Kevin Magra

Edoardo Domenichini

Antonio Francés-Monerris

Cristina Cebrian

Marc Beley

Mohamed Darari

Mariachiara Pastore

Antonio Monari

Xavier Assfeld

Stefan Haacke

Philippe C Gros
email: philippe.gros@univ-lorraine.fr

Impact of the fac / mer Isomerism on the Excited-State Dynamics of Pyridyl-carbene Fe(II) Complexes

Keywords: fac/mer isomerism, iron complexes, NHC, photophysics, TD-DFT, Minimum Energy Path, decay process

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

INTRODUCTION

The replacement of platinoids (Pt, Ru, Ir) by other more accessible metals in photoactive systems constitutes a great scientific and technological challenge. This accomplishment is crucial for the development of cheaper and more eco-friendly light-driven devices such as organic light-emitting diodes (OLEDs) [START_REF] Longhi | Iridium(III) Complexes for OLED Application[END_REF] or dye-sensitized solar cells (DSSCs). [START_REF] Hagfeldt | Dye-Sensitized Solar Cells[END_REF] As a matter of fact, in the DSSCs field, ruthenium complexes have dominated the scene and boosted the technological development thanks to their optimal electrochemical and photophysical properties, such as an efficient coverage of the solar spectrum and a fast electron transfer via long-lived metal-to-ligand charge transfer (MLCT) excited states. [START_REF] Vos | Ruthenium Polypyridyl Chemistry; from Basic Research to Applications and Back Again[END_REF][START_REF] Pastore | Electronic and Optical Properties of Dye-Sensitized TiO2 Interfaces[END_REF] However, the low abundance, high price and non-negligible toxicity of ruthenium definitely represent a major limit in view of large-scale technological applications. Thus, the quest for sustainable alternatives has pushed the scientific community to develop analogous iron-based compounds, the latter being a cheaper, more abundant and less toxic element, as it was pioneered by Ferrere two decades ago. [START_REF] Ferrere | New Photosensitizers Based upon [Fe(L) 2 (CN)[END_REF][START_REF] Ferrere | Photosensitization of TiO 2 by [Fe II (2,2'-Bipyridine-4,4'-Dicarboxylic Acid) 2 (CN) 2 ]: Band Selective Electron Injection from Ultra-Short-Lived Excited States[END_REF] Nevertheless, the population of low-energy metal-centered (MC) states in typical iron polyimine complexes leads to the ultra-fast deactivation of the MLCT states initially accessed upon light excitation of the complex and as a consequence to the loss of the suitable photophysical properties. [START_REF] Creutz | Lifetimes, Spectra, and Quenching of the Excited States of Polypyridine Complexes of Iron(II), Ruthenium(II), and Osmium(II)[END_REF][START_REF] Gawelda | Ultrafast Nonadiabatic Dynamics of [Fe II (Bpy) 3 ] 2+ in Solution[END_REF] Hence, the development of iron-based optically active devices will necessarily imply the synthesis and production of systems presenting long-lived MLCT states. In turn, this task is a veritable scientific challenge and necessitates to achieve a thorough understanding of the fine photophysical process dictating the MLCT deactivation and most importantly the non-trivial coupling between electronic and vibrational effects. In this respect, the fine comprehension of the fundamental factors tuning the excited state relaxation and of their subtle interplay is fundamental.

Considerable success in increasing the excited-state lifetime of organometallic iron complexes has been achieved in the very last years thanks to the use of N-heterocyclic carbenes (NHC) as ligands. Indeed, the combined strong -donating and weak-tomoderate -accepting character of NHC induces a higher splitting of the iron d orbitals, resulting in a destabilisation of the MC states over the MLCT manifold and consequently a hampering of the deactivation pathways. For instance, the use of tridentate pyridyldicarbenes ligands allowed achieving a remarkable [START_REF] Vos | Ruthenium Polypyridyl Chemistry; from Basic Research to Applications and Back Again[END_REF] MLCT lifetime of 9 ps for complex C0 (Fig. 1). [START_REF] Liu | Towards Longer-Lived Metal-to-Ligand Charge Transfer States of Iron(II) Complexes: An N-Heterocyclic Carbene Approach[END_REF] Confirming the subtle interplay between different structural and chemical effects, our group also showed [START_REF] Duchanois | NHC-Based Iron Sensitizers for DSSCs[END_REF] that the presence of a carboxylic group on the central pyridyl ring is non-innocent and almost doubled the [START_REF] Vos | Ruthenium Polypyridyl Chemistry; from Basic Research to Applications and Back Again[END_REF] MLCT lifetime up to 16.5 ps. [START_REF] Duchanois | An Iron-Based Photosensitizer with Extended Excited-State Lifetime: Photophysical and Photovoltaic Properties: An Iron-Based Photosensitizer with Extended Excited-State Lifetime[END_REF] Switching from imidazolylidenes to benzimidazolylidenes moieties in the chromophoric ligand further increased the excited state lifetime, attaining a record lifetime of 26 ps. [START_REF] Liu | A New Record Excited State 3 MLCT Lifetime for Metalorganic Iron(II) Complexes[END_REF] This experimental observation has also been rationalized by molecular modelling revealing, for the first time, an inversion of the MLCT/MC energy levels for an iron complex. In addition, the inclusion of carboxylic group allowed the sensitization of TiO2 surface and the production of the first working DSSC device based on iron NHC, although with a low efficiency of light-to-electricity conversion. [START_REF] Duchanois | An Iron-Based Photosensitizer with Extended Excited-State Lifetime: Photophysical and Photovoltaic Properties: An Iron-Based Photosensitizer with Extended Excited-State Lifetime[END_REF][START_REF] Pastore | Interfacial Charge Separation and Photovoltaic Efficiency in Fe( II )-Carbene Sensitized Solar Cells[END_REF] An alternative strategy to increase the ligand-field splitting, and hence the MLCT lifetime, is to employ ligands that reduce the angular strain around the metal centre upon coordination. [START_REF] Abrahamsson | A 3.0 Μs Room Temperature Excited State Lifetime of a Bistridentate Ru II -Polypyridine Complex for Rod-like Molecular Arrays[END_REF][START_REF] Hu | A Luminescent Pt(II) Complex with a Terpyridine-like Ligand Involving a Six-Membered Chelate Ring[END_REF][START_REF] Vezzu | Highly Luminescent Tridentate N ∧ C*N Platinum(II) Complexes Featured in Fused Five-Six-Membered Metallacycle and Diminishing Concentration Quenching[END_REF][START_REF] Mengel | A Heteroleptic Push-Pull Substituted Iron(II) Bis(Tridentate) Complex with Low-Energy Charge-Transfer States[END_REF][START_REF] Chábera | Fe II Hexa N -Heterocyclic Carbene Complex with a 528 Ps Metal-to-Ligand Charge-Transfer Excited-State Lifetime[END_REF][START_REF] Kjaer | Luminescence and Reactivity of a Charge-Transfer Excited Iron Complex with Nanosecond Lifetime[END_REF] This effect is invoked to justify the different photophysical behaviour of the angularly strained [Ru(tpy)2] 2+ complex (tpy = 2,2':6',2''-terpyridine) that experiences an ultra-fast non-radiative via MC states population, [START_REF] Sauvage | Ruthenium (II) and Osmium (II) Bis(Terpyridine) Complexes in Covalently-Linked Multicomponent Systems: Synthesis, Electrochemical Behavior, Absorption Spectra, and Photochemical and Photophysical Properties[END_REF] and the weakly distorted bidentate homologue [Ru(bpy)2] 2+ (bpy = 2,2'-bipyridine) [START_REF] Campagna | Photochemistry and Photophysics of Coordination Compounds: Ruthenium[END_REF] that is instead strongly luminescent at room temperature since the deactivation channels are not accessible. [START_REF] Rillema | Structure of Tris(2,2′-Bipyridyl)Ruthenium( II ) Hexafluorophosphate[END_REF] As a consequence, the selection of tridentate or bidentate coordinating ligands can have a deep impact on the photophysical properties of the final complex.

However, the photophysical phenomena taking place in transition metal complexes are extremely complicated also due to the high density of excited states and their high coupling. Therefore, many different relaxation channels may compete, and a simple rationalization based on very intuitive concepts, such as the ligand-field splitting, although useful, does not allow to catch all the subtle details of the processes. More elaborate modelling strategies, involving the full exploration of the potential energy surfaces (PESs) of the involved states are needed to complete the picture.

Recently, and as a way to influence the angular strain, we have reported the first example of bidentate-liganded iron complexes containing pyridylcarbene ligands C1. [START_REF] Francés-Monerris | Synthesis and Computational Study of a Pyridylcarbene Fe(II) Complex: Unexpected Effects of Fac / Mer Isomerism in Metal-to-Ligand Triplet Potential Energy Surfaces[END_REF] The asymmetric nature of the employed ligand led to different geometrical isomers. In fact, C1 was obtained as a mixture of mostly the meridional (mer) isomer with a concomitant fraction of the facial (fac) isomer in a 14:1 ratio. A thorough molecular modelling of the different singlet, triplet and quintet excited states PES involved in the photoresponse evidenced totally different landscapes for the two isomers. In particular, the low-lying triplet PES of the mer isomer, leading to the MC population and deactivation, was found to be much steeper than the one of the corresponding fac isomer. [START_REF] Francés-Monerris | Synthesis and Computational Study of a Pyridylcarbene Fe(II) Complex: Unexpected Effects of Fac / Mer Isomerism in Metal-to-Ligand Triplet Potential Energy Surfaces[END_REF] This aspect is intimately linked to the larger stabilization of the 3 MC state in the mer arrangement as compared to the fac one. Altogether, our molecular modelling results indicate a much faster deactivation for the mer isomer. This result represented, to the best of our knowledge, the first hypothesis on the possibility of tuning and controlling the excited states life-time by the effects of fac/mer isomerism. As such, it also represents a fundamental aspect of the inherent photophysics of organometallic compounds that should be precisely investigated and whose implication can go far beyond the production of photoactive iron complexes.

However, the two isomers could not be separated, hence precluding the possibility to perform time-resolved spectroscopy to obtain the two separate life-times. To overcome this problem, we propose a different strategy based on the precise control of the geometrical arrangement of the ligands to selectively obtain only one of the two isomers. This leads to the synthesis of a new C3-symmetric hemicage ligand L2 based on bidentate pyridine-imidazolylidene moieties, subsequently used to prepare the corresponding pure fac iron(II) complex C2 (Fig. 1). In this contribution we report the full characterization of both C1 and C2 including molecular modelling and time-resolved spectroscopy, thereby allowing for the quantification and the rationalisation of the fac/mer isomerism on the properties of these iron-based compounds. Most importantly, while globally confirming the difference in relaxation times of the two isomers, we also show how this effect is due to a quite complex interplay between two decay pathways that are, in turn, affected differentially by the PES topologies. This work represents the first direct observation of different decay characteristic times in fac and mer isomers of iron compounds. As such, it opens the way to a novel strategy that can be synthetically pursued to achieve the precise tuning of important photophysical properties of materials.

RESULTS AND DISCUSSION

Synthesis and characterization

The preparation of pyridylimidazolium salts precursors L1 and L2 was achieved by reacting 2-(1H-imidazol-1-yl)pyridine 1 with the appropriate haloalkane. In the case of L1, the quarternisation reaction was performed with methyl iodide in acetonitrile at 85ºC (Scheme 1). [START_REF] Raba | Facile and Scalable Preparation of 2-Imidazolylpyridines[END_REF][START_REF] Barbante | Electrochemiluminescent Ruthenium (II) N -Heterocyclic Carbene Complexes: A Combined Experimental and Theoretical Study[END_REF] As for L2, tris(2-chloroethyl)amine 2 was selected as the alkylating agent. Temperatures as high as 150 °C and isopropanol as solvent were required to attain the triple-fold substitution. It is noteworthy to mention the dramatic effect of the microwave heating in both substitution reactions, which were performed in only 1h while similar reactions performed under classical thermal conditions have been previously reported to last up to 7 days. [START_REF] Soukup-Hein | Evaluating the Use of Tricationic Reagents for the Detection of Doubly Charged Anions in the Positive Mode by ESI-MS[END_REF] The target complexes C1 and C2 were obtained following a recently reported protocol by our group. Upon in situ generation of the carbene species by deprotonation of the pyridylimidazolium salts in the presence of t-BuOK, the resulting ligand is then coordinated to iron via FeCl2 metal precursor in DMF (Scheme 1). It should be noted the importance of adding the base at the end in order to reduce possible detrimental sidereactions, e.g. carbene degradation or iron precipitation. [START_REF] Soukup-Hein | Evaluating the Use of Tricationic Reagents for the Detection of Doubly Charged Anions in the Positive Mode by ESI-MS[END_REF][START_REF] Wang | Formation of Stable Iron/Cobalt NHC Complexes via Unexpected Ring Opening and in Situ Generation of a Tridentate Ligand[END_REF] In the case of tripodal ligand L2, the protocol required more diluted conditions in order to reduce the formation of oligomeric side-products. Successful coordination was confirmed in both cases by analysing the variation of the chemical shifts in 1 H-NMR spectra. As expected, the procarbenic proton was absent in the final complexes. Moreover, the coordination of the three pyridine rings in C2 was nicely confirmed by the noticeable shift change of the alpha pyridine hydrogens, excluding a possible coordination of the central amine moiety (See Fig. S1). As aforementioned, C1 was obtained as a non-separable mixture of mer and fac isomers in a 14:1 ratio. This remarkably high ratio might be attributed to the steric requirements of the ligand, that would particularly disfavour a facial arrangement, along with the employed reaction conditions. [START_REF] Dabb | Mer and Fac Isomerism in Tris Chelate Diimine Metal Complexes[END_REF][START_REF] Dabb | Mer and Fac Isomerism in Tris Chelate Diimine Metal Complexes[END_REF][START_REF] Ragni | Emitting Iridium Complexes: Synthesis, Photophysics and Phosphorescent Devices[END_REF] Nevertheless, the identification and characterisation of both isomers was carried out unambiguously by NMR analysis due to their differences in signal intensity and pattern. Concerning C2, the three bidentate units are magnetically equivalent, which correlates well with the inherent C3 symmetry of the complex. It is worth noting that the constrained geometry causes the four hydrogens of the ethylene units to be non-equivalent. Despite all our attempts, we were unsuccessful in growing quality crystals suitable for diffraction XRay analysis so we have resorted to high-level molecular modelling to get the molecular structures of C0, C1 and C2. The ground state equilibrium geometry of the tridentate and bidentate compounds has been obtained by molecular modelling (Fig. 2) and the details are reported (Table S2). The redox properties of both complexes were analyzed using cyclic voltammetry, and full results are provided in SI (Table S1). In comparison with the Fe II /Fe III oxidation potential of the tridentate complex C0 (0.71 V), less positive values were obtained for C1 (0.65 V) and C2 (0.61 V). These latter values were much lower compared with [Fe(bpy)3] 2+ (1.05 V/SCE) confirming the induction of important ligand-field effects.

The complexes were also characterized by UV-vis spectroscopy (Fig. 3 and Table S1).

For all the systems, the absorption spectrum, shows three bands in the 200-700 nm region. Usually, the intense bands near 250-300 nm can be assigned to intraligand (IL) [*] transitions. In the lower energy region, the two less intense, broader bands are attributed to MLCT transitions as previously reported for C0, 9 C1, [START_REF] Francés-Monerris | Synthesis and Computational Study of a Pyridylcarbene Fe(II) Complex: Unexpected Effects of Fac / Mer Isomerism in Metal-to-Ligand Triplet Potential Energy Surfaces[END_REF] and other related Fe(II) complexes. The band in the 450-500 nm area is clearly blue shifted by 30 nm for C1 and C2 compared with C0.

The optical properties have also been modelled by TD-DFT allowing also to characterize the nature of the electronic excited states. Figure S11 displays the calculated UV-Vis spectrum of C2, which shows an intense band centred at  ~443 nm, in reasonably good agreement with the experimental recordings (see Fig. 3). Note that the small red shift of C2 with respect to C1 observed in the experimental band maxima (see Fig. 3) is also captured by our theoretical protocol, since the convoluted spectrum of the 1:14 fac/mer-C1 mixture has an absorption band peaking at ~415 nm. Interestingly, C2 has additional electronic transitions from the nitrogen atom of the 'bridge' amino group to the * orbitals of the pyridyl-carbene ligands (nNL). The excitation energies lie in the 350 -450 nm range, although the associated oscillator strengths are small as compared to the MLCT transitions (see Fig. S13). However, certain MLCT/nNL mixing, also enhanced by the symmetry breaking induced by the tertiary amine 'bridge', might be responsible for the absorption intensity loss displayed by C2 in that region, with respect to the more efficient absorption of C1.

Excited-state computational studies

As already stated, important differences in the excited states PES landscapes have been found between fac and mer isomers. [START_REF] Francés-Monerris | Synthesis and Computational Study of a Pyridylcarbene Fe(II) Complex: Unexpected Effects of Fac / Mer Isomerism in Metal-to-Ligand Triplet Potential Energy Surfaces[END_REF] Hence, it is necessary to prove that the C2 complex represents a good model for a pure fac-FeNHC complex and does not alter this energetic landscape.

The PESs and the associated geometrical distortions that drive the C2 decay to the ground state have been modelled using DFT and TD-DFT methods. Given the ultrafast population of the triplet manifold determined for this compounds and other Fe(II) derivatives in the literature, [START_REF] Gawelda | Ultrafast Nonadiabatic Dynamics of [Fe II (Bpy) 3 ] 2+ in Solution[END_REF][START_REF] Chábera | Fe II Hexa N -Heterocyclic Carbene Complex with a 528 Ps Metal-to-Ligand Charge-Transfer Excited-State Lifetime[END_REF][START_REF] Monat | Femtosecond Excited-State Dynamics of an Iron(II) Polypyridyl Solar Cell Sensitizer Model[END_REF][START_REF] Bressler | Femtosecond XANES Study of the Light-Induced Spin Crossover Dynamics in an Iron (II) Complex[END_REF][START_REF] Zhang | Tracking Excited-State Charge and Spin Dynamics in Iron Coordination Complexes[END_REF][START_REF] Kjaer | Solvent Control of Charge Transfer Excited State Relaxation Pathways in [Fe(2,2′-Bipyridine) (CN) 4 ] 2[END_REF] it is thus expected that the triplet states can be accessed already at the Franck-Condon or S1 ( 1 MLCT) equilibrium geometry, taking also into account the structural similarity of both geometries (see Table S3), as it is also analysed in our previous report. [START_REF] Francés-Monerris | Synthesis and Computational Study of a Pyridylcarbene Fe(II) Complex: Unexpected Effects of Fac / Mer Isomerism in Metal-to-Ligand Triplet Potential Energy Surfaces[END_REF] Fig. 4 tracks the low-lying singlet, triplet and quintet states along the key structures involved in the decay process, namely the S0 min, S1 ( 1 MLCT) min, T1 ( 3 MC) min and Q1 min geometries. As in the case of fac-C1, [START_REF] Francés-Monerris | Synthesis and Computational Study of a Pyridylcarbene Fe(II) Complex: Unexpected Effects of Fac / Mer Isomerism in Metal-to-Ligand Triplet Potential Energy Surfaces[END_REF] the triplet PESs of C2 evolve adiabatically from MLCT to MC nature as the system approaches to the T1 ( 3 MC) min structure. The natural transition orbitals (NTOs) isosurfaces for the lowest-lying singlet and triplet states are shown in Figure S14. Coherently with what observed for fac-C1 and mer-C1, [START_REF] Francés-Monerris | Synthesis and Computational Study of a Pyridylcarbene Fe(II) Complex: Unexpected Effects of Fac / Mer Isomerism in Metal-to-Ligand Triplet Potential Energy Surfaces[END_REF] the main coordinate driving the process is the enlargement of the Fe-N1 bond, which has a bond distance of 2.82 Å at the T1 ( 3 MC) min geometry (Fig. 4a). Further Fe-N1 stretching is required to reach the T1-S0 singlet-triplet crossing (STC) at 3.05 Å (Fig. 4b). On the other hand, the T2-S0 STC area is achieved at larger Fe-N1 elongations. It shall be noted that the Fe-N1 enlargement has a small impact on the triplet PESs but greatly destabilizes the ground state, giving rise to relatively planar triplet surfaces and unveiling the absence of significant energy barriers throughout the decay process.

Interestingly, the T1 ( 3 MC) min geometry lies at a relative energy of 1.43 eV, similar to the 1.35 eV determined for fac-C1 and higher in energy with respect to mer-C1 (1.13 eV). [START_REF] Francés-Monerris | Synthesis and Computational Study of a Pyridylcarbene Fe(II) Complex: Unexpected Effects of Fac / Mer Isomerism in Metal-to-Ligand Triplet Potential Energy Surfaces[END_REF] It can also be readily seen that the T1-Q1 triplet-quintet crossing (TQC) is less accessible from the T1 ( 3 MC) min structure as compared to the S0-T1 STC, since an energy barrier has to be surmounted in order to populate the quintet state, whereas the access to the S0-T1 STC is barrierless. Therefore, we may conclude that the PES landscape of C2 can be considered globally equivalent to the one of fac-C1 and is significantly different from the one of the mer isomer.

Femtosecond transient absorption spectroscopy

In order to understand the photophysics of C1 and C2, their solutions in ACN are studied using transient absorption spectroscopy (TAS), under excitation with ≈50 fs pulses at 400 nm. In Fig. 5A, some relevant spectra at different time delays are reported for the fac-only C2. The negative signal is the ground state bleach (GSB), i.e this band mirrors the depletion of the population of the ground state induced by the pump. Such signal is broad, from 300 up to 475 nm, but two main peaks are detected at 427 nm and another, less intense and narrow, at 364 nm. The first one, at 0.3 ps overlaps with the inverse Steady State Absorption SSA, (blue dash-dot line) in the region 410-450 nm, while outside of this interval, a broad positive signal, due to excited state absorption (ESA), reduces the negative intensity. Indeed, the smaller GSB amplitude as compared to the SSA for λ< 380 nm (25% reduction at 0.3 ps) is due to a hidden ESA for these wavelengths. ESA appears as a dominant positive signal for λ> 475 nm and even 460 nm for longer delay times (> 5 ps). This band is broad, extending from 475 to beyond 650 nm at 0.3 ps, narrows down and blue-shifts for longer delay times. This is highlighted when the A spectra are normalized at the ESA peak (Fig. S19).

Spectral evolution stops only after 20 ps, when a ≈50 nm narrow spectrum with a maximum at 475 nm remains. Note that these A spectra are significantly different from the ones we reported for tridentate-liganded complexes of NHC-Fe(II). [START_REF] Duchanois | NHC-Based Iron Sensitizers for DSSCs[END_REF][START_REF] Duchanois | An Iron-Based Photosensitizer with Extended Excited-State Lifetime: Photophysical and Photovoltaic Properties: An Iron-Based Photosensitizer with Extended Excited-State Lifetime[END_REF][START_REF] Liu | A New Record Excited State 3 MLCT Lifetime for Metalorganic Iron(II) Complexes[END_REF] Here, the extinction coefficients of the ESA transitions are 4, or even ≈10 (cf. C1 below), times smaller than for the S0 -> 1 MLCT transitions dominating the GSB, while for the tridentate-liganded complexes, the ratio is not larger than 3. In addition, tridentateliganded complexes display additional positive ESA bands below 450 nm, which are not observed, or probably hidden in the GSB signal for C1 and C2, here.

The result of the continuous spectral shifts and narrowing is a strong wavelength dependence of the kinetic traces in the ESA region (Fig. 5A, and S20, S21). While 1 is attributed to electronic relaxation from the optically excited [START_REF] Longhi | Iridium(III) Complexes for OLED Application[END_REF] MLCT state and subsequent vibrational relaxation, the occurrence of two distinct excited state lifetimes is unusual for NHC-Fe(II) complexes. Indeed, previous reports agreed in the description of a sequential relaxation process attributing one single ps lifetime 2 to the population of the [START_REF] Vos | Ruthenium Polypyridyl Chemistry; from Basic Research to Applications and Back Again[END_REF] MLCT state (Fig. 7A):

𝑀𝐿𝐶𝑇 ⎯⎯ 𝑀𝐿𝐶𝑇 𝑀𝐿𝐶𝑇 → 𝑀𝐶𝐿𝑇 → 𝑆 .
In the present case, for C2 containing only the fac isomer, the question arises, whether 2 reflects an internal conversion between two excited state A and B, the latter decaying in a second stage with 3 (Fig. 7B), or whether states A and B decay in parallel with these two lifetimes (FigC). A partial answer comes from an inspection of the time-resolved A spectra. Indeed, 3

is associated with the long-lived quasi-static narrow ESA component dominating the spectra for t≥ 10 ps, characterising state B (Fig. 5). In Fig. S19B, we display the results of a subtraction approach that removes the smoothed and normalized A*(t=15.0 ps) spectrum from the normalized A* at earlier times (the '*' denotes normalisation to the max. ESA signal). To a good degree of approximation, one may consider these double difference spectra tA*(t)-A*(t=15.0 ps), as the time-dependent spectra of the earlier time populations, associated with 1 ( 1 MLCT) and 2 (state A). For delays t> 0.5 ps, the ESA part of trepresentative of state A, is much broader than the ESA of state B. It displays only a slight dynamic blue-shift, so that a quasi-isobestic point appears at 485 nm, for t> 1.6 ps, with the GSB part of t.

In Fig. 8, we summarize the results of the 3-exp. fits (see SI for details) performed for 20 single wavelengths. As anticipated above, at longer wavelengths (>550 nm) the ESA band is dominated by the amplitudes of 1 (1MLCT) and 2 (state A), A1 and A2, while A3 (state B) is negligible. τ1 has an average value of 0.4±0.1 ps. It appears both in the ESA and GSB decay and is related to intersystem crossing from the 1 MLCT state and subsequent intramolecular vibrational cooling in states A and B. As such it appears as a rise time for the ESA in the 500-540 nm range (A1<0). In the bleach region (<450 nm), τ2 and τ3 are only slightly wavelength dependent with average values of 4±1 ps and 18±3 ps, respectively, while τ2 effectively decreases down to ≈2.0 ps for the longest ESA wavelengths. In Table S4 all the parameters are reported, together with details for the fit procedure, used for meaningful convergence of the fits. The A3 amplitude associated with τ3, agrees with the 15 ps spectrum of Fig. S19B. In the range 440-600 nm, it has a positive amplitude, while below it is always negative (GSB). This decay associated amplitude spectrum demonstrates that the decay of state B leads to filling of the ground state (GS), in agreement with schemes A and B of Fig. A2, the amplitude of τ2 agrees with the evolution described in Figs. S19: The spectral shape of A2 is consistent with the long-wavelength ESA component revealed after subtraction of the 15 ps spectrum (Fig. S19B). Most importantly, A2 has a significant amplitude (30-40% larger than a3) throughout the entire GSB region.

The spectral shape of A2 allows us to discriminate between the parallel and sequential excited state relaxation schemes (Fig. 7, A andB). In the sequential scheme, the population moves from state A to state B without any ground state bleach recovery. The 4 ps component should thus appear as a rise time (neg. A2) for the ESA of B in the 450-500 nm range. This is partly true since A2 is negative for < 470 nm, but not for the wavelengths of maximum ESA of state B (470-490nm). Hence, a clear indication of state A giving rise to population of state B is lacking. More importantly is the observation of the GSB recovery occurring dominantly with τ2 for < 470 nm. This is in contradiction to the sequential relaxation scheme, but fully in agreement with the parallel one. Indeed, both the decays of the population of A and B lead to ground state recovery.

As far as C1 is concerned, even though this complex contains only 6.7% of the fac isomer, the transient absorption data show, at first sight, a high resemblance with those of C2 (Fig. 5B). The main differences are in the smaller ratio of ESA to GSB amplitude, which is 1:10, and in a more drastic evolution (dynamic shift and spectral narrowing) of the ESA in the early 2-5 ps. The ESA and GSB kinetics are significantly faster than for C2, as can be seen from Fig. 9, and from Figs. S24 and S26. Like C2, a detailed visual inspection of the kinetic traces in semi-log scales reveals three distinct lifetimes (Fig. S24): 1 = 0.2-0.5 ps, 2= 2-3 ps, 3= 15-20 ps. Note that 2 is shorter for C1 than for C2.

A comparison of kinetic traces obtained for the 1:14 and a 1:8 mixture of fac/mer shows that they are identical (Fig. S25 and related discussion), which indicates that the contribution of the fac isomers is negligible in the mixtures. We will therefore attribute the following excited state dynamics to mer isomers only. A similar analysis of the normalised time-resolved spectra, as performed for C2, allows to identify two distinct ESA bands and thus excited state populations, corresponding to lifetimes 2 and 3 (Fig. S23). As suggested by Fig. 5B, the short wavelength ESA associated with 2 is blue-shifting and decaying on that same timescale, leading to strongly wavelength dependent ESA kinetics and values for 2, as can be readily seen from visual inspection (Fig. 9 top, Fig. S24 bottom). Global fitting is therefore not an option. Fig. S26 and S27 present the results of the 3-exp. fits carried out for individual wavelengths, and a detailed discussion is in the SI. Most importantly, the 2 component associated with the short-lived long-wavelength ESA, characterising state A in Fig. 7, is the dominating channel for ground state recovery (Fig. S27). Also, the decay of state B (lifetime 3 and amplitude A3), leads directly back into the ground state, since the latter is observed in the GSB recovery times, very clearly for (410-450 nm), where the signalto-noise ratio is sufficiently large. We therefore conclude, that, as for C2, the parallel relaxation scheme of two distinct excited state populations (Fig. 7C) also applies for C1.

If one wants to characterize the excited state lifetime by an average of the two populations, the experiments demonstrate that mer isomers have, in average, a 3-4 times shorter excited state lifetime than fac. It turns out that the faster overall decay of ESA and GSB of C1 with respect to C2 is mainly due to a larger relative amplitude for 2, and its shorter value (2.0-2.5 ps). Fig. S23 shows that, in the GSB region, the ratio of A2/A3 is ≈7:1, while for C2, this ratio is ≈4:3, on average. Other differences are in the smaller ESA amplitude and slightly red-shifted ESA spectra for mer with respect to fac.

Overall interpretation of the excited-state decay

With the computational results of the excited state manifold in mind, the first obvious question concerns the assignment of states A and B and their location in the excited state landscape (Fig. 4). We can unambiguously rule out that the metal-centered quintet 5 T2 participates in the relaxation scheme since the latter is characterised by a sharp (10-15 nm broad) excited state absorption in the 340-360 nm range for tridentate ligands. [START_REF] Duchanois | NHC-Based Iron Sensitizers for DSSCs[END_REF][START_REF] Liu | A New Record Excited State 3 MLCT Lifetime for Metalorganic Iron(II) Complexes[END_REF] No sign of such an ESA is indicated by the A spectra (Fig. 5). Furthermore, the calculated PES for both isomers of C1 and C2 consistently rule out the possible population of the quintet state due to its high energy. As a consequence, and in extension to previous reports, [START_REF] Duchanois | NHC-Based Iron Sensitizers for DSSCs[END_REF][START_REF] Zimmer | The Connection between NHC Ligand Count and Photophysical Properties in Fe (II) Photosensitizers: An Experimental Study[END_REF] we conclude that "3" is the minimum number of carbene bonds necessary to avoid the quintet population and hence photo-induced spin crossover in NHC-Fe(II) complexes.

One initial motivation for the design of bidentate ligands was the prospect to produce NHC complexes with bite angles closer to 180°, preserving the octahedral coordination of Fe, and thus increasing the ligand field, with respect to tridentate ligands. So far, to the best of our knowledge, a heteroleptic tridentate-liganded complex with 3 carbene bonds has not been reported, making it difficult to identify a direct effect of the coordination geometry only. The present study definitely points to an important modification of the excited state energy landscape since, for both mer and fac isomers, we observe the co-existence of two excited state populations, while in all homoleptic tridentate-liganded Fe(II)-NHC complexes reported so far, a single excited state population was observed, [START_REF] Liu | Towards Longer-Lived Metal-to-Ligand Charge Transfer States of Iron(II) Complexes: An N-Heterocyclic Carbene Approach[END_REF][START_REF] Duchanois | NHC-Based Iron Sensitizers for DSSCs[END_REF][START_REF] Duchanois | An Iron-Based Photosensitizer with Extended Excited-State Lifetime: Photophysical and Photovoltaic Properties: An Iron-Based Photosensitizer with Extended Excited-State Lifetime[END_REF][START_REF] Liu | A New Record Excited State 3 MLCT Lifetime for Metalorganic Iron(II) Complexes[END_REF][START_REF] Zimmer | The Connection between NHC Ligand Count and Photophysical Properties in Fe (II) Photosensitizers: An Experimental Study[END_REF][START_REF] Chábera | Fe II Hexa N -Heterocyclic Carbene Complex with a 528 Ps Metal-to-Ligand Charge-Transfer Excited-State Lifetime[END_REF] always assigned to [START_REF] Vos | Ruthenium Polypyridyl Chemistry; from Basic Research to Applications and Back Again[END_REF] MLCT states.

The assignment of states A and B in terms of their electronic character is difficult on the sole basis of the TAS data. We can, however, safely exclude them to be singlet states since these would display stimulated emission, i.e. negative on the low-energy side of the GSB bands. Triplet states, as the 3 MLCT's reported for tridendate-liganded Fe(II)-NHCs, do not show sizeable stimulated emission since the radiative rate of the 3 MLCT-S0 transition has a ≈3 orders of magnitude smaller radiative rate than the allowed singlet-singlet transitions. [START_REF] Haacke | Ultrafast Excited State Dynamics of NHC-Fe(II) Complexes Designed for Light Harvesting (Conference Presentation)[END_REF] It is therefore most likely that the observed states A and B are located on the triplet excited state PES presented in the computational studies. TAS could potentially differentiate the MC or MLCT character on the basis of the ESA spectra, however a clear fingerprint differentiating one from the other has only been reported for the very specific case of [Fe(bpy)(CN)4] 2-, where the 3 MLCT/MC crossover goes along with a strong solvatochromism. [START_REF] Kjaer | Solvent Control of Charge Transfer Excited State Relaxation Pathways in [Fe(2,2′-Bipyridine) (CN) 4 ] 2[END_REF] A possible interpretation of the time-resolved spectroscopy results and in particular the existence of a parallel decay model with a branching ratio between the fast and slow component that is strongly dependent on the isomerism, can be formulated taking into account the results from molecular modelling reported here and in previous contributions. [START_REF] Francés-Monerris | Synthesis and Computational Study of a Pyridylcarbene Fe(II) Complex: Unexpected Effects of Fac / Mer Isomerism in Metal-to-Ligand Triplet Potential Energy Surfaces[END_REF] The slower decay from the first triplet state may be explained by a closer examination of the PES. Figure 10 displays a schematic representation of the two-state decay model.

Indeed, in the case of T1 the system will be trapped in the T1 minimum region where the S0 PES is higher in energy than T1 displaying a spin-crossover region. Hence, some vibrational cycles will be necessary to allow the crossing between the S0 and T1 states and subsequently transfer the population back to S0. This process is thus ascribed to the slower decay component, 3.On the other hand, a much larger region of the T2 PES lies at higher energies with respect to S0, having a much smaller spin-crossover area and thus being able to funnel the population to the available low-lying S0 state in a much more efficient way, leading thus to shorter triplet excited-state lifetimes (3 component).

The difference in the branching ratio between the two paths in the case of fac and mer isomers can also be rationalized in terms of the different shape of the PES (Fig. 10). In the case of mer (C1) one can observe an initially steep PES; furthermore, T1 is significantly lower in energy than T2 at the Franck-Condon region. The situation is totally different for the fac (C2) isomer where the PESs are globally flatter and the two triplet states are degenerated at Franck-Condon area. As such, in the case of the mer isomer a large population of T2 should be expected in an ultrafast time scale, driving the system rapidly towards the central regions of the PES. [START_REF] Francés-Monerris | Synthesis and Computational Study of a Pyridylcarbene Fe(II) Complex: Unexpected Effects of Fac / Mer Isomerism in Metal-to-Ligand Triplet Potential Energy Surfaces[END_REF] On the other hand, in the case of fac, and in the 1 time-scale, i.e. close to the Franck-Condon region, the population of the triplet manifold will distribute more equally between T1 and T2 since the PESs are flatter and accessible T1/T2 energy degeneracy areas are predicted at the surroundings of the mentioned Franck-Condon region. Remarkably enough, the steeper PES calculated for the mer isomer is also in agreement with the shorter value observed for the 2 component of this isomer as compared to the 2 measured for fac.

Theoretical determinations of the triplet ESA spectra support the present interpretation.

Computational methods

The computational methods employed in this work have been fully detailed elsewhere [START_REF] Francés-Monerris | Synthesis and Computational Study of a Pyridylcarbene Fe(II) Complex: Unexpected Effects of Fac / Mer Isomerism in Metal-to-Ligand Triplet Potential Energy Surfaces[END_REF] and only a brief description will be provided here. All computations have been performed with the GAUSSIAN 09 (D01 revision) software package. [START_REF] Frisch | Gaussian 09, Revis. D.01[END_REF] The S0 and Q1 equilibrium geometries of C2 have been obtained with the standard DFT/B3LYP method, whereas optimizations and MEPs of the T1 state have been performed using the unrestricted DFT/HCTH407 method (hereafter, DFT/HCTH). [START_REF] Boese | A New Parametrization of Exchange-Correlation Generalized Gradient Approximation Functionals[END_REF] On the other hand, the S1 and T2 states have been optimized at the TD-DFT/HCTH level. All energies discussed in the present work have been obtained with the TD-DFT/HCTH method. The GAUSSIAN 09 standard optimization thresholds have been used in all minimizations except for the S0 equilibrium structures of C0, fac-C1, mer-C1 and C2 shown in Fig. 4 and Table 1. In these cases, very tight optimization thresholds (opt=verytight) have been used to provide highly accurate geometrical parameters. The NTOs 38 have been obtained by post-processing the GAUSSIAN output using the Nancy_EX code. [START_REF] Etienne | Toward a Quantitative Assessment of Electronic Transitions' Charge-Transfer Character[END_REF][START_REF] Etienne | New Insight into the Topology of Excited States through Detachment/Attachment Density Matrices-Based Centroids of Charge[END_REF] The photochemical landscape displayed in Fig.

Femtosecond transient absorption spectroscopy

In the transient absorption setup, an amplified 5kHz Ti: sapphire laser generates 30 fs pulses to pump a commercial optical parametric amplifier (TOPAS: Light Conversion), from which 60 fs pulses at 400 nm are derived to excite the samples. The white light continuum probe beam is generated in a 2 mm thick CaF2 crystal and split in two: the probe that is sent through the sample, and a reference beam is used for measuring and compensating the white-light intensity fluctuations. The polarization of the probe beam is set at magic angle (54.7°) with respect to the pump.

A 1 mm path length cuvette in fused silica contains the complexes dissolved in CH3CN.

Time-resolved spectra are acquired in the range 350-650 nm as a function of pump-probe delay, by a Peltier-cooled CCD with 220Hz acquisition rate. A solvent-only sample is measured, and the data are processed to (i) remove the background at negative delay times, (ii) the solvent Raman signal and the coherent interactions of the pump and probe in the cell, (iii) to correct for the group velocity dispersion of the probe beam, characterized in the solvent-only data set. The temporal resolution is characterized by the 70±5 fs FWHM of the solvent Raman response. For more details see Ref [START_REF] Roland | Sub-100 Fs Charge Transfer in a Novel Donor-Acceptor-Donor Triad Organized in a Smectic Film[END_REF] . The multi-exponential fit procedure is described in the SI.

CONCLUSIONS

In the present contribution, we report the synthesis and the detailed experimental and computational characterization of an original iron-carbene bidendate compound. The complex is obtained with a 100% fac isomerism, in contrast to previous conventional synthetic procedures which only lead to mixtures dominated by the mer isomer. The excited-state lifetimes of the pure fac compound have been compared with those of the mer isomer. It is shown that, as it was the case for tridentate-liganded complexes reported before, the inclusion of carbene moieties increases the MLCT lifetime from the subpicosecond range to a maximum of about 20 ps.

By producing the constrained fac-only bidentate-liganded iron-carbene complex we have confirmed large differences in the photophysics of bidentate-liganded and tridentate-liganded complexes. Our transient absorption spectroscopy measurements and the computational results unveil crucial differences in the photophysics of the fac and mer isomers. We show that bidentate-liganded iron-carbene complexes decay to the ground state via a parallel two-state mechanism composed by a fast and a slow component. The former component is ascribed to the population of the T2 state whereas the latter is ascribed to the T1 state. Differences in the excited-state lifetime are interpreted based on a large spin-crossover region in T1 as compared to T2, which will act as a more efficient trap for the triplet state slowing down its decay.

While in the case of the fac isomer the two pathways appear as equally probable, a preference for the fastest decay is observed in the mer dominated mixture, hence globally leading to a faster decay for the latter.

Our results clearly confirm the peculiarity of the photophysics of iron bidentate-liganded compounds, especially the crucial role of fac/mer isomerism in driving the overall decay process, illustrating how the simple diabatic picture relying in the competition between well separated MC and MLCT states is largely insufficient to provide a full coherent explanation of the time resolved photophysics.

ASSOCIATED CONTENT

NMR, spectroscopic and electrochemical data for complexes as well as additional computational and photophysical details can be found in the Supporting Information.

Figure 1 .

 1 Figure 1. Complexes studied in this work. In all cases, counter ion is PF6 -.

Scheme 1 .

 1 Scheme 1. Overall synthesis of pro-carbene ligands L1 and L2, together with final iron(II) complexes C1 and C2. Counterion is PF6 for all charged compounds.

Figure 2 .

 2 Figure 2. Atom labelling and ground-state equilibrium geometries for a) C0, b) fac-C1, c) mer-C1 and d) C2. The optimization procedures have been conducted with very tight convergence thresholds

23

 23

Figure 3 .

 3 Figure 3. UV-Vis spectra of C0, C1 and C2 in in air-equilibrated CH3CN solution

Figure 4 .

 4 Figure 4. a) Photochemical landscape of C2. Arbitrary nuclear coordinate values are used to label the structures obtained along the decay path. The nuclear coordinates 0, 4.0, 31.5 and 67.0 correspond to the S0 min, S1 min ( 1 MLCT), T1 min ( 3 MC) and Q1 min, respectively. T1 and T2 energies at the T2 min ( 3 MC) structure (nuclear coordinates = 24.5) are also shown. b) Energies of the most relevant states mapped upon Fe-N1 bond stretching by means of relaxed scan calculations on the T1 surface starting from the T1 min ( 3 MC) geometry of C2. All energies are relative to the S0 minimum. STC = singlet-triplet crossing, TQC = triplet-quintet crossing.

Figure 5 .

 5 Figure 5. Transient absorption spectra at relevant time of (A) fac-only C2 and (B) C1 fac/mer 1/14 in CH3CN after excitation at 400 nm. Data at 390-415 nm are not shown (pump laser scattering). Dash-dotted lines are the respective inverted and scaled ground state absorption spectra. Note the different scales for positive and negative signals.

Figure 6 .

 6 Figure 6. Kinetic traces (dots) and relative fits (line) of C2 in different wavelength regions: ESA in the top panel, main GSB in the centre and short wavelength GSB in the bottom.

Figure 7 .

 7 Figure 7. Excited state relaxation schemes in the presence of a single dominant ps excited state (A), or for the co-existence of 2 states labelled A and B (schemes B and C), as found for the bidentate-liganded C1 and C2 here.

Figure 8 .

 8 Figure 8. Results of single wavelength 3-exp. fits of C2. Left: Decay times vs wavelength. Right: Decay time associated amplitudes vs wavelength. Values are from table S4. '*' denote lifetime values kept fixed for fit convergence.

Figure 9 .

 9 Figure 9. Kinetic traces (dots) and relative fits (line) of C1 in different wavelength regions: ESA in the top panel, main GSB in the centre and short wavelength GSB in the bottom.

Fig. 10 .

 10 Fig. 10. Schematic interpretation of the 2 and 3 excited-state components on the basis of the singlet and triplet PES topologies of the fac and mer arrangements.

  photochemical landscape displayed in Fig.4 has been built by combining MEP calculations (relaxing the T1 state) starting from the S1 ( 1 MLCT) min (nuclear coordinate = 4.0) and the Q1 min geometry (nuclear coordinate = 67.0). Both MEP computations ended up at the T1 ( 3 MC) min structure (nuclear coordinate = 31.5), identified in a separated calculation using the standard optimization algorithm. The Tamm-Dancoff approximation 41 has been used in all TD-DFT calculations and the 6-31+G(d,p) basis set has been used throughout. The solvent effects (acetonitrile) have been included in all optimization and single-point computations by means of the polarizable continuum model (PCM) method, employing the GAUSSIAN 09 default settings.

ACKNOWLEDGMENT

The project is funded by the French Agence Nationale de la Recherche (ANR-16-CE07-0013-02). The Nancy team is grateful to S. Parant for UV spectroscopy and F. Lachaud for mass spectrometry. A.F.-M. acknowledges the Région Grand Est government (France) for the financial support. The Strasbourg group acknowledges financial support through the Labex NIE.


Arthur Woods

H Arthur Woods

Sylvain Pincebourde

Michael E Dillon

John S Terblanche

Trends in Ecology and Evolution Extended phenotypes: buffers or amplifiers of climate change?

Keywords: Evolution, microclimate, niche construction, plasticity, temperature evolution, microclimate, niche construction, plasticity, temperature

Historic approaches to understanding biological responses to climate change have viewed climate as something external that happens to organisms. Organisms, however, at least partially influence their own climate experience by moving within local mosaics of microclimates. Such behaviors are increasingly being incorporated into models of species distributions and climate sensitivity. Less attention has focused on how organisms alter microclimates via extended phenotypes --phenotypes that extend beyond the organismal surface, including structures that are induced or built. We argue that predicting the consequences of climate change for organismal performance and fitness will depend on understanding the expression and consequences of extended phenotypes, the microclimatic niches they generate, and the power of plasticity and evolution to shape those niches.

2

Abstract

Historic approaches to understanding biological responses to climate change have viewed climate as something external that happens to organisms. Organisms, however, at least partially influence their own climate experience by moving within local mosaics of microclimates. Such behaviors are increasingly being incorporated into models of species distributions and climate sensitivity. Less attention has focused on how organisms alter microclimates via extended phenotypes --phenotypes that extend beyond the organismal surface, including structures that are induced or built. We argue that predicting the consequences of climate change for organismal performance and fitness will depend on understanding the expression and consequences of extended phenotypes, the microclimatic niches they generate, and the power of plasticity and evolution to shape those niches.

Extended phenotypes and organismal responses to climate change

Among biologists, a rough consensus has emerged about the biological effects of climate change [1]. The consensus in part reflects widespread agreement about how climates around the globe are changing [2]. It also reflects a shared view of causality in which organisms react physiologically and behaviorally to externally imposed shifts in climate. This philosophy is exemplified by the use of species distribution models (see Glossary) and environmental niche models to forecast climate-related vulnerability [3,4], and by mechanistic models that explicitly consider organismal performance in relation to variable abiotic conditions (primarily temperature) and organismal tolerance limits [5]. Such models have been extended recently by agent-based models [6] that explicitly include behavioral plasticity [7], and by integral projection models that integrate information on genetics, population structure, individual states, stochasticity, and climate to predict evolutionary trajectories [8].

Here we propose that the predictive utility of these models may be enhanced by considering an underappreciated component of climate-organism interactions --that organisms alter their experience of climate via their extended phenotypes. Extended phenotypes refer to traits that extend beyond an organism's surface into the environment surrounding it, as articulated from the point of view of genes by Dawkins [9] and of organisms by Turner [10].

Extended phenotypes are remarkably common [11] (Figure 1). They include externally induced or built structures such as burrows and nests of mammals and birds, the nests of termites and ants, organisms which account for a large fraction of animal biomass on earth, and the many structures induced or constructed by arthropods in different phases of their life cycle, including galls, feeding galleries, burrows, above-ground nests, pupation structures, and reproductive structures. Extended phenotypes also emerge when organisms exchange energy and materials with their local environments [12] or modify the structure or texture of local materials [10,13]. Importantly, many species use structures built by other species, with varying levels of maintenance or modification of the structure required, and in some instances, substantial energetic consequences (e.g. [14])the link between extended phenotype and ecosystem engineering is tight.

Depending on size, materials, and structure, extended phenotypes may buffer or amplify the effects of climate change, and any such effects may be age-or time-dependent. The consequences of extended phenotypes for the organisms that build them will depend critically on the kinds and magnitudes of plasticity that extended phenotypes exhibit, on heritabilities of underlying behavioral and physiological traits, and on how plasticity shapes selection on the entire organism -extended phenotype system. Because temperature in terrestrial systems is the focus of most published studies, our ideas below reflect this bias. Nevertheless, climate change is multifactorial (i.e., involving diverse concurrent stressors) [2], and the general ideas apply also to other physical factors and to marine and freshwater biomes.

The origin of microclimatic niches

Individuals live in localized sets of conditions, their microclimates, which have extents that scale to body size [15]. It is well known that organisms can modify their microclimatic experience by moving among local patches [7,16,17]. Moving even a few millimeters or centimeters, for example, may be enough to alter body temperatures by 5 -10 °C for small leafdwelling arthropods [18], for intertidal gastropods [19], or for spiders exploiting vertical temperature gradients under rocks into the soil [20]. The spatial configuration of such local mosaics, and the capacity of organisms to move within them, can be critically important for avoiding thermal stress [18,21,22] and thus for coping with more frequent high-temperature events during climate warming [23].

Both movement and extended phenotypes are forms of niche construction, which refers broadly to the effects that organisms have on their local habitats and microclimates [24][25][26]. A key idea in niche construction is that altered conditions constructed in part by the organisms themselves feedback to affect their own performance, ecology, and evolution. In this sense, extended phenotypes and niche construction are complementary: calling an external structure or condition 'an extended phenotype' emphasizes the role of the organism in building or shaping it, whereas calling it 'niche construction' emphasizes its feedback onto the performance and fitness of the organism [12].

The microclimatic niches produced by extended phenotypes determine how individuals experience climate variation and climate change (Figure 1, Box 1). In particular, they determine how much warming is transferred into the extended phenotype (the structure) and to what extent plasticity in structure, use of materials, or design can alter organismal performance via changes in the construction of the microclimate. Indeed, shifts in climate that switch a buffering structure into an amplifying structure, with effects that cannot be combatted via plasticity, may trap organisms and populations in destructive microclimatic extremes of their own making.

Conversely, effective buffering via plasticity can minimize the negative effects of climate change. We expect also that the structure and function of extended phenotypes will vary with climatic gradients (e.g., latitude, altitude). For example, we predict that extended phenotypes from cold climates will generally function to warm microclimates above local ambient air temperatures or to prevent heat loss from organisms, whereas extended phenotypes from warm climates will exhibit cooling functions. These expectations appear to be fulfilled by intra-and interspecific variation in the morphology of bird nests, which show strong correlations with climatic conditions [27]. Zebra finches, for example, build larger, warmer nests in cool conditions [28], while the dome (a roof of grass built over the nest cup) may shade eggs and chicks and keep them from overheating in very hot conditions [29].

Extended phenotypes as amplifiers or buffers

An extended phenotype can alter environmental fluctuations depending on the dynamics of the heat budget [15], with outcomes reflecting two biophysical rules. First, size matters. In the same way that larger ectotherms have longer time constants for heating and cooling [30], so do larger structures that animals build. Larger constructed niches dampen thermal fluctuations, filtering out high-frequency fluctuations and therefore short-term extremes in macroclimatic conditions. Thus, e.g., large bird nests or insect mounds often heat and cool slowly enough that their inhabitants escape daily extremes. Larger structures also allow for more complex internal plumbing that can facilitate air movements used to stabilize temperature or ventilate gases (e.g., in bee hives or termite mounds) (see [31]). Such advantages, however, can disappear on longer time scales: the larger thermal inertia of large structures may trap inhabitants in high heat conditions as average temperatures continue to climb. In addition, organisms with larger or more elaborate extended phenotypes give up freedom of movement and scope for plasticity via behavioral flexibility. Lastly, heat tolerance interacts with body size in complex ways. In general, small animals are more resistant to short-term heat extremes but less resistant to long-term exposure [32], and acclimation capacity typically increases with body size [33]. Consequently, size directly modifies an organism's experience of climate [17] and sets the bounds within which the organism can modify that experience using behavior and physiology.

Second, how much a structure amplifies or buffers its residents (Figure 2A) will depend on how it alters components of their heat budgets [34], including conduction, radiation, convection, and evaporation. Extended phenotypes often are buffering because they are coupled by conduction to much larger, more thermally stable objects (large rocks, tree trunks, or the Earth itself, i.e., underground). As with animal surfaces [35], the color and structure of extended phenotypes have complex effects on radiative exchange. Silk tents built by caterpillars, and also galls, rolled leaves, and leaf mines, may to differing degrees reflect or absorb both solar and long wavelength radiation, altering local microclimates [36,37]. Diverse bird species alter the positioning of nests to manipulate radiative load in relation to latitude, altitude, and season [38] (Table 1). Plovers (Charadrius melodus), for instance, ensure that eggs experience cooler temperatures by laying them near white pebbles, which reflect more solar radiation [39]. Bird nest placement and materials also alter convective and evaporative heat exchange (reviewed by [40]; Table 1). Construction materials that maintain high humidity minimize water loss by eggs and also provide evaporative cooling [38], a benefit in the heat but also a potential liability when temperatures fall. For all of these reasons, amplifiers are rather rare in the animal kingdom (Table 1).

Flexible behaviors and the plasticity of extended phenotypes

Broadly speaking, behavior is likely to be a major compensatory process for coping with climate change [41][42][43]. Because extended phenotypes often (but not always) reflect behaviors of their builders, we expect extended phenotypes to show extensive plasticity in response to local conditions and cues (Figure 2B). Some of the best examples of behavioral plasticity that underlies niche construction are from termite mounds and bird nests (Figure 1, Table 1). In Africa, subterranean termite mounds extend well above the ground surface, and mound architecture is altered via collective behaviors so that colony temperature, humidity, and gas concentrations remain within narrow ranges [10,44]. In addition, many birds adjust nest design and placement in response to predictable variation in environmental conditions [40,45]. In zebra finches (Taeniopygia guttata), however, nest plasticity reflects temperature only during the first reproductive event, with second nests built like the first nests regardless of ambient temperature [28]. Thus, although some extended phenotypes clearly are modified by behavioral plasticity, limited data preclude generalizations about whether behavioral plasticity will be sufficient to contend with ambient climate warming.

Likewise, the mechanistic roles of plasticity in physiological traits underlying extended phenotypes are seldom documented [46] (Table 1). For example, the silk structures of tent caterpillars interact strongly with incoming radiation [36]. It is unknown, however, whether the temperature history of caterpillars drives plasticity in silk biochemistry or whether local microclimatic conditions alter the properties of extruded silk [47]. Such effects are likely, however, and may have cascading feedbacks on tent temperature. Similarly, the structure of insect galls emerges from crosstalk between insect physiology and plant biochemistry [48]. Gall morphology varies along climatic gradients, raising the possibility that the crosstalk is modulated by temperature [49]. Lastly, plasticity in physiological traits underlying extended phenotypes is likely to be intimately linked to plasticity in behavior: we expect higher physiological plasticity when behavioral flexibility is low and environments vary predictably, such as in sessile organisms (analogous to behavioral versus physiological thermoregulation; [46]).

A key problem is to understand how the plasticity of extended phenotypes depends on the modularity of the underlying behaviors and physiologies used to construct them. For example, nest-building and other behaviors require a complex sequence of specific behaviors, which may be highly correlated and even traded-off against one another. But in general, how much are different behaviors in a sequence free to vary? In a rare investigation of this question, Royauté et al. [50] found overall no or only weak positive correlations among diverse nestbuilding behaviors in alfalfa leaf-cutting bees (Megachile rotundata). Similar studies across diverse taxa, from divergent climates, are urgently needed to derive generalities about the plasticity (or a lack thereof) of extended phenotypes, and any direct or indirect link(s) of these to climate change.

Evolutionary responses of organisms and their extended phenotypes

To examine evolutionary trajectories of organisms and their extended phenotypes, it is useful to divide phenotypes into those internal to the organism, including physiological tolerances, versus the extended phenotype along with the organismal traits responsible for inducing, building, or maintaining it. As climates change, which phenotypes will come under more sustained selection? Which will evolve more readily? The outcome (Figure 2) will depend on whether the extended phenotype is amplifying or buffering, on patterns of plasticity if any, and on relative levels of genetic variation between internal and extended phenotypes.

For the few extended phenotypes that are amplifiers, novel climatic conditions are likely to generate novel microclimates. These may often be stressful and will lead to selection either on the extended phenotype (selecting for modified construction that provides more suitable microclimates) and underlying patterns of plasticity, or on organismal tolerances (selecting for greater tolerance). The outcome will depend on how radically climate changes and on preexisting patterns of plasticity. For example, both leaf mines and silk tents likely evolved in part to buffer their builders from cold. As they absorb solar radiation, they warm substantially above air temperature, which can provide critical boosts to rates of feeding, growth, and development.

The offsetting risk on hot days, which will occur more frequently with climate warming, is overheating, which we expect to exert strong selection on the builder's upper thermal limits (Figure 2C). In such populations, climate change in effect will drive a change in sign of the primary problem: from too little heat to too much, thereby shifting the buffer into an amplifier.

Consequently, these lineages appear unlikely to exhibit adaptive plasticity in construction that could be modified rapidly by strong selection [51], such that selection will fall most strongly on organismal tolerances. The question then becomes whether upper thermal limits have heritabilities high enough to support rapid evolution. Although estimating such heritabilities accurately is difficult, meta-analyses comparing ectotherms, endotherms, and plants suggest substantial conservation of upper limits across taxa [52,53] and only low to moderate heritabilities [54].

For buffering extended phenotypes, by contrast, we predict fundamentally different evolutionary trajectories and more resilience in the face of climate change, at least in the short term. Because the plastic physiologies and behaviors of buffering are already in place, they may be more readily extended by strong selection ([55]; but see [56]) (Figure 2D). Furthermore, some complex behaviors resulting in extended phenotypes have known genetic bases [57] and some extended phenotypes show significant heritabilities [58], as do reaction norms for behavior more generally [59]. We thus predict that selection by extreme climatic events (like high temperatures) will shape primarily the extended phenotype and its patterns of plasticity rather than traits internal to the organism (tolerances). This argument invokes a kind of Bogert effect for extended phenotypes --in which behavioral flexibility or plasticity blunts selection on physiology [60,61]. Indeed, a recent meta-analysis compiling data on more than one thousand selection gradients found significantly weaker selection on traits subjected to constructed versus non-constructed niches [62]. Nevertheless, because climates are changing rapidly [2], buffering capacities may eventually be exceeded, revealing cryptic genetic variation for tolerances. In this sense, buffering extended phenotypes may act as evolutionary capacitors --that allow genetic variation in physiological tolerances to build up, which in turn can facilitate rapid evolution if and when the limits of buffering are reached (see [63]). These issues lie squarely within a broader discussion about the degree to which plasticity facilitates or inhibits evolutionary change [41,43,55,60,[64][65][66].

Concluding remarks

Since the publication almost 40 years ago of The Extended Phenotype [9], the idea of extended phenotypes has gained traction in many areas of biology [10,25]. The concept is stimulating because it blurs the line between genes and environment [67]. By way of extended phenotypes, genes and organisms project their phenotypes into their local environments. In doing so, however, they modify those environments --such that the environment becomes an experience that they participate in constructing. In this age of climate change, studies on the roles of constructed microclimates, and how plasticity and evolution can shape them, are critical to resolving whether extended phenotypes will buffer or amplify climate change. Progress will require focused studies on a broad array of interlocking aspects of the biology of extended phenotypes (see Outstanding Questions).

Acknowledgements: Thanks to Jackson Birrell, Zac Cheviron, James Frakes, Marty Martin, Tom Martin, David McDonald, Kate Wilsterman, and the evolutionary physiology reading group at the University of Montana for discussion and critical comments on an earlier version of the manuscript. We also thank two anonymous reviewers for constructive comments that substantially clarified the manuscript. Mines provide high humidities and are warmer than ambient air when the mine is illuminated with sunlight (buffer in cold conditions, amplify in warm) (see [68,69]). In winter, nests of the squirrel Sciurus vulgaris provide enough insulation that metabolic heat can raise nest temperatures 20 -30 °C above ambient air temperatures (buffer; see [70]). Goldenrod (Solidago) galls are induced by larvae of the fly Eurosta solidaginis. Direct sunlight can raise gall temperatures substantially over air temperature (amplifier; see [71]). Mounds of the African termite, Macrotermes michaelseni, harness solar and metabolic heat and ambient wind to drive internal gas flows, stabilizing internal gas levels and temperatures (buffer; see [72,73]). Larvae of dung beetles in the genus Onthophagus feed, grow, and develop within underground balls of dung constructed by their mothers, benefitting from dampened temperature fluctuations [74] and from structural and microbial/nutritional modifications to their dung balls (buffer; see [75]). In direct sunlight, communal silk tents built by caterpillars can reach temperatures far above ambient air temperature (buffer in cold climates, amplifier in warm). Temperature mosaics across sunny and shady sides are also used for behavioral thermoregulation (buffer; see [76]).

Figure legends

Forest canopies stabilize temperatures below the canopy and into the ground, which supports growth of fine roots and mycorrhizae (buffer; see Box 1). Great tits construct heavier, more insulated nests when the weather is colder in the week before clutch initiation (buffer; [77]). . Thus, the forest canopy and its moderating influences are a collective extended phenotype of the trees, and their climatic effects are a form of thermal niche construction (Figure 1). These effects are beneficial for many forest-dwelling species [89] and likely also feedback on the performance of mature trees and hence evolution.

For example, growth rates of fine roots, which are critically important components of carbon Microclimate. Climatic conditions at small spatial scales surrounding individual organisms or colonies. Relevant conditions include air temperature and humidity, wind speed, surface temperature, and radiative environment, including both longwave radiation from the sun, sky, and nearby objects and shortwave radiation from the sun. Microclimatic conditions can differ strongly from nearby macroclimates and often show much higher spatial diversity and more rapid temporal fluctuations (e.g., in response to clouds passing in front of the sun).

Modularity.

A system or network property that describes the degree to which different compartments can be decoupled into separate clusters. Trait modularity influences the degree to which interconnected sets of functional traits may evolve independently of one another.

Because extended phenotypes often reflect behaviors of their builders, understanding the relative modularity of suites of behaviors will be important for predicting patterns of plasticity and evolution of extended phenotypes.

Figure 1 .

 1 Figure 1. Examples of extended phenotypes and the microclimatic niches that they produce. Extended phenotypes determine how organisms experience climate (painting by Bayla Arietta). Larvae of many insects create leaf mines as they consume internal leaf tissues.

Figure 2 .

 2 Figure 2. Plasticity of extended phenotypes and consequences for selection. (A) Organisms experience microclimates, e, stemming from extended phenotypes, which are influenced by, but often quite different from, local macroclimates, E. Extended phenotypes can amplify or buffer variation in local climates, with many possible patterns. (B) Round symbols

Ecological inheritance .

 inheritance Extragenetic inheritance by offspring of conditions or microhabitats established by the niche-constructing activities of their parents (or earlier ancestors).Bogert effect. Behavioral flexibility allows organisms to avoid extremes and therefore dampen selection on morphological and physiological traits. Such an effect can slow rates of morphological and physiological evolution.Ecosystem engineering. Often derived from extended phenotypes, and similar to niche construction, but with more pervasive effects on whole communities in space and time.Extended phenotype. Phenotypes of organisms that extend beyond their integuments into the environment.Heat budget. An accounting of gains and losses of heat through different pathways (often measured in joules per second[Watts], sometimes per unit surface area), including conduction, convection, radiation, metabolism, and evaporation/condensation. When an ectotherm achieves a steady-state (stable) temperature, gains and losses of heat are balanced.Metabolic heat. Heat produced by the sum of all of chemical reactions occurring inside a living organism. Heat output occurs because chemical transformations are inefficient, and rate of heat output can be used as a measure of metabolic rate.

Table 1 .

 1 Examples across various taxa illustrating well-known extended phenotypes that shape local thermal environments, many of which also exhibit plasticity (legend: T, temperature).

	Extended	Traits	Thermal function	Level of plasticity	Reference
	phenotype
	Mammals
	Squirrel nest	Quantity and quality	[Cold buffer]	Unknown	[70]
	(Sciurus vulgaris)	of materials	Insulation during
			winter
	Birds
	Blue and great	Mass, insulative	[Cold Buffer]	Heavier, better	[77]
	tit nests	properties	Insulation from cold	insulated nests in
	(Cyanistes caeruleus, Parus			cold
	major)
	Open nest of	Size, quantity of	[Cold Buffer]	Larger nests in	[78]
	yellow warbler	material	Insulation from cold	arctic vs temperate
	(Setophaga petechial)			zones
	Roofed nest of	Number of strings	[Cold/heat Buffer]	Fewer strings at	[28]
	zebra finches	in nest	Insulation property:	elevated T to
	(Taeniopygia guttata)		within narrow range maintains nest T	decrease insulation
	Ground nest of	Optical properties	[Heat Buffer]	Unknown	[39]
	Piping plovers	of materials	Reflective materials
	(Charadrius melodus)		provide cooler nest for eggs
	Reptiles
	Burrow of	Depth of burrow	[Heat buffer]	Unknown	[79]
	tortoise (Gopherus		Insulation from heat
	polyphemus)
	Insects
	Mound of	Morphology:	[Cold/heat buffer]	Construction	[80]
	termites (several	volume, sphericity	Insulation from both	adjusted depending
	species)		cold and heat	on internal T
	Hive of honey	Self-shielding of the	[Heat buffer]	Limit overheating of	[81]
	bees (Apis	workers	Insulation from heat	the honey comb by
	mellifera)			increasing the
				number of workers
				at its surface
	Tent of	Thickness of silk	[Cold buffer] Heat	Thicker tents	[82]
	Thaumetopoea	tent	absorption to keep	generate higher
	caterpillars (T.		winter nest warm	internal T
	pityocampa)
	Tent of	Thickness of silk	[Cold/heat buffer]	Thicker tents	[36]
	Eriogaster		Insulation from solar	provide better
	caterpillars (E.		energy to better	thermoregulatory
	lanestris)		regulate nest T	control
	Leaf mine of the	Optical property of	[Heat amplifier]	Unknown	[69]
	apple tentiform	surface and lower	Provides warm
	leaf miner	transpiration	environment when
	(Phyllonorycter blancardella)		exposed to radiation

The collective extended phenotype of forest trees creates a buffered thermal niche

  We view extended phenotypes as a continuum of structures from simple to complex, and involving little to great amounts of organismal time and resources. Extended phenotypes may also result in a continuum of microclimate effects that may not scale with organismal effort, or (vice versa) small changes in traits may lead to large microclimatic effects. Although some examples of extended phenotypes are fairly straightforward and well accepted, some extensions of this logic are contentious[85]. Here, we consider the special case of collective extended phenotypes produced by multiple, possibly unrelated individuals. Such collective

	extended phenotypes do not follow the more direct mappings of genotype to phenotype
	discussed in the main text. Nevertheless, they can have profound, community-wide effects on
	climatic conditions, and those effects emerge from the collective actions of the individuals
	comprising those communities, with consequences for all community members (ecosystem
	engineering). The evolutionary trajectories of such traits may reflect 'diffuse evolutionary
	processes' such as those commonly invoked in coevolutionary theory.
	In temperate regions, old-growth forest canopies buffer temperature extremes such that
	understory air temperatures are lower than atmospheric temperatures during hot periods and
	higher during cool periods, including in winter [86,87]. Consequently, soil temperature in mature
	forests is buffered compared to soils in open areas [88]

  cycles[90], depend on soil temperature[91] among other factors. Higher soil temperatures promote fine root growth up to some optimum temperature[92,93], which varies among taxa[91], especially in the superficial soil layers. For mature forest trees, however, the optimal soil temperature usually is lower than atmospheric temperature[94], and even modest warming may slow growth[95]. In addition, in temperate zones, fine root biomass of forest trees peaks in summer[94], when buffering by canopy cover is strongest[86]. We propose that perturbations to canopy cover in summer may, via shifts in soil temperature, have cascading effects on fine root biomass, thereby contributing to the overall response of forest to global change. Along with other abiotic variables linked to climate change, soil temperature also influences interactions between trees and mycorrhizae [96], which provide many benefits to trees [97]. The net effects of warming on plant-mycorrhizae associations is difficult to predict. In general, climate change appears to have a positive impact on colonization by mycorrhizae [98] but also to alter the dominant types of mycorrhizae (arbuscular vs ectomyccorhizal) associated with trees [99] and soil fungus communities more generally [100]. It remains unclear whether global change is directly altering mycorrhizal communities or whether global tree declines are driving the shifts [101]. The mechanisms by which extended phenotypes of mature trees influence forest floor thermal processes remains poorly documentedyet the thermal buffer should combine with other facilitating processes linked to soil structure for instance. What is needed now are integrative studies that connect the buffering ability of forest canopies, soil temperature patterns, root performance across soil temperature patterns, and mycorrhizae-tree interactions in their natural thermal contexts in the field. A recent global analysis of forest buffering ability [87] and current calls for large scale monitoring of soil temperatures [88], are key points of entry for integrating the extended phenotypes of trees into the biology of climate change.

	Glossary Box
	Behavioral thermoregulation. Achieving desired body temperature by moving throughout
	locally available mosaics of operative temperature.

Niche construction. The process of organisms or colonies or communities altering their local microclimates by means of their activities or efforts to construct objects/artifacts. Nest/Tent/Shelter/Burrow Animal-built structures or spaces used by animals to seek refuge for some part of their life cycle, or to overwinter, that may be considered as forms of extended phenotypes.

Mechanistic model. A biological process-based model in which organismal traits are used to infer population dynamics, geographic distribution or environmental constraints. While these models have become increasingly sophisticated and computationally intensive, covering diverse aspects of organism-environment relationships, to our knowledge none/few studies have explicitly examined the impact of variation in the extended phenotype on organismal traits and fitness in a climate change context.

Species distribution model.

Correlative approach to estimating expected distributions of organisms (usually assumed to approximate the fundamental niche) whereby point locations of organisms are associated with suites of environmental variables at those locations to infer suitable conditions for species persistence. C. Extended phenotype evolved to bu er builder from cold conditions. D. Extended phenotype evolved to bu er builder from hot conditions. How rapidly will extended phenotypes evolve, and will selection shape the capacities and tolerances of the organisms themselves or the behaviors and physiologies used to construct extended phenotypes? Answers will require estimating heritabilities of extended phenotypes and the mechanisms underlying them, assessing the strength of selection on these components, and analyzing evolutionary trajectories in related taxa using comparative methods. Does plasticity in extended phenotypes facilitate or inhibit evolutionary change? In the shortterm, adaptive plasticity may blunt selection on underlying physiological tolerances. Plasticity, however, also provides coordinated, pre-existing axes of variation that selection may amplify rapidly.

How important are the effects of extended phenotypes on temperature versus on other aspects of microclimates? Extended phenotypes often play multiple roles, including protection from predators, enhanced respiratory gas control, water savings, as well as altering temperatures Outstanding Questions experienced. A key question is how important temperature effects are compared to others, or how they interact with others, and whether responses to selection imposed by warming climates is constrained by other functions.

Highlights

All organisms modify their local conditions by exchanging materials and energy with their environments. Many organisms also induce or build external structures around themselves, which are considered extended phenotypes.

External structures play central roles in determining the microclimates that organisms experience. Depending on their materials, architecture, and location, they may buffer or amplify local climate variability and macroclimatic change. To date, however, no general frameworks are available for predicting the microclimatic effects of these structures.

The importance of extended phenotypes during climate change will depend on whether they exhibit adaptive plasticity and how rapidly they evolve in response to climate variability.

Highlights