Juliette Blanc
email: juliette.blanc@ifsttar.fr

Emmanuel Chailleux

Pierre Hornych

Chris Williams

Zahra Sotoodeh-Nia

Laurent Porot

Simon Pouget

François Olard

Jean-Pascal Planche

Davide Lo Presti

Jean- Pascal Planche

Ana Jimenez

Del Barco

From Laboratory Mixes Evaluation to Full Scale Test: Fatigue Behavior of Bio-Materials Recycled Asphalt Mixtures

Keywords: Bio-materials, Reclaimed asphalt, Accelerated pavement test, Monitoring, Fatigue test

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Tested materials

The project aimed at maximising the use of RA with bio-based materials. For this purpose asphalt mixtures were made with 50 % of RA and three different, but complementary, innovative technologies [START_REF] Chailleux | BioRePavation: innovation in bio-recycling of old asphalt pavements, comparison between EU and US mix design specification systems[END_REF]):

 Mix1 was a bio-based rejuvenator, SYLVAROAD™ RP1000 Performance Additive, from Kraton Chemical used to treat the RA. It has been specifically designed to increase the RA content up to 100 % or reuse adverse, very hard, quality RA.  Mix 2 was a bio-binder, Biophalt®, from Eiffage Travaux Publics designed for a total replacement of bituminous binder in recycling technique.  Mix 3 was a bio-based additive from Iowa State University: an Epoxidized Soybean Soyate (EMS) aimed to compatibilise the virgin binder and aged binder from the RA.

A new type of base course mix (GB5® type) was used, based on aggregate packing optimization concept by maximizing their interlock [START_REF] Pouget | GB5® Mix Design: A New Approach for Aggregate Grading Optimization for Heavy Duty Flexible Pavements[END_REF]. The properties of the three GB5-type mixes were compared with a reference high modulus asphalt mix (EME2), commonly used in France for base layer.

Figure 1 presents the final grading curves of the four different materials (produced in plant), in comparison with the theoretical grading curve of the GB5® type material. The EME2 presented a coarser grading than the other materials.

Fig. 1 GB5® and EME 50% RA mixtures' gradation

Fatigue (standard EN 12967-22) and complex modulus characteristics of the mixes produced in plant are given in Table 1. All mixes exhibited high modulus values, exceeding 14 GPa. The fatigue performance of the tested mixes, measured in lab, was significantly below the reference mix.

Characteristics of the IFSTTAR accelerated pavement testing facility

The fatigue carrousel of IFSTTAR is an outdoor road traffic simulator designed to study the behaviour of real scale pavements under accelerated heavy traffic. The fatigue carrousel has a diameter of 40 meters and four loading arms, which can each carry loads up to thirteen tons, at a speed reaching 100 km/h (Figure 2). Two months of testing can represent up to 20 years of moderate traffic (150 heavy trucks/day).

During loading, a lateral wandering of the loads can be applied to simulate the lateral distribution of loads of real traffic [START_REF] Nguyen | Review of glass fiber grid use for pavement reinforcement and APT experiments at IFSTTAR, International journal of road materials and pavement design[END_REF].

Fig. 2 The IFSTTAR accelerated pavement testing facility

Characteristics of the experimental structures

Four different pavement structures, built with the materials described above, Mix1, Mix2, Mix3 and EME2, were tested. The thicknesses of the bituminous layer for EME2, Mix1, Mix2 and Mix3 were respectively 102mm, 86mm, 95mm and 91mm, for a targeted nominal thickness of 90 mm. The subgrade includes a stone bed (50/120 mm), under an unbound granular (UGM) layer. The bearing capacity of the subgrade was measured at different positions on each structure by means of dynamic plate load test, which gave values between 63 and 86 MPa for the stone bed, and between 103 and 111 MPa on top of the UGM layers.

The bituminous mixes were produced and the pavements were built on the 30 th and 31 st of May 2017 by Eiffage.

The full scale experiment included two phases: The first phase was performed between July and September 2017, to evaluate rutting resistance under a 65 kN dual wheel load, with a speed of 43km/h and a narrow transversal wandering (+/-26 cm). This first phase is not detailed here, but is presented in [START_REF] Blanc | Full-scale validation of bio-recycled asphalt mixtures for road pavements[END_REF].

The second phase was performed between November 2017 and March 2018, to evaluate the fatigue resistance of the pavements, also under 65 kN load, with a speed of 70 km/h and a higher transversal wandering (+/-52 cm). Before the end of this phase, after the application of 1 million load cycles, no surface damage was observed on the four test sections. Therefore, it was decided to continue the experiment, and apply 400 000 additional load cycles, with a higher load (75 kN).

During the fatigue test, the pavement surface temperature were mainly comprised between 4 °C and 14°C. The mean surface temperature was 8.8°C, and the mean temperature in the middle of the bituminous layers was 9.0°C. These relatively low temperatures were adequate for testing fatigue resistance of the mixes.

Pavement instrumentation and monitoring

Each tested section was instrumented with strain gauges and temperature sensors:  2 longitudinal asphalt strain gauges (TML KM-100HAS) were placed at the bottom of the bituminous layer, in the middle of the wheel path.  8 Temperature sensors, placed at 4 different depths.

The following measurements were performed on each section throughout the experiment:

 Strain gauge measurements, performed approximately every 20 000 loads.  Temperature measurements, performed continuously during the experiment, at time intervals of 10 minutes.  FWD tests: at 0, 500 000 and 1 million loads, and then at 1.1, 1.2, 1.3 and 1.4 million loads).

 Visual crack monitoring (before loading, and every 100 000 loads)

3 Performance of the tested structures

Visual crack monitoring

During the experiment, pavement cracking was evaluated by visual inspection on all the sections, and cracks were marked with paint, to facilitate their identification.

On the APT, the extent of cracking is defined as the percentage of the length of the pavement affected by cracks. For longitudinal cracks, the "cracked length" corresponds to the measured length of the cracks. For transversal cracks, a length of 50 cm is conventionally attributed to each crack (Figure 3).

Fig. 3 Evaluation of the level of cracking

Figure 4 presents the evolution of the extent of cracking, as a function of the number of applied loads, on the four pavement sections. On the EME2 section, the first cracks appeared after 900 000 load cycles. Until 1.4 million loads, cracking increased regularly on this section, reaching 28 % at the end of the test.

On the Mix3 section, the first cracks were observed after 1 000 000 load cycles, and then cracking increased regularly, reaching 10 % at the end of the test. On the Mix1 and Mix2 sections, no cracks were visible until the end of the test.

Crack patterns observed on the EME2 and Mix3 sections at the end of the test are presented on Figure 5. On these sections, cracks developed as follows: first, very fine isolated transversal cracks appeared (marked in white on Figure 5). Then, under traffic, these cracks started to open, and fines started to come out. Other thin transversal cracks developed nearby. The cracks marked in blue appeared at 1.1 million loads, in orange, at 1.2 million loads and in pink at 1.3 million loads. It can be seen that most cracks are transversal. This type of fatigue cracking is frequently observed on the IFSTTAR APT, under dual wheel loading [START_REF] Hornych | The LCPC's ALT facility contribution to pavement cracking knowledge[END_REF]). The cracking was believed to be bottom-up. Loaded at 6.5 tons Loaded at 7.5 tons slightly higher thickness of the EME2 layer. These results differed from those obtained in the laboratory, which indicated that the EME2 material had the best fatigue resistance (Table 1).

Extent of cracking (%) = Li : lengths of zones with cracks

FWD measurements

FWD measurements were performed at 0, 500 000 loads, 1 million loads, and then every 100 000 loads, until the end of the experiment. The maximum deflections measured during each successive test campaign, on each pavement section are presented on figure 6. The temperature corresponding to each series of measurements is indicated, but no temperature correction was applied to the measurements. Figure 6 shows that:

 Between 0 and 500 000 loads, deflections decreased, probably due to post-compaction, and also to a decrease of temperature, and were quite similar on all pavement sections.  At 1 million loads, deflections started to increase significantly at some points, on the EME2 and Mix3 sections, indicating damaged zones. On the EME2 section, these high deflections coincided with the appearance of cracks. However, on the Mix3 section, high deflections were observed before the apparition of any cracks.

Cracks

The first cracks appeared only at 1.1 million loads. Thus, the FWD measurements were able to detect damage, before the apparition of any surface cracks.  During the next measurements, deflections continued to increase in the damaged zones (EME2 and Mix3 sections) along with the cracks.  On the Mix1 and Mix2 sections, only a small increase of deflections was observed until the end of the experiment, but no important deflection peaks, and no cracking was observed on these sections.

Strain gauge measurements

Each section was instrumented with 2 longitudinal strain gauges, placed at the bottom of the bituminous layers during construction. For each section, measurements were made approximately every 20 000 loads. Maximum strains under the passage of the 4 arms of the APT were determined, and the mean of these four values was calculated. The evolution of the maximum longitudinal strains, for each section, is presented on Figure 7. At the beginning of the fatigue experiment, the longitudinal strains at the bottom of the asphalt layer were of the same level on the 4 pavement sections (between 100 and 130 strains, depending on temperature). On the Mix1 and Mix2 sections, the longitudinal strains presented no significant evolution until the end of the experiment. On the Mix3 and EME2 sections, however, longitudinal strains started to increase after about 250 000 loads, and then increased progressively until the end of the experiment, or until the gauges failed (with a higher increase on the EME2 section). This strain increase was probably linked with damage of the asphalt material. It is interesting to notice that the strains started to increase well before cracks appeared on the surface of the pavements, which tends to indicate a bottom-up crack propagation.

The strain measurements at the bottom of the asphalt layers were consistent with the other measurements, indicating that damage developed first on the EME2 section, then on the Mix3 sections, and that no significant damage affected the Mix1 and Mix2 sections.

Conclusions

A full scale accelerated test were performed at IFSTTAR, to validate the performance of the three mixes tested in the project. These mixes were tested simultaneously with a reference EME2 material.

All the measurements made in the full scale test were very consistent. The test results lead to the conclusion that the 3 mixes with bio-materials present a better fatigue performance in situ than the reference EME2 mix, which is considered in France as a high performance base layer material. These results differ from those of laboratory fatigue tests, which indicated the best fatigue resistance for the EME2 material. This confirms that these innovative mixes, with bio binder or bioadditives and 50% of reclaimed asphalt, can be used successfully for pavement construction.

Fig. 4 Fig. 5

 45 Fig. 4 Extent of cracking, in percent, on the four sections

Fig. 6

 6 Fig. 6 Evolution of deflections on the four pavement sections (FWD measurements, central geophone, plate loaded at 65 kN).

Fig. 7

 7 Fig. 7 Evolution of maximum longitudinal strains during the fatigue test

Table 1

 1 Mechanical properties of the plant manufactured mixes (compacted in lab)

			Stiffness parameters at	Fatigue parameters
		Void (%)	(15°C, 10Hz)
			E*(MPa)	 (°)	strain	b
	EME2	4.3	16 770	10.3	126	-0.178
	Mix1	2.9	14 540	15.8	115	-0.190
	Mix2	3.3	16 200	16.7	100	-0.176
	Mix3	4.5	16 360	12.2	109	-0.156
	2 Accelerated pavement test

Acknowledgements

This project has received funding from the European Union's Seventh Framework Programme for research, technological development and demonstration under grant agreement n°607524. BioRePavation is co-funded by Funding Partners of The ERA-NET Plus Infravation and the European Commission.


Simona Mattioli Autumn

Simona Mattioli

Muscle chickens' changes in the last 70 years

take into account many aspects (behaviors, welfare, physiology, health status, performance, quality)

Outdoor adaptability criteria

Extensive Rearing System (ERS) must optimize a production system that promotes biodiversity, environmental sustainability and food safety (National Organic Standards Board, 1995) Broiler Percentages for each genotype are represented in pie charts. The splines depict tibia shape configuration corresponding to opposite patterns of behavior. Only foraging was significantly correlated to range visits in all periods, even before range access!!

  th -27 th October 2023, Assisi, Italy (B) Shape variation along PC1 and PC2 was represented by splines relative to positive and negative extremes of the axes. For shape variation along PC1, tibiae of two extreme individuals were reported.(A) Principal component analysis plot of chicken classified on the basis of walking activity.• walking (W)• not-walking (NW) • walking naked neck (W-NN) behavior described as a percentage of time spent in two main activities (Walking W and Not Walking-NW).

*

  Schematic representation of the exploitation of the outdoor run by the different genotypes on the basis of behavioral observations or GPS movement detection in different as percentage, normal and density curves of Activity index in three commercial lines. Commercial lines: L1 = active commercial line; L2 = sedentary commercial line; L3 = Fast Growing Ross 308. The blu lines indicate normal distribution; The red lines indicate kernel density estimation. Failla et al., 2021 -Poultry Science KINETIC ACTIVITY Simona Mattioli Autumn School 25 th -27 th October 2023, Assisi, Italy

  Cartoni Mancinelli et al., 2022 -Scientific report Simona Mattioli Autumn School 25 th -27 th October 2023, Assisi, Italy Mattioli et al., -phD dissertation Simona Mattioli Autumn School 25 th -27 th October 2023, Assisi,

  feather condition, TI) Performances ROC curve, indicated that the DWG was a moderately accurate predictor of adaptability and that, when DWG was ≥ 38.6 g/d, it predicted a low adaptability with a sensitivity of 87% and a specificity of 73%. Moreover, for each 1 g/d increase in DWG, the odds of having a low adaptability increased by 42%. grass per day as the two other strainsThe S757N chicken outdoor excretions of N and P per day of outdoor access were about 200% and 30% greater than those of dual-purpose and JA757 chickens, respectively Environment and range use ? Claire Bonnefous et al., Autumn School 25 th -27 th October 2023, Assisi, Italy Claire Bonnefous et al., Autumn School 25 th -27 th October 2023, Assisi, Italy Is range use a personality trait ? al., Autumn School 25 th -27 th October 2023, Assisi, Italy Range use relationship with indicators at slaughter Indicators of later range use : behavioural indicators. Ferreira et al, 2021

Health status Antioxidants intake n-3 ALA HUFA acetyl-CoA ALA HUFA diet anabolica Catabolica anabolica (D6) Energy expenditure

	TREINED Walking: EXERCISE IN FAST vs SLOW-GROWING What means
	In vivo Oxidation ↑lipid/protein oxidation of meat ↓Shelf-life of meat ↓technological quality activity Kinetic	Kinetic activity FG 1 , pre E IN VIVO Foraging		Antioxidants PUFA n-3 ↑ animal welfare ↑ meat antioxidants ↓ meat oxidation ↓ technological quality .1 .2 .3 .4 (desease) FG 1 , post E Trained and not trained animals?
	Antioxidants	SG 2 , pre E ROMs	Energy expenditure	SG 2 , post E LC-PUFA energy compensation consumption for Antioxidants	.1 .2 ROMs .3 .4
	0	10	20	30	0	10	20	30
		TBARS 4 Activity index day		↓ lipid % in meat ↓ healthy PUFA ROMS 5 ↓ fat deposit
								Failla et al., 2021 -Poultry Science Mattioli et al., 2014 -Poultry Science
	Simona Mattioli
	Autumn School 25 th -27 th October 2023, Assisi, Italy

Simona Mattioli

Autumn School 25 th -27 th October 2023, Assisi, Italy Simona Mattioli Autumn School 25 th -27 th October 2023, Assisi, Italy

Meat Cuts morphology

Simona Mattioli Autumn School 25 th -27 th October 2023, Assisi,

Italy Fiber Slow-growing Fast-growing Pectoralis major

	Muscle fiber
	and genetic		R (1) w (2)	4 96	0 100
	strain	Biceps femoris	R w	56 44	37 63
					Broiler Leghorn
	FG			Fibers	VSG
				(n/microscopic	49.99 a 140.15 b
				field)
				Capillaries
				(n/microscopic	24.56 b	22.35
				field)

Branciari et al., 2009 -Journal of Animal Science Simona Mattioli Autumn School 25 th -27 th October 2023, Assisi, Italy Muscle anomalies in broilers Ross; PM. Inflammatory infiltrates composed mainly by lymphocytes, plasma cells and macrophages. Ross; PM. with muscle fibers necrosis (black arrows) and giant fiber (red arrow) Sforna et al., 2017 -Italian Journal of Animal Science a Simona Mattioli Autumn School 25 th -27 th October 2023, Assisi, Italy

This project has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No 816172 Claire

	State behaviour recorded during focal sampling
	Standing	0.01	-0.01	-0.02	-0.15
	Resting	-0.17	0.06	-0.08	0.00
	Sleeping	-0.24	0.06	0.02	0.11
	Locomotion	0.26	0.09	-0.01	0.06
	Foraging	0.29	-0.02	0.17	0.04
	Drinking & Eating	-0.17	-0.12	-0.02	0.06
		Variables of the social motivation test
	Latency to exit	-0.09	-0.04	-0.08	-0.02
	Latency to arrive to the zone close to conspecifics	-0.13	0.05	-0.10	0.09
	Number of pecks	0.03	-0.14	-0.06	-0.09
		Variables of the multivariate test
	Latency to make a first step	-0.09	0.05	0.07	0.00
	Foraging	0.17	0.04	0.00	0.22
	Number of times the
	chicken walked behind a	0.05	-0.03	-0.04	-0.05
	wooden panel
	Time in the outer circle	0.02	0.20	0.00	-0.01

Bonnefous et al., Autumn School 25 th -27 th October 2023, Assisi, Italy

Indicators of later range use : behavioural indicators

	JA757	S757N	White Bresse Dual-purpose
	Thank for your attention

Pulcini et al., 2022 -Animals


Georges Beainy

Tiphaine Cerba

Franck Bassani

Mickaël Martin

Thierry Baron

Jean-Paul Barnes

Suppression of self-organized surface nanopatterning on GaSb/InAs multilayers induced by low energy oxygen ion bombardment by using simultaneously sample rotation and oxygen flooding

Time of flight secondary ion mass spectrometry (ToF-SIMS) is a well-adapted analytical method for the chemical characterization of concentration profiles in layered or multilayered materials. However, under ion beam bombardment, initially smooth material surface becomes morphologically unstable. This leads to abnormal secondary ion yields and depth profile distortions. In this contribution, we explore the surface topography and roughening evolution induced by O 2 + ion bombardment on GaSb/InAs multilayers. We demonstrate the formation of nanodots and ripples patterning according to the ion beam energy. Since the latter are undesirable for ToF-SIMS analysis, we managed to totally stop their growth by using simultaneously sample rotation and oxygen flooding. This unprecedented coupling between these two latter mechanisms leads to a significant enhancement in depth profiles resolution.

Introduction

Recent progress in materials science necessitates knowledge and control of matter at the nanometer scale. Nowadays, given the complexity of the devices in terms of chemical composition as well as in dimension, their accurate characterization has become difficult and sometimes challenging. For this purpose, Time of Flight Secondary Ion Mass Spectrometry (ToF-SIMS) has become a crucial tool that is well adapted analytical method for the chemical characterization of concentration profiles in layered or multilayered materials. However, the reliability and accuracy of the interface characterization may be affected by methodological factors, which alter the depth resolution such as: macroscopical or microscopical initial roughness of the material and ion bombardment induced surface topography and roughening. In fact, material surface of initially smooth monocrystals becomes morphologically unstable under ion bombardment. This instability leads to the formation of self-organized nanostructures on the surface of the material such as nanoscale ripples [START_REF] Flamm | Evolution of surface topography of fused silica by ion beam sputtering[END_REF], [2], [START_REF] Datta | Atomic force microscopy study of 60-keV Ar-ion-induced ripple patterns on Si(100)[END_REF], [START_REF] Chason | Roughening instabilityand evolution of the Ge(001) surface during ion sputtering[END_REF] and dots [5], [6], [7], [8]. These nanostructures on semiconductors surface have attracted much interest due to their potential applications in low-dimensional devices, particularly as ordered quantum nanodots for optoelectronics and quantum devices [9], [2], in magnetic storage technology [10] and for selective attachment of specific molecules [11]. On the other hand, extensive efforts in the form of experimental as well as theoretical studies have been devoted to understand the fundamental processes of ripples and dots formation on surfaces subjected to energetic ion bombardment [12]. The pioneering works by Bradley and Harper [13] referred to as BH model was the first successful theoretical approach to explain the ripple formation mechanism on an amorphous material. Later, other models have been also proposed which account for different experimental behaviors such as the presence or absence of saturation for the ripple amplitude, ripple orientation, ripple dynamics as well as the existence or not of kinetic roughening [START_REF] Cuerno | Dynamic scaling of ion-sputtered surfaces[END_REF], [START_REF] Park | Dynamics of ripple formation in sputter erosion: Nonlinear phenomena[END_REF].

While the formation of morphological features can be used in various applications, they are extremely undesirable in many occasions especially for depth profiling in ToF-SIMS. This kind of roughening brings about degradation of depth resolution. Therefore, improvement of the interface depth resolution in sputter depth profiling is a very important topic. Accordingly, the aim of this work was to study surface topography and roughening induced by low energy range 0.25 to 2 keV O 2 + ion bombardment, corresponding to the typical depth profiling regime of the new generation of SIMS instruments. The objects in our experiments consist of GaSb/InAs multilayers deposited on silicon substrate because of their interest for application in opto-and micro-electronics [16], [START_REF] Walther | Proc. SPIE[END_REF], [START_REF] Kim | [END_REF]. Our overall objective is to try to examine the conditions under which depth profiling of such material is optimal and reliable.

Experimental

The investigated sample in this work was grown by Metalorganic Chemical Vapor Deposition (MOCVD) which can provide heterostructures with very abrupt interfaces. It consists of a stack of GaSb/InAs layers deposited on 300 mm (001)-Si substrate as shown in Fig. 1. The growth of GaSb layer was performed on (001)-Si substrate with an offcut angle of 0.11° along [110] direction. The substrate was first deoxidized in a Siconi TM pre-clean chamber under an NF 3 /NH 3 remote plasma. Prior to the growth, the substrates were heated above 800°C under H 2 atmosphere in the growth chamber to promote double step silicon surface structuration [19]. Subsequently, we used a two-step process to grow GaSb on Si. A nucleation layer between 10 nm and 40 nm was grown at 1 nm/min in the 350-450°C temperature range. The growth was then interrupted and the temperature was increased above 600°C under H 2 atmosphere. A GaSb epilayer was then grown with a V/III ratio around 1 at 30 nm/min. The InAs layer was deposited following a process flow published in previous work [20]. The top GaSb layer was then grown directly on InAs at 1 nm/min in the 350-450°C temperature range.

All experiments were carried out on a ToF-SIMS V from IONTOF. The samples approximately 1 × 1 cm 2 in size were cut from standard 300 mm wafers. Controlled irradiation was carried out at room temperature by O 2 + ions with ion energies of 0.25, 0.50, 1 and 2 keV. The ion bombardment was performed at a 45° incidence angle with a constant (for each ion energy) ion beam current density, beam raster size (300 × 300 µm 2 ) and sputtering depth. Sputtering was performed throughout the layers until reaching the bottom GaSb layer up to 105 nm from the surface of the sample (see figure 1.b). After ion irradiation the surface morphology was investigated by atomic force microscopy (AFM) in tapping mode. All measurements were conducted in air by using silicon tips.

Results and discussion

The surface morphology of the sample before irradiation was studied using atomic force microscopy (AFM). The surface exhibits monoatomic steps as it can be seen from figure 2. The Root-Mean-Square (RMS) roughness extracted from a 1×1 µm 2 area of the sample is 0.68 nm. Basically, the depth resolution depends on the primary ion beam (type, energy, incident angle) and the substrate. Therefore, the sample was analyzed by ToF-SIMS using oxygen ion beam with different energy in order to understand its effect. Hereinafter, we will first discuss the formation of the nanopatterns under ion beam bombardment to move on later to their suppression as well as the improvement of depth profile resolution.

A. Patterns formation under ion beam bombardment: transition from nanodots to ripples

Figure 3 shows the GaSb bombarded surface with oxygen ions at 45° incidence angle and different ion beam energies. One can observe the evolution of the surface topography until the formation of well oriented and long range ordered ripples pattern as the energy of the ion beam increases. For low incident beam energies 250 eV and 500 eV, nanodots are already being formed on the sputtered surface as shown in Fig. 3.(a) and 3.(b). Increasing ion beam incident energy up to 1 keV leads to a more advanced state of corrugation with larger amplitude and more enhanced ordering. Finally, clearly regular and large rippled structure is formed at 2 keV incident beam energy. The observed pattern at 2 keV presents larger lateral sizes and heights compared to the other previous incident energies. These results demonstrate a transition from an isotropic (e.g. phase separation) to an anisotropic mechanism (e.g. ballistic or stress effects) as the energy is increased. Thus, while the formation of nanodots or ripples was highlighted as incidence angle dependence in the literature, here we prove that it is also reliant on ion beam energy. Moreover, the wave vector of the formed ripples is perpendicular to the incident beam direction. The impact of primary ion beam energy on the surface topography formation can be seen in table 1. Higher surface topography is formed when increasing the impact energy up to 2 keV (RMS=5.35 nm). The smoothest surface was obtained for 250 eV impact energy (RMS=1.45 nm). On the other hand, the wavelength and the amplitude of the nanopatterns become greater with the increase of the incident beam energy as it can be deduced from table 1. All of these observations imply that the topography of the GaSb/InAs surface changes from a less positionally ordered nanodots to a more ordered ripples chain structure aligned perpendicular to the ion beam direction. According to these results, the most convenient sputter regime for the GaSb depth profiling is the 250 eV oxygen ion beam energy. Highly the impact energy highly the surface topography and deterioration of depth resolution occurs. The effect of the primary energy on the depth resolution of GaSb/InAs material is similar to that observed on boron doped layers in Si where the best depth resolution in Si material is obtained at the lowest primary energy [21] and SiGe [22].

B. Patterns suppression and enhancement of depth profile resolution

Many different macro topography formations on materials surface induced by ion bombardment were already observed, such as ripples, cones, pyramids, and terraces… The formation of ripples and changes of ion yield under oxygen sputtering has been reported for many III/V materials such as GaAs, GaSb and InAs [START_REF] Nakagawa | Scanning tunneling microscopy study of the ripple formation on sputtered surfaces[END_REF]. Nevertheless, it was not the case for GaP material under oxygen ion bombardment with the energy of 10.5 keV and the incidence angle of 37°. While the formation of morphological features can be used in various applications, they are undesirable for depth profiling in ToF-SIMS. In fact, this kind of roughening brings about degradation of depth resolution. One method to optimize depth resolution is oxygen flooding inside the chamber where gaseous oxygen is injected close to the sample's surface through a capillary. The oxygen flooding is used to saturate the analysis chamber with oxygen and more precisely at the vicinity of sample surface in order to create a completely oxidized layer at its surface prior to sputtering and to maintain it during the sputtering process. When this complete oxidation condition is achieved, maximum yield enhancement and optimized depth resolution and to suppress ripple formation. Thus, in the second part of our experiments, we performed ToF-SIMS analysis under oxygen flooding.

Figure 4.b shows the AFM image on the GaSb surface bombarded at 250 eV under oxygen leakage. As we notice, formation of small nanodots is observed similarly to the sample bombarded at 250 eV without oxygen flooding (figure 4.a). However, we can note a significant improvement in the surface roughness (RMS=0.57 nm) with respect to the sample analyzed without oxygen leakage (RMS=1.45 nm) as reported in table 2. Thus, we demonstrate that oxygen flooding is an effective method to enhance surface topography in GaSb/InAs multilayers. However, we still have formation of nanopatterns that can deteriorate depth resolution. Thus, in order to try to suppress totally the surface topography formed, we performed depth profiling under sample rotation which is an effective method to improve depth resolution profiling by smoothing the surface during ion beam sputtering [START_REF] Bradley | [END_REF]. Figure 4.c shows the obtained AFM image with sample rotation. The latter leads to a smooth and flat surface compared to without rotation. No formation of nanopatterns is evidenced. An improvement in the RMS is clearly observed as represented in table 2. Furthermore, in figure 4.d, we performed the experiment under simultaneously O 2 flooding and sample rotation. The latter procedure leads to more improvement in the RMS (see table 2). Figure 5 shows the superposition of the In and Ga depth profiles obtained at 250 eV under O 2 + sputtering for three cases: a) with sample rotation and without O 2 flooding, b) with O 2 flooding and without sample rotation, c) with sample rotation and O 2 flooding. The depth resolution of upslope of In and Ga profiles is similar. However, higher depth resolution of downslope (3 nm/decade for In and 4 nm/decade for Ga) using sample rotation and O 2 flooding compared to other cases is observed (8 nm/decade for In and 6 nm/decade for Ga for case b) and 6 nm/decade for In and 5 nm/decade for case a)) for Ga. We demonstrate that under these conditions the preservation of the initial surface topography is achieved.

Conclusion

In this study, we investigated the impact of O 2 + sputtering of GaSb/InAs multilayers by a correlative study using ToF-SIMS and AFM. We highlighted the formation of nanopatterns which evolve with incident beam energy. A continuous improvement of depth resolution by lowering down the O 2 + beam impact energy to 250 eV is observed. Moreover, we demonstrated that in order to obtain a smooth sputtering of GaSb/InAs, the optimal condition is the use of O 2 flooding and sample rotation which allows suppression of surface nanopatterning induced by the incident ion beam thus leading to higher depth profiles resolution.

Figure 1 :

 1 Figure 1: Structure of GaSb/InAs multilayers grown on Si substrate.

Figure 2 :

 2 Figure 2: 1×1 µm 2 AFM image of surface topography before ion irradiation. RMS = 0.68 nm.

Figure 3 :

 3 Figure 3: 1×1 µm 2 images of sample bombarded with oxygen ions at 45° incidence angle for different ion beam energies; a) 250 eV, b) 500 eV, c) 1 keV and d) 2 keV.

Figure 4 :

 4 Figure 4: 1×1 µm 2 of sample bombarded under 250 eV oxygen ions at 45° incidence angle. (a) no O 2 flooding and no rotation, (b) O 2 flooding, (c) with rotation (d) with O 2 flooding and sample rotation.

Figure 5 :

 5 Figure 5: Ga and In depth profiles of the GaSb/InAs multilayers.

Table 1 :

 1

		Initial topography	Under 250 eV O 2 + sputtering	Under 500 eV O 2 + sputtering	Under 1 keV O 2 + sputtering	Under 2 keV O 2 + sputtering
	RMS (1×1 µm 2 ) in nm	0.68	1.45	3.37	4.03	5.35
	Nanopatterns
	wavelength (λ)	X	25	33	40	52
	in nm
	Nanopatterns
	amplitude (A)	X	4	5	7	9
	in nm

Roughness measurements of GaSb/InAs multilayers under oxygen bombardment at different energies using TOF-SIMS.

Table 2 :

 2 Roughness measurements of GaSb/InAs multilayers under 250 eV oxygen bombardment with O 2 flooding and with sample rotation using TOF-SIMS.

					Under 250 eV
		Under 250 eV O 2 + sputtering	Under 250 eV O 2 + sputtering with O 2	Under 250 eV O 2 + sputtering with sample	+ sputtering with O 2 flooding and O 2
			flooding	rotation	sample
					rotation
	RMS (1×1 µm 2 ) in nm	1.45	0.57	0.46	0.39

Acknowledgments

This work has been supported by ANR-13-NANO-0001 MOSINAS project. The measurements were performed at the nanocharacterization platform (PFNC) of the CEA Grenoble.


Rebecca E Koch

Katherine L Buchanan

Casagrande Stefania

Crino Ondi

Damian K Dowling

Geoffrey E Hill

Wendy R Hood

Mckenzie Matthew

Mariette Mylene

Noble Daniel

Pavlova Alexandra

Seebacher Frank

Sunnucks Paul

Udino Eve

Craig R White

Salin Karine

Stier Antoine

Integrating Mitochondrial Aerobic Metabolism into Ecology and Evolution

Keywords: bioenergetics, metabolic rate, mitochondrial efficiency, mitochondrial, uncoupling reactive oxygen, species life-history trade-off

Biologists have long appreciated the critical role that energy turnover plays in understanding variation in performance and fitness among individuals. Whole-organism metabolic studies have provided key insights into fundamental ecological and evolutionary processes. However, constraints operating at subcellular levels, such as those operating within the mitochondria, can also play important roles in optimizing metabolism over different energetic demands and time scales. Herein, we explore how mitochondrial aerobic metabolism influences different aspects of organismal performance, such as through changing adenosine triphosphate (ATP) and reactive oxygen species (ROS) production. We consider how such insights have advanced our understanding of the mechanisms underpinning key ecological and evolutionary processes, from variation in life-history traits to adaptation to changing thermal conditions, and we highlight key areas for future research.

Energy metabolism drives ecological processes

Aerobic respiration is a principal source of energy for most eukaryotes. Energy metabolism controls the amount of energy uptake from the environment and the relative allocation of this energy to life-history traits. Classical ecological approaches for quantifying energy flows have estimated metabolic rate by measuring whole-organism gas exchange [START_REF] Ruiz | A microcalorimetric approach for investigating stoichiometric constraints on the standard metabolic rate of a small invertebrate[END_REF][START_REF] Pettersen | Metabolic rate covaries with fitness and the pace of the life history in the field[END_REF][START_REF] Glazier | Is metabolic rate a universal "pacemaker" for biological processes?[END_REF], which has provided a powerful foundation for understanding variation in individual performance, population growth rate, and community structure [START_REF] Brown | Toward a metabolic theory of ecology[END_REF]. However, respiration at the wholeorganism level ultimately derives from energy flow at the subcellular level [START_REF] Delhaye | Interspecific correlation between red blood cell mitochondrial ROS production, cardiolipin content and longevity in birds[END_REF]. Aerobic respiration in the mitochondria is a central process by which organisms transfer chemical energy locked in substrates into the chemical bonds of adenosine triphosphate (ATP), the main form of chemical energy fueling cellular processes. The efficacy with which mitochondria execute this energy transfer can be a major determinant of organismal fitness [START_REF] Ballard | Review: Can diet influence the selective advantage of mitochondrial DNA haplotypes?[END_REF][START_REF] Heine | Mitochondrial behaviour, morphology, and animal performance[END_REF][START_REF] Burton | Cytonuclear genomic interactions and hybrid breakdown[END_REF][START_REF] Hill | Assessing the fitness consequences of mitonuclear interactions in natural populations[END_REF][START_REF] Melvin | Intraspecific variation in survival and mitochondrial oxidative phosphorylation in wild-caught Drosophila simulans[END_REF].

Investigating variation in mitochondrial measurements provides an opportunity for integrating how mechanistic constraints and trade-offs at the cellular level may shape fundamental evolutionary and ecological patterns [START_REF] Delhaye | Interspecific correlation between red blood cell mitochondrial ROS production, cardiolipin content and longevity in birds[END_REF][START_REF] Salin | Variation in the link between oxygen consumption and ATP production, and its relevance for animal performance[END_REF][12][START_REF] Hood | Life history trade-offs within the context of mitochondrial hormesis[END_REF][START_REF] Casagrande | Increased glucocorticoid concentrations in early life cause mitochondrial inefficiency and short telomeres[END_REF][15]. Such constraints and trade-offs can involve interrelationships between ATP, reactive oxygen species (ROS; see Glossary) production, and heat generated during cellular respiration-relationships that can be flexibly modulated in response to changing needs, but may also face physiological constraints (see below) [START_REF] Gyllenhammer | Developmental programming of mitochondrial biology: a conceptual framework and review[END_REF][START_REF] Salin | Differences in mitochondrial efficiency explain individual variation in growth performance[END_REF]. Teasing apart these relationships has the potential to offer valuable insights into the nature of the physiological changes that enable organisms to adapt and to optimize function across a broad range of conditions.

Here, we suggest that characterizing energy flow via measurements of mitochondrial aerobic metabolism can provide new insights into differences in energy turnover and physiological performance among and within individuals, across time, and in response to environmental change (Figure 1) [START_REF] Mahalingam | Chronic cold exposure induces mitochondrial plasticity in deer mice native to high altitudes[END_REF][START_REF] Scott | The mitochondrial basis for adaptive variation in aerobic performance in high-altitude deer mice[END_REF]. Meeting energetic demands at the subcellular level while preventing the accumulation of potentially damaging byproducts under changing conditions requires careful orchestration of biochemical processes in the mitochondria [START_REF] Treberg | Comparing electron leak in vertebrate muscle mitochondria[END_REF].

The effectiveness of mediating such processes is driven by both genes and environments (see Online Supplementary Material Box S1), linking mitochondrial processes not only to individual fitness, but also to local adaptation (Figure 1) [START_REF] Hill | Mitonuclear coevolution as the genesis of speciation and the mitochondrial DNA barcode gap[END_REF]. Our aim is to discuss how the study of mitochondria may yield new insights into evolutionary and ecological sciences. We will firstly summarize mitochondrial traits and their quantification, and then secondly discuss the implications of these processes for ecology and evolution.

Mitochondrial traits and their quantification

Mitochondria have a wide range of functions (e.g. immune signaling pathways, apoptosis, epigenetics [START_REF] Angajala | Diverse roles of mitochondria in immune responses: novel insights into immuno-metabolism[END_REF][23][START_REF] Wallace | Energetics, epigenetics, mitochondrial genetics[END_REF]), but we focus predominantly on the central mitochondrial process of oxidative phosphorylation (OXPHOS) and on how in vitro mitochondrial measurements can be interpreted in relation to animal performance and fitness. OXPHOS is the specific component of mitochondrial aerobic respiration that harnesses potential energy from an electrochemical gradient to drive the phosphorylation of ADP into ATP, consuming oxygen in the process. Specifically, electrons harvested from food-derived substrates flow through the protein complexes of the electron transport chain (ETC) to fuel the pumping of protons from the mitochondrial matrix into the intermembrane space (Figure 2). This creates a disequilibrium in both charge and proton concentration across the inner membrane (a source of potential energy called the protonmotive force). Protons passing down this gradient through ATP synthase power the phosphorylation of ADP into ATP (Figure 2) [25,[START_REF] Stier | How to measure mitochondrial function in birds using red blood cells: a case study in the king penguin and perspectives in ecology and evolution[END_REF]. OXPHOS fluctuates in response to changes in substrate availability and composition, ATP demand, and environmental conditions. Flexibility in the activity of the ETC, the build-up of protonmotive force, and the movement of protons across the inner mitochondrial membrane sets the rate of ATP production and affects metabolic heat generation as well as mitochondrial ROS production (Figure 2).

Mitochondrial ROS are formed when electrons escape the ETC and react directly with oxygen, and some ROS are always generated during mitochondrial aerobic metabolism. The contribution of ROS to oxidative stress plays a prominent role in many ecological and evolutionary hypotheses, such as theories of aging [START_REF] Balaban | Mitochondria, oxidants, and aging[END_REF][START_REF] Harman | Aging: A Theory Based on Free Radical and Radiation Chemistry[END_REF][START_REF] Salmon | Update on the oxidative stress theory of aging: Does oxidative stress play a role in aging or healthy aging? Free Radic[END_REF] and of reproductive costs [START_REF] Speakman | Oxidative stress as a cost of reproduction: Beyond the simplistic trade-off model[END_REF][START_REF] Zhang | Current versus future reproduction and longevity: A re-evaluation of predictions and mechanisms[END_REF][START_REF] Monaghan | Oxidative stress as a mediator of life history trade-offs: Mechanisms, measurements and interpretation[END_REF]. While ROS serve important signaling functions and are not inherently harmful [33],

excess ROS have the potential to cause oxidative damage and lead to cellular dysfunction.

Rate of mitochondrial ROS generation may increase due to altered functionality of the ETC, such as mild dysfunction from oxidative damage, mutation, replication error, or genetic incompatibilities between genes encoding subunits of OXPHOS (mito-nuclear interactions;

Box S1) [START_REF] Hood | An ecologist's guide to mitochondrial DNA mutations and senescence[END_REF][START_REF] Hill | Mitonuclear Ecology[END_REF][START_REF] Lane | Mitonuclear match: Optimizing fitness and fertility over generations drives ageing within generations[END_REF]); we can consider this structural variation as variation in mitochondrial "quality" (Figure 3). In contrast, mitochondrial ROS production also changes inherently with changing of protonmotive force (Figures 2,3); specifically, more ROS are generated during high protonmotive force, such as may occur when demand for ATP is low (Figure 2B) [START_REF] Jastroch | Mitochondrial proton and electron leaks[END_REF]. This point is key because it highlights the misconception that increased metabolic rate will inherently increase rate of ROS production. Rather, ROS production increases with the buildup of protonmotive force, which can be countered either by a higher rate of ATP production (coupled with demand) [START_REF] Speakman | Oxidative stress as a cost of reproduction: Beyond the simplistic trade-off model[END_REF]38,[START_REF] Korshunov | High protonic potential actuates a mechanism of production of reactive oxygen species in mitochondria[END_REF] or a higher proton leak-protons flowing back to the mitochondrial matrix without passing through ATP synthase, dissipating energy as heat (Figure 2) [START_REF] Korshunov | High protonic potential actuates a mechanism of production of reactive oxygen species in mitochondria[END_REF].

Proton leak across the inner mitochondrial membrane can occur through two means: passive proton leak (movement of protons directly through the membrane, largely influenced by protonmotive force and membrane composition [40]) and inducible proton leak (regulated movement of protons through protein channels that span the inner mitochondrial membrane [START_REF] Brand | The efficiency and plasticity of mitochondrial energy transduction[END_REF]). Either form of proton leak is essentially "uncoupling" proton movement from the phosphorylation of ADP into ATP. Proton leak decreases rate of ATP synthesis relative to mitochondrial respiration rate, but it is not necessarily a dysfunction; rather, proton leak can be beneficial for respiratory function by lowering protonmotive force and thereby reducing mitochondrial ROS production (Figure 2) [START_REF] Brand | Uncoupling to survive? The role of mitochondrial inefficiency in ageing[END_REF]. Inducible proton leak can also be an important means to generate extra metabolic heat for thermoregulation in endotherms (e.g. UCP1 in the brown adipose tissue of small mammals [START_REF] Nedergaard | UCP1: the only protein able to mediate adaptive nonshivering thermogenesis and metabolic inefficiency[END_REF]), in addition to heat already produced during ATP synthesis.

Because oxygen consumed during aerobic respiration can be uncoupled from ATP synthesis through proton leak (Figure 2), two individuals (or, more specifically, two mitochondria) with the same rate of oxygen consumption can have quite different rates of ATP synthesis. Indeed, the proportion of oxygen consumed that leads to ATP synthesis versus the proportion that leads to proton leak (e.g. as captured in measures of mitochondrial efficiency; Box 2, Figure 3) can vary considerably across species, individuals, and time (Box 3) [START_REF] Salin | Variation in the link between oxygen consumption and ATP production, and its relevance for animal performance[END_REF][START_REF] Speakman | Uncoupled and surviving: Individual mice with high metabolism have greater mitochondrial uncoupling and live longer[END_REF]. Such variation can be due to strategic adjustment of mitochondrial processes to best respond to current conditions(e.g. increasing mitochondrial efficiency to enhance energy savings during prolonged fasting [45]; Figure 3). However, such variation may also be indicative of structural differences in, for example, the ETC-differences that could constrain ATP production capacity during times of high demand (Figure 3). Only limited research to date has focused on the evolutionary implications of within-individual variation in mitochondrial aerobic respiration, across tissues and life stages.

Understanding the interconnections between mitochondrial oxygen consumption, ATP and ROS production, and proton leak is critical for understanding physiological tradeoffs operating at the cellular level that may influence animal phenotype and performance (Box 1). The importance of these interactions has been recognized for decades, including their relevance to the evolution of body size [46], thermal biology [START_REF] Seebacher | Is endothermy an evolutionary by-product?[END_REF], and more recently, even sexually selected displays [START_REF] Hill | Plumage redness signals mitochondrial function in the house finch[END_REF]. Exploring these processes-particularly with recently developed techniques that allow for non-terminal and even repeated sampling (Box 2)offers ecologists insights into the long-term costs and constraints of the metabolic processes that underlie life-history strategies. An important outstanding area of research with relevance to both ecological and evolutionary questions is how and why individuals differ in their mitochondrial phenotype-parameters such as the efficiency with which ATP is produced from mitochondrial aerobic metabolism, or density of mitochondria within cells (Figure 3; Box 1)-and the higher-order effects of such subcellular variation on broader aspects of organismal performance.

While a full overview of the practical and methodological considerations required to collect informative mitochondrial measures is beyond the scope of this review, we provide a brief overview of several of the most well-established methods by which mitochondrial performance can be quantified in Box 1 (though none are without limitations), and important considerations for experimental design in Box 2. In the following section, we seek to translate the broad applications of the study of mitochondrial aerobic metabolism to evolutionary and ecological processes.

Applications of mitochondrial aerobic metabolism to ecological and evolutionary research

Mitochondrial processes underlie whole-organism metabolic rate

An understanding of mitochondrial aerobic metabolism can enhance our understanding of variation in whole-organism traits. Whole-organism metabolic rates have been found to vary severalfold among species and individuals [49,50], and variation in metabolic rate can be a functional determinant of variation in fitness and fitness-related traits [START_REF] Auer | Resting vs. active: a meta-analysis of the intra-and interspecific associations between minimum, sustained, and maximum metabolic rates in vertebrates[END_REF][START_REF] Mathot | The covariance between metabolic rate and behaviour varies across behaviours and thermal types: meta-analytic insights[END_REF][START_REF] Pettersen | Understanding variation in metabolic rate[END_REF]. Field and laboratory studies have identified strong relationships between whole-organism metabolic rate and survival and reproductive success, but the strength and form of selection varies among species and ecological contexts [START_REF] Pettersen | Understanding variation in metabolic rate[END_REF]. Variation in mitochondrial aerobic metabolism is likely an important underlying source of the observed among-individual variation in wholeorganism metabolic rate and is likely to influence the relationship between metabolic rate and fitness [START_REF] Speakman | Uncoupled and surviving: Individual mice with high metabolism have greater mitochondrial uncoupling and live longer[END_REF]54,[START_REF] Gaudry | Inactivation of thermogenic UCP1 as a historical contingency in multiple placental mammal clades[END_REF]. Almost all measurements of metabolic rate are made through the measurement of O2 or CO2 fluxes (e.g. the data for thousands of species summarized in [56]).

Such measurements are important estimates of overall energy flow, but as described above, variation at the mitochondrial level (e.g. uncoupling) alters the proportion of that energy that ultimately results in ATP, heat, or ROS (Figures 2 and3) [START_REF] Salin | Variation in the link between oxygen consumption and ATP production, and its relevance for animal performance[END_REF].

Exploring variation in mitochondrial aerobic metabolism can therefore help better pinpoint the significance of variation (or lack thereof) in energy flow at the whole-organism level. For example, two animals with the same whole-organism metabolic rate can differ in energy available as ATP (e.g. if they differ in mitochondrial efficiency; Box 1); similarly, two animals that differ in whole-organism metabolic rate can have the same energy available as ATP, but different levels of heat or ROS generation (Figures 2 and3). Differences in strategy or quality at the mitochondrial level thereby can alter the relationships between whole-organism metabolic rate and physiological performance or life-history trait expression.

Measurements of mitochondrial aerobic metabolism (e.g. mitochondrial efficiency; Box 1, 2) offer a means to further contextualize measurements of whole-organism energy flow by providing information on whether that energy may be put toward ATP or instead toward heat or ROS production-alternatives that have ramifications for the hypotheses we formulate for how metabolic rate relates to fitness. Combining measurements of fitness with measurements of mitochondrial aerobic metabolism (e.g. from repeated, non-terminal sampling; Box 2) and whole-organism metabolic rate will provide an opportunity to grow our understanding of the functional bases of variation.

Can variation in mitochondrial aerobic metabolism explain life-history trade-offs?

The study of metabolism has also played a central role in explaining fundamental life-history trade-offs, such as that between growth rate and lifespan. Applying an understanding of mitochondrial aerobic metabolism and the relationships between metabolic rate, ATP production, and ROS production offers a new tool for further exploring such life-history trade-offs. The "oxidative stress life-history hypothesis," for example, proposes that energydemanding phases of life that necessitate increased metabolism, such as growth or reproduction, come at the cost of increased generation of mitochondrial ROS, which in turn would have ultimate consequences to longevity (Figure 1) [START_REF] Costantini | Understanding diversity in oxidative status and oxidative stress: The opportunities and challenges ahead[END_REF]58]. However, as we describe above, increased metabolism does not necessarily increase the production of mitochondrial ROS-and can actually have the opposite effect (Figure 2); so, it is perhaps unsurprising that empirical evidence for this hypothesis is currently mixed (reviewed in [START_REF] Zhang | Current versus future reproduction and longevity: A re-evaluation of predictions and mechanisms[END_REF][START_REF] Costantini | Understanding diversity in oxidative status and oxidative stress: The opportunities and challenges ahead[END_REF][START_REF] Speakman | Oxidative stress and life histories: Unresolved issues and current needs[END_REF]). More comprehensive comparisons of mitochondrial and organismal respiration across species, individuals, and tissues has the potential to provide new insights into longstanding questions regarding major physiological and life-history patterns, such as trade-offs surrounding whole-organism metabolic rate [54] or growth [START_REF] Barneche | Warming increases the cost of growth in a model vertebrate[END_REF]. For example, an individual could potentially maintain growth rate during times of food scarcity without altering oxygen consumption by instead increasing efficiency of ATP production (Box 1)-but such increases in coupling between electron flow and ATP synthesis at lower metabolic rates may come at the potential cost of the increases in mitochondrial ROS production (Figures 2 and3) [START_REF] Salin | Differences in mitochondrial efficiency explain individual variation in growth performance[END_REF][START_REF] Salin | Decreased mitochondrial metabolic requirements in fasting animals carry an oxidative cost[END_REF][START_REF] Salin | Individuals with higher metabolic rates have lower levels of reactive oxygen species in vivo[END_REF]. Mitochondrial processes can therefore provide testable predictions for how and why individuals differ in performance when whole-organism measures do not fully explain observed variation.

A second hypothesis more specifically considers how regulation of protonmotive force to adjust ROS production may itself be a key to life-history trade-offs. This "uncoupling to survive" hypothesis proposes that proton leak is carefully controlled to decrease protonmotive force and thereby to regulate mitochondrial ROS generation (Figures 2 and3) [START_REF] Brand | Uncoupling to survive? The role of mitochondrial inefficiency in ageing[END_REF][START_REF] Speakman | Uncoupled and surviving: Individual mice with high metabolism have greater mitochondrial uncoupling and live longer[END_REF][START_REF] Brookes | Mitochondrial H + leak and ROS generation: An odd couple[END_REF]64]. Studies have tested this hypothesis by experimentally manipulating uncoupling and/or by comparing uncoupling levels to life-history trait performance and oxidative stress [START_REF] Casagrande | Increased glucocorticoid concentrations in early life cause mitochondrial inefficiency and short telomeres[END_REF][START_REF] Da Silva | Mild mitochondrial uncoupling in mice affects energy metabolism, redox balance and longevity[END_REF][START_REF] Salin | Alteration of mitochondrial efficiency affects oxidative balance, development and growth in frog (Rana temporaria) tadpoles[END_REF][START_REF] Stier | Short Communication: Long-term intake of the illegal diet pill DNP reduces lifespan in a captive bird model[END_REF]. For example, the administration of an experimental mitochondrial uncoupler to captive zebra finches (Taeniopygia guttata) caused wholeorganism metabolic rate to increase, likely in response to reduced ATP production efficiency caused by the treatment, but did not alter oxidative stress. However, finches receiving the uncoupling treatment had decreased reproductive investment, which supports the potential existence of a trade-off between possible benefits of uncoupling and maximizing current fitness [68]. Further studies are needed to better understand how uncoupling varies in wild animals that are balancing ATP and ROS production, lifespan, and reproduction across constantly varying environmental conditions.

Mitochondrial aerobic metabolism and temperature: thermoregulation, climatic adaptation, and acclimation

Uncoupling also has important ramifications for thermoregulation, as proton leak can be an important endogenous heat source. In developing chicken embryos, for example, the transition from ectothermy to endothermy is accompanied by increased mitochondrial uncoupling [START_REF] Walter | Endothermy in birds: underlying molecular mechanisms[END_REF], suggesting an important role of proton leak in mechanisms of thermoregulation. In addition, a particular uncoupling protein in mammals (UCP1) has been found to drive heat production through non-shivering thermogenesis in the brown adipose tissue of many (but not all) cold-adapted mammals [START_REF] Nedergaard | UCP1: the only protein able to mediate adaptive nonshivering thermogenesis and metabolic inefficiency[END_REF][START_REF] Gaudry | Inactivation of thermogenic UCP1 as a historical contingency in multiple placental mammal clades[END_REF]. Increased proton leak may also potentially be a means to generate extra metabolic heat while decreasing ROS production [71]; for example, a study subjecting zebra finches to acute cold stress found that untreated birds had increased oxidative damage to DNA, while birds treated with an experimental mitochondrial uncoupler showed no such increases [START_REF] Stier | Chronic mitochondrial uncoupling treatment prevents acute cold-induced oxidative stress in birds[END_REF]. Such examples provide insight into the potential for mitochondrial measurements to provide new mechanistic explanations for the potential costs or benefits of different means of endogenous heat production.

Mitochondria are not only important to regulating internal temperature, but also to responding to changes in external temperature-on the scales of both long-term adaptation and short-term acclimation. For example, a study of wild mayfly larvae (Baetis and Drunella spp.) found that high-and low-elevation-adapted populations differed in multiple measures of mitochondrial aerobic metabolism when tested under common conditions [START_REF] Havird | Powerhouses in the cold: Mitochondrial function during thermal acclimation in montane mayflies[END_REF]. This study demonstrates the potential for the evolution of mitochondrial phenotypes (over generations) to prevailing climatic conditions, with implications for local population adaptation. On shorter-term scales, mitochondrial aerobic metabolism also appears to be key to organisms' response to varying temperatures, such as may occur during seasonal acclimation (e.g. [74]) or short-term thermal stress (e.g. [START_REF] Jimenez | Rapid changes in cell physiology as a result of acute thermal stress House sparrows, Passer domesticus[END_REF]). Thermal conditions can themselves have fundamental thermodynamic effects on reaction rates in the mitochondria as cellular temperature changes, and organisms may respond by changing mitochondrial efficiency or density (potentially facing trade-offs in the process; e.g. [76,[START_REF] Chung | Mechanisms and costs of mitochondrial thermal acclimation in a eurythermal killifish (Fundulus heteroclitus)[END_REF]). Importantly, the limits of mitochondria to respond to changing conditions are expected to be a key determinant of thermal limits [START_REF] Havird | Powerhouses in the cold: Mitochondrial function during thermal acclimation in montane mayflies[END_REF][START_REF] Chung | Mechanisms and costs of mitochondrial thermal acclimation in a eurythermal killifish (Fundulus heteroclitus)[END_REF][START_REF] Harada | Variation in thermal tolerance and its relationship to mitochondrial function across populations of Tigriopus californicus[END_REF], with implications for how populations will respond to a changing climate.

Concluding remarks

There is a growing awareness among ecologists and evolutionary biologists that mitochondrial phenotype can play a central role in shaping fundamental characteristics of organisms. At the heart of this awareness is an understanding that selection acts on individual variation, and yet we know relatively little about how selection acts on mitonuclear genotype and mitochondrial phenotype. To understand how variation in mitochondrial aerobic metabolism has contributed to shaping selection on organisms, we need to understand how mitochondrial phenotype constrains and/or enables response to environmental change. Fruitful lines of research include quantifying individual variation in mitochondria, including individual consistency in responses to changing environmental conditions, and tissue-specific responses for which fitness is dependent on managing periods of intense energy demands (e.g. rapid growth or migration). As interest in mitochondrial biology expands, there emerges an increasing need for a common foundation in the best practices for studying mitochondrial aerobic metabolism, grounded in a careful understanding of mitochondrial biology. Such a foundation will promote a more unified understanding of how cellular processes may shape the evolution of life-history traits, and support new research avenues that probe the broader ecological and evolutionary implications (see Outstanding Questions).

Figure Captions

Figure 1. Mitochondria link energy flow through the environment to organism life-history, impacting meta-population and community dynamics. Environmental factors, such as temperature and water availability, affect net primary productivity (NPP) (A), and thus food availability. Organisms ingest food (B) and liberate the organic molecules that are absorbed, transporting them to tissues and cells. Carbohydrates, fats, and proteins are each independently catabolized and the energy stored within their chemical bonds is captured to produce ATP through aerobic respiration. The final steps take place in the electron transport chain (ETC) within the inner membrane of the mitochondria. Protonmotive force across the inner mitochondrial membrane is used to fuel ATP production through ATP synthase (C), but can also be dissipated through proton leak; this decreases ATP production efficiency and increases the amount of energy released as heat (D). Mitochondria are also a source for reactive oxygen species (ROS), which can damage proteins, lipids, and nucleic acids, potentially causing mitochondrial and cellular dysfunction and affecting life-history traits (E).

Mitochondrial and nuclear genes coding for enzymes in the ETC (F) interact with environmental factors to play critical roles in determining the efficiency of mitochondria in producing ATP, heat, and ROS. Mitochondrial aerobic metabolism is therefore important to determining the amount of ATP available to fuel metabolic processes underlying cellular and tissue maintenance, activity, growth, and reproduction-all critical underpinnings of an organism's life-history repertoire (G). Changes in life-history traits at the individual level "funnel" up to affect the dynamics of populations, and ultimately, meta-populations and communities (H).

Figure 2

Simplified diagrams illustrating relationships among ATP demand, protonmotive force, mitochondrial ROS production, and proton leak. Here, we illustrate electron movement through the ETC, ATP synthesis from ADP via ATP synthase, and proton movement across the inner mitochondrial membrane through the ETC (i), ATP synthase (ii), or proton leak (iii). The top region of each graphic represents the intermembrane space, and the bottom region represents the mitochondrial matrix. The three panels represent conditions of high ATP demand (A), low ATP demand (B), and low ATP demand with increased inducible proton leak (C). In (A), high demand for ATP prevents the build-up of protonmotive force as protons are quickly passed through ATP synthase, which limits mitochondrial ROS production. In (B), decreased ATP demand has increased protonmotive force because proton movement through ATP synthase has decreased; under these conditions, electrons "escape" the ETC at higher rates, forming ROS. In (C), proton leak-both basal and induced (black bars across the membrane represent upregulated protein channels)-prevents the build-up of protonmotive force and thereby controls ROS generation even while demand for ATP is low, producing heat in the process. The differences between (B) and (C) are key to the concept of how the strategic adjustment of proton leak may be beneficial, but also underlie trade-offs; for example, greater proton leak in (C) compared to (B) decreases mitochondrial efficiency, but also decreases mitochondrial ROS production even when ATP demand is low.

Figure 3

Illustration of within-and among-individual variation in mitochondrial aerobic

metabolism. The shading of each region represents whether oxygen is put toward ATP production (slashed region), proton leak (filled gray region), or ROS production (unshaded region). In (A), the filled circles represent possible measurements of an animal at rest (i) or the same animal in a state of high activity (ii), informed by data in [START_REF] Roussel | Threshold effect in the H2O2 production of skeletal muscle mitochondria during fasting and refeeding[END_REF]. In a highly active state (ii), the high demand for ATP causes ATP synthase to rapidly use up the proton gradient, so protonmotive force and rate of proton leak become relatively low. In contrast, the slowing of ATP synthase activity when an active animal shifts to rest (i) causes an increase in membrane potential. The open circles represent the in vitro measurements of OXPHOS (iii) or LEAK (iv) respiration (Box 1), though these extremes would not occur in vivo; it is worth noting that using these extremes to calculate the proportion of oxygen resulting in proton leak (i.e. RCR; Box 1) would result in an misleading estimate of the actual proportions of oxygen resulting in leak or ATP production at either during either high or low activity [80,[START_REF] Salin | The RCR and ATP/O indices can give contradictory messages about mitochondrial efficiency[END_REF]. In (B), within-and among-individual variation in mitochondrial efficiency is illustrated by tracing hypothetical relative proportions of different endpoints of mitochondrial oxygen consumption during states of high or low demand for ATP.

Mitochondria with high efficiency (v) will produce more ATP at a given level of oxygen consumption than those with low efficiency (vi and vii). However, higher efficiency mitochondria can also generate higher levels of ROS under states of low ATP demand (v, right column), so decreased efficiency may hypothetically be an adaptive "strategy" to lower ROS (e.g. shift from v to vi). In contrast, lower efficiency can also be caused by decreased mitochondrial "quality" (e.g. due to mild dysfunction from damage or mito-nuclear incompatibility), potentially both reducing ATP production and increasing ROS generation (vii). Note that the relative proportions allocated to ROS are inflated to be more visually clear.

Box 1: The biological implications of methodological choices (1) Sample type and preparation

Mitochondrial aerobic metabolism can be measured in vitro from isolated mitochondria, permeabilized cells/tissues, or intact cells [START_REF] Brand | Assessing mitochondrial dysfunction in cells[END_REF]. While isolated mitochondria provide precise control of cellular conditions (i.e. substrates and ADP availability), they lack cellular context. Intact cells have a preserved cellular and mitochondrial network and are able to inform on endogenous mitochondrial aerobic metabolism, but lack control of substrates and ADP availability. Permeabilized cells/tissues share some of the advantages and drawbacks of both isolated mitochondria and intact cells [START_REF] Brand | Assessing mitochondrial dysfunction in cells[END_REF]. Isolated mitochondria are considered a better choice for mechanistic studies, while intact cells have a greater ecological relevance [START_REF] Brand | Assessing mitochondrial dysfunction in cells[END_REF].

(2) Mitochondrial aerobic metabolism parameters Different mitochondrial aerobic metabolism parameters can be measured (Table I); the two main categories are respiration rates (e.g. OXPHOS and LEAK respiration) and proxies of mitochondrial efficiency (e.g. P/O, the amount of ADP used or ATP produced per atom of oxygen consumed; or, respiratory control ratio (RCR), the ratio of oxygen used for OXPHOS to oxygen used for proton leak). Although RCR (OXPHOS/LEAK respiration) is widely used, it has several mathematical and interpretation limitations; a related metric has recently been proposed to alleviate these limitations, called OXPHOS Coupling Efficiency (abbreviated here as OxCE). OxCE reflects the proportion of total mitochondrial oxygen consumption being allocated to ATP synthesis (1-LEAK/OXPHOS respiration) [80,[START_REF] Doerrier | High-resolution fluorespirometry and OXPHOS protocols for human cCells, permeabilized fibers from small biopsies of muscle, and isolated mitochondria[END_REF]. Yet, both RCR and OxCE are biased by the fact that the proton leak estimated when no ATP synthesis occurs (i.e. LEAK respiration) over-estimates the proton leak occurring when active ATP synthesis takes place (Fig. 3A). Respiration rates indicate potential metabolic capacity levels, while efficiency proxies indicate how efficient mitochondria are at producing ATP [START_REF] Salin | The RCR and ATP/O indices can give contradictory messages about mitochondrial efficiency[END_REF][START_REF] Brand | Assessing mitochondrial dysfunction in cells[END_REF]. Both are likely to be relevant for organismal performance, but the in vivo relevance of such in vitro measurements must be carefully considered.

Box 2: Designing experimental tests of mitochondrial aerobic metabolism

Measures of mitochondrial aerobic metabolism tend to be relatively low-throughput and most have to be measured in freshly collected biological samples (especially efficiency proxies) within a few hours of sampling ( [START_REF] Krajčová | High resolution respirometry to assess function of mitochondria in native homogenates of human heart muscle[END_REF], but see [89,90]). Such constraints may limit the number of animals sampled and require that tissue collection be organized strategically. In addition, the sampling approach (e.g. choice of tissue, frequency of sampling), the mitochondrial properties to assess (e.g. mitochondrial respiration rates, efficiency, density;

Box 1), and the experimental conditions (e.g. assay temperature [START_REF] Treberg | Comparing electron leak in vertebrate muscle mitochondria[END_REF], substrates [91]) should each be chosen with care based on the focus and constraints of a given study.

Since mitochondrial aerobic metabolism varies considerably among tissues (e.g. [START_REF] Salin | Differences in mitochondrial efficiency explain individual variation in growth performance[END_REF]54]), conclusions based on a single tissue should always be considered within context, and extrapolation to additional tissues without validation should be avoided. When selecting which tissue to measure, researchers should consider both the physiology that underlies the question being evaluated and the practical and ethical constraints of destructive sampling.

For example, a study of ornamental red coloration in male house finches (Haemorhous mexicanus) measured the performance of mitochondria in the liver because the process of converting dietary yellow to red feather pigments is thought to occur specifically within the liver in that species [START_REF] Hill | Plumage redness signals mitochondrial function in the house finch[END_REF]. When studies focus on small species, sample collection often must be destructive, making repeated sampling of an individual impossible. In larger animals, it may be possible to biopsy organs such as skeletal muscle [START_REF] Stier | How to measure mitochondrial function in birds using red blood cells: a case study in the king penguin and perspectives in ecology and evolution[END_REF], but the consequences of such invasive procedures should be carefully considered. Beyond ethical considerations, it is important to consider how the stressors animals experience prior to death or during sampling can impact their physiology [START_REF] Hill | Changes in concentrations of circulating heat-shock proteins in House Finches in response to different environmental stressors[END_REF].

Evaluating mitochondrial aerobic metabolism from blood cells has emerged over the last decade as a potential alternative to circumvent some of these issues. This approach using blood cells is becoming more prevalent in biomedical studies (e.g. [START_REF] Sjövall | Mitochondrial respiration in human viable platelets-Methodology and influence of gender, age and storage[END_REF]), and is starting to be implemented in ecological studies on non-model and wild animals [START_REF] Casagrande | Increased glucocorticoid concentrations in early life cause mitochondrial inefficiency and short telomeres[END_REF]15,[START_REF] Stier | How to measure mitochondrial function in birds using red blood cells: a case study in the king penguin and perspectives in ecology and evolution[END_REF][94][95][96].

Several

Glossary

Electron transport chain (ETC): A series of protein complexes in the inner mitochondrial membrane that transfer electrons via oxidation/reduction reactions. These reactions are coupled to the pumping of protons out of the mitochondrial matrix to create a protonmotive force.

LEAK respiration: Oxygen used to offset proton leak. Also called state 4 respiration rate.

Mitochondrial aerobic metabolism:

The metabolic (anabolic and catabolic) reactions that occur within the mitochondria and that involve direct and indirect use of oxygen. This comprises activity of the electron transport chain, protonmotive force, ATP production, and ROS generation.

Mitochondrial efficiency: Efficiency of mitochondria to convert food-derived energy substrate into ATP. Efficiency is often estimated in vitro with P/O ratios and respiratory control ratios (RCR).

Mito-nuclear interactions:

Functional associations between the products of the mitochondrial and nuclear genomes that can affect mitochondrial aerobic metabolism.

Oxidative phosphorylation (OXPHOS):

Phosphorylation of ADP into ATP in the presence of oxygen. Conversion of ADP to ATP is performed by ATP synthase, and uses the energy provided by the protonmotive force.

OXPHOS respiration: Oxygen used when mitochondria are actively producing ATP. Also called state 3 respiration rate.

Oxidative stress: Imbalance between the production of pro-oxidants (such as ROS) and the ability of a biological system to neutralize them through antioxidant defenses, leading to oxidative damage to biomolecules.

Proton leak: Protons that flow back across the inner mitochondrial membrane into the matrix outside of ATP synthase. Proton leak may be passive, particularly when membrane potential is high, or inducible (and regulated through transmembrane proteins).

Protonmotive force:

The potential energy stored across the inner mitochondrial membrane that is established by pumping protons from the mitochondrial membrane to the intermembrane space; the protonmotive force involves both the chemical gradient formed by the difference in proton concentration, and the electrical gradient formed by the difference in charge. Evidence that mito-nuclear interactions matter for the evolutionary trajectories of natural populations comes from the study of both laboratory and wild populations. In the lab, biologists can engineer strains of organisms carrying precise combinations of mito-nuclear genotype and quantify the fitness consequences. Many such studies have reported that epistatic interactions between mitochondrial and nuclear genotype shape the expression of key life-history traits as well as mitochondrial measurements [START_REF] Brown | Toward a metabolic theory of ecology[END_REF]. In the wild, emerging evidence indicates mito-nuclear evolution can be adaptive and could potentially drive reproductive isolation, promoting speciation [START_REF] Pettersen | Metabolic rate covaries with fitness and the pace of the life history in the field[END_REF][START_REF] Delhaye | Interspecific correlation between red blood cell mitochondrial ROS production, cardiolipin content and longevity in birds[END_REF][START_REF] Ballard | Review: Can diet influence the selective advantage of mitochondrial DNA haplotypes?[END_REF][START_REF] Heine | Mitochondrial behaviour, morphology, and animal performance[END_REF][START_REF] Burton | Cytonuclear genomic interactions and hybrid breakdown[END_REF]. Hybridization between such populations can result in mito-nuclear mis-match, leading to lower metabolic performance [START_REF] Hill | Assessing the fitness consequences of mitonuclear interactions in natural populations[END_REF]. Hypotheses about mitonuclear interactions in evolution can be tested via predictions based on knowledge of the genomic control of mito-nuclear phenotypes and their evolutionary relevance [START_REF] Melvin | Intraspecific variation in survival and mitochondrial oxidative phosphorylation in wild-caught Drosophila simulans[END_REF]. We present three examples to illustrate the range of emerging findings. OXPHOS proteins [START_REF] Hood | Life history trade-offs within the context of mitochondrial hormesis[END_REF]. This is accompanied by positive selection in nuclear genes whose products are predicted to interact functionally with mitochondrial-encoded gene products.

Reactive oxygen species (ROS

Populations are locally exposed to different temperatures in the wild, and at least some of the mito-nuclear genetic variation is thought to contribute to the differences in thermal sensitivity among populations [START_REF] Casagrande | Increased glucocorticoid concentrations in early life cause mitochondrial inefficiency and short telomeres[END_REF]. This is associated with intrinsic reproductive isolation between populations.

Box S1 :

 S1 Mitochondrial and nuclear gene interactions affect evolutionary processesMitochondria retain their own diminutive genome (in bilaterian animals comprising 13 proteincoding genes, 22 tRNAs, and 2 rRNAs). Gene products encoded by this mitochondrial genome must co-function with the products of nuclear genes to form the machinery that regulates bioenergetic functions critical to organismal fitness. Mitochondrial aerobic metabolism may therefore evolve with the key unit of selection being the joint mitochondrial-nuclear genotype[START_REF] Ruiz | A microcalorimetric approach for investigating stoichiometric constraints on the standard metabolic rate of a small invertebrate[END_REF][START_REF] Pettersen | Metabolic rate covaries with fitness and the pace of the life history in the field[END_REF][START_REF] Glazier | Is metabolic rate a universal "pacemaker" for biological processes?[END_REF]. Accordingly, epistatic mito-nuclear interactions might represent the molecular components under selection.

( 1 )( 2 )( 3 )

 123 Within population mito-nuclear epistasis is associated with ecologically relevant phenotypes Within a single admixed population of killifish (Fundulus heteroclitus), individuals bearing different mitochondrial genetic haplotype differ in their complements of hundreds of nuclear loci of mitochondrial function [11]. This variation is associated with the modulation of cardiac mitochondrial respiration. Mito-nuclear interactions drive climate-associated differentiation Eastern yellow robins (Eopsaltria australis) occupying different climates harbor deeply divergent mitochondrial genetic haplotypes [12]. Populations bearing the haplotypes show strong nuclear divergence only for a genome region enriched for genes of mitochondrial aerobic metabolism. This region and the mitochondrial genome show signatures of selective sweeps and adaptive mito-nuclear co-introgression, suggesting that adjustments to mitochondrial metabolism may be one component by which these populations adapted to local climate. Nuclear compensation for mitochondrial evolution promotes reproductive isolation and lineage divergence Tigriopus californicus copepod populations show extreme divergence in mitochondrial-encoded

Table I :

 I Logistical considerations when measuring mitochondrial aerobic metabolism parameters For instance, two groups of animals could exhibit similar respiration rates expressed per unit of tissue, but one group may require more mitochondria to reach the same respiratory capacity; contextualizing measures in this way is important to reveal hidden complexity, which in turn can suggest the possibility of further underlying patterns to explore (e.g.

		Permeabilized	Feasible	Feasible	Not feasible	Not feasible	Feasible
		cells/tissues
		Intact cells	Feasible	Feasible	Not feasible	Not feasible	Not feasible
	Start-up effort required	Low	Low	Low	High	High
	Able to measure ATP production?	No	No	No	Yes	Yes
	Relative processing time per	Low to moderate	Low to	Low to	Very high	Moderate
	sample			moderate	moderate
	Cost (equipment + consumables)	Low to moderate	Low to	Low to	Low to	High
				moderate	moderate	moderate
	Key methodological reference	[84]	[84,85]	[84,85]	[84,85]	[86]
	Respiration rates increased mitochondrial density to compensate for dysfunction, at the possible cost of RCR or OxCE ADP/O ATP/O ratio
	Equipment required increased ROS generation [87]).	Oxygraphy	Oxygraphy	Oxygraphy	Oxygraphy + Colorimetry	Oxygraphy + Fluorimetry
	Mitochondrial	Isolated	Feasible	Feasible	Feasible	Feasible	Feasible
	preparation	mitochondria

(3) Normalization of mitochondrial respiration rates Ratios like P/O, RCR, and OxCE are unitless, but mitochondrial respiration rates (e.g. OXPHOS or LEAK respiration rates) need to be normalized relative to tissue/mitochondrial content in order to be compared. Normalizing respiration rates of isolated mitochondria is usually done by expressing oxygen consumption per amount of mitochondrial protein content. Normalizing mitochondrial respiration rates from permeabilized and intact cells can either be achieved using mass of tissue or number of cells, or a proxy of mitochondrial abundance/density, and this choice has critical consequences on data interpretation

[80]

.

Speakman, J.R. (2005) Body size, energy metabolism and lifespan. J. Exp. Biol. 208,

Acknowledgements

This manuscript arose from discussions at a workshop hosted by Deakin University. We thank Damien Roussel for discussion of technical details related to this manuscript, and three anonymous reviewers for excellent feedback. Funding from the Australian Research Council supported the writing of this manuscript.

Supplementary Material Integrating Mitochondrial Aerobic Metabolism into Ecology and Evolution

Rebecca E. Koch 1 , Katherine L. Buchanan 2 , Stefania Casagrande 3 , Ondi Crino 2 , Damian K. Dowling 1 , Geoffrey E. Hill 4 , Wendy R. Hood