J Hooper

Lucia Cardo

James S Craig

Lazaros Melidis

Viktoriia Sadovnikova

Ross Egan

Aditya Garai

Florian Burkert

Louise Male

Nikolas J Hodges

Michael J Hannon
email: m.j.hannon@bham.ac.uk

David Bardelang
email: david.bardelang@univ-amu.fr

Simin Liu
email: liusimin@wust.edu.cn

Catherine A J Hooper

Douglas F Browning

Roselyne Rosas

Fillipe V Rocha

Mauro A Lima

Rotaxanating Metallo-supramolecular Nano-cylinder Helicates to Switch DNA Junction Binding

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

Rotaxanating metallo-supramolecular nano-cylinder helicates to switch DNA junction binding.

Catherine A.J. Hooper, Lucia Cardo, James S. Craig, Lazaros Melidis, Viktoriia Sadovnikova, Ross Egan, Aditya Garai, Florian Burkert, Louise Male, Nikolas J. Hodges, Douglas F. Browning, Roselyne Rosas, Fengbo Liu, Fillipe V. Rocha, Mauro A. Lima, Simin Liu, * David Bardelang, * and Michael J. Hannon. * ABSTRACT: A new class of rotaxane is created, not by encapsulating a conventional linear thread, but rather by wrapping a large cucurbit [10]uril macrocycle about a three-dimensional, cylindrical, nanosized, self-assembled supramolecular helicate as the axle. The resulting pseudo-rotaxane is readily converted into a proper interlocked rotaxane by adding branch points to the helicate strands that form the surface of the cylinder (like branches and roots on a tree trunk). The supramolecular cylinder that forms the axle is itself a member of a unique and remarkable class of helicate metallo-drugs that bind Y-shaped DNA junction structures and induce cell death. While pseudo-rotaxanation does not modify the DNA-binding properties, proper, mechanically-interlocked rotaxanation transforms the DNA-binding and biological activity of the cylinder.

■ INTRODUCTION

Drawing on nature for inspiration, supramolecular chemistry has created a variety of complex, often exotic, nanoscale structures. Mechanically linked molecules have been of particular interest, and rotaxanes have been formed by threading molecular strands through a variety of different classes of macrocycle. 1- 3 Applications of rotaxanes have ranged from protecting peptides from rapid enzymatic degradation, to creating materials with switchable surface properties. [START_REF] Stoddart | Mechanically Interlocked Molecules (MIMs)-Molecular Shuttles, Switches, and Machines (Nobel Lecture)[END_REF][2][START_REF] Zhang | Molecular machines with bio-inspired mechanisms[END_REF][START_REF] Woltering | Photochemical Unmasking of Polyyne Rotaxanes[END_REF][START_REF] Danon | Triply Threaded [4]Rotaxanes[END_REF][START_REF] Lewis | Metal ions in the synthesis of interlocked molecules and materials[END_REF][7][START_REF] Pairault | Rotaxane-based architectures for biological applications[END_REF] Yet the threads used, to date, have been principally linear, 1-dimensional or 2-dimensional, organic, covalent molecules.

An alternative, but equally exciting, aspect of supramolecular chemistry, has been to take the designs and lessons of supramolecular chemistry and to apply them back to the biology that provided the initial inspiration for the field. Indeed selfassembled metallo-supramolecular helicates [START_REF] Ayme | From Coordination Chemistry to Adaptive Chemistry[END_REF][START_REF] Albrecht | From Hierarchical Helicates to Functional Supramolecular Devices[END_REF] are enjoying a resurgence of interest for their application in biology. [START_REF] Cardo | Non-covalent Metallo-Drugs: Using shape to target DNA and RNA junctions and other nucleic acid structures[END_REF][12][START_REF] Allison | Ruthenium-Containing Linear Helicates and Mesocates with Tuneable p53-Selective Cytotoxicity in Colorectal Cancer Cells, Angew[END_REF][START_REF] Li | Mitochondrial-DNA-Targeted IrIII-Containing Metallohelices with Tunable Photodynamic Therapy Efficacy in Cancer Cells[END_REF][START_REF] Kumar | structure, stability and antimicrobial activity of a ruthenium(II) helicate derived from a bis-bidentate "click" pyridyl-1,2,3-triazole ligand[END_REF][START_REF] Domarco | Self-assembled Pt2L2 boxes strongly bind G-quadruplex DNA and influence gene expression in cancer cells[END_REF][START_REF] Eskandari | A Triangular Platinum(II) Multinuclear Complex with Cytotoxicity Towards Breast Cancer Stem Cells[END_REF][START_REF] Hrabina | Optically Pure Metallohelices That Accumulate in Cell Nuclei, Condense/Aggregate DNA, and Inhibit Activities of DNA Processing Enzymes[END_REF][START_REF] Adamski | Full characterization and cytotoxic activity of new silver(I) and copper(I) helicates with quaterpyridine New[END_REF][START_REF] Qin | Metallo-supramolecular Complexes Enantioselectively Eradicate Cancer Stem Cells in Vivo[END_REF] They offer quite large nanoscale surfaces of defined topography, of comparable scale to key structures found in biomolecules. We have used these external surfaces to access unique DNA-and RNA-recognition properties, [START_REF] Hannon | Intramolecular DNA coiling mediated by a metallo supramolecular cylinder[END_REF][START_REF] Meistermann | Intramolecular DNA coiling mediated by metallo-supramolecular cylinders: differential binding of P and M helical enantiomers[END_REF][START_REF] Ducani | Noncovalent DNA-Binding Metallo-Supramolecular Cylinders Prevent DNA Transactions in vitro[END_REF] demonstrating with X-ray crystal structures the remarkable binding of cylindrical metallohelicates in the heart of DNA and RNA 3-way junction structures, [START_REF] Oleksi | Molecular recognition of a threeway DNA junction by a metallo-supramolecular helicate[END_REF][START_REF] Phongtongpasuk | Binding of a Designed Anti-Cancer Drug to the Central Cavity of an RNA Three-Way Junction[END_REF][START_REF] Boer | Towards functionalizable DNA frames: Self assembly of two-component 3D DNA arrays through induction of DNA three-way junction branch points by supramolecular cylinders[END_REF][START_REF] Cerasino | DNA Three-Way Junction with a Dinuclear Iron(II) Supramolecular Helicate at the Center: A NMR structural Study[END_REF][START_REF] Malina | Recognition of DNA Three-Way Junctions by Metallosupramolecular Cylinders: Gel Electrophoresis Studies[END_REF] where the nucleic acid bases form perfectly stacked aromatic interactions [START_REF] Martinez | Rethinking the term "pi-stacking[END_REF] with the external aromatic surfaces of the metallo-helicates. Nucleic acid junction structures are exciting targets found both in biology and nucleic acid nanoscience. [START_REF] Duskova | Identification of three-way DNA junction ligands through screening of chemical libraries and validation by complementary in vitro assays[END_REF][START_REF] Duskova | DNA junction ligands trigger DNA damage and are synthetic lethal with DNA repair inhibitors in cancer cells[END_REF][START_REF] Van Rixel | Induction of a Four-Way Junction Structure in the DNA Palindromic Hexanucleotide 5′-d(CGTACG)-3′ by a Mononuclear Platinum Complex[END_REF][START_REF] Chien | Structural Basis for Targeting T:T Mismatch with Triaminotriazine-Acridine Conjugate Induces a U-Shaped Head-to-Head Four-Way Junction in CTG Repeat DNA[END_REF][START_REF] Brogden | Ligand bridging of the DNA Holliday junction: molecular recognition of a stacked-X four-way junction by a small molecule[END_REF][START_REF] Zhu | Fe II 4L4 Tetrahedron Binds to Nonpaired DNA Bases[END_REF] Our junction-binding cylinders arrest the proliferation of cancer cells and the HIV virus. [START_REF] Hotze | A supramolecular triple-stranded iron cylinder with unprecedented DNA binding action is a potent cytostatic and apoptotic agent without exhibiting genotoxicity[END_REF][START_REF] Cardo | Metallo supramolecular cylinders inhibit HIV-1 TAR-TAT complex formation and viral replication in cellulo[END_REF][START_REF] Pope | Issues surrounding standard cytotoxicity testing for assessing activity of non-covalent DNA-binding metallo-drugs[END_REF][START_REF] Cardo | Arginine conjugates of metallo-supramolecular cylinders prescribe helicity and enhance DNA junction binding and cellular activity[END_REF] Given that the supramolecular nano-cylinder helicates thread so beautifully into and through junctions and bulges in nucleic acids we were intrigued to see whether we could push the boundaries of supramolecular assembly to similarly thread such self-assembled nanoscale metallo-supramolecular helicates through synthetic macrocycles to create new types of rotaxanes and pseudo-rotaxanes in which the thread was not a simple chain but a three dimensional, self-assembled, metallo-cylinder. Since the cylinders recognise DNA-junction structures through their external surfaces, we anticipated that wrapping another molecule around this region of the cylinder might also interfere with (and ultimately control) the DNA-binding. Rotaxanation in DNA-binding agents has not been explored previously.

We report now, for the first time, the wrapping of a synthetic macrocycle around the external surface of a self-assembled metallo-supramolecular nanostructure to create a pseudo-rotaxane, subsequent conversion to a proper (mechanically -interlocked) [2]-rotaxane, and the effect of rotaxanation on the biomolecular recognition and biological properties of the metallosupramolecular cylinders. Examples of larger threads in rotaxanes include pseudo-rotaxanes created by clipping macrocycles around carbon nanotubes, [START_REF] Pérez | Putting Rings around Carbon Nanotubes[END_REF][START_REF] Barrejón | Carbon Nanostructures in Rotaxane Architectures[END_REF] a rotaxane with a spherical fullerene in the centre, [START_REF] Xu | Concave-Convex π-π Template Approach Enables the Synthesis of[END_REF] and DNA rotaxanes [START_REF] Ackermann | A double-stranded DNA rotaxane[END_REF] but this work is distinct in representing (to the best of our knowledge) the first example of a proper interlocked rotaxane assembled around a three-dimensional metal-assembled supramolecular helicate as axle. An interesting report of rotaxanation of a helical peptide has also appeared, but in that case the small rotaxane ring wraps just around its linear chain (primary structure) rather than encapsulating a helical secondary structure. [START_REF] Moretto | A Rigid Helical Peptide Axle for a [2]Rotaxane Molecular Machine[END_REF] The large three-dimensional cylindrical surface that our metallo-supramolecular helicate axle affords also brings another opportunity: rather than attaching a branched organic unit at each end of the thread as stopper we now clip unbranched units onto each thread of the helicate axle exploiting the metal-coordination and supramolecular structure to achieve the branching effect which mechanically constrains the ring. We show that the rotaxanation allows us to switch the DNA-binding properties and thus the biological activity of the cylinder.

■ RESULTS AND DISCUSSION

The first challenge was to identify a suitable 'ring' that might be wrapped about these metallo-supramolecular helicates. They are tetracations formed by wrapping three organic ligand strands about two metal centres (Fig. 1). [START_REF] Cardo | Non-covalent Metallo-Drugs: Using shape to target DNA and RNA junctions and other nucleic acid structures[END_REF] They are cylindrical in shape, and around 2 nm in length and 1 nm in diametermuch greater than the dimensions normally associated with rotaxanation or encapsulation -but we identified that cucurbit [10]uril (Fig. 1) might offer a cavity of suitable dimensions. [START_REF] Yang | Cucurbit[10]uril-based chemistry[END_REF][START_REF] Liu | Cucurbit[10]uril[END_REF] Figure 1: The ligands used in this study together with the structure of the [Ni2L3] 4+ cylinder (CSD-NITBIB) and cucurbit [10]uril (from CSD-LAZPIM and illustrating the flexibility and potential ellipsoidal distortions CB [10] can accommodate in response to guests).

Cucurbit[n]urils (CB[n]s) are a class of macrocycles that are attracting much attention for both host-guest chemistry and rotaxanation studies. [START_REF] Yang | Cucurbit[10]uril-based chemistry[END_REF][START_REF] Liu | Cucurbit[10]uril[END_REF][START_REF] Shetty | Can we beat the biotin-avidin pair?: cucurbit[7]uril-based ultrahigh affinity host-guest complexes and their applications[END_REF][START_REF] Isaacs | Stimuli Responsive Systems Constructed Using Cucurbit[n]uril-Type Molecular Containers[END_REF][START_REF] Barrow | Cucurbituril-Based Molecular Recognition[END_REF][START_REF] Assaf | Cucurbiturils: from synthesis to high-affinity binding and catalysis[END_REF][START_REF] Murray | The aqueous supramolecular chemistry of cucurbit[n]urils, pillar[n]arenes and deep-cavity cavitands[END_REF] They are pumpkin-shaped molecules with a rigid hydrophobic cavity flanked by two identical openings (portals) whose rims are each lined with n carbonyl groups. CB [5,6,7] are ideal sizes for traditional small organics and CB [6,7,8] have been used with organic, linear threads and chains to create rotaxanes. [START_REF] Isaacs | Stimuli Responsive Systems Constructed Using Cucurbit[n]uril-Type Molecular Containers[END_REF][START_REF] Cheng | A pH-driven ring translocation switch against cancer cells[END_REF] Other applications have included using the host guest chemistry to attach proteins to surfaces or fluorophores, and for drug delivery. [START_REF] Shetty | Can we beat the biotin-avidin pair?: cucurbit[7]uril-based ultrahigh affinity host-guest complexes and their applications[END_REF][START_REF] Villarroel-Lecourt | Encapsulation of Chemotherapeutic Drug Melphalan in Cucurbit[7]uril: Effects on Its Alkylating Activity, Hydrolysis, and Cytotoxicity[END_REF] The largest of the class, with an untwisted cavity, is CB [10] (Fig. 1) which can accommodate multiple or larger molecules [START_REF] Yang | Cucurbit[10]uril-based chemistry[END_REF][START_REF] Liu | Cucurbit[10]uril[END_REF] as demonstrated by its use to capture both a Stoddart 'blue-box' (bis-bipyridinium) macrocycle and its methoxybenzene guest to create a Russiandoll style assembly. [START_REF] Gong | From Packed "Sandwich" to "Russian Doll": Assembly by Charge-Transfer Interactions in Cucurbit[10]uril[END_REF] Binding interactions include hydrophobic effects in the heart of the cavity and ion-dipole interactions at the rims. [START_REF] Yang | Cucurbit[10]uril-based chemistry[END_REF][START_REF] Liu | Cucurbit[10]uril[END_REF][START_REF] Shetty | Can we beat the biotin-avidin pair?: cucurbit[7]uril-based ultrahigh affinity host-guest complexes and their applications[END_REF][START_REF] Isaacs | Stimuli Responsive Systems Constructed Using Cucurbit[n]uril-Type Molecular Containers[END_REF][START_REF] Barrow | Cucurbituril-Based Molecular Recognition[END_REF][START_REF] Assaf | Cucurbiturils: from synthesis to high-affinity binding and catalysis[END_REF][START_REF] Murray | The aqueous supramolecular chemistry of cucurbit[n]urils, pillar[n]arenes and deep-cavity cavitands[END_REF][START_REF] Cheng | A pH-driven ring translocation switch against cancer cells[END_REF][START_REF] Villarroel-Lecourt | Encapsulation of Chemotherapeutic Drug Melphalan in Cucurbit[7]uril: Effects on Its Alkylating Activity, Hydrolysis, and Cytotoxicity[END_REF][START_REF] Gong | From Packed "Sandwich" to "Russian Doll": Assembly by Charge-Transfer Interactions in Cucurbit[10]uril[END_REF] While lower order cucurbiturils have relatively rigid cavities, CB [10] is more flexible. It is observed to be ellipsoidal in its crystal structure (cavity diameter 1.13-1.24 nm; portal diameter 0.95-1.06 nm) in complex with 4,6-bis(4-(ammoniomethyl)phenylamino)-1,3,5-triazen-2(1H)-one, [START_REF] Yang | Cucurbit[10]uril-based chemistry[END_REF][START_REF] Liu | Cucurbit[10]uril[END_REF] and it is observed to also complex platinum terpyridyls [START_REF] Kuang | Enhancement of metal-metal interactions inside a largecavity synthetic host in water[END_REF] and porphyrins 56 which implies further distortion is possible. The previous use of CB [10] for rotaxanation is restricted to a report of a kinetic rotaxane -a pseudo-rotaxane in which the thread is encapsulated at room temperature but can enter and leave at elevated temperatures. [START_REF] Yu | uril-Based[END_REF] To initiate our studies we first explored whether the supramolecular cylinders would bind to CB [10] to give a tube threaded through a ring. CB [10] itself is an insoluble compound, but rapidly dissolves on addition of an aqueous solution of [M2L3] 4+ cylinders (M= Fe, Ru, Ni). Electrospray mass spectrometry shows peaks corresponding to [M2L3•CB10] 4+ (Fig. S1) and proton NMR spectroscopy of [Fe2L3] 4+ with CB [10] confirms formation of a 1:1 complex (Fig. 2). DOSY NMR reveals that a single species is formed, of greater hydrodynamic radius than the free cylinder, and involving both cylinder and CB [10]. The cylinder retains its two-fold and three-fold symmetry in the complex. The proton NMR resonances corresponding to the central CH2 of the cylinder and the adjacent phenylene protons experience upfield shifts, consistent with a shielded location in the CB [10] cavity, while the imine proton and the pyridine H3 are shifted downfield, consistent with encountering the carbonyls at the rim of the macrocycle. NOEs are observed from these two cylinder protons (Him and H3) to the CH2 proton on the rim of the CB [10] macrocycle (Fig. S2). The NMR is unambiguous confirmation that the cylinder sits symmetrically (on the NMR timescale) in the centre of the macrocycle threading through from one side to the other as a pseudo-rotaxane, with the macrocycle sitting over the central diphenylmethanes of the cylinder as desired. To retain its threefold symmetry within the ten-fold symmetric CB [10], the cylinder must be rapidly spinning or rocking about the metal-metal axis while within the macrocycle. DFT calculations and MD simulations reproduce this solution structure, with the DFT structure showing axial deviation between the host and guest main symmetry axes at the energy minimum (Fig. 2d) and the MD showing the rotation ( Fig 6). Use of a small excess of cylinder (Fig. S3) in the NMR allows free and bound cylinder peaks to be observed indicating that exchange is slow on the NMR timescale, however on mixing [Fe2L3] 4+ with [Ni2L3•CB10] 4+ and immediately injecting into an electrospray mass spectrometer a mixture of [Fe2L3•CB10] 4+ and [Ni2L3•CB10] 4+ was observed, the ratio of which remained unchanged over a further hour of sampling (Fig. S4). Together these observations indicate that the exchange takes place over a timescale of milliseconds to seconds, which is consistent with measurements on other cucurbiturils with different guests. [START_REF] Masson | Kinetics Inside, Outside and Through Cucurbiturils[END_REF] The proton NMR spectrum of [Ru2L3] 4+ with CB [10] shows analogous upfield and downfield shifts in the resonances and the paramagnetically shifted proton NMR of [Ni2L3] 4+ with CB [10] also shows the binding (Figs. S5 andS6).

While the ruthenium(II) and nickel(II) [M2L3] 4+ cylinders have excellent stability, the iron(II) cylinders can experience some hydrolytic degradation over long periods, [START_REF] Cardo | Non-covalent Metallo-Drugs: Using shape to target DNA and RNA junctions and other nucleic acid structures[END_REF][START_REF] Cardo | Metallo supramolecular cylinders inhibit HIV-1 TAR-TAT complex formation and viral replication in cellulo[END_REF] though they are stabilised by binding to DNA. [START_REF] Hannon | Intramolecular DNA coiling mediated by a metallo supramolecular cylinder[END_REF][START_REF] Meistermann | Intramolecular DNA coiling mediated by metallo-supramolecular cylinders: differential binding of P and M helical enantiomers[END_REF] Encapsulation of the cylinder within the CB [10] macrocycle similarly enhances the cylinder stability; an experiment with 0.9 equivalents of CB [10] revealed no degradation of the bound cylinder in aqueous solution over a period of 194 days (Fig. S7). The insolubility of unbound CB [10] prevents accurate measurements of binding affinity with guests, however an NMR dilution experiment showed no unbound cylinder (Fig. S8) implying the binding constant is greater than 10 7 M -1 , and NMR competition studies show that the cylinder binds more strongly than both methyl viologen and memantine hydrochloride.

To explore the effect of the encapsulation on the cylinder's DNA-recognition properties we investigated the binding of the nickel cylinder•CB [10] complex (as representative of the three complexes) to DNA 3-way junctions (3WJ) 24-28, 30-31 using gel electrophoresis. The assay (Fig. 3a) uses a 3WJ formed from three different complementary DNA strands. The 3WJ structure is (entropically) unstable at room temperature in absence of the cylinder and the DNA exists as three single strands. When cylinder binds, the 3WJ is stabilised and observed in the gel. Radiolabelling one strand allows a simple on/off gel-shift binding assay that is readily visualised. Figure 3a shows that the cylinder•CB [10] complex stabilises the 3WJ and the effect of the cylinder•CB [10] complex is the same as the cylinder alone, though higher loadings are required. This indicates that the cylinder can leave the CB [10] cavity and bind to the anionic DNA 3WJ cavity; while binding of an intact cylinder•CB [10] complex to the 3WJ cannot be excluded this would be expected to give a different gel shift. The cylinder binding to the 3WJ is in competition with the binding to the CB [10]; Adding small amounts of solid CB [10] or of the soluble methylviologen CB [10] complex to the pseudorotaxane leads to small reductions in 3WJ formation (Fig. S9). In these experiments, the binding energy to the 3WJ also incorporates the energetic cost of assembling the 3WJ from the DNA single strands. Given that the 3WJ is not observed in absence of cylinder at room temperature, the binding constant of cylinder to the 3WJ structure itself must be (significantly) greater than that to CB [10].

We also studied the binding to calf-thymus DNA (a polymeric genomic DNA), using circular and flow linear dichroism spectroscopies (Fig. 3b,c). The effects are again the same as the free cylinder binding: The circular dichroism confirms binding of cylinder (an induced CD signal is observed in the cylinder spectroscopy between 300-400 nm) and retention of a B-DNA structure (typical signature between 200-300 nm). The CD spectra are the same as those observed with cylinder alone (Fig S10 ), although at high loading some scattering is observed consistent with release of CB [10] which (uncomplexed) will be insoluble. The flow linear dichroism experiments probe the orientation of ct-DNA in a Couette flow cell, with the magnitude of the peak at 260 nm reflecting the orientation. [START_REF] Hannon | Intramolecular DNA coiling mediated by a metallo supramolecular cylinder[END_REF][START_REF] Meistermann | Intramolecular DNA coiling mediated by metallo-supramolecular cylinders: differential binding of P and M helical enantiomers[END_REF] Cylinders are known to coil ct-DNA (leading to a loss of orientation); [START_REF] Hannon | Intramolecular DNA coiling mediated by a metallo supramolecular cylinder[END_REF][START_REF] Meistermann | Intramolecular DNA coiling mediated by metallo-supramolecular cylinders: differential binding of P and M helical enantiomers[END_REF] the spectra on addition of the cylinder•CB [10] complex confirm the same DNA-coiling effects and are analogous to those observed with free cylinder (Fig S10). We conclude that while the CB [10] does bind the cylinder in solution to form the pseudorotaxane, the cylinder would prefer to bind to anionic DNA. For the 3WJ, which is not pre-formed in solution, the extra energy of forming the structure means that there is competition between binding CB [10] or 3WJ. When presented with a polymeric DNA anion the cylinder leaves the CB [10] cavity and binds its preferred partner. Preferential binding to the anionic DNA over the neutral CB [10] cavity is unsurprising, given the anticipated electrostatic contribution to that binding energy, though it contrasts with spermine that preferentially binds CB [7] over DNA. 59 Consistent with the in vitro DNA binding, when we treat cells (A2780 ovarian and MDA-MB-231 breast cancer lines) with the cylinder•CB [10] complex, we observe very similar inhibitions of cell proliferation (as assessed by an MTT assay) to those with the corresponding free cylinder (Figs. 3d andS11). There is a very slight enhancement in activity for the ruthenium cylinder but a slight drop in activity for the iron and nickel cylinders. Uptake studies (assessed by ICP-MS) indicate that the encapsulation inside CB [10] has little effect on the extent of overall uptake (Fig. 3e). Cucurbiturils are frequently used as drug delivery agents and platinum metallo-drugs (e.g. oxaliplatin) encapsulated inside cucurbit [7]uril show some enhanced effects [START_REF] Wu | Host-Guest Interactions between Oxaliplatin and Cucurbit[END_REF][START_REF] Chen | Supramolecular Chemotherapy: Cooperative Enhancement of Antitumor Activity by Combining Controlled Release of Oxaliplatin and Consuming of Spermine by Cucurbit[7]uril[END_REF][62][START_REF] Jeon | Novel Molecular Drug Carrier: Encapsulation of Oxaliplatin in Cucurbit[7]uril and Its Effects on Stability and Reactivity of the Drug[END_REF][START_REF] Wheate | Improving Platinum(II)-Based Anticancer Drug Delivery Using Cucurbit[n]urils[END_REF][START_REF] Bali | Cucurbituril binding of trans-[{PtCl(NH3)2}2(µ-NH2(CH2)8NH2)] 2+ and the effect on the reaction with cysteine[END_REF] due to better uptake and/or pharmacology (less degradation and side reactions with thiols). The lack of dramatic enhancement for the cylinder perhaps reflects the already excellent cell uptake of cylinders [START_REF] Hotze | A supramolecular triple-stranded iron cylinder with unprecedented DNA binding action is a potent cytostatic and apoptotic agent without exhibiting genotoxicity[END_REF][START_REF] Cardo | Metallo supramolecular cylinders inhibit HIV-1 TAR-TAT complex formation and viral replication in cellulo[END_REF] and the lack of competing side reactions. The DNA-binding studies indicate that whether the cylinders have entered alone or in complex with CB [10], their effects when they reach the target DNA are expected to be the same. Consistent with this, a dinuclear ruthenium complex (from Keene, Collins et al.) [START_REF] Li | Probing the Pharmacokinetics of Cucurbit[7, 8 and 10]uril: And a Dinuclear Ruthenium Antimicrobial Complex Encapsulated in Cucurbit[10]uril[END_REF][START_REF] Sun | Eukaryotic Cell Toxicity and HSA Binding of [Ru(Me4phen)(Bb7)] 2+ and the Effect of Encapsulation in Cucurbit[10]uril[END_REF] that also binds non-covalently to DNA, and that can partially insert (but is too large to thread) into CB [10], similarly showed only small effects of complexation on its in cellulo behaviour (though the DNA-binding of that agent is different and the complex only mildly cytotoxic).

Having established that pseudo-rotaxanation with CB[10] was readily achieved and that it placed the macrocycle in the correct position over the central section of the cylinder but that it had little effect on the biological properties due to competitive de-threading, we next explored if we could lock the macrocycle onto the cylinder as a proper rotaxane. Traditional rotaxanes are formed by attaching two bulky stoppers, one at each end of the thread. By using a 3-dimensional helicate as the axle we open up an alternative possibility to create the branching needed to lock the ring onto the structure, by attaching multiple (smaller) groups that protrude out of the cylindrical surface at the ends of the helicate axle. To explore this we modified the design of the cylinder, replacing the pyridines with 2-imidazole units. The 2-imidazole offers a position to which we can add further functionalisation, through alkylation of the non-coordinated nitrogen in the imidazole ring, and we selected 2-picolyl as the alkylating group. Thus we introduce three branch points onto the cylinder surface at each end. This new design approach is shown in Fig. 4.

We first prepared two non-rotaxanated cylinders, [Ni2L'3] 4+ and [Ni2L"3] 4+ , analogous to the parent cylinders but bearing the 2-imidazole and 2-imidazole-N-picolyl groups in place of the pyridines. They are readily prepared from the condensation of the 2-imidazole and 2-imidazole-N-picolyl carboxaldehdyes Figure 4: Our synthetic strategy to create a rotaxanated cylinder with bis-1,4-diaminophenylmethane and subsequent coordination to nickel(II). While the corresponding iron(II) complexes were also prepared, [Fe2L'3] 4+ had a lower solution stability than the corresponding nickel(II) complex and so we focused our studies on the nickel complexes. The X-ray crystal structures of the [M2L"3] 4+ cations (M= Ni, Fe) were obtained (as the hexafluorophosphate salts) and confirmed the expected structure (Figs. 4 andS12), with the retention of the triple-helical cylindrical structure, the retention of the central core of the cylinder structure which is crucial for its binding to both DNA 3-way junctions and CB [10], and now the introduction of the picolyl units sticking out of the helix and in such a position that they should not interfere with the CB [10] binding site but would potentially sterically prevent it from threading/dethreading.

We then explored whether we could post-synthetically transform [Ni2L'3] 4+ into the alkylated [Ni2L"3] 4+ by reaction with alkylating agent 2-(bromomethyl)pyridine and Hunig's base in acetonitrile; heating at reflux overnight afforded the desired hexa-alkylated product, [Ni2L"3] 4+ , as confirmed by mass spectrometry and (paramagnetic) proton NMR spectroscopy. Small amounts of the penta-functionalised cylinder were also observed by mass spectrometry. To access the rotaxane we then repeated the same reaction in the presence of CB [10]. While acetonitrile did not prove to be a suitable solvent, reaction in aqueous methanol at reflux overnight, afforded the rotaxane as a cream precipitate in ~60% isolated yield. The mass spectrum (Fig. 5 andS14) shows a dominant peak corresponding to [Ni2L"3•CB10] 4+ (correct mass and isotope distribution). Smaller peaks corresponding to penta-alkylated rotaxanes are also observed but only a tiny trace peak corresponding to unrotaxanated [Ni2L"3] 4+ indicating that the rotaxane (as opposed to a pseudo-rotaxane) is formed in which the ring is now constrained by the picolyl groups to remain on the cylinder. The (paramagnetically shifted) proton NMR spectrum shows the [10] and the more restricted motion caused by the picolyl groups for [Ni2L"3] 4+ ; in the 1µs timescale the rotaxanated cylinder does not rotate. Hydrogens are omitted for clarity. Supplementary videos of the simulations are also available. There is a tendency for the CB [10] ring to undergo fluxional distortions during the simulation, including a buckling of the ring at the CH2 groups that create a heart-shaped ring and allowing it to close up around the cylinder. This is also seen at very low frequency (<0.1%) in simulations of the free CB [10] but is more prevalent in the rotaxane. The paramagnetic broadening in the 1 H NMR does not allow this feature to be confirmed experimentally but reflects the greater flexibility of CB [10] compared to lower order cucubit[n]urils. See also Figs S18 andS22.

presence of peaks corresponding to the cylinder and the CB [10] (Figs. 5 andS11). The cylinder peaks in the rotaxane are further broadened than those observed in the free [Ni2L"3] 4+ cylinder; consistent with this, MD simulations (Figs. 4 and6) indicate that the picolyl groups restrict the spinning motion of the cylinder about the metal-metal axis while rotaxanated inside the CB [10]. The rotaxanated complex [Ni2L"3•CB10] 4+ was mixed with an equimolar amount of [Fe2L3] 4+ and heated under reflux for 24 hours in 10% methanol: water. The rotaxane was unchanged and no Fe2L3 pseudo-rotaxanes were observed by mass spectrometry; this contrasts with the analogous mixing experiments with pseudo-rotaxanes and is strong evidence of proper rotaxane formation.

Rotaxanation would also be expected to enhance the inherent stability of the cylinder and to explore this we treated the picolyl functionalised cylinder [Ni2L"3] 4+ and the rotaxane [Ni2L"3•CB10] 4+ with edta. Edta binds strongly to Ni 2+ in water (log k >18) [START_REF] Harris | Quantitative Chemical Analysis[END_REF] and is able to remove nickel from [Ni2L"3] 4+ over a period of hours at room temperature. The rotaxanation does provides additional stabilisation, and the rotaxanated cylinder is competitive with edta for nickel binding (Fig. S21) with significant amounts rotaxane still present even after treatment with 10 equivalents of Na2(edta).

Multiply-threaded rotaxanes are structures of considerable interest; [START_REF] Mcgonigal | Multiply threaded rotaxanes[END_REF][START_REF] Danon | Triply Threaded [4]Rotaxanes[END_REF][START_REF] Cheng | En Route to a Molecular Sheaf: Active Metal Template Synthesis of a [3]Rotaxane with Two Axles Threaded through One Ring[END_REF] Considering only the ligands of the helicate the rotaxane might be viewed as a [4]rotaxane with two metal bound to the three strands. However, mass spectrometric analysis demonstrates that on demetallation the ligands are no longer sterically constrained in the CB [10] cavity and dethread (Fig. S21). The metallo-supramolecular assembly of the triple-helicate is thus crucial to the design and rotaxane preparation and we consider a [2]rotaxane nomenclature to be more appropriate for this particular supramolecule, though it might be possible to access [4]rotaxanes by this approach in the future using bulkier groups.

The non-rotaxanated [Ni2L'3] 4+ and [Ni2L"3] 4+ cylinders bind DNA analogously to the original pyridyl cylinders (Figs. 7 and S15-17). PAGE gel electrophoresis experiments confirms that both are able to bind and stabilise DNA 3-way junctions, just as the original pyridyl cylinders do, with [Ni2L'3] 4+ less effective than the parent (perhaps due to imidazole deprotonation) and [Ni2L"3] 4+ slightly more so (perhaps because of its larger surfaces which may extend into the 3WJ grooves). Circular dichroism spectra confirm that both cylinders bind calf thymus DNA (inducing a CD signal in the cylinder spectroscopy) which retains a B-DNA structure, and the linear dichroism demonstrates that both cylinders give rise to DNA-coiling as observed with pyridyl-based cylinders. Cell proliferation (MTT) assays confirmed that the functionalised (but non-rotaxanated) cylinder [Ni2L"3] 4+ exerts a cytotoxic effect in cells (IC50, 72 h, MDA-MB-231 55±10 µM; SKOV3 52±5 µM). Thus, as anticipated, making changes to the ends of these cylinders does not, of itself, prevent the key DNA-binding and biological activities.

The contrast with the rotaxane [Ni2L"3•CB10] 4+ is striking (Fig. 7). The rotaxane can no longer stabilise the DNA 3WJ in the gel electrophoresis experiment. It also no longer coils DNA (rather a small increase in DNA orientation is observed). Thus wrapping the CB [10] macrocycle around the central phenylene units has switched off the DNA junction binding and DNA coiling as anticipated. The greater DNA orientation observed in a Couette cell in the linear dichroism experiment indicates some form of DNA binding occurs, while the circular dichroism confirms that the DNA remains in its B form. Molecular dynamics simulations (Fig. S17) suggest that the rotaxane may associate through a partial entry into the grooves, and that this leads to some small elongation of the DNA. Association with the bases at the ends of the DNA strand is also seen in the simulations together with some capacity to bridge across two pieces of DNA that could also lead to enhanced orientation in a Couette cell.

Just as the rotaxanation switches off the key DNA binding features of the cylinders, it also switches off the biological action with the rotaxane showing no cytotoxic effect in cell proliferation (MTT) assays (IC50, 72 h >>140 µM) in MDA-MB-231 (breast) cell lines (Fig. S11).

■ CONCLUSIONS

We have demonstrated a new approach to design of rotaxanes by employing cylindrical metallo-supramolecular helicates as the axle, with the ring held in place by branch points which emanate from the strands of the helix. These intriguing new agents are assembled by combining two designs on which the field of supramolecular chemistry was founded: the metallo-helicates (pioneered by Lehn) [START_REF] Ayme | From Coordination Chemistry to Adaptive Chemistry[END_REF] and rotaxanes (Stoddart and Sauvage). [START_REF] Stoddart | Mechanically Interlocked Molecules (MIMs)-Molecular Shuttles, Switches, and Machines (Nobel Lecture)[END_REF]2 The metallo-cylinders (a class of triple-helicates) used as the rotaxane axle, offer unique and unprecedented DNA binding properties; their activity has stimulated a great interest in designing metallo-supramolecular drugs. [START_REF] Cardo | Non-covalent Metallo-Drugs: Using shape to target DNA and RNA junctions and other nucleic acid structures[END_REF]12 Pseudo-rotaxanation is not sufficient to modify the bio-activity of these cylinders in vitro or in cellulo, but by proper rotaxanation those properties are modified.

Switching the activity of a metallo-drug on rotaxanation represents an unconventional approach: indeed incorporation of metallo-drugs into proper rotaxanes is rare and restricted to mononuclear platinum [START_REF] Wang | Pt-rotaxanes as cytotoxic agents[END_REF] and ruthenium 73 drugs attached to the terminal stopper of a rotaxane, with the rotaxane part essentially behaving as an attached organic vector. Organic drugs such as paclitaxel have also been attached as a rotaxane stopper [START_REF] Barat | A mechanically interlocked molecular system programmed for the delivery of an anticancer drug[END_REF] while Papot and Leigh have used a potentially bioactive peptide as a linear thread in a hydrogen-bond assembled rotaxane and combined it with a sophisticated enzyme-cleavage rotaxane degradative release. [START_REF] Fernandes | Rotaxane-Based Propeptides: Protection and Enzymatic Release of a Bioactive Pentapeptide[END_REF][START_REF] Fernandes | Second generation specific-enzyme-activated rotaxane propeptides[END_REF] Our results raise the tantalising possibility of creating systems in which a triggered rotaxanation or de-rotaxanation (de-threading) inside a cell might be used to switch a metallo-drug action on or off. This might be applicable to a range of different molecular and metallo-supramolecular designs and we are actively exploring the further potential of this approach.

Figure 2 :

 2 Figure 2: 500 MHz 1 H NMR spectra of the [Fe2L3] 4+ cylinder (0.5mM) (a) alone and (b) with 1 equivalent of CB[10] in D2O illustrating the shifts caused by binding. (c) DOSY NMR spectra (stacked) showing the change in diffusion coefficient on binding consistent with formation of a single species of increased hydrodynamic radius. (d) DFT model of the cylinder + CB[10] also showing the experimentally observed NOEs from cylinder H-im & H-3 to a CH2 proton on the rim of the CB[10].

Figure 3 :

 3 Figure 3: (a) PAGE gel showing that the [Ni2L3•CB10] 4+ complex stabilises DNA 3WJ in the same way as [M2L3] 4+ cylinders alone. (* = radiolabel) (b) CD and (c) LD spectra of ct-DNA (100μM DNA concentration; 10 mM sodium chloride; 1 mM sodium cacodylate) with [Ni2L3•CB10] 4+ . R values represent DNA base pairs/complex. The spectra show binding and DNA coiling and importantly are analogous to those obtained with free [Ni2L3] 4+ (Fig. S8) (d) MTT cell survival assay (72 hours treatment of A2780 cells), demonstrating that [Ni2L3•CB10] 4+ and [Ni2L3]4+ have similar effects on cell survival (triplicate independent repeats; two way ANOVA analyses). (e) Cell uptake studies (triplicate) demonstrating that CB[10] does not affect the cell uptake. The greater uptake of the Ru (over Ni) cylinder is consistent with previous observations in 1G5 (Jurkat T-) cells.[START_REF] Cardo | Metallo supramolecular cylinders inhibit HIV-1 TAR-TAT complex formation and viral replication in cellulo[END_REF]

37

 37 Figure 3: (a) PAGE gel showing that the [Ni2L3•CB10] 4+ complex stabilises DNA 3WJ in the same way as [M2L3] 4+ cylinders alone. (* = radiolabel) (b) CD and (c) LD spectra of ct-DNA (100μM DNA concentration; 10 mM sodium chloride; 1 mM sodium cacodylate) with [Ni2L3•CB10] 4+ . R values represent DNA base pairs/complex. The spectra show binding and DNA coiling and importantly are analogous to those obtained with free [Ni2L3] 4+ (Fig. S8) (d) MTT cell survival assay (72 hours treatment of A2780 cells), demonstrating that [Ni2L3•CB10] 4+ and [Ni2L3]4+ have similar effects on cell survival (triplicate independent repeats; two way ANOVA analyses). (e) Cell uptake studies (triplicate) demonstrating that CB[10] does not affect the cell uptake. The greater uptake of the Ru (over Ni) cylinder is consistent with previous observations in 1G5 (Jurkat T-) cells.[START_REF] Cardo | Metallo supramolecular cylinders inhibit HIV-1 TAR-TAT complex formation and viral replication in cellulo[END_REF]

Figure 5 :

 5 Figure 5: (a) Mass Spectrum of the [Ni2L"3•CB10] 4+ rotaxane (b) 1 H NMR Spectrum (0-10ppm shown only) of the [Ni2L"3•CB10] 4+ rotaxane and comparison with imidazole pseudo-rotaxane [Ni2L'3•CB10] 4+ showing presence of CB[10] and the alkylpyridine groups on the modified cylinder.

Figure 6 :

 6 Figure 6: Combined and superimposed views of Molecular Dynamics simulations of the pseudo-rotaxanated [Ni2L'3•CB10] 4+ (left) and proper rotaxanated [Ni2L"3•CB10] 4+ (right) complexes showing the free rotation of the [Ni2L'3] 4+ cation in the CB[10] and the more restricted motion caused by the picolyl groups for [Ni2L"3] 4+ ; in the 1µs timescale the rotaxanated cylinder does not rotate. Hydrogens are omitted for clarity. Supplementary videos of the simulations are also available. There is a tendency for the CB[10] ring to undergo fluxional distortions during the simulation, including a buckling of the ring at the CH2 groups that create a heart-shaped ring and allowing it to close up around the cylinder. This is also seen at very low frequency (<0.1%) in simulations of the free CB[10] but is more prevalent in the rotaxane. The paramagnetic broadening in the 1 H NMR does not allow this feature to be confirmed experimentally but reflects the greater flexibility of CB[10] compared to lower order cucubit[n]urils. See also Figs S18 and S22.

Figure 7 :

 7 Figure 7: (a) PAGE gel showing that the [Ni2L'3] 4+ and [Ni2L"3] 4+ complexes stabilise DNA 3WJ but the rotaxanated [Ni2L"3•CB10] 4+ cylinder does not. (b) CD and (c) LD spectra of ct-DNA (100μM DNA concentration; 10 mM sodium chloride; 1 mM sodium cacodylate) with [Ni2L"3] 4+ and (d) the corresponding CD and (e) LD spectra of ct-DNA with [Ni2L"3•CB10] 4+ . R values represent DNA base pairs/complex.

■ ACKNOWLEDGMENTS

This work was funded by the EPSRC Physical Sciences for Health Centre (EP/L016346/1), the EU DNAREC Marie Curie Training Site (MEST-CT-2005-020842), an EU Marie Curie Fellowship (H2020-MSCA-IF-2018-844145), FAPESP (2019/11242-1) and the University of Birmingham. Simulations used the Bluebear and Castles HPC facility (U. Birmingham). 77 FB was supported by a student exchange from LMU Munich. We thank the Centre for Chemical and Materials Analysis (U. Birmingham), the EPSRC UK National Crystallography Service at the University of Southampton for the collection of the crystallographic data, 78 Dr. Mirela Pascu (U. Birmingham) for some initial experiments (on L') and Dr. Tim Barendt, Prof. Zoe Pikramenou (both U. Birmingham), Prof Alexandre Martinez (ENS Lyon) and Prof Benoît Colasson (U. Paris Descartes) for helpful discussions.

■ ASSOCIATED CONTENT

Supporting Information

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacs.xxxxxxxxx.

Author contributions

# These authors contributed equally: Catherine Hooper and Lucia Cardo


Foteini Spagopoulou
email: foteini.spagopoulou@gmail.com

Céline Teplitsky

Stéphane Chantepie

Martin I Lind

Lars Gustafsson

Celine Teplitsky

Alexei A Maklakov

Silver-spoon upbringing improves early-life fitness but promotes reproductive ageing in a wild bird

Keywords: ageing, condition-dependence, disposable soma theory, early-life conditions, senescence, "silver-spoon" theory, brood size manipulation

Introduction

Ageing, a physiological deterioration with advancing age, is a complex and highly variable trait that is widespread across the tree of life [START_REF] Jones | Diversity of ageing across the tree of life[END_REF][START_REF] Shefferson | The Evolution of Senescence in the Tree of Life[END_REF]) and affects organismal fitness in nature [START_REF] Bouwhuis | The forms and fitness cost of senescence: age-specific recapture, survival, reproduction, and reproductive value in a wild bird population[END_REF][START_REF] Nussey | Senescence in natural populations of animals: Widespread evidence and its implications for biogerontology[END_REF]. Theory maintains that ageing evolves because selection gradients on traits decline with age [START_REF] Medawar | An unresolved problem of biology[END_REF][START_REF] Williams | Pleiotropy, natural selection, and the evolution of senescence[END_REF][START_REF] Hamilton | The moulding of senescence by natural selection[END_REF], favouring alleles that increase early-life fitness even at the cost of negative pleiotropic fitness effects in late-life. While a large body of evidence supports these theoretical predictions on the population level, very little is known about the individual variation in ageing rates.

There is a general agreement in the field that early-life environmental conditions can shape the life-history of an adult organism and explain much of the individual variation in ageing rates [START_REF] Monaghan | Early growth conditions, phenotypic development and environmental change[END_REF][START_REF] Marshall | Lifetime fitness consequences of early-life ecological hardship in a wild mammal population[END_REF][START_REF] Cooper | Ageing with a silver-spoon: A meta-analysis of the effect of developmental environment on senescence[END_REF], but there is little consensus regarding the direction of the effect. Good environmental conditions during development can result in lifelong positive effects on physiology, reproduction and longevity [START_REF] Lindström | Early development and fitness in birds and mammals[END_REF]van de Pol et al. 2006a;[START_REF] Nussey | Environmental conditions in early life influence ageing rates in a wild population of red deer[END_REF][START_REF] Hayward | Influence of early-life nutrition on mortality and reproductive success during a subsequent famine in a preindustrial population[END_REF][START_REF] Cooper | Ageing with a silver-spoon: A meta-analysis of the effect of developmental environment on senescence[END_REF] ("silver-spoon" hypothesis -Grafen 1990). Alternatively, organisms raised in good natal environments can invest heavily in growth and early-life reproduction, resulting in accelerated senescence in late-life [START_REF] Hunt | High-quality male field crickets invest heavily in sexual display but die young[END_REF][START_REF] Bonduriansky | Reproductive ageing and sexual selection on male body size in a wild population of antler flies (Protopiophila litigata): Ageing and sexual selection[END_REF][START_REF] Hooper | Ontogenetic timing as a condition-dependent life history trait: High-condition males develop quickly, peak early, and age fast[END_REF][START_REF] Marshall | Lifetime fitness consequences of early-life ecological hardship in a wild mammal population[END_REF]) ("live-fast die-young" hypothesis - [START_REF] Promislow | Living fast and dying young: A comparative analysis of life-history variation among mammals[END_REF]. These studies suggest that organisms can allocate resources to reproduction and somatic maintenance depending on their early-life environmental conditions, and that high latent costs of increased early-life growth and reproduction, under favourable conditions, can result in accelerated ageing [START_REF] Adler | Phenotypes optimized for early-life reproduction exhibit faster somatic deterioration with age, revealing a latent cost of high condition[END_REF][START_REF] Hooper | Ontogenetic timing as a condition-dependent life history trait: High-condition males develop quickly, peak early, and age fast[END_REF].

Previous studies emphasised the role of sexual selection in the evolution of resource allocation strategies that can result in rapid ageing under favourable natal conditions. Males, for example, are predicted to invest in costly secondary sexual traits and risky behaviours, trading-off with investment in somatic maintenance and late-life performance [START_REF] Hunt | High-quality male field crickets invest heavily in sexual display but die young[END_REF][START_REF] Bonduriansky | Reproductive ageing and sexual selection on male body size in a wild population of antler flies (Protopiophila litigata): Ageing and sexual selection[END_REF][START_REF] Nussey | Environmental conditions in early life influence ageing rates in a wild population of red deer[END_REF][START_REF] Hooper | Ontogenetic timing as a condition-dependent life history trait: High-condition males develop quickly, peak early, and age fast[END_REF]. However, recent studies also show that early-life investment in reproduction trades-off with late-life fitness in females in natural populations (reviewed in [START_REF] Lemaître | Early-late life trade-offs and the evolution of ageing in the wild[END_REF] and in the laboratory [START_REF] Travers | Live fast die young life history in females: evolutionary trade-off between early life mating and lifespan in female Drosophila melanogaster[END_REF]. This suggests that "silver-spoon" females could allocate more resources to early-life reproduction and suffer from faster senescence later in life, while females experiencing early-life stress could reduce their investment in early-life reproduction and delay senescence. Interestingly, mild stress results in reduced early but improved late breeding performance in captive female Zebra Finches (Taeniopygia guttata) [START_REF] Marasco | Environmental conditions shape the temporal pattern of investment in reproduction and survival[END_REF]). Overall, it is possible that two related processes shape the expression of age-specific life-histories in response to differences in earlylife conditions. First, poor conditions in early-life constrain the amount of resources that can be allocated to reproduction, which delays the reproductive peak. Second, the cumulative effect of investment in early-life reproduction under good early-life conditions accelerates senescence. For example, a recent model specifically suggested that the cost of reproduction decreases the variance in lifetime reproductive success and organisms that start to reproduce earlier will also senesce earlier [START_REF] Coste | Trait level analysis of multitrait population projection matrices[END_REF]. However, whether females do indeed make similar reproductive decisions under natural conditions remains largely unknown.

We studied the long-lasting effects of natal environment on age-specific survival and reproduction in female Collared Flycatchers (Ficedula albicollis) from a natural population on the Swedish island of Gotland. Competition is an important aspect of early environment and earlier studies have shown that experimental increase of brood size results in reduced body mass at fledgling and increased offspring mortality [START_REF] Alatalo | Phenotypic Selection on Heritable Size Traits: Environmental Variance and Genetic Response[END_REF][START_REF] Tinbergen | Nestling weight and survival in individual great tits ( Parus major )[END_REF][START_REF] De Kogel | Long-term effects of brood size manipulation on morphological development and sex-specific mortality of offspring[END_REF][START_REF] Sendecka | Age-dependent reproductive costs and the role of breeding skills in the Collared flycatcher[END_REF]. We experimentally manipulated the brood size of hundreds of nesting pairs across 26 breeding seasons (i.e. 1983-2009) to create either artificially highcompetition (increased) or low-competition (reduced) broods. The nests were paired by matching hatching dates and two nestlings were transferred from a nest to the other for the reduced and enlarged broods or exchanged between nests for the control broods. Individuals from nests with a reduced brood size experience a "silverspoon" upbringing and grow to a larger body size, a proxy for body condition, at fledgling [START_REF] Voillemot | Effects of brood size manipulation and common origin on phenotype and telomere length in nestling collared flycatchers[END_REF], allowing us to test directly whether experimentally improved developmental conditions decelerate or accelerate reproductive and actuarial ageing. To our knowledge, this is the first study that directly investigates the consequences of individual heterogeneity in early-life condition for ageing in an experimentally manipulated natural population.

Methods

Study species and study area

Collared flycatchers (Ficedula albicollis) are small (~ 13g), migratory, insectivorous, cavity-nesting passerines, that breed in deciduous and mixed forests of eastern and central Europe (and southwestern Asia). Females incubate four to eight eggs (mean ± sd = 6.3 ± 0.65) during a 14-day period and rear a single brood each year.

Juveniles are fed by both parents and fledge 14 to 18 days after hatching (for further details on population and methods see [START_REF] Gustafsson | Collared flycatcher[END_REF]).

Isolated from their main breeding grounds, additional breeding populations have been established on the Swedish islands of Gotland and Öland in the Baltic Sea.

The collared flycatcher population on the island of Gotland (57° 10´, 18° 20´) has been well studied since 1980. Each year, new individuals (i.e. both adults and offspring) are measured and ringed, to allow individual identification throughout their lifetime and enable the assessment of yearly reproductive success. The females are caught at the nest during incubation or the chick provisioning period and the males only during the chick provisioning period. Further details of the breeding region, and ecology have been collected using standard methods [START_REF] Gustafsson | Collared flycatcher[END_REF][START_REF] Pärt | Breeding dispersal in the collared flycatcher (Ficedula albicollis): Possible causes and reproductive consequences[END_REF]).

Collared flycatchers in general and the long-term studied Gotland population in specific, are particularly suitable for the study of life history trade-offs in nature due to their high degree of site fidelity, limited dispersal and preference for nest boxes over natural cavities, which allows accurate age and survival estimation [START_REF] Pärt | Breeding dispersal in the collared flycatcher (Ficedula albicollis): Possible causes and reproductive consequences[END_REF].

Treatments and data selection

Since 1983, multiple brood size manipulation experiments have been conducted in the Gotland population [START_REF] Gustafsson | The costs of reproduction in the collared flycatcher Ficedula albicollis[END_REF][START_REF] Nordling | Reproductive effort reduces specific immune response and parasite resistance[END_REF][START_REF] Doligez | Costs of reproduction: assessing responses to brood size manipulation on life-history and behavioural traits using multi-state capture-recapture models[END_REF][START_REF] Sendecka | Age-dependent reproductive costs and the role of breeding skills in the Collared flycatcher[END_REF], to examine various aspects of parental investment, the costs of increased reproductive effort and the trade-off between reproductive investment and immune function. To investigate the effects of early-life natal conditions on individual life histories, we collected data from individuals raised in manipulated nests, where brood size was increased or reduced by two offspring.

In addition, we accounted for possible translocation effects by including a control treatment, in which offspring were swapped between nests without alteration of the initial total number (Fig. 1). The distribution of brood size after treatment was flatter but the number of nestlings remained in the range of the natural variation. The average brood size (mean ± S.E.) for reduced broods was 4.33 ± 0.05, for enlarged broods was 8.31 ± 0.04 and for control broods was 6.18 ± 0.05. We monitored the entire life-history of 943 collared flycatchers, which were raised in such manipulated nests, but eventually focused on females, due to the degree of male extra-pair paternity (~15% -Sheldon & Ellegren 1999) that makes the process of estimating male reproductive values challenging.

The experimental reduction of the brood size resulted in increased body mass at fledgling (15.12 ± 0.12, χ 2 = 10.69, P < 0.01, Fig. 2A), while there was no effect of body size measured as tarsus length (19.56 ± 0.05, χ 2 = 0.52, P = 0.77, Supplementary Fig. S1). This suggests that brood reduction indeed improved natal environment and body condition of the offspring, rather than simply their structural size. In support of these findings, previous studies showed that experimental increase of brood size results in reduced body mass at fledgling and increases offspring mortality [START_REF] Alatalo | Phenotypic Selection on Heritable Size Traits: Environmental Variance and Genetic Response[END_REF][START_REF] Tinbergen | Nestling weight and survival in individual great tits ( Parus major )[END_REF][START_REF] De Kogel | Long-term effects of brood size manipulation on morphological development and sex-specific mortality of offspring[END_REF][START_REF] Sendecka | Age-dependent reproductive costs and the role of breeding skills in the Collared flycatcher[END_REF]).

Reproductive and survival data were collected for 514 females born between 1983 and 2009. 122 females were raised in nests with reduced broods ("low competition"), 215 females were raised in controlled nests ("control") and 177 females were raised in nests with increased broods ("high competition"). For each female, we recorded their reproductive success, as the number of recruits (i.e.

offspring that returned to Gotland to breed the following years, after overwintering in Africa; Fig. 1) during each reproductive season. Females failing to breed on a given year were attributed a reproductive success of 0. This individual-based monitoring provides access to age-specific reproductive success (annual number of recruits), lifetime reproductive success (sum of recruits produced over lifetime) and the rate-sensitive individual fitness λ ind , which encompasses the number of offspring, as well as their timing [START_REF] Brommer | Reproductive timing and individual fitness[END_REF][START_REF] Lind | Sex-specific tradeoffs with growth and fitness following life-span extension by rapamycin in an outcrossing nematode, Caenorhabditis remanei[END_REF] and is analogous to the intrinsic rate of population growth [START_REF] Stearns | The Evolution of Life Histories[END_REF].

Over the years, some of our focal females (N = 205) were involved in additional experiments addressing different questions [START_REF] Nordling | Reproductive effort reduces specific immune response and parasite resistance[END_REF][START_REF] Doligez | Costs of reproduction: assessing responses to brood size manipulation on life-history and behavioural traits using multi-state capture-recapture models[END_REF][START_REF] Pitala | The effects of experimentally manipulated yolk androgens on growth and immune function of male and female nestling collared flycatchers Ficedula albicollis[END_REF]. We made the hypothesis that these experiments do not have a strong impact on survival, and kept all the females for survival analyses, so the data set included the 514 monitored females. In turn these experiments directly influence reproductive success. When investigating reproductive success, we excluded the current and/or future reproductive values of each affected bird, depending on the nature of these additional experimental manipulations and the effect they may have on the female's reproduction. Due to these experiments, the full data set for agespecific reproductive success included 783 observations of 435 females. The data set for lifetime reproductive success restricted to females that were never manipulated during their lifetime included 534 observations of 307 females (i.e. we excluded 205 females that participated in the additional experiments and 2 more females that had missing values in their life histories) .

The age-specific reproductive patterns were similar in both the full (N females = 435) and the restricted dataset N females = 307), so we only present results for the full data set. When analysing lifetime reproductive success and the rate-sensitive individual fitness (λ ind ), any female who had been involved in an experiment affecting breeding success during her lifetime should be excluded from the dataset, thus we only used the restricted dataset.

Statistical analysis

Reproductive success analysis:

Age-specific reproductive success

To analyse the effects of the brood size manipulation on the age-specific reproduction, we used both a frequentist and a Bayesian framework, which yielded similar results. Thus, we describe below the frequentist approach, but see Supplementary Methods for further details on the Bayesian approach. We constructed a generalised linear mixed-effects model (GLMM) with Poisson distribution and log link function. We accounted for the non-independence of data by including individual identity, nest of origin identity and year of annual reproduction as random effects. Furthermore, we included an observation-level random effect to control for overdispersion [START_REF] Harrison | Using observation-level random effects to model overdispersion in count data in ecology and evolution[END_REF]. As fixed effects, we included the treatment (increased, reduced and control broods), as well as a linear and a quadratic term of age (Age and Age 2 ), to test for non-linear variation of reproductive value with age. In addition, we included the Age of First Reproduction (AFR) and Age at Last Reproduction (ALR) in our models, to control for selective appearance and selective disappearance [START_REF] Van De Pol | Age-dependent traits: A new statistical model to separate within-and between-individual effects[END_REF]. All continuous predictor variables were mean centered and standardised (mean = 0 and SD = 1). Centering to the mean allows to give a natural meaning to the intercept, because the intercept is the predicted value when all fixed effects are set to 0. Furthermore, scaling to unit variance allows to obtain estimates of coefficients that can be compared across variables i.e. an interpretation of relative effect sizes. We initially run a complete model including all fixed effects and up to three-way interactions when biologically meaningful (Full model, Eq. 1).

𝐴𝐴𝐴𝐴𝐴𝐴 𝑖𝑖𝑖𝑖 = 𝜇𝜇 + 𝛼𝛼 1 𝑇𝑇 𝑖𝑖 + 𝛼𝛼 2 𝐴𝐴 𝑖𝑖𝑖𝑖 +
Where the breeding success of female 𝑖𝑖 in year 𝑗𝑗 is explained by several fixed effects: early life conditions (treatment, 𝑇𝑇 𝑖𝑖 ), age 𝐴𝐴 𝑖𝑖𝑖𝑖 and age-squared 𝐴𝐴 𝑖𝑖𝑖𝑖 2 of female 𝑖𝑖 in year 𝑗𝑗, age at first (𝐴𝐴𝐴𝐴𝐴𝐴 𝑖𝑖 ) and last breeding (𝐴𝐴𝐴𝐴𝐴𝐴 𝑖𝑖 ) of female 𝑖𝑖, and their interactions. To take into account the non-independence of data collected on the same female and the same year, we included female identity (𝑖𝑖𝑖𝑖𝑖𝑖 𝑖𝑖 ) and year (𝑦𝑦𝑦𝑦𝑦𝑦𝑦𝑦 𝑖𝑖 )

as random effects. The final term in equation 1 is the residual error term 𝜀𝜀 𝑖𝑖𝑖𝑖 .

We then evaluated which interaction to retain based on the conservative Akaike Information Criterion for small sample sizes (AIC c ). Three of these models had the smallest AIC c values (with a difference of less than 2 points), indicating best model fit (models 17, 24 and 31; for models details and differences see Supplementary

Material and Supplementary Table S4). They all gave similar outcomes and we therefore only present here the most comprehensive model (model 17) that includes most variables, but see supplementary material for the output of the other two models (Supplementary Table S5).

All analyses were performed in R version 3.3.1 (R Development Core Team 2017). Statistical models were fitted using the lme4 package (v.1.1-14 -Bates et al. We found no evidence of zero-inflation. We used the package AICcmodavg (v. 2.1-0

2015) and the

-Mazerolle 2016) to compute the Akaike's Information Criterion for small sample sizes (AICc). To test for significance of model terms we used the Anova function of the car package (v. 2.1-3) with Type III Wald chi-square tests. Post-hoc comparisons were performed with the emmeans function in the emmeans package (v. 1.4.4).

Lifetime reproductive success

For the lifetime number of recruits, we tested the treatment effect using a GLMM with Poisson distribution and log link function. We included nest of origin identity and year of birth as random effects to control for cohort effects (Supplementary Table S2).

Finally, for the analysis of rate-sensitive individual fitness, we first estimated λ ind by solving the Euler-Lotka equation of age-specific recruit number and survival for each individual using the lambda function in the popbio package (v 2.4.4 -Stubben & Milligan 2007). Then we tested for the effect of treatment on the log of λ ind in a linear mixed model (LMM) including the nest of origin and year of birth were used as random factors to control for cohort effects (Supplementary Table S3).

minimum age of reproduction (Supplementary Methods). We performed four parallel simulations that each model for 1 200 000 iterations, used a burn-in of 200 000

iterations and took a model sample each 500 iterations. The model parameters likely converged on the most optimal mode and showed low serial autocorrelations (<5%)

and robust posterior distributions (with N= 2 000). For the model comparison, we used the lowest deviance information criterion (DIC), which is similar to the Akaike Information Criterion (AIC) in non-Bayesian models. Comparison of the DIC values [START_REF] Millar | Comparison of hierarchical bayesian models for overdispersed count data using DIC and Bayes' factors[END_REF] revealed that the Logistic model with the bathtub shape (Eq. 2) is the most appropriate mortality function (Supplementary Table S9). Mortality rate:

𝜇𝜇 0 (𝑥𝑥|𝒃𝒃, 𝒂𝒂, 𝑐𝑐) = 𝑦𝑦 𝑎𝑎 0 -𝑎𝑎 1 𝑥𝑥 + 𝑐𝑐 + 𝑒𝑒 𝑏𝑏 0 +𝑏𝑏 1 x 1+𝑏𝑏 2 𝑒𝑒 𝑏𝑏 0 𝑏𝑏 1 �𝑒𝑒 𝑏𝑏 1 x -1�
(2)

The bathtub structure used in the BaSTA package adds a declining Gompertz To assess the differences among the posterior distributions of our brood size manipulation treatments we used the Kullback-Leibler divergence calibration (KLDC) [START_REF] Kullback | On information and sufficiency[END_REF][START_REF] Mcculloch | Local model influence[END_REF][START_REF] Karabatsos | Bayesian nonparametric model selection and model testing[END_REF][START_REF] Boonekamp | Reproductive effort accelerates actuarial senescence in wild birds: an experimental study[END_REF][START_REF] Atema | Costs of long-term carrying of extra mass in a songbird[END_REF][START_REF] Rodríguez-Muñoz | Slower senescence in a wild insect population in years with a more female-biased sex ratio[END_REF], which show the mean Kullback-Leibler discrepancies (KL) between two distributions. Values closer to 0.5 imply that there is minimal difference among the distributions and values closer to 1 imply major differences [START_REF] Kullback | On information and sufficiency[END_REF][START_REF] Mcculloch | Local model influence[END_REF]. We considered a KLDC value > 0.85 to indicate a substantial difference between the two posterior distributions that are compared (Fig. 5).

Results

While the high-competition treatment decreased body mass at fledgling when considering all fledglings (Voillemot et al. 2012, overall: 15.02 ± 0.09, χ 2 = 36.35, P < 0.01, high-competition vs control: post hoc Tukey: P = 0.015; Fig. 2B), this effect disappears when restricting the analysis to recruited females (overall: 15.12 ± 0.12, χ 2 = 10.73, P < 0.01, high-competition vs control: post hoc Tukey:

P = 0.989, Fig 2A).
Overall, the quality of the natal environment had no significant effect on lifetime reproduction (χ 2 = 0.99, P = 0.610; Fig. 3C, Supplementary Table S2) or

individual fitness λ ind (χ 2 = 1.07, P = 0.585; Fig. 3D, Supplementary Table S3) in this study. In terms of age-specific reproductive success, females from the lowcompetition treatment had a high success during the early years of their life, reaching a peak in their second year of breeding (Fig. 3A-B, Table 1). In contrast, females raised in the high-competition treatment started with a lower reproductive success, but steadily increased their performance reaching a peak in their third year and enjoyed a later onset of senescence (Fig. 3A-B, Table 1). More specifically, while the raw data suggest that individuals from high-competition nests maintained high reproductive performance in their fifth year (Fig. 3A), taking into account selective appearance (measured as age at first reproduction) and disappearance (measured as age at last reproduction) reveals a reproductive decline after year four (Fig. 3B). Nevertheless, high-competition females showed a delayed reproductive peak and started to senesce later than their counterparts from low-competition nests for whom senescence started a year earlier. Females raised in control nests started at intermediate level of reproduction, maintained relatively steady reproductive performance during the first three years of their life and declined at the fourth year.

Natal environment also had profound effects on age-specific mortality rates (Fig. 4, see Table S11 for age specific survival probabilities and reproductive rates).

Low-competition females had substantially lower initial acceleration in mortality rate (smaller early-life acceleration parameter b 1 with KLDC > 0.85 -Fig. 4, Fig. 5 and Supplementary Table S10) than both control and high-competition females, suggesting that "silver-spoon" natal environment is beneficial for survival. However, while the mortality rates of control and high-competition females strongly decelerated in late-life and reached a plateau around four years of age, the mortality rate of lowcompetition females continued to increase and reached a higher plateau around the fifth year (smaller late-life deceleration parameter b 2 with KLDC > 0.85 -Fig. 4, Fig. 5 and Supplementary Table S10).

Discussion

We found that females experiencing a low-competition natal environment fledge at higher body mass, start to reproduce at a higher rate, have an earlier reproductive peak and have a lower acceleration of mortality rate than their counterparts raised in high-competition nests. However, low-competition females also show earlier signs of reproductive senescence and suffer from increased mortality rate in the last years of their lives. Thus, our results provide an experimental demonstration of conditiondependent ageing in a natural population. Specifically, these findings support the hypothesis that high-condition females raised in "silver-spoon" natal environment allocate their resources to early-life reproduction, but pay a cost in terms of accelerated ageing in late-life.

There has been a sustained interest in how resource availability affects agespecific life histories but, remarkably, both theoretical and empirical studies to date are focused exclusively on the role of sexual selection in driving rapid ageing in highcondition males (i.e. "live-fast die-young" hypothesis) [START_REF] Kokko | Good genes, old age and life-history trade-offs[END_REF][START_REF] Radwan | Comparison of life-history traits of the two male morphs of the bulb mite, Rhizoglyphus robini[END_REF][START_REF] Hunt | High-quality male field crickets invest heavily in sexual display but die young[END_REF][START_REF] Bonduriansky | Reproductive ageing and sexual selection on male body size in a wild population of antler flies (Protopiophila litigata): Ageing and sexual selection[END_REF][START_REF] Bonduriansky | Sexual selection, sexual conflict and the evolution of ageing and life span[END_REF][START_REF] Preston | Sexually extravagant males age more rapidly[END_REF][START_REF] Adler | Phenotypes optimized for early-life reproduction exhibit faster somatic deterioration with age, revealing a latent cost of high condition[END_REF][START_REF] Hooper | Ontogenetic timing as a condition-dependent life history trait: High-condition males develop quickly, peak early, and age fast[END_REF][START_REF] Hooper | Sexual competition and the evolution of condition-dependent ageing[END_REF]. This is, in part, because of the striking absence of studies showing that high-condition females may also trade-off early-life reproduction for accelerated ageing (but see [START_REF] Travers | Live fast die young life history in females: evolutionary trade-off between early life mating and lifespan in female Drosophila melanogaster[END_REF]. Here, we used an experimental manipulation of early-life environment to show that "silver-spoon" (i.e. low-competition) females indeed benefit from elevated early-life reproductive success, but suffer from an earlier onset of reproductive ageing and increased mortality late in life. Thus, our findings call for a re-appraisal of the idea that "live-fast die-young" strategy applies primarily to males when faced with increased sexual competition [START_REF] Bonduriansky | Sexual selection, sexual conflict and the evolution of ageing and life span[END_REF][START_REF] Hooper | Sexual competition and the evolution of condition-dependent ageing[END_REF].

Furthermore, our results suggest, that we should look beyond a simple negative correlation between net reproduction and longevity when investigating the long-term fitness consequences of early-life conditions. Specifically, in this study "silver-spoon" females did not live shorter, nor reproduced more, but rather had different age-specific mortality and reproduction rates. It is possible that stochastic natural environment masks the effect of our brood size manipulation on total reproduction of the recruits and that the addition of several more years of data collection could show that high-condition females actually have higher fitness. At this point, however, we can only state that a "silver-spoon" early-life environment results in increased early-life but reduced late-life performance. High-competition females fledge at lower body mass then low-competition birds and the control birds but those that survive to come back and reproduce have the same body mass as control birds.

This suggests that high-competition treatment resulted in increased mortality of birds that had low body mass as fledglings.

Our results are in line with the "disposable soma" theory of ageing, which maintains that early-life investment in growth and reproduction results in reduced late-life fitness due to an increased amount of unrepaired cellular damage [START_REF] Kirkwood | The evolution of ageing and longevity[END_REF][START_REF] Kirkwood | Evolution of ageing[END_REF][START_REF] Kirkwood | The Evolution of Senescence in the Tree of Life[END_REF]. However, such patterns can also emerge directly from upregulation of nutrient-sensing molecular signalling pathways in "silver-spoon" females that received more food during development, resulting in increased cellular hypertrophy and accelerated cellular senescence in late life [START_REF] Gems | Genetics of longevity in model organisms: Debates and paradigm shifts[END_REF][START_REF] Maklakov | Evolution of ageing as a tangle of trade-offs: energy versus function[END_REF]. Future research should focus on understanding the physiological and molecular underpinnings of the phenomenon that improved early-life conditions lead to detrimental effects in late-life.

  MCMCglmm package (v.2.23 -Hadfield 2010). For generalized linear mixed models (Poisson error structure), we ensured an appropriate fit using the DHARMa package (v. 0.1.2) for diagnostic tests of model residuals[START_REF] Hartig | DHARMa: residual diagnostics for hierarchical (multi-level/mixed) regression models[END_REF]).

  function and a constant to the basic logistic mortality function. Therefore, the two alpha parameters (a 0 and a 1 ) describe the exponential decline that may be observed very early in life and the constant (c) describe the lowest point of the mortality function. The beta parameters (b 0 , b 1 and b 2 ) describe different parts of the logistic increase in mortality rates with age (i.e. initial exponential increase in mortality that decelerates until it reaches a plateau). More specifically, b 0 is the age-independent or baseline mortality, b 1 describes the initial exponential increase in mortality with age and b 2 describes the degree of deceleration in mortality with age and the level of the asymptote.

Figure 1 .

 1 Figure 1. The graphical outline of the experimental design. The experimental brood size

Figure 2 .

 2 Figure 2. A) Body mass at fledgling (means ± SE) of the focal birds raised in the three

Figure 3 .

 3 Figure 3. Reproduction of the three manipulated natal environments (Means ± SE). A) Age-

Figure 4 .

 4 Figure 4. Survival (A) and Mortality (B) curves with 95% confidence intervals for each

Figure 5 .

 5 Figure 5. Values of the Kullback-Leibler divergence calibration (KLDC), which compare

  𝛼𝛼 3 𝐴𝐴 𝑖𝑖𝑖𝑖 2 + 𝛼𝛼 4 𝐴𝐴𝐴𝐴𝐴𝐴 𝑖𝑖 + 𝛼𝛼 5 𝐴𝐴𝐴𝐴𝐴𝐴 𝑖𝑖 + 𝛼𝛼 6 𝑇𝑇 𝑖𝑖 𝐴𝐴 𝑖𝑖𝑖𝑖 + 𝛼𝛼 7 𝑇𝑇 𝑖𝑖 𝐴𝐴 𝑖𝑖𝑖𝑖 2 + 𝛼𝛼 8 𝑇𝑇 𝑖𝑖 𝐴𝐴𝐴𝐴𝐴𝐴 𝑖𝑖 + 𝛼𝛼 9 𝑇𝑇 𝑖𝑖 𝐴𝐴𝐴𝐴𝐴𝐴 𝑖𝑖 + 𝛼𝛼 10 𝐴𝐴 𝑖𝑖𝑖𝑖 𝐴𝐴𝐴𝐴𝐴𝐴 𝑖𝑖 + 𝛼𝛼 11 𝐴𝐴 𝑖𝑖𝑖𝑖 2 𝐴𝐴𝐴𝐴𝐴𝐴 𝑖𝑖 + 𝛼𝛼 10 𝐴𝐴 𝑖𝑖𝑖𝑖 𝐴𝐴𝐴𝐴𝐴𝐴 𝑖𝑖 + 𝛼𝛼 11 𝐴𝐴 𝑖𝑖𝑖𝑖 2 𝐴𝐴𝐴𝐴𝐴𝐴 𝑖𝑖 + 𝛼𝛼 12 𝑇𝑇 𝑖𝑖 𝐴𝐴 𝑖𝑖𝑖𝑖 𝐴𝐴𝐴𝐴𝐴𝐴 𝑖𝑖 + 𝛼𝛼 13 𝑇𝑇 𝑖𝑖 𝐴𝐴 𝑖𝑖𝑖𝑖 2 𝐴𝐴𝐴𝐴𝐴𝐴 𝑖𝑖 + 𝛼𝛼 12 𝑇𝑇 𝑖𝑖 𝐴𝐴 𝑖𝑖𝑖𝑖 𝐴𝐴𝐴𝐴𝐴𝐴 𝑖𝑖 + 𝑖𝑖𝑖𝑖𝑖𝑖 𝑖𝑖 + 𝑦𝑦𝑦𝑦𝑦𝑦𝑦𝑦 𝑗𝑗 + 𝜀𝜀 𝑖𝑖𝑗𝑗

Proc. R. Soc. B Biol. Sci., 285, 20172442.

Acknowledgments

We thank everyone involved in collecting the flycatcher data over the years. This work has been supported by the ERC Starting Grant AGINGSEXDIFF 260885 and the ERC Consolidator Grant GermlineAgeingSoma 724909 to A.A.M. The long-term field study have been supported continuously by grants from the Swedish Research Council (VR) to L.G.

Survival analysis:

To investigate how early-life developmental conditions affect mortality rates of female collared flycatchers, we performed capture-mark-recapture (CMR) survival analysis on our data [START_REF] Lebreton | Modeling Survival and Testing Biological Hypotheses Using Marked Animals: A Unified Approach with Case Studies[END_REF]. Age-specific mortality trends was inferred using the Bayesian Survival Trajectory Analysis framework (BaSTA package, v. 1.9.5) (Colchero et al. 2012). BaSTA is a powerful package to fit agespecific mortality function on CMR data where information about the exact age of death is missing (right-censored data) [START_REF] Colchero | Bayesian inference on age-specific survival for censored and truncated data[END_REF].

The survival analyses were performed on individual encounter histories. Models assume no dispersal, which could bias the effect of natal environment on actuarial senescence. Indeed, collared flycatchers show high site fidelity and very limited dispersal [START_REF] Pärt | Breeding dispersal in the collared flycatcher (Ficedula albicollis): Possible causes and reproductive consequences[END_REF]. Given that the year of birth is known for all individuals, our CMR analyses correspond to the classical Comark-Jolly-Seber (CJS) framework [START_REF] Lebreton | Modeling Survival and Testing Biological Hypotheses Using Marked Animals: A Unified Approach with Case Studies[END_REF]. To ensure that the CJS assumptions about trap-dependency and transience are met and models adequately fit the data, we first performed a goodness of fit analysis with the U-care Program [START_REF] Choquet | U-CARE: Utilities for performing goodness of fit tests and manipulating CApture-REcapture data[END_REF].

Then, we found the best model that describes the recapture rate (see Supplementary Methods for all details). The probability to recapture individuals in our data was found to be constant and equal to 60% (Supplementary Table S10).

We explored actuarial age-specific mortality rates using Weibull, Gompertz and Logistic functions, with either a simple shape or one of the more complex Makeham [START_REF] Pletcher | Model fitting and hypothesis testing for age-specific mortality data[END_REF] or bathtub [START_REF] Siler | A competing-risk model for animal mortality[END_REF] shapes. The nine previous models were compared to a model without senescence (exponential model with a simple shape). The age-specific mortality curves were fitted starting from one year old, the

Competing Interests statement

The authors declare no competing interests.