding season Alpine tundra Subalpine forest Prairie grasslands

9. 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)

10. = anti-testosterone antibody Y Y Y Y Y Y Y Y Y Y YYYYYY YYY Y YY YYYY YY = hormone in serum sample = labeled hormone of known amount YY Y Y Color Y Y How do you measure hormones?

11. Hypothesis: seasonal variation in T expression Mountain Mice Prairie Mice TT T T

12. 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

13. Alpine tundra Subalpine forest Prairie grassland

14. Hemolytic Complement Activity by Altitude CH50 Altitude 00.20.40.60.811.21.41.6 Prairie Grasslands Subalpine Forest Alpine Tundra

15. anti-KLH IgG (% plate positive) Alpine Tundra Prairie Grasslands 0 20 40 60 80 Acquired Immune Response (KLH)

16. 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.

17. 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

18. 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”).