1. Evolutionary Biology
Download icon

Convergent changes in muscle metabolism depend on duration of high-altitude ancestry across Andean waterfowl

  1. Neal J Dawson  Is a corresponding author
  2. Luis Alza
  3. Gabriele Nandal
  4. Graham R Scott
  5. Kevin G McCracken
  1. Department of Biology, McMaster University, Canada
  2. Department of Biology University of Miami, United States
  3. University of Alaska Museum and Institute of Arctic Biology, University of Alaska Fairbanks, United States
  4. Centro de Ornitología y Biodiversidad - CORBIDI, Peru
  5. Department of Marine Biology and Ecology, Rosenstiel School of Marine and Atmospheric Sciences, University of Miami, United States
  6. Human Genetics and Genomics, Hussman Institute for Human Genomics, University of Miami Miller School of Medicine, United States
Research Article
Cite this article as: eLife 2020;9:e56259 doi: 10.7554/eLife.56259
6 figures, 2 tables and 3 additional files

Figures

Enzyme pathway diagram illustrating where we observed differences in metabolic enzyme activity and myoglobin content in high-altitude waterfowl compared to their close low-altitude relatives.

In addition to the observed increases in myoglobin content, increases in the activities of hexokinase, ATP synthase, HOAD, and complex II (succinate dehydrogenase), and decreases in activities of pyruvate kinase, lactate dehydrogenase, creatine kinase and complex IV (cytochrome c oxidase), we observed no changes in activity for the enzymes citrate synthase, isocitrate dehydrogenase, malate dehydrogenase, complex I (NADH-ubiquinone oxidoreductase), and adenylate kinase.

Figure 2 with 1 supplement
Simplified phylogenetic tree, generated using maximum parsimony and constrained to the same topology as the global waterfowl phylogeny published by Gonzalez et al., 2009.

(see Figure 2—figure supplement 1). Branch lengths are measured as the total number of nucleotide substitutions in the 5’ end of the mtDNA control region.

Figure 2—figure supplement 1
Phylogeny of the waterfowl based on Gonzalez et al.

Gonzalez et al., 2009 showing the placement of seven high-altitude waterfowl lineages in this study.

Metabolic enzyme activities for (A) lactate dehydrogenase (LDH), (B) pyruvate kinase (PK), (C) creatine kinase (CK), (D) hexokinase (HK), and (E) 3-hydroxyacyl-CoA dehydrogenase (HOAD), measured in the pectoralis of high- and low-altitude waterfowl.

The diagonal represents the line of equality (x = y). Values are shown as mean ± SEM U/g tissue (n = 8–12). High-altitude values are significantly different overall from the corresponding low-altitude values when p<0.05 in Wilcoxon’s Signed-Rank Tests, which were carried out including (*) and excluding (†) ruddy ducks.

Mitochondrial enzyme activities for (A) Complex II, (B) Complex IV, and (C) ATP synthase measured in the pectoralis of high- and low-altitude waterfowl.

The diagonal represents the line of equality (x = y). Values are shown as mean ± SEM U/g tissue (n = 8–12). High-altitude values are significantly different overall from the corresponding low-altitude values when p<0.05 in Wilcoxon’s Signed-Rank Tests, which were carried out including (*) and excluding (†) ruddy ducks.

Myoglobin content measured in the pectoralis of high- and low-altitude waterfowl.

The diagonal represents the line of equality (x = y). Values are shown as mean ± SEM mg/g tissue (n = 8–12). High-altitude values are significantly different overall from the corresponding low-altitude values when p<0.05 in Wilcoxon’s Signed-Rank Tests, which were carried out including (*) and excluding (†) ruddy ducks.

Changes over evolutionary time at altitude of (A) ATP synthase activity and (C) hexokinase activity measured in thepectoralis of seven high- and low-altitude waterfowl pairs.

Values are given as the mean ± SEM U/g tissue (n = 8–12). * - Significantly different activity in high-altitude ducks compared to low-altitude ducks (two-factor ANOVA followed by the Bonferroni post-tests; p<0.05). (B) ATP synthase and (D) Hexokinase activities in each high-altitude taxon plotted against the t/site value between each high-low pair.

Tables

Table 1
Seven species of Andean ducks showing classification level, ΦST, time since divergence (t/site), and the approximate time (T) ago in years they became established at high altitude based on coalescent analysis.

ΦST and t/site were calculated using previously published mtDNA sequences. T in years was calculated using the substitution rate published by Peters et al., 2005 of 4.8 × 10−8 substitutions/site/year.

Cinnamon tealYellow-billed pintailRuddy duckCrested duckPuna teal (H)
Silver teal (L)
Speckled tealAndean goose (H)
Magellan goose (L)
NewNewNewIntermediateEstablishedEstablishedEstablished
SubspeciesPopulationsSubspeciesSubspeciesSpeciesSubspeciesSpecies
ΦST = 0.07ΦST = 0.05ΦST = 0.38ΦST = 0.85ΦST = 0.93ΦST = 0.77ΦST = 1.0
t/site =
0.000143116
t/site =
0.00052227
t/site =
0.000806087
t/site =
0.003174242
t/site =
0.017886364
t/site =
0.019886364
t/site =
0.04547956
T (years) =
2982
T (years) =
10,898
T (years) =
16,793
T (years) =
66,130
T (years) =
372,633
T (years) =
414,219
T (years) =
947,491
Capture range
HA = 3812 m
LA = 0–13 m
Capture range
HA = 3812 m
LA = 3 m
Capture range
HA = 3812 m
LA = 480–507 m
Capture range
HA = 4281–4655 m
LA = 760–1050 m
Capture range
HA = 3812 m
LA = 410–485 m
Capture range
HA = 4209–4657 m
LA = 760–1050 m
Capture range
HA = 4368–4806 m
LA = 0–27 m
HA (n = 8)
LA (n = 8)
HA (n = 8)
LA (n = 10)
HA (n = 6)
LA (n = 10)
HA (n = 12)
LA (n = 10)
HA (n = 11)
LA (n = 10)
HA (n = 11)
LA (n = 10)
HA (n = 12)
LA (n = 8)
Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional
information
Chemical compound, drugGlucoseSigma
G8270
D-(+)-Glucose
≥99.5% (GC)
Enzyme assay reagent
Chemical compound, drugATPSigma
A2383
Adenosine 5’-triphosphate disodium salt hydrate
Grade I,≥99%, from microbial
Enzyme assay reagent
Chemical compound, drugMgCl2Sigma
M8266
Magnesium Chloride anhydrous,≥98%Enzyme assay reagent
Chemical compound, drugNADP+BioShop Canada NAD007B-NADP, Disodium trihydrate,>95%Enzyme assay reagent
Chemical compound, drugG6PDHRoche
10127655001
Glucose-6-Phosphate Dehydrogenase (G6P-DH) grade I, from yeastEnzyme assay reagent
Chemical compound, drugLDHRoche
10127876001
L-Lactate Dehydrogenase (L-LDH) from rabbit muscleEnzyme assay reagent
Chemical compound, drugPEPSigma
P7002
Phosphoenolpyruvic acidtrisodium salt hydrate
≥97% (enzymatic)
Enzyme assay reagent
Chemical compound, drugADPSigma
A5285
Adenosine 5’-diphosphate monopotassium salt dehydrate bacterial,≥95%, powderEnzyme assay reagent
Chemical compound, drugPyruvateSigma
P2256
Sodium pyruvate
ReagentPlus,≥99%
Enzyme assay reagent
Chemical compound, drugNADHBioShop Canada NAD002NADH ß-NICOTINAMIDE ADENINE REDUCEDEnzyme assay reagent
Chemical compound, drugOxaloacetateSigma
O4126
Oxaloacetic acid
≥97% (HPLC)
Enzyme assay reagent
Chemical compound, drugAcetyl CoABioShop Canada ACO201ACETYL COENZYME A, Trilithium SaltEnzyme assay reagent
Chemical compound, drugDTNBSigma
D218200
5,5’-Dithiobis(2-nitrobenzoic acid)
ReagentPlus, 99%
Enzyme assay reagent
Chemical compound, drugIsocitrateSigma
I1252
DL-Isocitric acid trisodium salt hydrate
≥93%
Enzyme assay reagent
Chemical compound, drugCoQ10Sigma
C9538
Coenzyme Q10,
≥98% (HPLC)
Enzyme assay reagent
Chemical compound, drugRotenoneSigma
R8875
Rotenone,
≥95%
Enzyme assay reagent
Chemical compound, drugBSASigma
A6003
Bovine Serum Albumin lyophilized powder, essentially fatty acid free,≥96% (agarose gel electrophoresis)Enzyme assay reagent
Chemical compound, drugKCNSigma
60178
Potassium cyanide
BioUltra,≥98.0% (AT)
Enzyme assay reagent
Chemical compound, drugSuccinateSigma
S2378
Sodium succinate dibasic hexahydrate
ReagentPlus,≥99%
Enzyme assay reagent
Chemical compound, drugDCPIPSigma
D1878
2,6-Dichloroindophenol sodium salt hydrate,
BioReagent
Enzyme assay reagent
Chemical compound, drugDUBSigma
D7911
Decylubiquinone,
≥97% (HPLC)
Enzyme assay reagent
Chemical compound, drugCytcCH2Sigma
C7752
Cytochrome c from equine heart
≥95% based on Mol. Wt. 12,384 basis
Enzyme assay reagent
Chemical compound, drugOligomycinSigma
O4876
Oligomycin from Streptomyces diastatochromogenes
≥90% total oligomycins basis (HPLC)
Enzyme assay reagent
Chemical compound, drugHKRoche
11426362001
Hexokinase (HK)Enzyme assay reagent
Chemical compound, drugAcetoacetyl CoASigma
A1625
Acetoacetyl coenzyme A sodium salt hydrate
Cofactor, for acyl transfer
Enzyme assay reagent
Chemical compound, drugCreatineSigma
C3630
Creatine monohydrate,
≥98%
Enzyme assay reagent
Chemical compound, drugPKRoche
PK-RO
Pyruvate Kinase (PK) from rabbit muscleEnzyme assay reagent
Chemical compound, drugKH2PO4P5378Potassium phosphate monobasic,
ReagentPlus
Assay buffer reagent
Chemical compound, drugEGTASigma
E4378
Ethylene glycol-bis(2-aminoethylether)-N,N,N’,N’-tetraacetic acid,
≥97.0%
Assay buffer reagent
Chemical compound, drugEDTASigma
EDS
Ethylenediaminetetraacetic acid
BioUltra, anhydrous, ≥99% (titration)
Assay buffer reagent
Chemical compound, drugTriton-X 100Sigma
X100
Triton X-100
laboratory grade
Assay buffer reagent
Software, algorithmGeneiousBiometters Ltd., Auckland, NZUsed for sequence alignment
Software, algorithmPAUPVersion 4, Sinauer Associates, Sunderland, Massachusetts, USAUsed to generate branch lengths
Software, algorithmMESQUITEhttps://www.mesquiteproject.org/Used to analyze phylogenetic contrasts
Software, algorithmPDAP modulehttp://mesquiteproject.org/pdap_mesquite/Used to analyze phylogenetic contrasts
Software, algorithmIMhttps://bio.cst.temple.edu/~hey/softwareUsed to calculate divergence

Data availability

- Mitochondrial DNA sequences for Anas puna and Anas versicolor were deposited in GenBank under accession numbers MN734269-MN734345 (details in Supplementary file 2). All data generated or analysed during this study are included in the manuscript and supporting files.

Additional files

Supplementary file 1

Supporting data tables, statistical analyses, and methodology.

(a) Maximal activities (µmol/g tissue/min), body mass (g) and myoglobin (Mb; mg/g tissue) concentration in pectoralis muscle. (b) Two-factor ANOVA results of maximal activities (µmol/g tissue/min), mass (g) and myoglobin (Mb; mg/g tissue) concentration in pectoralis muscle. (c) Two-factor ANOVA results of maximal activities (µmol/g tissue/min), mass (g) and myoglobin (Mb; mg/g tissue) concentration in pectoralis muscle excluding data for ruddy ducks from the subfamily Oxyurinae. (d) Test of covariance for enzyme activity (µmol/g tissue/min) or myoglobin content (Mb; mg/g tissue) and body mass (g). (e) Test of covariance for enzyme activity (µmol/g tissue/min) or myoglobin content (Mb; mg/g tissue) and body mass (g) excluding data for ruddy ducks from the subfamily Oxyurinae. (f) Correlation analyses of phylogenetic independent contrasts of bird mass (g), myoglobin (Mb) content (mg/g tissue), or enzyme activity (µmol/g tissue/min) versus altitude (m). (g) Correlation analyses of phylogenetic independent contrasts of bird mass (g), myoglobin (Mb) content (mg/g tissue), or enzyme activity (µmol/g tissue/min) versus altitude (m) excluding data for ruddy ducks from the subfamily Oxyurinae. (h) Assay conditions for enzymatic measurements. (i) List of GenBank gene accession numbers for mtDNA control region used in the construction of the phylogenetic tree. (j) Maximal activities (µmol/g tissue/min) in pectoralis muscle from surface, intermediate and deep tissue sampling locations.

https://cdn.elifesciences.org/articles/56259/elife-56259-supp1-v4.docx
Supplementary file 2

Complete list of accession numbers, databases and source references used to create the phylogenetic tree.

https://cdn.elifesciences.org/articles/56259/elife-56259-supp2-v4.xlsx
Transparent reporting form
https://cdn.elifesciences.org/articles/56259/elife-56259-transrepform-v4.pdf

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)