Hodaya Klimovsky

Omer Shavit

Carine Julien

Ilya Olevsko

Mohamed Hamode

Yossi Abulafia

Hervé Suaudeau

Vincent Armand

Martin Oheim

Adi Salomon
email: adi.salomon@biu.ac.ilmartin.oheim@u-paris.fr

Characterizing nanometric thin films with far-field light

Ultra-thin, transparent films are being used as protective layers on semiconductors, solar cells, as well as for nano-composite materials and optical coatings. Nano-sensors, photonic devices and calibration tools for axial super-resolution microscopies, all rely on the controlled fabrication and analysis of ultra-thin layers. Here, we describe a simple, non-invasive, optical technique for simultaneously characterizing the refractive index, thickness, and homogeneity of nanometric transparent films. In our case, these layers are made of the biomimetic polymer, My-133-MC, having a refractive index of 1.33, so as to approach the cytosol for biological applications. Our technique is based on the detection in the far field and the analysis of supercritical angle fluorescence (SAF), i.e., near-field emission from molecular dipoles located very close to the dielectric interface. SAF emanates from a 5-nm J-aggregate emitter layer deposited on and in contact with the inspected polymer film. Our results compare favorably to that obtained through a combination of atomic force and electron microscopy, surface-plasmon resonance spectroscopy and ellipsometry. We illustrate the value of the approach in two applications, (i), the measurement of axial fluorophore distance in a total internal reflection fluorescence geometry; and, (ii), axial super-resolution imaging of organelle dynamics in a living biological sample, cortical astrocytes, an important type of brain cell. In the later case, our approach removes uncertainties in the interpretation of the nanometric axial dynamics of fluorescently labeled vesicles. Our technique is cheap, versatile and it has obvious applications in microscopies, profilometry and optical nano-metrology.

INTRODUCTION

Ultra-thin polymer films play crucial roles as transparent spacer materials for the characterization of evanescent fields [START_REF] Li | Ca 2+ -Supplying Black Phosphorus-Based Scaffolds Fabricated with Microfluidic Technology for Osteogenesis[END_REF][START_REF] Lambacher | Direct Characterization of the Evanescent Field in Objective-Type Total Internal Reflection Fluorescence Microscopy[END_REF] , as transparent microwells [START_REF] Nakahara | Fabrication of High-Aspect-Ratio Amorphous Perfluorinated Polymer Structure for Total Internal Reflection Fluorescence Microscopy[END_REF] , or as scaffolds in microfluidics [START_REF] Gleason | Nanoscale Control by Chemically Vapour-Deposited Polymers[END_REF][START_REF] Leroux | Conducting Polymer Electrochemical Switching as an Easy Means for Designing Active Plasmonic Devices[END_REF] Thin films are also essential building blocks for optical coatings, organic/inorganic [START_REF] Gleason | Nanoscale Control by Chemically Vapour-Deposited Polymers[END_REF] or photonic devices [START_REF] Leroux | Conducting Polymer Electrochemical Switching as an Easy Means for Designing Active Plasmonic Devices[END_REF][START_REF] Abad | The Influence of UV Radiation and Ozone Exposure on the Electronic Properties of Poly-3-Octyl-Thiophene Thin Films[END_REF] , nano composites [START_REF] Loste | Transparent Polymer Nanocomposites: An Overview on Their Synthesis and Advanced Properties[END_REF][START_REF] Nagendra | C-Perpendicular Orientation of Poly(L-Lactide) Films[END_REF] , and for transparent nano-and micro-structures [START_REF] Nakahara | Fabrication of High-Aspect-Ratio Amorphous Perfluorinated Polymer Structure for Total Internal Reflection Fluorescence Microscopy[END_REF][START_REF] Eto | Membrane-Coated 3D Architectures for Bottom-up Synthetic Biology[END_REF] . The fabrication of such ultra-thin polymer films goes along with their detailed characterization in terms of refractive index (RI), thickness ∆, (and, related, smoothness), as well as their uniformity and optical homogeneity. These different parameters are typically being quantified through a multitude of measurements involving a range of techniques, which include ellipsometry, stylus profilometry, atomic force microscopy (AFM), scanning electron microscopy, to verify the quality of the coatings.

In the current work, we show that -instead of characterizing thin transparent layers through a combination of complicated, expensive and time-consuming techniques -much of the same information can be retrieved through an all-optical readout, in a single measurement, on a standard inverted microscope equipped with a high numerical-aperture (NA) objective. This can be achieved by depositing a thin fluorophore layer on top of the transparent nanometric film to be characterized, and by measuring the fluorophore radiation pattern by aid of a phase telescope (Bertrand lens). The later shifts the focus from the front focal plane, i.e., the usual sample plane (SP), to the objective's back focal plane (BFP), located inside the objective lens. BFP imaging allows us to collect the angular distribution (k-space representation, Fourier image). Quantitative BFP-image analysis thus reveals information that is otherwise lost [START_REF] Novotny | Principles of Nano-Optics[END_REF][START_REF] Cancądo | Observing the Angular Distribution of Raman Scattered Fields[END_REF] and it allows us monitoring subtle changes in the fluorophore radiation pattern in real time. Our technique is based on the fact that fluorophores located in a non-homogenous medium close to an interface display a directional emission different from the classical dipole emission in free space. Moreover, if these molecular fluorophores are located close enough for their near field to interact with the interface, they emit into angles otherwise forbidden by Snell's law -a phenomenon known as supercritical angle fluorescence (SAF) or leakage radiation [START_REF] Verdes | Supercritical Angle Fluorescence (SAF) Microscopy[END_REF][START_REF] Oheim | Supercritical Angle Fluorescence Microscopy and Spectroscopy[END_REF] . We exploit that this radiation pattern is further modified by changes in the proximity of the fluorophores to the interface, but also by the RI and homogeneity of the transparent film.

We [START_REF] Brunstein | Near-Membrane Refractometry Using Supercritical Angle Fluorescence[END_REF] and others [START_REF] Ferdman | Ultrasensitive Refractometry via Supercritical Angle Fluorescence Article[END_REF] have shown that the precise position of the intensity maximum at the emission critical angle 𝜗c provides a sensitive measurement of the RI of the medium in contact with the flurophores, while the relative intensity emitted into supercritical-and under critical angles encodes the fluorophore distance from the interface [START_REF] Bourg | Direct Optical Nanoscopy with Axially Localized Detection[END_REF][START_REF] Axelrod | Evanescent Excitation and Emission in Fluorescence Microscopy[END_REF][START_REF] Niederauer | Direct Characterization of the Evanescent Field in Objective-Type Total Internal Reflection Fluorescence Microscopy[END_REF][START_REF] Brunstein | Decoding the Information Contained in Fluorophore Radiation Patterns[END_REF] . By collecting the information from the extreme periphery of the objective's back pupil, in the region corresponding to 𝜗NA > d𝜗 > 𝜗c (where 𝜗c are the critical angle given by Snell's law and 𝜗NA the limiting polar angle set by the NA, respectively), we can retrieve in the far-field the information originating from the evanescent wave emitted by the dipoles close to the interface.

In the present work, we first report of fabrication of a set of test samples in which a 5-nm homogenous layer of fluorophores (H6TPPS) is embedded in a thin polymer transparent film with RI of 1.33, at controlled axial distances from the surface, over a range of 10 to 340 nm. By imaging the BFP rather than the sample plane, we retrieve information from this near-field region. We measure the axial distance of the fluorophores at the interface, the RI of the transparent thin film and detect its local defects, when present. Our transparent medium has a refractive index of 1.33 (My-133-MC polymer) to mimic a cell environment for biological ap- plications. Working with this polymer, and fabricating thin homogenous films is a challenge by itself and it is important for biological studies.

Our fabricated samples and SAF-based thin-film characterization have a large range of potential applications in nanotechnology, material science, molecular plasmonics, physics and nanobiology. In the current work, we describe its use for axial optical nano-metrology, and we report on an application for biological axially resolved nanoscopy. The here presented approach is easy to implement and it presents a significant advance over existing techniques, in terms of speed, cost and ease. We expect it to become a routine step in thin-film and 2-D material characterization, and a meaningful complement to evanescent-wave and pointspread function engineering based axial optical techniques, where it can considerably enlarge the information content compared to that of fluorescence-intensity images alone.

RESULTS!AND DISCUSSION

Fabrication of samples with controlled ultra-thin polymer films of RI = 1.33

My-133-MC is a commercial, transparent polymer having a RI of 1.33, similar to that of the aqueous environment of the cytosol of a biological cell [START_REF] El Arawi | Advanced Quantification for Single-Cell Adhesion by Variable-Angle TIRF Nanoscopy[END_REF] . My-133-MC cures upon exposure to ambient moisture. Using this polymer, we assembled transparent layered structures through a combination of spin-coating and plasma surface-activation techniques. Fig. 1a shows a typical cross-sectional image, illustrating the characteristic nano-layered architecture of the resulting sandwiches. They exhibit, from bottom to top, a borosilicate substrate (BK-7 optical grade coverslip), a flat My-133-MC spacer layer of controlled nm-thickness; a 5-nm thin fluorescent-emitter layer and, on top, another transparent index-matched polymer-capping layer. This capping layer ensures that the dye molecules are exposed to a homogenous RI microenvironment. In the particular sandwich shown, we deposited additional conductive layers at the very bottom and on top, only for the purpose of electron microscopy. On the focused-ion beam (FIB) cross-section (left), we observe a remarkable homogeneity, smoothness and flatness of the different component layers (see also Fig. S1 in the Supplementary Material Online).

As an emitter we used a far-red emitting porphyrin derivative, (H6TPPS) J-aggregate, studied thoroughly by us before [START_REF] Weissman | Fabrication of Dipole-Aligned Thin Films of Porphyrin J-Aggregates over Large Surfaces[END_REF][START_REF] Efremushkin | Molecular Plasmonics: Strong Coupling at the Low Molecular Density Limit[END_REF] . TPPS aggregates retained their bulk fluorescence properties when sandwiched between the two polymer layers, with neither a change in peak absorption (S2 at 491 nm, and S1 at 700 nm) nor in peak fluorescence (720 nm), Fig. 1b. From the measured optical density, we can estimate an equivalent dye concentration of "0.1M within the thin layer. Using our previously developed nitrogen-flow technique [START_REF] Weissman | Fabrication of Dipole-Aligned Thin Films of Porphyrin J-Aggregates over Large Surfaces[END_REF] a large uniform dye coverage onto surfaces up to several mm 2 can be obtained, Fig. 1c. We deposited these fluorescent films of about 5 nm thickness onto different surfaces, either the bare substrate, or an intermediate polymer layer, resulting in a well-defined nm fluorophore height ∆ above the substrate.

Spin coating allowed us to form a homogenous spacer of MY-133-MC and to systematically control its thickness, Fig. 1d. Different angular frequency and polymer concentration resulted in transparent layers of variable and precisely controllable thickness, ranging from a few to hundreds of nanometers. We used stylus profilometry and AFM for measuring the layer thickness, ∆, Fig. 1e. The resulting spacer layers had a RMS (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. roughness (Rq) of the order of 1-2 nm, independent of ∆. Ellipsometry confirmed a RI of about 1.33, as specified for My-133-MC polymer, Fig. 1f. Taken together, through a combination of nano-fabrication and -characterization techniques we assembled and experimentally validated smooth, flat and uniform nano-layered structures alternating transparent and fluorescent layers. With their transparency and RI close to that of a biological cell, they feature a thin, homogenous and uniform dye layer, at a precisely controlled distance from the glass substate's surface and can serve as a calibration tool for various axial super-resolution microscopies [START_REF] Lambacher | Direct Characterization of the Evanescent Field in Objective-Type Total Internal Reflection Fluorescence Microscopy[END_REF][START_REF] Oheim | Calibrating Evanescent-Wave Penetration Depths for Biological TIRF Microscopy[END_REF] .

Thin-layer refractometry using far-field light

The radiation pattern of an emitter changes when a fluorophore approaches an interface. Near the boundary, the evanescent near-field emission component of a molecular dipole can couple to the interface and become propagative. By analogy to the excitation evanescent field in TIRF that requires supercritical illumination angles, this near-surface emission must propagate at supercritical angles, forbidden according to Snell's law for far-field dipole emission. As a consequence, near-interface fluorophores emit an important power fraction into the higher-index medium (n2), predominantly beyond the emission critical angle 𝜗c=asin(n1/ n2). Analyses of supercritical angle fluorescence on pupil plane images can retrieve 𝜗c and from that the RI of the local environment of the fluorophores, n1 = f/rc, where rc is the equivalent critical radius and f the focal length of the objective ( see Fig. We implemented BFP imaging on a custom combined total internal reflection and supercritical angle fluorescence (TIR-SAF) microscope built from optical bench components (Fig. S2). Our instrument allows for the continuous selection of the polar illumination angle at the sample, between 𝜗#0° (epifluorescence, EPI) and about ±74° (TIRF). We limited the scan angle to this value so as not to clip the laser beam by the measured effective NAeff of 1.465 (~ 75.7°). Before each experiment, we systematically ran a test protocol validating alignment, setup performance and the absence of coverslip tilt (Fig. S3). In the following experiments, the emission from a 5-nm thin J-agg layer was bright enough for recording highly contrasted BFP images on a standard sCMOS camera with reasonable acquisition settings (P = 40 µW at the objective and 100-ms integration time), Fig. 2a. The recorded BFP images revealed a radially symmetric emission, indicative of non-preferentially oriented dipoles. They also showed the expected characteristic intensity transition at the emission critical angle 𝜗c with an intensity maximum just above 𝜗c, and an outer boundary corresponding to NAeff of the used objective [START_REF] Brunstein | Near-Membrane Refractometry Using Supercritical Angle Fluorescence[END_REF][START_REF] Gregor | Measuring Large Numerical Apertures by Imaging the Angular Distribution of Radiation of Fluorescing Molecules[END_REF] . Line profile intensities, but not peak positions and measured RI values, were sensitive to coverslip tilt (Fig. S4). The deposit of My-133-MC polymer on top of a dye-coated BK-7 substate (∆ = 0 nm), changed the measured BFP intensity profile and RI from 1.047 ± 0. In order to characterize the uniformity of the produced My-133-MC polymer layers over larger spatial scales and to independently confirm our SAF-based RI measurements, we studied the polymer films by nanoplasmonics. Surface plasmons are evanescent waves that strongly depend on the dielectric function of the medium in contact. To avoid tedious phase-matching and to couple free propagating light to plasmonic modes, we turned to plasmonic structures that scatter the incoming light and overcome the k-vector mismatch by additional momentum from the reciprocal lattice. We milled periodic nano-hole arrays in a thin Au-film deposited on the BK-7 glass substrate. Under white light illumination we observed on a dark-field microscope structural colors that varied among plasmonic hole arrays according to their different periodicities, P, i.e., inter-hole spacing (340, 380 and 420 nm) [START_REF] Novotny | Principles of Nano-Optics[END_REF][START_REF] Efremushkin | Molecular Plasmonics: Strong Coupling at the Low Molecular Density Limit[END_REF][START_REF] Salomon | Molecule-Light Complex: Dynamics of Hybrid Molecule-Surface Plasmon States[END_REF] Taken together, our ellipsometric (fig. 1f), SAF-derived (fig. 2) and plasmonic RI measurements (fig. 3) confirm a RI of ultra-thin My-133-MC layers close to that of water, validating this transparent polymer as a suitable optical medium for diverse biomimetic applications in fluorescence microscopy and in biophotonics.

Detec7ng imperfec7ons of the polymer thin layers

We filtered the polymer solution to remove air bubbles and obtain flawless, uniform and homogenous My-133-MC polymer films. Nevertheless, BFP images sometimes revealed a conspicuous double-ring structure featuring two intensity transitions, one at RI = 1.33 and another one at RI = 1, Fig. 4a (left half image).

The inner ring was dimmer compared to the outer one, see red arrow on panel (a), and the diameter of the outer ring corresponded to that observed with the majority of produced polymer samples (right). We reasoned that such a double-ring pattern could result if some -but not all -fluorophores were exposed to air rather than the polymer. Such a non-homogeneous dye environment could result from cavities, cracks or airbubbles in the polymer (Fig. S6). Indeed, a post-hoc FIB cross-section on these samples confirmed the presence of µm-sized bubbles in the polymer layer, Fig. 4b. The observed bubble size is probably an overestimate given that SEM images were acquired in vacuum. Imperfections could equally be observed by the light transmitted through the plasmonic hole arrays on which the polymer layers were deposited (Fig. S7), lending further plausibility to our interpretation. Dual RIs can be also appear when cracks or dimples were present. Thus, the information contained in the fluorophore radiation pattern does not only permit precise thin-layer refractometry but it also reveals imperfections, the detection of which requires otherwise more involved, lengthy and expensive experiments. The time-series in Fig. 4c furthermore illustrates that our technique permits time-resolved measurements and in operando refractometry, an important feature for sensors and thinfilm devices. Based on this SAF-based BFP-image refractometry, we retained only samples that passed the quality gate of a single-ring BFP pattern, and we next investigated how changes in the thickness of the spacer layer, i.e., the fluorophore height above the substrate, modified the radiation pattern.

Measuring axial nanometric distances from SAF/UAF ra7os

Capitalizing on our ability to fabricate controlled thin nanolayers, we next studied sandwiches with different fluorophore-substrate distances Δ. We prepared otherwise identical sandwiches featuring the TPPS emitter layer at Δ = 0, 5, 15, 25, 32, 29, 75 and at 340 nm, respectively, three of which are schematized on Fig. 5a. We used TIRF, the focused laser spot is seen in the periphery of the BFP image for the incoming and back-reflected beam, to excite only near-interface fluorescence. As expected upon evanescent-wave fluorescence excitation, the intensity dropped with increasing Δ, Fig. 5b. While the evanescent-wave penetration depth can be calculated from the local RI of the medium (n1 = 1.33 for My-133-MC) and the incidence angle of the illuminating beam at the laser wavelength (that can be fitted from the laser spot observed on the BFP image [START_REF] Vézy | Axial Nanoscale Localization by Normalized Total Internal Reflection Fluorescence Microscopy[END_REF] ), the actual depth in a given experiment, on the particular microscope is much less certain. How deep the excitation light actually penetrates into the sample is difficult to know and is modified by sample, surface and objective imperfections [START_REF] Oheim | Calibrating Evanescent-Wave Penetration Depths for Biological TIRF Microscopy[END_REF] , which altogether make the quantitation of TIRF intensity images and the measurement of axial fluorophore height from single-angle TIRF data problematic.

We therefore turned to a ratiometric measurement by plotting supercritical (SAF) vs. undercritical fluorescence (UAF) emission components, R = ISAF/IUAF, as a function of fluorophore height Δ, Fig. 5c. Because the light passing through the supercritical zone of the BFP originates entirely from the be the SAF component [START_REF] Axelrod | Evanescent Excitation and Emission in Fluorescence Microscopy[END_REF][START_REF] Axelrod | Selective Imaging of Surface Fluorescence with Very High Aperture Microscope Objectives[END_REF][START_REF] Axelrod | Fluorescence Excitation and Imaging of Single Molecules near Dielectric-Coated and Bare Surfaces: A Theoretical Study[END_REF] . UAF on the other hand, is almost constant, with the exception of a small surface effect at distances very close to the interface. Advantageously, the SAF/UAF fluorescence ratio normalizes for fluorophore concentration, laser intensity and other factors that linearly influence the measured signal. We segmented background-subtracted BFP images into undercritical and supercritical regions, based on the previously measured NAeff and RI and the integrated intensities over these regions. Fig. 5c graphs the evolution with Δ of the SAF/UAF ratio R = ISAF/IUAF, along with simulations for different fluorophore orientations, using the constant total-radiated power model from Hellen & Axelrod [START_REF] Axelrod | Fluorescence Emission at Dielectric and Metal-Film Interfaces[END_REF] . We see a good agreement with theory, except for the values very close (Δ <15 nm) to the interface, where the observed ratios were systematically lower than the predicted one. While we can exclude systematic errors in the thickness of the spacer layers even at that scale from our earlier electron-microscopy controls, one possible explanation is that surface quenching affects SAF stronger than UAF, or that fluorophore orientation is modified in a surface-distance dependent manner, e.g., by surface charges. Alternatively the objective might not be as efficient in capturing high-angle (SAF) as capturing low-angle (UAF) light. In fact, theory assumes a flat collection efficiency over the entire solid angle covered by the NA, which might not be the case for the used objective lens (as already observed others, see, e.g., [START_REF] Oheim | Two-Photon Microscopy in Brain Tissue: Parameters Influencing the Imaging Depth[END_REF] ). Might that as it be, a systematically lower fractional SAF also explains why the best fit (grey trace) corresponds to theory (blue), up to a constant scaling factor. Another reason for this offset could be a generally higher UAF fraction than expected: for example, if My-133-MC polymer was even slightly autofluorescent, this would lead to a relatively higher UAF component (resulting from the spacer and capping layers) and be detectable under the low-light conditions of the TIRF experiment. Yet, fluorescence spectroscopy of bulk of polymer did not reveal any detectable autofluorescence. Of course, the objective itself or the immersion oil could fluoresce and contribute UAF, despite a detection above 720 nm.

Taken together, even with the note of caution for very small values of Δ, SAF/UAF ratiometry allows a reliable, quantitative analysis of the thickness of nanometric layers in the sub-100-nm range, with a precision much higher than that of TIRF-intensity measurements alone. (1987) for dipole orientations as indicated by the line dash. Grey through line is the same curve as for a dipole oriented in the incidence plane, scaled to best fit and suggesting a lower collected fraction of SAF compared to UAF (seen main text). The shaded region of the first surface-proximal 15 nm was omitted from fit. Error bars were measured Rq roughness (for x) and generated (for y) by allowing for +/-0.05 RI-unit uncertainty, respectively.

Biological axial nanoscopy using SAF/UAF emission ra7os

One example of biological nanoscopic imaging that would benefit from a precise axial fluorophore localization is the analysis of single-vesicle mobility during regulated exocytosis. These experiments were pioneered in TIRF studies of the of the late 1990 [START_REF] Steyer | A Real-Time View of Life within 100 Nm of the Plasma Membrane[END_REF][START_REF] Steyer | Docking and Exocytosis of Single Secretory Granules in Live Chromaffin Cells[END_REF][START_REF] Oheim | The Last Few Milliseconds in the Life of a Secretory Granule[END_REF][START_REF] Loerke | Super-Resolution Measurements with Evanescent-Wave Fluorescence-Excitation Using Variable Beam Incidence[END_REF][START_REF] Oheim | Simple Optical Configuration for Depth-Resolved Imaging Using Variable-Angle Evanescent-Wave Microscopy[END_REF] and have widely been used in the cell-and neurobiology community since. Exocytosis involves the transport, docking and release of tiny, membrane-delimited and cargo-loaden compartments ('vesicles') that are formed and filled inside the cell, transported to the cell's outer membrane and that, upon a stimulus -often a rise in the intracellular free calcium concentration ([Ca 2+ ]i), fuse with the outer membrane to release their contents, see Fig. 6a. In such experiments, it would be important to know if a vesicle is close to the membrane or just a bit above, if it moves towards it or away from it, or if it simply loses fluorescent content by leakage or bleaching -questions that cannot be easily answered from an intensity TIRF recoding, due to the unknown evanescent-wave penetration depth and the confounding effects of dye concentration and fluorophore distance on single-vesicle brightness. Unfortunately, the fast dynamics of the process of exocytosis does neither permit to capture z-stacks of images for axial localization at higher precision, nor to acquire a variable-angle series of TIRF images for a 'tomographic' reconstruction of vesicle distance.

We therefore studied, in cultured mouse cortical astrocytes labelled with an amphiphilic styryl pyridinium dye [START_REF] Li | FM Dyes Enter via a Store-Operated Calcium Channel and Modify Calcium Signaling of Cultured Astrocytes[END_REF] , the radiation pattern emerging from small regions of evanescent-wave illuminated membrane, close to the coverslip. In astrocytes, a glial type of brain cells that are neighbors to most neuronal synapses, the green-yellow emitting non-membrane permeable dyes FM1-43 or FM3-25 [START_REF] Betz | Activity-Dependent Fluorescent Staining and Destaining of Living Vertebrate Motor Nerve Terminals[END_REF] show a behavior and uptake-mechanism different from that in neurons and are internalized to label larger vesicular compartments, called lysosomes [START_REF] Li | FM Dyes Enter via a Store-Operated Calcium Channel and Modify Calcium Signaling of Cultured Astrocytes[END_REF][START_REF] Kim | Chronic Activation of CB2 Cannabinoid Receptors in the Hippocampus Increases Excitatory Synaptic Transmission[END_REF] . On fluorescence images these lysosomes appear as diffraction-limited fluorescent spots in front of a diffuse background, Fig. 6b. On a timelapse series of TIRF images acquired close to the bottom of the cell, we can appreciate the movement of some lysosomes in and out of the evanescent field, while others dwell patiently close to the plasma membrane, Fig. 6c. Stimulation of the cell by application of extracellular adenosine triphosphate (ATP) activates purinergic receptors and increases the intracellular Ca 2+ concentration, leading to a loss of most near-membrane lysosomes. This interpretation is confirmed by analysing BFP images that were taken alternately with sample-plane images and that reproducibly displayed R values of 0.59-0.63 (0.56 ± 0.05, n = 15) for cells with neither exocytic events nor overt mobility, Fig. 6d, leftmost and left panels. Following the loss of near-membrane lysosomes, the measured SAF/ UAF ratios were consistently lower, R = 0.36 ± 0.06, explained by an average localization of the remaining dye molecules deeper within the cell, more distant from the interface. Clearly, the remaining FM dye molecules less capable of emitting SAF, Fig. 6d, second panel from right. The interest of concurrent sample-plane and quantitative BFP imaging is evident from the rightmost panel where R was 0.30 ± 0.02, despite the fact that a single lysosome was still present on the sampleplane image. While pure TIRF intensometry would suggest a membrane-proximal location, SAF/ UAF ratiometry revealed a R value of around 0.3 and hence an apparently greater distance from the basal cell membrane than suggest from the intensity reading alone. SAF/UAF ratiometry can thus eliminate unknowns like dye concentration, vesicle diameter or local illumination heterogeneities [START_REF] Axelrod | Light Scattering in TIRF Microscopy: A Theoretical Study of the Limits to Surface Selectivity[END_REF] and permit a more reliable interpretation of near-membrane fluorescence. By comparison with the ratios of fig. 5c, we can infer that the lysosomal dynamics occurs in nearmembrane region at the bottom of the cell, in a region closer than 100 nm to the coverglass. SAF/ UAF ratiometry thus allows the resolution of axial fluorophore dynamics within this range. One limitation of the R measurements in the current implementation of the microscope, however, is that they average fluororophore properties over the entire field-of-view (some 40 µm diagonal) . More resolved measurements can be expected when reducing the size of the illuminated field-of-view, an approach that we currently implement using Bessel-beam illumination with an evanescent needle of light [START_REF] Szederkenyi | Morphological and Molecular Diversity of Human Astrocytes in a Cerebral Organoid[END_REF] .

Very clearly, our proof-of-concept biological experiments illustrate the usefulness of concurrent sample-and back-focal plane imaging for interpreting and disambiguiting the dynamics of biological fluorescence, and they also illustrate the interest of fluorescence imaging with combined TIRF excitation and SAF detection [START_REF] Oheim | Supercritical Angle Fluorescence Microscopy and Spectroscopy[END_REF][START_REF] Brunstein | Eliminating Unwanted Far-Field Excitation in Objective-Type TIRF. Part II. Combined Evanescent-Wave Excitation and Supercritical-Angle Fluorescence Detection Improves Optical Sectioning[END_REF] in conjunction with a rapid, online BFP-image analysis. We expect our technique to have applications in various fields of "surface biology", like the dynamics of ER-PM junctions that link the endoplasmic reticulum to the plasma membrane, cellular adhesion sites, or biofilm growth, but also in surface chemistry and material science.

Conclusion

Taken together, the combination of nano-fabrication and -characterization techniques allowed us to assemble and validate controlled, smooth and uniform nanometric thin fluorescent transparent layers of precisely defined thickness and having a RI close to that of a biological cell. Our sandwich samples can serve, e.g., as a nanoscopic axial ruler for axial super-resolution microscopies and they provide a valuable reference for interpreting the axial dynamics of intracellular processes in the near-membrane space of live cells, cultured on a glass coverslip. We demonstrate that combined sample-plane and quantitative BFP imaging provides unique insight into fluorophore properties and dynamics. Particularly, we show that SAF based refractometry and SAF/ UAF ratioing can be used for exploring the physical and chemical microenvironment of fluorophores and can be used for time-lapse in situ imaging of fluorophore dynamics at or close to a dielectric interface.

BFP imaging is easily implemented on a standard inverted microscope and it allowed us to retrieve axial fluorophore information in the sub 100-nm range, SAF/UAF ratios provide complementary information to fluorescence intensity. In fact, the analysis of the fluorophore radiation pattern gives access to the RI of the fluorophore-embedding medium, the fluorophore height (or film thickness) and orientation of emitters, with the only condition that they are sufficiently close for their nearfield and SAF emission component to be modulated by the surface proximity. As a simple and reliable far-field optical technique, the SAF-based thin-film characterization is an interesting alternative to more complex, time-consuming and expensive techniques. We see additional potential in the field of molecular plasmonics, or polaritons in chemistry, where the nanofabricated samples and the proposed k-space imaging technique togehter offer a powerful tool for studying strong coupling between emitters and plasmonic system [START_REF] Efremushkin | Molecular Plasmonics: Strong Coupling at the Low Molecular Density Limit[END_REF][START_REF] Salomon | Molecule-Light Complex: Dynamics of Hybrid Molecule-Surface Plasmon States[END_REF][START_REF] Weissman | Strong Coupling between an Inverse Bowtie Nano-Antenna and a J-Aggregate[END_REF][START_REF] Salomon | Strong Coupling between Molecular Excited States and Surface Plasmon Modes of a Slit Array in a Thin Metal Film[END_REF] . In fact, for the here reported controlled nanolayers, the interaction between molecules and plasmons is very well defined, and so is the coupling (Rabi splitting) that is not averaged over emitters at different distances. Finally, we also expect axialnanoscopic rulers to become a standard for various fields areas of nano-metrology. With their transparency and RI close to that of a biological cell, they feature a thin, homogenous and uniform dye layer, at a precisely controlled distance from the glass substate$s surface and can be used as calibration standards for axial optical sectioning, super-resolution and fluorohpore localisation techniques.

Finally, the robust SAF/UAF ratios can instruct axial measurements from unknown samples, e.g., to disambiguate biological recordings from TIRF microscopes that have been challenging to interpret.

EXPERIMENTAL PROCEDURES

Materials. We chose My-133-MC (MyPolymers LTD, Ness Ziona, Israel) for its RI close to that of water and two fluorinated (Hydro-Fluoro-Ether, HFE) compounds as solvents for this polymer, Novec™ 7500 or 7100 (3M TM , Minnesota, USA). For the fluorescent emitter layer, we opted for H6TPPS4 (meso-tetra 4-sulfonatophenyl porphyrin) (Sigma-Aldrich, #88074) in its J-aggregate form due to its far-red/near-IR emission remote from autofluorescence and most biological fluorophores (peak absorption/emission wavelength, λabs/ λem = 488/720 nm [START_REF] Weissman | Fabrication of Dipole-Aligned Thin Films of Porphyrin J-Aggregates over Large Surfaces[END_REF] ).

Thin-film polymer deposition. #1 BK-7 glass coverslips (Menzel Gläser, Braunschweig, Germany) were cleaned in 1/100 Hellmanex/double-distilled-deionized water (DI water, 18.2 mΩ) and sonicated (20 min, 40 °C). They were then rinsed thoroughly with DI water, ethanol, and DI water again (30 s each). Substrates were dried with a N2 flow and immediately used thereafter. MY-133-MC polymer solution was filtered and thin films were spin-coated (Laurell, WS-650) and cured under ambient conditions (RT, 20-22°C) during at least 6 hours. My-133-MC polymer resin was applied either pure (undiluted) or diluted in HFE-7500.

Thin dye-layer deposition. H6TPPS4 was dissolved in DI water and aqueous nitric acid (65% w/w, Sigma-Aldrich) to reach pH~ 1. The thin film-deposition procedure is described elsewhere [START_REF] Weissman | Fabrication of Dipole-Aligned Thin Films of Porphyrin J-Aggregates over Large Surfaces[END_REF] . Absorption and fluorescence spectra were recorded both from films and in solution, using a home-built microspectrofluorimeter built around an Olympus inverted microscope coupled to a spectrophotometer (IsoPlane SCT-320, Princeton Instruments) and equipped with a charge-coupled device camera (CCD, PIXS1024b, Princeton Instruments).

Nanoplasmonics. Plasmonic structures were milled in our in-house clean rooms, using a Gallium focused-ion beam (FIB, Helios NanoLabDualBeam 600, FEI) into previously deposited 200-nm thin silver films. The fabrication process was entirely calibrated for milling depth, beam size, and beam deflection. The integrated scanning electron microscope (SEM) enabled the in operando imaging of the fabricated structures as shown.

Thin-film characterization. Film thickness and roughness were measured using a Stylus Profiler (DektakXT, Bruker) in at least 6 different areas. RMS surface roughness (Rq) was calculated using Mountains8, Digital Surf. Results were cross-validated on the same specimens with AFM (Nanoscope, Veeco). The AFM images are shown as pseudo-colored height maps, from which Rq was calculated. RIs were measured by spectroscopic ellipsometry (J.A. Woollam, M-2000), nanoplasmonics and SAF (see below). For ellipsometry, we constructed a model for the My-133-MC polymer, based on the known wavelength and thickness of the deposited layers, obtained by SEM (See Fig. S1).

Combined TIR-SAF imaging. Optical microscopy was performed on a custom set-up assembled from optical bench components (see Fig. S2 as well as Supporting Experimental Procedures for details). Briefly, the beam of a 488-nm laser (Coherent Sapphire SF 488-50) was cleaned up with a 488-nm notch filter, attenuated with neutral density filters, spatially filtered and expanded to "2" diameter. It was then scanned by a rotary mirror and tightly focused in the BFP of a ×100/1.46NA objective (a Plan-Apochromat, oil DIC M27, ZEISS). Images were acquired either upon evanescent-wave excitation (with the spot positioned in the extreme periphery of the objective's BFP) or upon EPI excitation (spot on axis, at 0, 0) using the laser powers and exposure times as indicated. Fluorescence was collected through the same objective, filtered by a ultra flat (2-mm) long-pass 488LPXR dichroic mirror (AHF Analysentechnik), RET493LP (Semrock) and ET520LP (Chroma) long-pass filters. Band-pass filters were housed in independent filter wheels and used as stated in the figure legends). The filtered fluorescence was imaged on a back-illuminated sCMOS camera (PCO edge4.2bi). The effective pixel size in the sample plane was (30 ± 1 nm/px, allowing for a 3-to 4-fold binning. A Bertrand lens mounted on a motorized flipper (Thorlabs) allowed shifting the focus to the objective's BFP (3.36 µm/px on BFP images). SAF/UAF ratio image analysis. SP and BFP fluorescence images were subtracted with their respective dark images. BFP images were segmented into SAF and UAF regions in a multi-step process: we first found the center of the BFP pattern and the number of pixels corresponding to the limiting radius of the NA (rNA). For this, we binarized the image and searched the white disk on the black background by an object recognition algorithm implemented in MATLAB. Next, we calculated the pixel radii corresponding to the critical angle of the pattern (rc) by imposing an a priori RI estimate from independent measurements, rc = rNA⋅RI/NA. Finally, we generated error bars by allowing for a positive and negative deviation (dRI) according to rc (±) = rNA⋅(RI±dRI)/NA. Together, the center coordinates, rNA and rc enable us to segment the image into three areas: background, SAF and UAF. With the background subtracted, we integrated the intensity over each area and calculate the SAF/UAF ratio R = ISAF/IUAF, where I is the respective integral over SAF and UAF areas.

Repeating this integration for each dRI gave us an estimate of the dependence of SAF/UAF ratio on the accuracy of the RI estimate. For the error bars shown we opted for dRI = ±0.05.

Cell culture, staining, and microscopy. Cortical astrocytes were prepared from newborn mice, cultured and labeled previously described [START_REF] Li | FM Dyes Enter via a Store-Operated Calcium Channel and Modify Calcium Signaling of Cultured Astrocytes[END_REF] (See Supporting Experimental Procedures for details). We systematically applied 100 µM ATP at the end of the recordings to test for cell viability and raise the intracellular Ca 2+ . Labelling and the overall aspect of the cell culture was confirmed over a larger field on a custom upright microscope fitted for EPI and prism-type TIRF microscopy [START_REF] Van | Screening by Imaging: Scaling up Single-DNA-Molecule Analysis with a Novel Parabolic VA-TIRF Reflector and Noise-Reduction Techniques[END_REF] . Fluorescence was excited at 488-and 568-nm wave-(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

length from a polychromatic tuneable light-source (TILL Photonics). After appropriate filtering, fluorescence was detected on a EMCCD camera (Photometrics). METAMORPH and ImageJ software were used for acquisition and image analysis.

For combined TIR-SAF imaging, the same cells were transferred to the home-built inverted TIR-SAF microscope [START_REF] Brunstein | Eliminating Unwanted Far-Field Excitation in Objective-Type TIRF. Part II. Combined Evanescent-Wave Excitation and Supercritical-Angle Fluorescence Detection Improves Optical Sectioning[END_REF][START_REF] Van | Screening by Imaging: Scaling up Single-DNA-Molecule Analysis with a Novel Parabolic VA-TIRF Reflector and Noise-Reduction Techniques[END_REF][START_REF] Van $t Hoff | A Programmable Light Engine for Quantitative Single Molecule TIRF and HILO Imaging[END_REF][START_REF] Brunstein | Eliminating Unwanted Far-Field Excitation in Objective-Type TIRF. Part I. Identifying Sources of Non-evanescent Excitation Light[END_REF] and a tiny sub-region of the cell's footprint containing FM-loaded lysosomes imaged. Equipment and imaging parameters were similar to that described above.

FIG. 1

 1 FIG. 1 Multi-layered sandwich of transparent and fluorescent nanometric films with controlled properties. (a) schematic representation (left) and FIB cross-section (right) of a fabricated sandwich, alternating metallic (gold), My-133-MC polymer (light blue) and dye (green) layers. Metal layers were only added for FIB imaging. Note the thin fluorescent (4-nm) layer of H6-TPPS J-aggregate, and the well-defined layering. (b), absorbance (blue) and fluorescence (red) spectra of TPPS films sandwiched between polymer spacer and capping layers. (c), fluorescence micrograph of the dye layer showing a uniform concentration over large scale. (d), varying spin-coating parameters, ultra-thin layers of thicknesses down to a few nm thickness could be produced. Thickness was measured by Stylus profilometry, (e), that also provided a RMS roughness estimate of 1-2 nm. (f), Ellipsometry of the polymer layers, showing a RI around 1.33 in the visible range.

15 FIG. 2 .

 152 FIG. 2. Supercritical-angle fluorescence (SAF) -based measurement of the refractive-index of ultra-thin transparent layers. (a) Schematic illustration of the sample (inset) and observed radiation patterns by BFP imaging of a 5-nm TPPS Jaggregate layer homogeneously deposited directly on the BK-7 substrate and left in air (top), or else covered a 3-µm thick My-133-MC polymer capping layer (bottom). Fluorescence excitation by TIRF. Image contrast inverted for better clarity (b), radial cross-sections of the images in (a), were largely independent of azimuthal 𝜑. Thin, pale traces are profiles measured at 𝜑 = 0°, 45°, 90° and 135°, respectively. Solid lines are azimuthal averages and are shown as polar plots in (c), revealing the characteristic directional lobes at supercritical angles. Note the change in emission pattern for the polymer-covered dye layer (blue). From the emission critical angle (grey in (b)), we obtained the refractive indices (RIs) of air and My-133-MC polymer, (d). Violin plots show population distributions over n = 129 (air) and 34 (polymer) measurements, respectively.

  FIG. 3 RI homogeneity in ultra-thin polymer layers. (a), dark-field image upon white-light illumination and, (b), electron micrographs of a single plasmonic hole array (left) as well as a larger plasmonic sample comprising several patterns with different periodicities P (in nm, right). (c), Normalized transmitted intensity vs. wavelength upon white-light illumination, showing similar responses of the plasmonic hole array when covered with water (through line) or My-133-MC polymer (dashed). Vertical lines highlight identical peak positions for the plasmonic modes either at interface with water or My-133-MC, respectively.

  Fig. 3c shows transmission spectra of those arrays upon white-light illumination. The same peaks appeared for plasmonic structures covered with either water or My-133-MC polymer, indicating the same RI for both media. Compared to the localized SAF-based RI measurement limited by the field-of-view of the x100 objective (Fig 2), this plasmonic approach intrinsically probes different locations of the polymer layer over >100 µm, indicating that the deposited My-133-MC layers have a homogenous RI over larger length-scales.

Fig. 4c shows

  Fig. 4c shows such an example for which a marked RI change was observed upon wetting the polymer surface. Starting from an air-dominated environment, pipetting a drop of polymer onto the surface changed the

Figure 5 .

 5 Figure 5. SAF-based axial profilometry. (a), schematic representation of a subset of three prepared test slides, featuring a thin TPPS J-aggregate emitter layer sandwiched at different fluorohpore heights Δ between a flat and homogenous My-133-MC spacer and capping layer. (b), corresponding example BFP images after background subtraction and scaled to equal min-max intensity for direct comparison. (c), plot of the SAF/UAF intensity ratio R of supercritical vs. undercriticalangle fluorescence emission components vs. fluorophore height Δ. Symbols are measurements from segmented BFP images as in (b), thin lines are theoretical values calculated using the constant-radiated power dipole of Hellen & Axelrod

Figure 6 .

 6 Figure 6. SAF-based disambiguation of TIRF image data. (a), schematic representation of the intracellular steps characteristic for calcium (Ca 2+ ) -regulated exocytosis. FM dye (green) is a membrane-resident (brown) amphiphilic fluorophore that is internalized into cortical astrocytes and labels a tiny intracellular vesicular compartment called lysosome. These organelles approach the basal plasma membrane of cells cultured on a glass coverslip and are released in a Ca 2+ -dependent manner (arrow). (b) epifluorescence (EPI) image showing a diffuse cytosolic labeling as well as bright spots representing individual lysosomes (arrowheads). (c), zoom on the rectangular region shown in blue on (b). The timelapse image series acquired at 1 Hz shows two lysosomes, "1" and "2". While 2 (solid arrowhead) is present from the beginning to the end of the recording, 2 arrives in frame #3, approaches the membrane and releases its content ("exocytosis") at the moment identified by an open arrowhead. The corresponding axial re-distribution of fluorophores is seen in (d), which examples of BFP images of ROIs (inset images) of basal membrane in cultured cortical astrocytes upon TIRF illumination, with either FM1-43 (left) or the spectrally identical but slightly more hydrophilic FM3-25 dye, right. Note the change in SAF intensity for ROIs showing membrane proximal lysosomes compared to void regions. The rightmost image is an exception because the SAF/UAF intensity ratio R dropped despite an organelle still visible, seen as a spot on the inset SP image. (e), Comparison of R values before (rest) and after (Ca 2+ ) stimulation, showing a systematic trend towards lower ratios, indicative of a loss of near-interface fluorophores consistent with exocytosis The red trace follows the trend but corresponds to the rightmost image on d, where the lysosome apparently moved back into deeper cytoplasmic regions, thus lowering ISAF and hence R.

  (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

	bioRxiv preprint	doi:	; https://doi.org/10.1101/2022.08.15.503956	this version posted August 15, 2022.	The copyright holder for this preprint

Acknowledgements

We thank Daniel Azulai for help with the polymer deposition and Naftali Kanovsky and Prof. Shlomo Margel for advice regarding the polymer. Thierry Bastien provided custom fine mechanics. This study was financed by the CNRS, University Paris Cité, and -mostly -by the European Union (H2020 Eureka! Eurostars, 'NanoScale', E!12848, https://nanoscale.sppin.fr, to MO and AS). AS acknowledges support from the French Ministry of Foreign Affairs and the French Embassy in Tel Aviv (Chateaubriand fellowship). The authors are grateful for mobility support from a Franco-Israeli CNRS-LIA, 'ImagiNano', and the FranceBio-Imaging large-scale national infrastructure initiative (FBI, ANR-10-INSB-04, Investments for the future).

The Oheim lab is a member of the C'Nano Excellence Network in Nanobiophotonics (CNRS GDR2972).


Daniel Sol
email: d.sol@creaf.uab.cat

Ignasi Bartomeus

César González-Lagos

Sandrine Pavoine

Urbanisation and the loss of phylogenetic diversity in birds

Keywords: global change, habitat loss, tolerance to environmental change, conservation of biodiversity, biological invasion

Despite the recognised conservation value of phylogenetic diversity, little is known about how it is affected by the urbanisation process. Combining a complete avian phylogeny with surveys along urbanisation gradients from five continents, we show that highly urbanised environments supported on average 450 million fewer years of evolutionary history than the surrounding natural environments. This loss was primarily caused by species loss and could have been higher had not been partially compensated by the addition of urban exploiters and some exotic species. Highly urbanised environments also supported fewer evolutionary distinctive species, implying a disproportionate loss of evolutionary history. Compared with highly urbanised environments, changes in phylogenetic richness and evolutionary distinctiveness were less substantial in moderately urbanised environments. Protecting pristine environments is therefore essential for maintaining phylogenetic diversity, but moderate levels of urbanisation still preserve much of the original diversity.

INTRODUCTION

Urbanisation is currently considered one of the most rapid and drastic alterations of natural ecosystems, causing an important impact on biodiversity and ecosystem services [START_REF] Mckinney | Urbanization, biodiversity and conservation[END_REF][START_REF] Aronson | A global analysis of the impacts of urbanization on bird and plant diversity reveals key anthropogenic drivers[END_REF]. Some well-documented consequences include a loss of species richness (e.g. [START_REF] Sol | Urbanisation tolerance and the loss of avian diversity[END_REF]) and an increase in biotic homogenization [START_REF] Lockwood | Taxonomic homogenization of the global avifauna[END_REF][START_REF] Mckinney | Urbanization as a major cause of biotic homogenization[END_REF]). Other consequences remain less well studied, however. One is the extent to which urbanisation alters phylogenetic diversity.

Phylogenetic diversity captures the shared ancestry of species both in terms of richness (i.e. amount of evolutionary history) and divergence (i.e. degree at which the species are phylogenetically-related; Vellend et [START_REF] Cadotte | Phylogenetic diversity promotes ecosystem stability[END_REF], few studies have quantified the phylogenetic consequences of converting natural habitats to cities, particularly at a global scale [START_REF] Morelli | Evidence of evolutionary homogenization of bird communities in urban environments across Europe[END_REF]. This is unfortunate considering that future projections predict an increase in urbanised surface by 1.2 million km 2 during the first 30 years of the 21st century [START_REF] Seto | Global forecasts of urban expansion to 2030 and direct impacts on biodiversity and carbon pools[END_REF].

The process of urbanisation may affect phylogenetic patterns in a number of ways. One is by reducing phylogenetic richness. As most species do not tolerate well human-driven alterations [START_REF] Sol | Urbanisation tolerance and the loss of avian diversity[END_REF], increased urbanisation may decrease phylogenetic richness in parallel with species loss. Yet other consequences are less obvious. If for instance the adaptations needed to thrive in cities are non-randomly distributed across the tree of life, the urbanisation process could lead to a disproportionate loss of evolutionary history [START_REF] Nee | Extinction and the Loss of Evolutionary History[END_REF]. This loss might result if more vulnerable species are evolutionary distinctive or unique, species lost are clustered within the phylogeny and the phylogeny is imbalanced [START_REF] Heard | Phylogenetically patterned speciation rates and extinction risks change the loss of evolutionary history during extinctions[END_REF][START_REF] Purvis | Nonrandom extinction and the loss of evolutionary history[END_REF][START_REF] Huang | How global extinctions impact regional biodiversity in mammals[END_REF][START_REF] Veron | Predicting loss of evolutionary history: Where are we? Biological reviews[END_REF]; see also Appendix S1). There is some evidence suggesting a loss of evolutionary distinctiveness in human-altered communities [START_REF] Fritz | Selectivity in mammalian extinction risk and threat types: A new measure of phylogenetic signal strength in binary traits[END_REF][START_REF] Frishkoff | Loss of avian phylogenetic diversity in neotropical agricultural systems[END_REF][START_REF] Morelli | Evidence of evolutionary homogenization of bird communities in urban environments across Europe[END_REF]), but whether this reflects a higher sensitivity of evolutionary distinct species to urbanisation is less clear. Urbanisation may alter the phylogenetic diversity of communities in yet another way, by facilitating the entrance of opportunistic species. While most species do not seem to tolerate well the extreme environmental alterations associated with urbanisation, and hence tend to avoid them, some perceive urban environments as ecological opportunities [START_REF] Blair | Land use and avian species diversity along an urban gradient[END_REF]. These so-called urban exploiters, some of which have an exotic origin, dominate urban communities despite being often absent or rare in the surrounding natural environments [START_REF] Case | Global patterns in the establishment and distribution of exotic birds[END_REF][START_REF] Møller | Urbanized birds have superior establishment success in novel environments[END_REF]. They thus provide a net gain in the regional phylogenetic richness. Despite this compensatory role, invasions by exotic and native urban exploiters can deeply alter the phylogenetic composition of urban community if they belong to different lineages than the species they replace [START_REF] Ricciardi | Distinctiveness magnifies the impact of biological invaders in aquatic ecosystems[END_REF][START_REF] Li | The effects of phylogenetic relatedness on invasion success and impact: Deconstructing Darwin's naturalisation conundrum[END_REF][START_REF] Sobral | Introductions do not compensate for functional and phylogenetic losses following extinctions in insular bird assemblages[END_REF]). This would be particularly true if, as the Darwin's naturalization hypothesis suggests, invaders are more successful in communities in which their close relatives are absent [START_REF] Duncan | Darwin's naturalization hypothesis challenged[END_REF][START_REF] Diez | Darwin's naturalization conundrum: dissecting taxonomic patterns of species invasions[END_REF][START_REF] Thuiller | Resolving Darwin's naturalization conundrum: a quest for evidence[END_REF]).

Here, we combine a full phylogeny of birds [START_REF] Jetz | The global diversity of birds in space and time[END_REF]) with information on wellsurveyed assemblages along urbanisation gradients from five continents to address three insufficiently understood questions: 1) How much phylogenetic richness, if any, is lost when natural habitats are urbanised? 2) Is this loss simply the result of species loss or also due to increasing species relatedness and a disproportionate loss of evolutionary distinctive species? And 3) does the presence of native and exotic urban exploiters help compensate the loss of phylogenetic richness? Given the obvious difficulties of conducting a before-after experiment, we addressed these questions by adopting a space-for-time substitution approach in which phylogenetic richness and divergence were compared between urbanised environments and the surrounding, little urbanised environments.

METHODS

Data

We compiled a global data set of species abundances per unit area or unit time for bird assemblages in 27 regions from Europe, North America, Australia, Africa and South America (Appendices S2 and S3). Information came from published studies and our own surveys [START_REF] Sol | Urbanisation tolerance and the loss of avian diversity[END_REF]. The data included 185,908 bird detections belonging to 1,219 species, 47 of which were exotic in at least one region.

For each region, spatially-defined bird assemblages were available for urbanised environments and the surrounding little urbanised environments (n = 161 assemblages; see data in Appendix S3). To define the degree of urbanisation, we differentiated [START_REF] Aronson | A global analysis of the impacts of urbanization on bird and plant diversity reveals key anthropogenic drivers[END_REF] highly urbanised environments, where buildings were densely packed and parks were small or absent, and ( 2 Although the same survey method was used for each habitat within a particular region, comparisons across habitats raise issues about detectability and sampling effort (Lahoz-Monfort et al. 2014). We tackled this by 1) testing whether certain methodological decisions could bias the results, 2) rarefying communities to compare phylogenetic richness with similar sampling effort, 3) taking into account the relative abundance of the species to reduce the influence of rare species, and 4) considering in the models geographic and human-related factors that could influence variation in phylogenetic diversity (see details in the supplementary Methods).

Phylogenetic information

Our analyses were based on a complete, dated phylogeny of birds [START_REF] Jetz | The global diversity of birds in space and time[END_REF]). We dealt with phylogenetic uncertainties in two ways. First, we built a maximum clade credibility phylogeny (CCP) with the TREEANNOTATOR software in BEAST [START_REF] Drummond | Bayesian phylogenetics with BEAUti and the BEAST 1.7[END_REF]) based on 200 phylogenies from the pseudoposterior distribution of 10,000 trees available in www.birdtree.org. Second, we repeated the most important analyses with 50 phylogenies randomly chosen from the same pseudoposterior distribution.

Loss of phylogenetic richness in urbanised environments

We used the phylogenies to estimate Faith's phylogenetic diversity (PD, hereafter) for each bird assemblage. PD was measured as the sum of the lengths of all those branches that were members of the corresponding minimum spanning path [START_REF] Faith | Conservation evaluation and phylogenetic diversity[END_REF]). To account for differences in sampling effort among habitats, we also calculated PD rarefied by randomly sampling individuals from the regional pool; the sample size was the minimum abundance among all assemblages within a region [START_REF] Cardoso | BAT -Biodiversity Assessment Tools, an R package for the measurement and estimation of alpha and beta taxon, phylogenetic and functional diversity[END_REF].

To further assess the influence of rare species on phylogenetic diversity measures [START_REF] Fine | Phylogenetic community structure and phylogenetic turnover across space and edaphic gradients in western Amazonian tree communities[END_REF], we then used a recently proposed phylogenetic diversity metric based on Hill numbers (PDw hereafter) that is sensitive to both species abundance and phylogenetic distances [START_REF] Chao | Phylogenetic diversity measures based on Hill numbers[END_REF]. We set the q parameter to 1, meaning that a species weight in the Hill number was proportional to its abundance.

To assess whether the loss of phylogenetic richness merely resulted from species loss, we standardized PD and PDw (PDI and PDIw, hereafter) by subtracting from the observed values the mean values expected under a null model in which taxa labels were shuffled across the tips of the regional phylogeny 999 times. This null model generates randomized phylogenetic relationships among taxa while maintaining the observed phylogenetic tree, species abundance distribution, and community structure. The effect size was then divided by the standard deviation of the randomized values. The more negative PDI and PDIw, the more phylogenetically clustered species were locally compared to the regional species pool.

All the above metrics were estimated for each assemblage with the R-packages "Picante" [START_REF] Kembel | Picante: R tools for integrating phylogenies and ecology[END_REF] and "BAT" [START_REF] Cardoso | BAT -Biodiversity Assessment Tools, an R package for the measurement and estimation of alpha and beta taxon, phylogenetic and functional diversity[END_REF] as well as our own scripts. Differences across urbanised and little urbanised environments were then evaluated by means of linear mixed models, using the Bayesian approximation implemented in the R-package 'MCMCglmm' [START_REF] Hadfield | MCMC methods for multi-response generalized linear mixed models: the MCMCglmm R package[END_REF]. As the response variables were continuous and exhibited "bell curve" shapes, we used models with a Gaussian structure of errors. Region was included as random effect to allow phylogenetic richness to be compared within urbanisation gradients. To account for spatial autocorrelation, region was nested within country and in some models we included geographic longitude and/or distance to Equator as fixed effects (see Appendix S4 for justifications). The survey method, sampling effort and whether detectability was or was not taken into account were also included as fixed effects, along with season, altitude, urbanisation period and urban area (see also [START_REF] Sol | Urbanisation tolerance and the loss of avian diversity[END_REF]. To check for model convergence, we ran the model twice with different starting values, sampling 1000 iterations from a total of 1100000. When the model did not adequately converge, we sequentially increased the total number of iterations and number of iterations passed before samples were stored. We further explored the influence of species loss on PD decline by means of Piecewise structural equation modelling [START_REF] Lefcheck | piecewiseSEM : Piecewise structural equation modeling in R for ecology , evolution, and systematics[END_REF].

Changes in phylogenetic composition with urbanisation

To quantify dissimilarity in phylogenetic composition among urbanised and the surrounding little urbanised environments, we estimated UniFrac β-diversity indices, a derivation of the Jaccard dissimilarity index that quantifies the proportion of shared branch length between pairs of assemblages [START_REF] Leprieur | Quantifying phylogenetic beta diversity: Distinguishing between "true" turnover of lineages and phylogenetic diversity gradients[END_REF]). We compared the indices for each combination of habitats by means of the MCMCglmm approach outlined above. Then, we separated 'True' turnover of lineages (turnover, hereafter) from phylogenetic diversity gradients (nestedness), following [START_REF] Leprieur | Quantifying phylogenetic beta diversity: Distinguishing between "true" turnover of lineages and phylogenetic diversity gradients[END_REF]. A predominance of turnover over nestedness would imply that β-diversity does not merely reflect a reduction of diversity but a different combination of phylogenetic lineages.

As we detected substantial turnover, we estimated the fraction of PD coming from exploiters (see definitions below), exotics and non-exploiters. To this purpose, we estimated the evolutionary distinctiveness (ED) of the three groups based on subtrees only containing the species of the assemblage. ED was calculated as the weighted sum of the edge lengths from the root to a tip of the tree, with the weights being 1/number of species that share that edge [START_REF] Isaac | Mammals on the EDGE: conservation priorities based on threat and phylogeny[END_REF]). Following [START_REF] Sol | Urbanisation tolerance and the loss of avian diversity[END_REF], we operationally defined an exploiter as a native species that was more abundant in the urbanised environment than expected by their abundance in the little urbanised surrounding environments (see [START_REF] Blair | Birds and butterflies along urban gradients in two ecoregions of the U.S. In: Biotic Homogenization[END_REF] for a more general definition). We also used a second, less restrictive classification in which we also considered exploiter those species that despite exhibiting abundances lower than expected by chance were still abundant in highly urbanised environment. We used a threshold of relative abundance > 5%, but the use of a higher threshold did not alter the conclusions.

Phylogenetic effects of urbanisation tolerance

Using the distinction between exploiter and non-exploiter, we investigated phylogenetic effects in the tolerance of species to urbanisation in two ways. First, we measured its phylogenetic signal by means of the D statistic (Fritz & Purvis 2010), as implemented in the R-package Caper [START_REF] Orme | Caper: Comparative Analyses of Phylogenetics and Evolution in R. R Packag[END_REF]. Second, we tested whether some avian families had more or less exploiters than expected by chance by means of randomizations, as described in the Supplementary methods. We illustrate the results with phylogenetic reconstructions based on the functions "contMap" and "plotSimmap" from the Phytools R-package [START_REF] Revell | Phytools: an R package for phylogenetic comparative biology (and other things)[END_REF]).

Evolutionary distinctiveness loss with urbanisation tolerance

To assess whether urban communities maintained less phylogenetically distinctive species, we again calculated the weighted sum of the edge lengths from the root to a tip of the tree [START_REF] Isaac | Mammals on the EDGE: conservation priorities based on threat and phylogeny[END_REF]). In this case, however, ED was estimated based on the entire phylogeny of 9,993 avian species (EDg, hereafter). EDg of each assemblage was estimated as the average value for all species present and modelled as a function of habitat with the Gaussian MCMCglmm approach outlined in previous sections. To help interpret differences in EDg, we also estimated evolutionary uniqueness (EU), measured as the length of the terminal branch of the species.

Using again the distinction between exploiter and non-exploiter, we asked whether vulnerable species fall within species poor, phylogenetically isolated clades by comparing EDg between urban exploiters and avoiders by means of a binomial MCMCglmm. Because being an exploiter does not only depend on the features of the species itself but also on how it interacts with other species from the region, we conducted the analyses at the assemblage level and included region and country as random factors in a nested structure together with the species identity and phylogeny.

RESULTS

Loss of native phylogenetic richness in urbanised environments

Highly urbanised environments supported on average 458.7 ± 346.8 million fewer years of evolutionary history than the surrounding little urbanised environments (pMCMC < 0.001; Figs. 1-2, S1-S2; Tables S1-S2). A decline in PD was also observed when considering the relative abundance of species, yet in this case the decline was less pronounced (Fig. 1, Tables S3). Compared with highly urbanised environments, the loss of PD in moderately urbanised environments relative to surrounding environments was less dramatic (211.4 ± 398.4 millions of years; Fig. 1, Tables S1-S3).

The observed decline in PD with urbanisation was robust to the effect of phylogenetic uncertainties (Fig. S3) and a set of potentially confounding variables such as urbanisation period, urbanised area and distance to the Equator (see Tables S1-S3).

Moreover, the decline was highly consistent regardless of the nature of the surrounding little-urbanised habitats (Fig. S4). Although the decline in PD was more accentuated in some regions than in others, we did not detect any clear latitudinal or longitudinal trend (Appendix S4). The amount of PD loss in a region was also unrelated to species richness or the duration and extension of the urban alterations (Appendix S4).

Mitigation of PD loss by the addition of native urban exploiters

Differences in phylogenetic β-diversity were particularly important in transitions from little to highly-urbanised environments (Table S4, Figs. 3,S5). In these transitions, there was a predominance of turn-over relative to nestedness (Figs. 3,S5), reflecting the existence of some species that were only present in urbanised environments. In many regions, exploiters explained a substantial fraction of total variation in PD (Fig. S6).

Native species richness as a driver of PD loss with urbanisation

The net loss of PD with urbanisation may result of two processes (see Appendix S1): species loss and increasing species relatedness [START_REF] Frishkoff | Loss of avian phylogenetic diversity in neotropical agricultural systems[END_REF]). We found clear evidence for a main role of species loss. Even when considering the gain in species due to the entrance of obligate native exploiters, native species richness in highly urbanised environments was on average over two times lower than that of the surrounding little urbanised environments (Fig. 2). Once phylogenetic richness was standardized by estimating PDI and PDIw, phylogenetic richness no longer decreased with urbanisation but increased (Fig. 2, Tables S5-S6). Piecewise structural equation modelling further confirmed the influence of species richness, showing that in the best supported causal scenarios habitat affected PD indirectly through its influence on species richness (Fig S7 ).

Phylogenetic effects in tolerance to urbanisation

Urban exploiters were more likely to be found in certain clades, as reflected by the existence of phylogenetic signal (Fig. S8). Although a wide array of clades across the avian phylogeny thrived in highly urbanised environments (Figs. 4 andS9), urban exploiters tended to be overrepresented in a few distantly-related families that exhibit relatively low EDg (Table S7; Fig. 4). Some of these families also contained highly successful invaders (Table S8).

Loss of evolutionary distinctiveness with urbanisation

Mean evolutionary distinctiveness (EDg) of native species assemblages tended to be lower in highly urbanised environments compared to little urbanised environments (pMCMC < 0.001 in all cases; Table S9, Fig. 5). This largely reflected the effect of evolutionary uniqueness (Table S10), a metric strongly correlated with EDg (Fig. 5).

The decrease in EDg with urbanisation was in part the result of a decline in species richness (Fig. S7). However, it also reflected a lower tolerance of evolutionary distinctive species to urbanization. Species with lower EDg were more likely to be urban exploiters (pMCMC < 0.001 in all cases; Tables S11-S12, Fig. 5). Although EDg increased at higher geographic longitudes (Tables S9-S10, see also [START_REF] Jetz | The global diversity of birds in space and time[END_REF], the loss of EDg with urbanisation was largely independent of this effect (Tables S11-12). The decline in EDg with urbanisation was more accentuated in regions with higher mean community evolutionary distinctiveness (Appendix S4). Other factors like species richness, urbanisation period, urban area, geographical longitude and distance to Equator had no effect on how much EDg was lost (Appendix S4).

Compensation of phylogenetic diversity by exotics in urbanised environments

Exotic species did not fully compensate for the loss of PD and EDg associated with urbanisation. Despite that exotic species tended to attain higher success in urbanised habitats (Table S8), the loss of PD and EDg with urbanisation was still substantial when including exotic birds in the analyses (Figs. S6, S10; Table S13). This in part resulted from the reduced number of exotic species present (range 0-17). In places where this number was high, like in New Zealand, the compensatory effect was more substantial (see Fig. S6). However, the low compensatory effect of exotic species also reflected that were not more evolutionary distinctive than the native species they replaced (Binomial MCMCglmm: P = 0.65 and 0.63, depending on the tolerance metric, Fig. 5).

DISCUSSION

Despite the growing concern over the impact of rapid human-induced environmental changes on biodiversity, assessments of how the conversion of natural habitats to cities affects phylogenetic diversity have been rare (but see [START_REF] Morelli | Evidence of evolutionary homogenization of bird communities in urban environments across Europe[END_REF]. Our comprehensive analysis contributes to fill this gap by showing that at a global scale urbanisation generally causes a significant decline in phylogenetic richness, an impact that varies across regions. The decline increased with the intensity of the urbanisation and was highly consistent regardless of the nature of the surrounding little urbanised habitats. Rarefactions suggest that the pattern was not merely the result of smaller community sizes in urbanised environments. However, phylogenetic richness loss was less pronounced when considering the relative abundance of species, perhaps reflecting phylogenetic redundancies and/or that the loss was partially compensated by an increase in abundance evenness across the phylogeny in urbanised environments.

By homogenizing the physical environment to meet the demands of human beings, cities may be creating filters that determine that only some lineages can persist in these new environments [START_REF] Mckinney | Urbanization as a major cause of biotic homogenization[END_REF]. Our results confirm this expectation, showing that tolerance to urbanisation was clustered within the phylogeny (see also [START_REF] Sol | Urbanisation tolerance and the loss of avian diversity[END_REF]). Thus, although a wide array of clades across the avian phylogeny thrived in highly urbanised environments, urban exploiters tended to be overrepresented in a few families. These include species-rich, widely distributed families like pigeons, crows, finches, swallows, and grackles, which are often represented by one or a few species in Even though urban exploiters were phylogenetically clustered, there was no evidence that this significantly contributed to the reduced phylogenetic richness of urban communities. Rather, most of the observed loss of phylogenetic richness associated with urbanisation was explained by species loss. Once standardized with values obtained from random expectations, phylogenetic diversity no longer decreased with urbanisation but increased. This implies that urban communities tended to have more phylogenetic richness than expected by the number of species present, notably when the intensity of urbanisation was high. The pattern may be interpreted as species in urbanised environments being more distantly related to other species, or at least equally distantly related, than species from natural, surrounding communities.

Despite sustaining a higher phylogenetic richness than expected by species richness, the average evolutionary distinctiveness of birds in highly urbanised environments tended to be lower compared to little urbanised environments. [START_REF] Morelli | Evidence of evolutionary homogenization of bird communities in urban environments across Europe[END_REF] also found that urbanisation caused an important loss of avian evolutionary distinctiveness in European cities. The loss of evolutionary distinctiveness was primarily driven by the loss of pendant edges, a pendant edge being the terminal branch that connects a species to the rest of the tree (see also [START_REF] Redding | Measuring evolutionary isolation for conservation[END_REF], and was higher in regions that held more evolutionary distinctive species. Therefore, it is possible that the loss of phylogenetic diversity has been sub-estimated by including in our analyses regions where most evolutionary distinctive species were extirpated a long time ago.

Unlike differences in phylogenetic richness, the decrease in the mean community evolutionary distinctiveness with urbanisation was not merely the result of a decline in species richness. Rather, it also reflected a lower tolerance of evolutionary distinctive species to urbanization, in line with the overrepresentation of threatened species in poorly diversified clades [START_REF] Bennett | Variation in extinction risk among birds: chance or evolutionary predisposition?[END_REF]. While the mechanisms remain unclear, one possible explanation is that these species belong to clades that have historically had difficulties to cope with environmental changes, and hence may have experienced high extinction rates and/or reduced speciation rates.

Simulations show that a reduction in evolutionary distinctiveness has little impact on phylogenetic richness if not accompanied by phylogenetic imbalance and by the phylogenetic clustering of species loss [START_REF] Veron | Predicting loss of evolutionary history: Where are we? Biological reviews[END_REF]). Thus, a loss of evolutionary distinctive species does not necessarily imply a loss of phylogenetic richness. Still, the finding that evolutionary distinctive species often exhibit lower tolerance to urbanisation suggests that transforming natural habitats to cities can have an impact on the tree of life (see also [START_REF] Frishkoff | Loss of avian phylogenetic diversity in neotropical agricultural systems[END_REF].

The observed loss of phylogenetic richness in cities could have been more important if it was not for the arrival of a number of urban exploiters, which dominated these communities despite being rare or absent in the surrounding little urbanised environments. As these "obligate" urban exploiters tended to be over-represented in a few distantly-related clades and often lacked close-relatives in the same community, their presence may contribute to explain why highly urbanised environments had a higher phylogenetic richness than expected by the number of species they sustained. Birds successfully thriving in urbanised environments therefore represent to some extent novel communities featuring a small number of distantly related taxa from little evolutionary distinctive clades. Our findings support thus claims that the ongoing global changes driven by human activities do not merely reduce biodiversity but also lead to the emergence of novel communities with different phylogenetic and functional properties [START_REF] Lurgi | Novel communities from climate change[END_REF].

Although exotic species tended to concentrate in urbanised habitats (see [START_REF] Sol | Why are exotic birds so successful in urbanized environments?[END_REF], their contribution to increasing phylogenetic richness was quite modest. This is because the number of exotics species within the studied regions was generally low.

Moreover, exotic species tended to belong to little evolutionary distinctive clades. Thus, the replacement of native species by exploiters and exotics can maintain or even increase taxonomic diversity, but nevertheless reduce part of the evolutionary history of the community [START_REF] Sobral | Introductions do not compensate for functional and phylogenetic losses following extinctions in insular bird assemblages[END_REF]; but see [START_REF] Garcia | Exotic birds increase generalization and compensate for native bird decline in plant-frugivore assemblages[END_REF]. As exotic birds are overrepresented in a few clades [START_REF] Blackburn | Establishment patterns of exotic birds are constrained by non-random patterns in introduction[END_REF], they should also contribute to increase phylogenetic homogenization of urbanised environments.

Our global analysis provides evidence that high levels of urbanisation reduce and alter phylogenetic diversity, favouring a few species with many close relatives at the expense of species from more evolutionary distinct clades. Phylogenetic diversity has intrinsic conservation value because it does not only take into account species richness but also captures genetic diversity, ecosystem functionality and evolutionary history [START_REF] Faith | Conservation evaluation and phylogenetic diversity[END_REF] Only families with 15 or more species are presented. For more details, see Table S7. S9 andS13). Below, relationship between EDg and EU across species (left) and differences in EDg native exploiter, native non-exploiter and exotic species (see Tables S11 andS12 and main text). In all cases, ED has been estimated based on the complete phylogeny of all extant bird species [START_REF] Jetz | The global diversity of birds in space and time[END_REF]. Letters indicate differences among habitats at P < 0.05.

a

  given region but by distinct species in different regions. In pigeons, for example, urban representatives include speckled pigeons (Columba guinea) in Africa, Collared Doves (Streptopelia decaocto) in Eurasia, Crested pigeons (Ocyphaps lophotes) in Australia, and Zenaida doves (Zenaida aurita) in the Caribbean. The homogenization of the physical environment of cities results thus in increased phylogenetic homogenization (McKinney 2006).

  ; Nee & May 1997; Purvis et al. 2000; Huang et al. 2011). While preserving little urbanised environments is crucial for maintaining phylogenetic diversity across the tree of life, our results are in line with Frishkoff et al. (2014) that moderate intensities of environmental alterations may protect against extreme loss of phylogenetic diversity.

Figure legends Figure 1 .

 legends1 Figure legends

Figure 2 .

 2 Figure 2. Differences in phylogenetic richness (PD, in million years), rarefied phylogenetic richness (PDrarefied), phylogenetic richness taking into account species relative abundance (PDw), species richness (SP), and standardized effect sizes of PD (PDI) and PDw (PDIw) among little urbanised (low), moderately-urbanised (mod) and highly-urbanised (high) environments for native species. Letters indicate differences among habitats at P < 0.05 (see Tables S1-3, S5-6 for full models), examined by changing the level of reference with which the other habitats are compared.

Figure 3 .

 3 Figure 3. UniFrac β-diversity index comparing differences between different habitats (above) and how is affected by turnover and nestedness (below). High = highlyurbanised, Mod = moderately-urbanised, Low = little urbanised; see TableS4for formal analyses.

Figure 4 .

 4 Figure 4. Left panel, family level variation in the likelihood to contain urban exploiters. Values close to 0 indicate that the family has more urban exploiters than expected by chance whereas values close to 1 suggest that the family has less exploiters than expected by chance. States of internal nodes are Maximum Likelihood reconstructions as implemented in the function "contMap" of Phytools (Revell 2011).

  Right panel, EDg averaged for all the species of each family and standardised by substracting the EDg averaged across all avian families. Confidence intervals represent ± standard deviations. Values below the vertical line indicate that the family has an EDg lower than the mean estimated for all the avian families.

Figure 5 .

 5 Figure 5. Above, community mean differences in evolutionary distinctiveness (EDg),among little urbanised (low), moderately-urbanised (mod) and highly-urbanised (high) environments, considering or not exotic species (see TablesS9 and S13). Below,

Fig. 1

 1 Fig. 1

  Fig.3

  Fig.4

  al. 2011; Tucker et al. 2016; Veron et al. 2016). Despite the increased appreciation of the intrinsic value of phylogenetic diversity, mainly due to its relevance for preserving evolutionary history (Nee & May 1997; Purvis et al. 2000; Huang & Roy 2013; Turley & Brudvig 2016) and ecosystem functioning

ACKNOWLEDGMENTS

We thank all authors who published the information on their intensive urban avian surveys, without which this study could not have been possible. We are also in debt with Joan Garcia-Porta for generating the CCP and Dario Moreira for helping assemble the dataset. We finally thank Phillip Clergeau, Mariana Villegas and Ivan Diaz for providing unpublished data, María Moirón, Gabriel Garcıa-Peña, Miquel Vall-llosera, Louis Lefebvre and three anonymous reviewers for insightful comments, Walter Jetz and co-workers for making available their phylogeny of birds, and the Rteam and package contributors for the R free software. This paper is part of the project CGL2013-47448-P from the Spanish Government to DS and IB. CG-L was supported by the grant PUC1203 MECESUP-Pontifica Universidad Católica de Chile.