DOI: 10.5061/dryad.m37pvmd9v
title: Data from: Mitonuclear interactions impact aerobic metabolism in hybrids and may explain mitonuclear discordance in young, naturally hybridizing bird lineages

description: Understanding genetic incompatibilities and genetic introgression between
 incipient species are major goals in evolutionary biology. Mitochondrial
 genes evolve rapidly and exist in dense gene networks with coevolved
 nuclear genes, suggesting that mitochondrial respiration may be
 particularly susceptible to disruption in hybrid organisms. Mitonuclear
 interactions have been demonstrated to contribute to hybrid dysfunction
 between deeply divergent taxa crossed in the laboratory, but there are few
 empirical examples of mitonuclear interactions between younger lineages
 that naturally hybridise. Here we use controlled hybrid crosses and high
 resolution respirometry to provide the first experimental evidence in a
 bird that inter-lineage mitonuclear interactions impact mitochondrial
 aerobic metabolism. Specifically, respiration capacity of the two
 mitodiscordant backcrosses (with mismatched mito-nuclear combinations)
 differ from one another, although they do not differ significantly from
 the parental groups or mitoconcordant backcrosses as we would expect of
 mitonuclear disruptions. In the wild hybrid zone between these subspecies
 the mitochondrial cline centre is shifted west of the nuclear cline
 centre, which is consistent with the direction of our experimental
 results. Our results therefore demonstrate asymmetric mitonuclear
 interactions that impact the capacity of cellular mitochondrial
 respiration and may help to explain the geographic discordance between
 mitochondrial and nuclear genomes observed in the wild.
description: <strong>Study species</strong>
 Long-tailed finches of each subspecies (<em>Poephila
 acuticauda acuticauda</em> and <em>P. a. hecki</em>)
 used in this study came from captive populations maintained at Macquarie
 University in Sydney, Australia. Wild caught <em>P. a.
 acuticauda</em> individuals were originally sourced from Mount House
 (17°02′S, 125°35′E) and Nelson’s Hole (15°49′S, 127°30′E) in Western
 Australia, and wild-caught <em>P. a. hecki</em> were sourced
 from October Creek (16°37′S, 134°51′E) in the Northern Territory[34,35].
 As individuals from these populations show no evidence of genomic
 admixture between subspecies[31,32] we hereafter refer to them as
 ‘parental’. <strong>mtDNA comparison between
 subspecies </strong> Changes in the mitochondrial
 genome are required but not sufficient for inter-lineage mitonuclear
 incompatibility. Even a single substitution in the mtDNA can lead to
 reproductive isolation (e.g., [36]). To identify substitutions with the
 potential to contribute to incompatibilities, we sequenced whole
 mitochondrial genomes from each long-tailed finch subspecies and counted
 the number of fixed differences and associated amino acid changes within
 13 protein coding genes between them. We assembled mitochondrial genomes
 using whole genome sequence (WGS) data from both long-tailed finch
 subspecies (<em>P. a. acuticauda, </em>N = 14; <em>P. a.
 hecki </em>N = 11) and their sister species the black-throated finch
 <em>P. cincta atropygialis </em>(N = 11; Table S1). We used
 bcftools (version 1.9)[37] consensus after calling and incorporating
 variants relative to the zebra finch reference genome (GCA_003957565.4) in
 each sample. Ambiguous positions were output with associated IUPAC
 codes[43]. We exclusively used WGS data derived from DNA extracted from
 muscle (rather than blood) to ensure that they were enriched for reads
 from mitochondrial DNA and not reads from the nuclear encoded mtDNA (NUMT)
 copy. The mitochondrial genome of the zebra finch reference was used as an
 additional outgroup to help assign substitutions to their lineage of
 origin. We used the MITOS2 WebServer[38] to extract specific sequences for
 each mtDNA encoded gene, including all 13 protein coding genes, 22 tRNA
 and 2 rRNA and the control region. Each gene was aligned using MAFFT
 (version 7) web-aligner[39] and we manually examined each alignment with
 Geneious prime (version 2023.1.2) to count and characterize the
 distribution of fixed differences between each of the three members of
 <em>Poephila</em> (Table S2). For each protein-coding gene, we
 calculated the average level of sequence divergence between taxa
 (D<sub>XY</sub>) using the program DnaSP (version 6
 [60]). We next evaluated the extent of non-synonymous
 change between long-tailed finch subspecies for each protein-coding gene.
 We generated lineage-specific consensus sequences for each gene and used
 the vertebrate mitochondrial genetic code to translate these to amino acid
 sequences. We again used data from the black-throated finch and zebra
 finch as outgroups to assign amino acid changes to the long-tailed finch
 subspecies of origin. We classified each amino acid substitution by any
 associated change in biochemical properties. Finally, we calculated the
 ratio of the rate of non-synonymous to the rate of synonymous nucleotide
 substitutions (dN/dS) between long-tailed finch subspecies for each
 protein-coding gene using the model M0 implemented in the program
 Easy-CodeML (v1.41)[40].
 <strong>Breeding</strong> Embryos
 were produced by rotating pairs of long-tailed finches through 20 outdoor
 aviaries (4.1 m long x 1.85 m wide x 2.24 m high), with one pair per
 aviary containing nest boxes and nesting material. Mealworms, greens, dry
 seed, and water were provided <em>ad libitum</em>. We required
 females to have been exclusively with the partner for 14 days minimum
 before any experimental eggs were collected, to avoid embryos being sired
 by stored sperm from a different male. Nest boxes were checked every day
 and eggs were collected the day they were laid, given a unique number
 using a permanent marker, and stored in a ‘soft box’ at cool room
 temperature (22<sup> o</sup>C) for up to seven days before
 being placed into an incubator (Brinsea Ovation 56 EX, Brinsea Products,
 Winscombe, U.K.). Eggs were incubated at 37.5<sup>o</sup>C and
 65% humidity for 12 days. On the 12<sup>th</sup> day (i.e.,
 the day before expected hatch) eggs were removed and measurements made.
 Working with embryos meant we eliminated the opportunity for the effects
 of extended parental care by parents of different genetic
 backgrounds. <strong>Crossing
 Design</strong> We produced backcrossed offspring
 by first breeding F<sub>1</sub> hybrid females with an
 unadmixed parental male of each subspecies, sequentially and in random
 order. Resulting offspring were defined as “mitoconcordant” backcrosses if
 their father’s lineage <em>did</em> match their mother’s
 mitotype and were defined as “mitodiscordant” backcrosses if father’s
 lineage <em>did</em> <em>not</em> match their
 mother’s mitotype (Figure 2). We note that in the literature these groups
 are often referred to as ‘maternal’ and ‘paternal’ backcrosses,
 respectively. Parental pairs of each subspecies were set up to breed at
 the same time and used as controls. Mitodiscordant
 backcrossing exposes incompatibilities by producing offspring who inherit
 most of their nuclear genome from a different lineage to that of their
 mitochondrial genome. This is particularly pronounced in the case of the
 long-tailed finch, as &gt;98.5% of fixed nuclear differences between
 subspecies are on the Z-chromosome[41]. In our cross design,
 mitodiscordant backcross offspring inherit Z chromosomes that have had no
 opportunity for recombination between subspecies. If male, they receive
 one Z chromosome from their hemizygous F<sub>1</sub> mother
 and a Z chromosome of matching subspecies identity from their father.
 Females have only a single Z chromosome inherited from their father.
 Mitodiscordant backcrosses have mismatched mitochondrial and nuclear
 genomes, but also have a low level of admixture on autosomes in their
 nuclear genome, which would otherwise be a potentially confounding source
 of hybrid dysfunction. For this reason, we included mitoconcordant
 backcross individuals as controls, as their level of autosomal admixture
 is the same as mitodiscordant backcrosses, but their mitochondria and Z
 chromosomes come from the same subspecies (Figure 2). It is worth noting
 that these several different types of backcrossed hybrids are also highly
 relevant to the wild hybrid zone, where we previously described that ~68%
 (243 of 357) of individuals had some level of admixture[32].
 The goal of our cross design was to create embryos in which the
 mitochondrial haplotype (i.e., mitotype) of the two subspecies was placed
 in three distinct contexts: 1) a nuclear genetic background exactly
 matching the mitotype lineage (parental crosses), 2) a nuclear genetic
 background mostly matching the mitotype lineage (mitoconcordant
 backcrosses), and 3) a nuclear genetic background mostly not matching the
 mitotype lineage (mitodiscordant backcrosses). The six resulting groups of
 embryos were thus 1. parental <em>acuticauda</em>
 (<em>acuticauda</em> mother
  <em>acuticauda</em>father), 2. parental
 <em>hecki</em> (<em>hecki</em> mother
  <em>hecki</em> father), 3. mitoconcordant backcross
 <em>acuticauda</em> (F<sub>1</sub> mother with
 <em>acuticauda</em> mtDNA  <em>acuticauda</em>
 father), 4. mitoconcordant backcross <em>hecki</em>
 (F<sub>1</sub> mother with <em>hecki</em> mitotype
  <em>hecki</em> father), 5. mitodiscordant backcross
 <em>acuticauda</em> (F<sub>1</sub> mother with
 <em>acuticauda</em> mtDNA  <em>hecki</em> father),
 and 6. mitodiscordant backcross <em>hecki</em>
 (F<sub>1</sub> mother with <em>hecki</em> mtDNA
  <em>acuticauda</em> father).  Mothers used in parental
 crosses only produced a single clutch while F<sub>1</sub>
 hybrid mothers used for backcrossing usually produced two clutches: one
 with an <em>acuticauda</em>father and one with a
 <em>hecki</em> father (however, 4 F<sub>1</sub>
 females only produced a clutch with one partner). All fathers were only
 used for a single clutch. <strong>Heart rate
 measurement</strong> After 12 days of incubation
 each egg was removed from the incubator and immediately placed on a Buddy
 digital egg monitor (Vetronic Services, Abbotskerswell, Devon, U.K.), to
 measure embryonic heart rate from within the egg (a proxy for whole body
 metabolic rate)[42]. A timer was started when the egg was removed from the
 incubator, and heart rate and corresponding time since removal were
 recorded at least twice over two minutes. We used these measurements to
 calculate the rate of cooling and the predicted heart rate halfway through
 the period (i.e., at 60 seconds). Five individuals where heart rate was
 only measured once were excluded from this dataset.
 <strong>High resolution respirometry
 strategy</strong> High resolution respirometry
 was performed with permeabilised tissue samples from these embryos using
 an Oroboros O2k (Oroboros Instruments, Innsbruck, Austria). High
 resolution respirometry allowed us to systematically stimulate, uncouple
 and inhibit certain combinations of mitochondrial complexes (Table 3;
 Figure S1). The biochemistry and genetic architecture underlying the core
 proteins of oxidative phosphorylation (OXPHOS) is highly conserved across
 eukaryotes and has been well described [17,85] with four protein complexes
 (CxI, CxII, CXIII and CxIV) establishing the proton gradient across the
 mitochondrial membrane, and CxV driving ATP synthesis (see also Figure 1).
 We measured the maximum capacity of OXPHOS system when fuelled by
 electrons either through complex I (CxI), or both CxI and CxII in tandem
 (OXP-I, OXP-I,II), and the maximum capacity of the ETS when accepting
 electrons from CxII, and both CxI and II in tandem (ETS-I,II, ETS,II).
 Comparing our experimental groups for these measures reveals whether CxI
 is compromised. We also assessed the maximum capacity for complex IV in
 isolation to consume O<sub>2</sub>, a measure not reliant on
 CxIII, which can reveal if CxIII is compromised. We also quantified the
 component of respiration that is not available to fuel ATP synthesis
 (LEAK-I,II). These measures enable us to test whether
 mitonuclear interactions impact mitochondrial aerobic metabolism in
 long-tailed finch hybrids. More specifically, they allow us to interrogate
 the source of any mitonuclear dysfunction, be it from the two complexes
 with amino acid differences between subspecies (CxI and III) or from
 translational issues related to fixed differences in the mtDNA encoded
 tRNAs or rRNAs [43-45]. <strong>High resolution
 respirometry technical steps</strong> After the
 embryonic heart rate was measured, embryos were used for high resolution
 respirometry. Embryos were euthanised via decapitation and then weighed
 (Mettler Toledo, PB303-S/FACT, Colombus, Ohio, USA). There was no
 significant difference between experimental groups in embryonic survival
 (X<sup>2</sup><sub>5</sub> = 4.1, p = 0.53; Figure
 S3B) or mass at day 12 of incubation
 (X<sup>2</sup><sub>5</sub> = 7.3, p = 0.20; Table
 5; Figure S3C). The head and body were then laterally cut in two, and the
 right-hand side was placed in 400 µl of phosphate buffered saline (PBS),
 homogenized with three strokes of a teflon-glass
 Potter-Elvehjem homogenizer (Wheaton, 5 mL), and stored at -80 for later
 analyses including DNA extraction for sex determination (described below).
 The left-hand side was weighed, suspended in a dilution of 100 mg/mL of
 MiR05 medium (0.5 mM Egtazic Acid (EGTA), 3 mM MgCl2, 50 mM
 K-lactobionate, 20 mM taurine, 10 mM KH2PO4, 20 mM Hepes, 110 mM sucrose,
 free fatty acid bovine serum albumin (1 g L-1), pH 7.1;[46,47]); and then
 homogenized and mechanically permeabilised using a teflon-glass
 Potter-Elvehjem homogenizer (Wheaton, 5 mL). All work was done on ice.
 This homogenate was centrifuged for 1 min at 100 g to pellet big tissue,
 and then 500ul of the supernatant was added to a chamber of an Oroboros
 O<sub>2</sub>k high resolution respirometer set at 37°C. The
 respirometer had two chambers and we always ran two samples in parallel.
 After two samples were loaded and the system had equilibrated (oxygen
 consumption was stable), 2 µl of digitonin (10 mg/mL) was added to ensure
 cells were fully permeabilised. We injected 5 µl pyruvate (5mM final), 5
 µl malate (2mM final), 10 µl glutamate (10mM final) as electron donors for
 CxI, and then measured oxidative phosphorylation when only CxI was
 receiving electrons (OXPHOS-I) by adding 5 µl adenosine diphosphate (ADP,
 1.25 mM final). We next measured coupled respiration capacity through both
 CxI and II (OXPHOS-I,II) by adding 25 µl succinate (10 mM final). We then
 determined O<sub>2</sub> consumption associated with proton
 leak when electrons were being received by both CxI and II (LEAK-I,II) by
 adding 5 µl oligomycin (10 uM final). We next estimated the maximum
 capacity of the electron transport system (ETS-I,II) by titrating the
 mitochondrial uncoupler carbonyl cyanide m-chlorophenyl hydrazine (CCCP;
 at 2 mM concentration) by first one injection of 3 µl, followed by 1 µl
 steps. We next estimated the ETS capacity when only receiving electrons
 through CxII (ETS-II) by adding 1 µl rotenone (0.5 uM final). We then
 measured non-mitochondrial O2 consumption by injecting 1 µl of antimycin A
 (2.5 uM final) to inhibit CxIII. Finally, we estimated the respiration
 capacity of CxIV directly by first adding 5 µl TMPD (0.5 mM final) and 5
 µl ascorbate (2 mM final), and then calculating the background chemical
 respiration rate by adding 50 µl azide (100 mM final). Across all runs the
 chambers were opened consistently at the same stages for reoxygenation.
 After each run 1 ml of the solution was taken from each respirometer
 chamber and frozen at -80 <sup>o</sup>C, and at the conclusion
 of all measures we performed a Pierce BCA protein quantification assay on
 this stored solution (ThermoFisher, Scientific, Waltham, MA, USA). Protein
 quantity was included as a covariate in statistical models (below) to
 account for slight differences in the amount of biological material in
 each sample. In most cases a different embryo sample was run in each
 chamber, but in 40 cases the same embryo was run in duplicate to assess
 how repeatable these measurements were (Table 3). When two different
 samples were run, the first was sitting in the respirometer for some time
 before the protocol was begun, and to account for any variability
 introduced by this we included a covariate in our models that was the
 length of time between when the embryo sample entered the chamber and when
 the measure was made. <strong>Molecular
 sexing</strong> We extracted DNA from the
 homogenized right-side of all embryos using a Gentra PureGene kit (Qiagen,
 Valencia, CA, USA) following manufacturer instructions. We sexed embryos
 by amplifying an intronic portion of the CHD gene that differs in size
 between the Z-linked and W-linked copy following published
 protocols[32,48]. Gel electrophoresis of the PCR product revealed the sex
 of the embryo as either male (one band, homozygous, ZZ) or female (two
 bands, heterozygous, ZW). <strong>mtDNA copy
 number</strong> We estimated mitochondrial
 density based upon mtDNA copy number for all samples using real-time
 quantitative PCR (qPCR) analysis following protocols established for
 passerine birds (Stier et al. 2019, 2022). While the amount of mtDNA in a
 mitochondrion can vary, mtDNA copy number is a reliable proxy of
 mitochondrial density. We estimated the relative mtDNA copy number by
 measuring the amount of mtDNA relative to the nuDNA for each sample by
 qPCR on a CFX96 Real-Time System (BIO-RAD). We used RAG1 as a
 representative single-copy nuclear gene (F: GCAGATGAACTGGAGGCTATAA, R:
 CAGCTGAGAAACGTGTTGATTC) and used COX1 as our representative mitochondrial
 gene (F: TCCTAGCCAACTCCTCAC, R: CCTGCTAGGATTGCAAAT). A complete nuclear
 copy of the mitochondrial genome (i.e., a NUMT) exists in Estrildid
 finches; for example, in the zebra finch reference genome
 (GCA_003957565.4) at chr2:72232845-72249691. To minimize the risk of NUMT
 interference in estimating mtDNA copy number, we designed our COX1 primers
 to perfectly match the mitochondrial sequence observed in
 <em>Poephila</em>. Moreover, our use of embryo tissue is less
 likely to result in amplification of the NUMT as gDNA from muscle tissue
 has a much lower nuclear:mitochondria ratio than gDNA from blood[49]. We
 further verified that COX1 amplification resulted in a single product of
 the expected size on an agarose gel. Following Stier et
 al. (2022), qPCR reactions had a total volume of 12 µl that included 6 ng
 of DNA, had PCR primers with concentrations of 200 nM and 6 µl of Absolute
 Blue qPCR Mix SYBR Green low ROX (Thermos Scientific). The qPCR conditions
 were: 15 mins at 95<sup>o</sup>C, 40 cycles of 15 s at
 95<sup>o</sup>C, 30 s at 58<sup>o</sup>C, 30 s at
 72<sup>o</sup>C. Samples were run in triplicate for each gene
 (RAG1 and COX1) across 5 plates, ten samples were pooled and included on
 every plate as a reference, and 20 samples were included on two separate
 plates. Amplicon efficiency was estimated from a standard curve of the
 reference between 1.5 to 24 ng (1.5, 3, 6, 12, 16, 24) run on one plate.
 The mean reaction efficiencies were 92.1% for RAG1 and 82.3% for COX1. To
 calculate relative mtDNA copy number for each sample we used the following
 formula:
 (1+Ef<sub>COX1</sub>)<sup>ΔCqCOX1</sup>/(1+Ef<sub>RAG1</sub>)<sup>ΔCqRAG1</sup>, with Ef as the amplicon efficiency, ΔCq as the difference in Cq between the reference and focal samples. Some samples were run twice on separate plates, with an inter-plate repeatability of 0.74 (95% C.I. [0.49, 0.88]), and based on triplicates intra-plate repeatability was 0.94 (95% C.I. [0.93, 0.95]). Seven samples were considered as outliers (Cq RAG1 &lt; Cq COI) and removed from analysis. <strong>Oxidative Damage assay</strong> It is possible that some aspect of mitochondrial function other than aerobic respiration is impaired, but that the respiration rate is maintained. For this reason, we also measured the damage induced by reactive oxygen species (ROS). High ROS levels resulting from mitochondrial dysfunction can damage macromolecules, cell components and structures[43,44] and ROS-induced DNA damage correlates with shorter lifespan in the closely related zebra finch[45]. 8-OHdG (8-hydroxy-2’-deoxyguanosine) is a predominant form of ROS-induced oxidative lesions to genomic DNA that has been widely used as a biomarker for oxidative damage[50], and has been associated with increased mitochondrial respiration in birds [51,52]. While 8-OHdG does not block DNA replication, it contributes to mutagenesis and can affect DNA transcription[53]. Here we quantified levels of 8-OHdG in the DNA extracted from embryos using a competitive immunoassay (300 ng DNA, EpiQuick 8-OHdG DNA Damage Quantification Direct Kit Colorimetric, Epigentek, USA) following the manufacturer recommendations. This was run on a subset of samples (N = 182) chosen blind to respiration capacity to have roughly balanced numbers from each experimental group (N = 28-33 samples per group). The intra-plate coefficient of variation based on duplicates was 6.3 + 0.83. The inter-plate coefficient of variation based on four samples repeated over all three plates was 22.7 + 4.05, so we included plate ID as random effect in our analyses. <strong>Statistical analyses</strong> For statistical analyses we used R version 4.1.2 (R Core Team 2020) using RStudio version 1.2.5033 (RStudio Team, 2020) for the graphical interface. We used the lme4 and lmerTest packages[54,55] to run linear mixed models to test whether our measures of respiration capacity differed between the six experimental groups (parental <em>acuticauda</em>, parental <em>hecki</em>, mitoconcordant and mitodiscordant backcross with <em>acuticauda</em> mtDNA, mitoconcordant and mitodiscordant backcrosses with <em>hecki</em> mtDNA). These measures were each included as the dependent variable in separate linear mixed models, in which the fixed effects were experimental group, embryo sex, protein quantity in the chamber, and the length of time after embryo entered the oroboros chamber when the measure was made. In each we tested for an interaction between experimental group and embryo sex, and if this was not significant the interaction term was removed (but left in the model if significant). In each model parental pair ID was included as a random effect, to account for multiple members of the same clutch being included. Where there was a significant effect of experimental group, we ran Tukey post-hoc comparisons using the emmeans package[56] to assess specifically which groups differed from one another. Model assumptions were checked using the DHARMa package in R[57]. We similarly ran linear mixed models to test whether heart rate, oxidative damage and mitochondrial copy number differed between our experimental groups, but in these cases the fixed effects were experimental group, embryo sex, and for the heart rate model embryo mass, and the random effect was still parental pair ID. Oxidative damage and mitochondrial copy number were log-transformed to better meet model assumptions. An interaction between experimental group and sex was also included for each model, but then removed if not significant. In the mitochondrial copy number and oxidative damage models, plate ID were additionally included as random effects.  Embryo ID was also initially included as a random effect in the mitochondrial copy number and oxidative damage models, but it explained close to zero variance so was removed for the final models, likely because very few individuals were measured twice (10 for mitochondrial copy number, 21 for oxidative damage). 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description: # Mitonuclear interactions impact aerobic metabolism in hybrids and may
 explain mitonuclear discordance in young, naturally hybridizing bird
 lineages
 [https://doi.org/10.5061/dryad.m37pvmd9v](https://doi.org/10.5061/dryad.m37pvmd9v) We have submitted our raw aerobic metabolism data (**aerobic_metabolism_data.csv**), embryonic heart rate data (**heart_rate.csv**), mitochondrial copy number data (**mitochondrial_copy_number.csv**), oxidative damage data (**oxidative_damage.csv**), and an R script for analyzing the datasets used in this study (**aerobic_metabolism_analyses_annotated_.R**). ## Descriptions ### aerobic\_metabolism\_data * *Egg*: Individual embryo ID number * *Dup*: An embryo that was measured multiple times as a technical replicate is marked 'DUP' * *Sex*: Sex of embryo * male * female * *OXPI*: Oxidative phosphorylation when only complex I (CxI) is receiving electrons (coupled respiration measurement) * *OXPI_II*: Coupled respiration capacity through both complex I (CxI) and complex II (CxII) (coupled respiration measurement) * *LEAK_II*: Oxygen consumption associated with proton leak when electrons are being received by both complex I (CxI) and complex II (CxII) (proton leak measurement) * *ETSI_II*: Maximum capacity of electron transport system (uncoupled respiration measurement) * *ETSII*: Capacity of electron transport system when only receiving electrons from complex II (CxII) * *CXIV*: Respiration capacity of complex IV (CxIV) (maximum respiration measurement) * *OXPI_tlength*: Measurement time for *OXPI* (seconds) * *OXPI_II_tlength*: Measurement time for *OXPI_II* (seconds) * *LEAKI_II_tlength*: Measurement time for *LEAK_II* (seconds) * *ETSI_II_tlength* : Measurement time for *ETSI_II* (seconds) * *ETSII_tlength*: Measurement time for *ETSII* (seconds) * *AsTM_tlength*: Measurment time for *AsTM* (seconds) * *EmbryoMass*: Mass of embryo (grams) * *PairID*: Breeding pair ID number combining maternal and paternal ID numbers * *FemID*: Maternal ID number * *PairType*: Experimental cross type * 01_acuM_acuN: Offspring of *acuticauda* parents (**mitoconcordant** parental) * 02_hecM_hecN: Offspring of *hecki* parents (**mitoconcordant** parental) * 03_acuM_acuN: Offspring of *acuticauda* mtDNA carrying F1 female and an *acuticauda* male (**mitoconcordant** backcross *acuticauda*) * 04_hecM_hecN: Offspring of *hecki* mtDNA carrying F1 female and a *hecki* male (**mitoconcordant** backcross *hecki*) * 05_acuM_hecN: Offspring of *acuticauda* mtDNA carrying F1 female and a *hecki* male (**mitodiscordant** backcross *acuticauda*) * 06_hecM_acuN: Offspring of *hecki* mtDNA carrying F1 female and an *acuticauda* male (**mitodiscordant** backcross *hecki*) * *ProtCont*: Protein quantity ### heart\_rate * *Egg*: Individual embryo ID number * *Sex*: Sex of embryo * male * female * *PairID*: Breeding pair ID number combining maternal and paternal ID numbers * *PairType*: Experimental cross type * 01_acuM_acuN: Offspring of *acuticauda* parents (**mitoconcordant** parental) * 02_hecM_hecN: Offspring of *hecki* parents (**mitoconcordant** parental) * 03_acuM_acuN: Offspring of *acuticauda* mtDNA carrying F1 female and an *acuticauda* male (**mitoconcordant** backcross *acuticauda*) * 04_hecM_hecN: Offspring of *hecki* mtDNA carrying F1 female and a *hecki* male (**mitoconcordant** backcross *hecki*) * 05_acuM_hecN: Offspring of *acuticauda* mtDNA carrying F1 female and a *hecki* male (**mitodiscordant** backcross *acuticauda*) * 06_hecM_acuN: Offspring of *hecki* mtDNA carrying F1 female and an *acuticauda* male (**mitodiscordant** backcross *hecki*) * *N_measures*: Number of heart rate measurements taken and averaged * *Int*: Time spent outside of incubator (seconds) * *y_at_60*: Predicted heart rate after 60 seconds of cooling (beats per minute) ### mitochondrial\_copy\_number * *Egg*: Individual embryo ID number * *Sex*: Sex of embryo * male * female * *PairID*: Breeding pair ID number combining maternal and paternal ID numbers * *PairType*: Experimental cross type * 01_acuM_acuN: Offspring of *acuticauda* parents (**mitoconcordant** parental) * 02_hecM_hecN: Offspring of *hecki* parents (**mitoconcordant** parental) * 03_acuM_acuN: Offspring of *acuticauda* mtDNA carrying F1 female and an *acuticauda* male (**mitoconcordant** backcross *acuticauda*) * 04_hecM_hecN: Offspring of *hecki* mtDNA carrying F1 female and a *hecki* male (**mitoconcordant** backcross *hecki*) * 05_acuM_hecN: Offspring of *acuticauda* mtDNA carrying F1 female and a *hecki* male (**mitodiscordant** backcross *acuticauda*) * 06_hecM_acuN: Offspring of *hecki* mtDNA carrying F1 female and an *acuticauda* male (**mitodiscordant** backcross *hecki*) * *Plate*: Plate ID * *mtCN*: Estimated mitochondrial copy number ### oxidative\_damage * *Egg*: Individual embryo ID number * *Sex*: Sex of embryo * male * female * *PairID*: Breeding pair ID number combining maternal and paternal ID numbers * *PairType*: Experimental cross type * 01_acuM_acuN: Offspring of *acuticauda* parents (**mitoconcordant** parental) * 02_hecM_hecN: Offspring of *hecki* parents (**mitoconcordant** parental) * 03_acuM_acuN: Offspring of *acuticauda* mtDNA carrying F1 female and an *acuticauda* male (**mitoconcordant** backcross *acuticauda*) * 04_hecM_hecN: Offspring of *hecki* mtDNA carrying F1 female and a *hecki* male (**mitoconcordant** backcross *hecki*) * 05_acuM_hecN: Offspring of *acuticauda* mtDNA carrying F1 female and a *hecki* male (**mitodiscordant** backcross *acuticauda*) * 06_hecM_acuN: Offspring of *hecki* mtDNA carrying F1 female and an *acuticauda* male (**mitodiscordant** backcross *hecki*) * *ngDNA*: Quantity of DNA (nanograms) * *ODav*: Primary measurement of 8-OHdG (8-hydroxy-2’-deoxyguanosine) * *Run*: Run ID * *OSperc*: Secondary measurement of 8-OHdG (8-hydroxy-2’-deoxyguanosine) ## Supplemental Tables Supplemental tables S1-S4 are available through an open access agreement with Molecular Ecology. ## Sequence Data * The WGS data used to assemble mitochondrial genomes is available via SRA under the BioProject accession PRJNA1101033. * The mitochondrial genome assemblies used in this project are available via GenBenk under the accessions PP372643-PP372678. ## Code/Software R is required to run *aerobic_metabolism_analyses_annotated_.R*. The script was created using R version 4.1.2 and was used to create plots in Figure 5 and supplemental Figures 2 and 3.