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Transcriptomic Variation and
Plasticity in Rufous-collared
Sparrows (Zonotrichia capensis)
Along an Altitudinal Gradient
Zachary A. Cheviron, Andrew
Whitehead and Robb T. Brumfield
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 highaltitude environments is largely unexplored.
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
Species Distribution and Sampling Sites
Digital range map created by
Ridgely et al. 2003 and
downloaded from InfoNatura
(2005).
- Higher altitudes indicated with darker colors.
- T, L, and H refer to Transplant, Low altitude, and High altitude
sampling sites, respectively.
Typical habitats along an elevational gradient
on the western slope of the Andes in Peru
Left: Agricultural fields along the coast (100 m elev.).
Middle: Arid montane scrub (2,000 m).
Right: Puna grassland (4,200 m).
Introduction
• 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
Objectives
•
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
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 lowaltitude populations at ~ 3800 m a.s.l.
Two high-elevation field sampling sites in
the central Andes during different seasons
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.
Sample Collection and Experimental Design
• 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
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
Results: Differential gene expression
among treatment groups
• 259 cDNAs represent 188 unique annotated transcripts
exhibited significant main effects of population of origin
– Almost all up-regulated in high-altitude individuals
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)
Expression levels
of 8 cDNAs
exhibited
significant main
effects of sampling
locality
(native alt vs.
transplanted alt)
77 cDNAs had significant interactions between population of origin and sampling
locality of these 77 cDNAs, 51 had significant population main effects
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
Hierarchical Clustering Analysis
333 genes in 4 clusters
Gene ontology
categories
Warmer colors = higher transcription
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
Plasticity patterns in gene expression for cDNAs
with significant population of origin effects
Plasticity pattern
No. of cDNAs
Percentage
Group means not significant
after multiple test correction
61
18.3
1. Convergence towards
intermediate expression
184
55.3
2. Convergence towards
native high altitude
86
25.8
3. Convergence towards
native low altitude
2
0.6
• cDNAs divided into plasticity patterns based on Tukey HSD results
• 99.2% cDNAs significantly different between native high- and
native low-altitude groups
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
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
Discussion
• 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
Gene ontology categories
(determined using gene databases)
Gene ontology category
Percentage of differentially
expressed transcripts
Metabolism
27.7
Protein synthesis
20.6
Signal transduction
9.4
Growth
7.3
Protein transport
6.1
Cell cycle
5.6
Oxidative stress response
3.9
Immune response
2.8
Protein catabolism
2.9
Hormone regulation
1.1
Stress response
1.1
Other
11.5
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
Differential gene expression between highand low-altitude populations
• 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 a 4, b 2, b 8, Fe-S
Complex II — succinate dehydrogenase
Complex III — cytochrome c
Complex IV — cytochrome c oxidase V1a
Complex 5 — F0 ATP synthase subunits d, f, and o, and F1 ATP synthase d
• Consistent with other examples of adaptation in fish, birds and rodents
Differential gene expression between high- and
low-altitude populations
• 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
• 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
–
–
–
–
a-globin producing genes
Preprocathepsin-D producing genes
Several other genes involved in nitric oxide
Suggests compensation for hypoxic stress as well
Take home message
Flexibility of transcriptomic response to
environmental stressors in rufous-collared
sparrows may play a role in explaining its
exceptionally broad altitudinal distribution.
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