DOI: 10.5061/dryad.0zpc86757
title: Experimental demonstration of functional divergence in mitochondrial metabolism between two finch subspecies subjected to a thermal challenge

description: Different environments and climates can influence physiological variation
 and species’ geographic ranges. It has been suggested that mitochondria
 may play an essential role in this adaptation. We measured mitochondrial
 metabolism in two geographically separate subspecies of a songbird – the
 Long-tailed Finch, Poephila acuticauda - before and after ten days of heat
 exposure at 40°C. There were significant differences between subspecies,
 with the Red-billed P. a. hecki having lower proton leak and energy
 production efficiency than the Yellow-billed P. a. acuticauda, independent
 of treatment. Mitochondrial metabolism increased significantly after the
 heat treatment in five out of six variables in both subspecies, with P. a.
 hecki showing steeper reaction norms in metabolic acclimation to 40°C for
 the electron transport system. The heat treatment did not significantly
 increase circulating corticosterone, and at an individual level
 corticosterone and mitochondrial metabolism were unrelated. Eight out of
 nine climatic variables differed between subspecies’ geographical ranges,
 but whether these or other ecological variables explain the pre- and
 post-heat divergent mitochondrial performance requires further research.
 The reduced ETS metabolic flexibility of P. a. acuticauda after heat
 exposure suggests that future increases in the frequency and intensity of
 heat waves may impose asymmetric physiological costs on the two
 subspecies.
description: The mitochondrial dataset was obtained using an
 O<sub>2</sub>k real-time respirometer (Oroboros Instruments).
 For details, see Materials and Methods.
description: # Experimental demonstration of functional divergence in mitochondrial
 metabolism between two finch subspecies subjected to a thermal challenge
 Dataset DOI: [10.5061/dryad.0zpc86757](10.5061/dryad.0zpc86757) ##
 Description of the data and file structure The data attached was collected
 for the manuscript titled "Experimental demonstration of functional
 divergence in mitochondrial metabolism between two finch subspecies
 subjected to a thermal challenge", and includes real-time
 respirometry measurements collected before and after a heat challenge,
 corticosterone levels measurements, genetic divergence estimation and
 climatological data of the area where these two finch subspecies (i.e.,
 *Poephila acuticauda acuticauda* and *P. a. hecki*) spread in the wild.
 ### Files and variables #### File: masterdb5.csv **Description:** This
 dataset contains 150 observations for the two subspecies of finches (i.e.
 the Yellow-billed *Poephila acuticauda acuticauda* and the Red-billed *P.
 a. hecki*). More precisely, there are 87 observations for the red beaks,
 corresponding to 46 unique birds, and 63 observations for the yellow
 beaks, corresponding to 34 unique birds. For each colour, the birds have
 been assigned to two groups, control (identified as 25C) and treated
 (identified as 40C). For the reds, we have 15 birds assigned to the
 control group and 31 assigned to the treated group (of which 26 have
 observations after the treatment). For the yellows, we have 11 birds
 assigned to the control group and 23 assigned to the treated group (of
 which 18 have observations after the treatment). ##### Variables *
 DATE: date indicating the date of the experiment (dd/mm/yy) *
 SAMPLE_ID: numeric value indicating the unique ID for each sample *
 BAND: factor indicating the unique bird ID (80 levels) * TREAT: factor
 indicating control group (25°C) and treatment group (40°C) *
 TREAT_TH: factor indicating whether the measurement has been taken before
 or after the treatment. In the case of the control group, the temperature
 is kept fixed at 25°C for both measurements. In the case of the treated
 group, the temperature is 25°C for the first measurement (Before), and
 40°C for the second measurement (After). * Bm: numeric value indicating
 the body mass (g) of the bird * AGE: approximate bird age (days from
 hatching date) * AGE_IN_YEARS: bird age (days rounded to years)  *
 AGE_GROUPED: birds age by hatching date (Y=young; M=medium; O=old) *
 COLOUR: birds' beak colour (Y=yellow; R=red) * CORT: numeric value
 indicating the corticosterone level (ng/ml) * BASAL_CORT: factor
 indicating whether the measurement has been taken before three minutes
 (Y=under 3 minutes; N=over 3 minutes) * CORT_PLATE: factor indicating the
 plate on which the corticosterone level has been measured (8 levels) *
 BASELINE: numeric value indicating the basal mitochondrial oxygen
 consumption (pmol O2s-1 mL-1) * P: numeric value indicating the basal
 mitochondrial oxygen consumption after a pyruvate injection (pmol O2s-1
 mL-1) * OMY: numeric value indicating the mitochondrial oxygen consumption
 related to proton leak (pmol O2s-1 mL-1) * U: numeric value indicating the
 oxygen consumption during the electron transport system (pmol O2s-1 mL-1)
 * PROT: numeric value indicating the sample protein content. * OXPHOS_2:
 numeric value indicating the oxidative phosphorylation (pmol O2s-1 mL-1).
 * OxCE: numeric value indicating the OxPhos coupling efficiency. *
 FCR: numeric value indicating the mitochondrial reserve capacity. *
 BLEEDING_TIME_SEC: numeric value indicating the bleeding time in seconds.
 #### File: VCFs.zip **Description:** To compare patterns of genetic
 divergence between the mitochondrial and nuclear genomes, we used the
 *PopGenome* package in R (Pfeifer et al., 2014). For the mitochondrial
 genome, we used a set of biallelic SNPs called from allopatric individuals
 of wild-caught *Poephila acuticauda acuticauda* (n = 14) and *P. a. hecki*
 (n = 11) (Methods described in McDiarmid et al., 2024). For the nuclear
 genome, we used a set of biallelic SNPs called from whole-genome
 re-sequencing of allopatric individuals of wild-caught acuticauda (n = 15)
 and hecki (n = 16) (Methods described in Hooper et al., 2024). We imported
 Variant Call Format (VCF) data for each of the 28 chromosomes greater than
 5 Mb in length into *PopGenome* and calculated 𝜋 – i.e., the average
 pairwise nucleotide diversity within a population – for *acuticauda* and
 *hecki* separately using the *diversity.stats* function and calculated Dxy
 – i.e., the average pairwise nucleotide diversity between populations –
 using the *diversity.stats.between* function. We then obtained diversity
 estimates per-site by dividing PopGenome results by the total number of
 non-repeat masked sites on each chromosome. We obtained our autosomal
 estimate of 𝜋 and Dxy by summing pairwise differences and accessible sites
 across each of the 27 autosomal chromosomes analysed. Finally, we
 calculated the absolute genetic divergence (da) for the autosomes, Z
 chromosome, and the mitochondrial genome as da = 𝑑𝑥𝑦 ― (𝜋𝑥 + 𝜋𝑦)/2. This
 measure gives an estimate of absolute genetic divergence, taking into
 account genetic variation both within and between subspecies. ##
 Code/software Mitochondrial respiration data were obtained using the O2k
 real-time respirometer graphical user interface, DataLab 7.4 (Oroboros
 Instruments). Data analysis was performed in R, using the following
 packages: tidyverse, lmerTest, patchwork, effects, stargazer, xtable,
 latex2exp, ICC, lme4, dplyr, ggplot2, PopGenome. The scripts used for
 mitochondrial data analysis are in the manuscript's Supplementary
 Materials, and the scripts for the genetic divergence estimation are in
 the VCFs.zip file attached to this repository.