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Transcriptomic Variation and Plasticity in Rufous-collared Sparrows (Zonotrichia capensis) Along an Altitudinal Gradient Zachary A. Cheviron, Andrew Whitehead.

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Presentation on theme: "Transcriptomic Variation and Plasticity in Rufous-collared Sparrows (Zonotrichia capensis) Along an Altitudinal Gradient Zachary A. Cheviron, Andrew Whitehead."— Presentation transcript:

1 Transcriptomic Variation and Plasticity in Rufous-collared Sparrows (Zonotrichia capensis) Along an Altitudinal Gradient Zachary A. Cheviron, Andrew Whitehead and Robb T. Brumfield

2 Introduction High elevation is metabolically challenging Constant energy production maintained despite: Reduced oxygen availability Increased thermal stress Genetic changes that alter transcript abundance are a possible route for adaptive evolution Adaptive role of transcriptional variation in high- altitude environments is largely unexplored.

3 Rufous-collared Sparrow (Zonotrichia capensis) Very broad, continuous distribution along Pacific slope of Peruvian Andes on altitudinal gradient Sea level to > 4600 m Gene flow reduced along altitudinal gradient Individuals at 4500 m have lower critical temperatures (temp at which metabolic resources must be used to maintain body temperature) Consistent with adaptation to cold, high-altitude habitats

4 Digital range map created by Ridgely et al. 2003 and downloaded from InfoNatura (2005). Species Distribution and Sampling Sites - Higher altitudes indicated with darker colors. - T, L, and H refer to Transplant, Low altitude, and High altitude sampling sites, respectively.

5 Left: Agricultural fields along the coast (100 m elev.). Middle: Arid montane scrub (2,000 m). Right: Puna grassland (4,200 m). Typical habitats along an elevational gradient on the western slope of the Andes in Peru

6 Acclimation to stressful environments often associated with up-regulation of hormones and proteins. – Correlated with changes at transcription level – Variation in protein expression accounts for many acclimation mechanisms on physiological timescales. – Similar regulatory changes may also contribute to adaptation over evolutionary timescales Introduction

7 Identify genes and biochemical pathways important for high-altitude stress compensation – Test for differences in gene expression between populations in native high- and low-altitude environments Distinguish expression differences that are plastic in response to environment, fixed between populations, or interact between population and environment – ‘common garden’ experiment: High- and low-altitude individuals transplanted to a single low-altitude site – Plastic transcriptional variation likely important in acclimation responses – Fixed transcriptional variation may be heritable and important in evolutionary adaptation Objectives

8 Sample Site Selection High-altitude site ~ 4150 m a.s.l Low-altitude site ~ 2000 m a.s.l. Below alt. where physiological acclimation begins High- and low-altitude populations genetically differentiated Population sampled at 2000 m genetically indistinguishable from populations sampled near sea level Phylo-geographic break between high- and low- altitude populations at ~ 3800 m a.s.l.

9 Left: Outside La Oroya, Peru (4,100 m) in June. Right: Near Lircay, Peru (4,300 m) in February. Rufous-collared Sparrows are non-migratory in this portion of their range, so seasonality adds physiological stress for high elevation individuals. Two high-elevation field sampling sites in the central Andes during different seasons

10 4 treatment groups with 4 individuals in each – High-altitude native and High-altitude transplant – Low-altitude native and Low-altitude transplant Native treatments – Individuals captured, housed at ambient temp 1 - 4 days – Tissues sampled at native altitude Transplanted high-altitude treatment – individuals captured at high altitude – transplanted to low altitude – housed at ambient temp for 7 days – tissues sampled Sample Collection and Experimental Design

11 Methods Microarrays – sample gene expression of many genes simultaneously – Used microarray chip from Zebra Finch – Achieved good binding with Rufous-collared Sparrow tissues Data Analysis – compared expression levels among native groups and altitudinal transplants

12 259 cDNAs represent 188 unique annotated transcripts exhibited significant main effects of population of origin – Almost all up-regulated in high-altitude individuals Results: Differential gene expression among treatment groups Expression levels of 8 cDNAs exhibited significant main effects of sampling locality (native alt vs. transplanted alt) 333 cDNAs exhibited significant linear model main effects of population of origin without an interaction effect. (74 unannotated or did not generate significant sequence matches in database searches) 77 cDNAs had significant interactions between population of origin and sampling locality of these 77 cDNAs, 51 had significant population main effects

13 Results: Hierarchical Clustering Analysis cDNAs cluster into 4 main groups based on transcription patterns Gene clusters 1 and 4 best defined high- and low-altitude sample clusters – Primarily genes involved in metabolic processes, esp. oxidative phosphorylation Gene clusters 2 and 3 – Genes relatively under-transcribed – Involved in protein synthesis

14 Hierarchical Clustering Analysis 333 genes in 4 clusters Gene ontology categories Warmer colors = higher transcription

15 Results: Plasticity in expression of cDNAs with population effects None differentially expressed in the common garden Suggests plasticity largely governs variation in transcriptomic profiles among populations native to different altitudes

16 Plasticity patterns in gene expression for cDNAs with significant population of origin effects Plasticity patternNo. of cDNAsPercentage Group means not significant after multiple test correction 6118.3 1. Convergence towards intermediate expression 18455.3 2. Convergence towards native high altitude 8625.8 3. Convergence towards native low altitude 20.6 cDNAs divided into plasticity patterns based on Tukey HSD results 99.2% cDNAs significantly different between native high- and native low-altitude groups

17 Plasticity patterns Pattern 1 (convergence towards intermediate expression) – Native high- and low-altitude individuals different – Transplanted birds not different from each other or either native group – Genes involved in metabolic processes oxidative phosphorylation citrate cycle pyruvate metabolism Pattern 2: (convergence toward native high-alt. expression levels in transplanted birds) – Included several transcripts involved in immune response signaling pathways – May be due to transplant-induced stress response

18 Discussion 188 unique annotated transcripts differentially expressed between populations at high and low altitude Almost all up-regulated in high-altitude birds These genes belong to relatively few gene ontology categories – oxidative phosphorylation – oxidative stress response – protein biosynthesis – signal transduction Many of these genes involved in cold and hypoxic stress response or are targets of natural selection at high altitude in a wide range of other vertebrates

19 Expression levels of differentially expressed genes were highly plastic No transcripts that were differentially expressed between individuals sampled at their native altitudes remained different in common environment Remarkable given short 1-week acclimation period Results suggest great deal of plasticity in transcriptomic profiles Discussion

20 Gene ontology categories (determined using gene databases) Gene ontology categoryPercentage of differentially expressed transcripts Metabolism27.7 Protein synthesis20.6 Signal transduction9.4 Growth7.3 Protein transport6.1 Cell cycle5.6 Oxidative stress response3.9 Immune response2.8 Protein catabolism2.9 Hormone regulation1.1 Stress response1.1 Other11.5

21 Differential gene expression between high- and low-altitude populations When challenged with cold stress, endotherms must increase metabolic heat production to maintain constant body temperature This response often mediated by an increase in metabolic rate, and thermogenic capacity has been shown to be under natural selection in high-altitude deer mice High-altitude rufous-collared sparrows have significantly greater cold tolerance than those from coastal populations Suggests cold adaptation could be mediated through increased metabolic thermogenic capacity

22 Mean minimum temperature at high-altitude site in Feb ~ 1 °C (~ 10 °C colder than low altitude site) Genes up-regulated in high-altitude birds involved in – ATP production (ADP, ATP translocases and ATP synthases) – Citric acid cycle (malate dehydrogenase and isocitrate dehydrogenase) – Oxidative phosphorylation – 5 major complexes of electron transport chain Complex I — NADH dehydrogenase  4,  2,  8, Fe-S Complex II — succinate dehydrogenase Complex III — cytochrome c Complex IV — cytochrome c oxidase V1a Complex 5 — F 0 ATP synthase subunits d, f, and o, and F 1 ATP synthase  Consistent with other examples of adaptation in fish, birds and rodents Differential gene expression between high- and low-altitude populations

23 Other genes upregulated with altitude – Two glycolytic enzymes – Several enzymes involved in minimizing ROS – 37 genes involved with protein synthesis – Several genes involved in ubiquitin-dependant protein catabolism Differential gene expression between high- and low-altitude populations

24 Transcriptomic profiles of high-altitude birds more similar to those in response to cold exposure than to hypoxia – Suggests that for rufous-collared sparrows, cold may impose a more severe physiological demand than hypoxia at high altitude Consistent with physiological studies suggesting increased cold resistance but not hypoxia resistance in high-altitude birds Number of genes involved in hypoxia response differentially expressed between high- and low-altitude individuals –  -globin producing genes – Preprocathepsin-D producing genes – Several other genes involved in nitric oxide – Suggests compensation for hypoxic stress as well Differential gene expression between high- and low-altitude populations

25 Flexibility of transcriptomic response to environmental stressors in rufous- collared sparrows may play a role in explaining its exceptionally broad altitudinal distribution. Take home message

26 Additional Resources Zachary Cheviron‘s Website: http://www.environment.ucla.edu/ctr/news/arti cle.asp?parentID=3137 Database websites: NCBI Gene database http://www.ncbi.nlm.nih.gov/sites/entrez?db =gene AmiGO http://amigo.geneontology.org


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