Alexandre Bancet

Claire Raingeval

Thierry Lomberget

Marc Le Borgne

Jean-François Guichou

Isabelle Krimm

Astex

Fragment Linking Strategies for Structure-Based Drug Design

where the linker is duplicated from a known inhibitor appears as an interesting strategy. To reach super-additivity, we propose to grow one of the fragments in order to minimize the distance between the two binders, and then link the resulting compounds using flexible alkyl-derived linkers.

INTRODUCTION

Fragment-based drug discovery (FBDD) has been acknowledged in the past two decades as a powerful tool for rational design of drug leads. [1][START_REF] Hajduk | A decade of fragment-based drug design: strategic advances and lessons learned[END_REF][START_REF] Chessari | From fragment to clinical candidate -a historical perspective[END_REF][START_REF] Murray | Structural biology in fragment-based drug design[END_REF][START_REF] Erlanson | Twenty years on: the impact of fragments on drug discovery[END_REF][START_REF] Mortenson | Fragment-to-lead medicinal chemistry publications in 2017[END_REF] The first step of the FBDD process consists in the identification of fragments that weakly bind the target protein, typically in the micromolar to millimolar affinity range.

Fragments are defined as low molecular weight (< 300 g/mol) and highly soluble organic molecules.

Jhoti and co-workers 7 proposed a rule of three to describe the physicochemical properties of the fragments, by analogy to Lipinski's rule of five. Due to the weak affinity of the fragments for their targets, biophysical techniques sensitive towards micromolar to millimolar affinities are required to identify hits; among them Nuclear Magnetic Resonance (NMR), X-ray crystallography and Surface Plasmon Resonance (SPR) are the most widely used. 1,[START_REF] Hajduk | A decade of fragment-based drug design: strategic advances and lessons learned[END_REF][START_REF] Chessari | From fragment to clinical candidate -a historical perspective[END_REF][START_REF] Erlanson | Twenty years on: the impact of fragments on drug discovery[END_REF] In a second, iterative and time-consuming step, the fragments that form high quality interactions are optimized into lead compounds exhibiting higher affinity and selectivity, through the so-called fragment growing, fragment merging or fragment linking strategies. As we will see in the examples discussed below, fragment linking may include growing or merging steps and distinguish between the three strategies may be ambiguous.

Vemurafenib, [START_REF] Bollag | Clinical efficacy of a RAF inhibitor needs broad target blockade in BRAFmutant melanoma[END_REF] the first drug originating from a fragment screen, was approved in 2011 by the Food and Drug Administration (FDA), and two other drugs (venetoclax [START_REF] Souers | ABT-199, a potent and selective BCL-2 inhibitor, achieves antitumor activity while sparing platelets[END_REF] and erdafitinib 10 ) were approved in 2016 and 2019, respectively. Vemurafenib and erdafitinib exemplify the growing approach, while venetoclax illustrates the linking method. To have an overview of the fragment-based development of therapeutic compounds, we report in Table 1 the 43 small-molecules currently in clinical trials (phase 1, 2 and 3) and the 3 FDA-approved compounds. We indicate the therapeutic target and the fragment-based strategy used. As reported in Table 1, most of the compounds in the clinic were obtained using the growing approach, where chemical groups are progressively added to the fragment hit to maximize the favorable interactions with the binding site residues. In some cases, fragment hits can be merged, as reported for the induced myeloid leukemia cell differentiation protein (MCL1) (Table 1). Here, we focus on the fragment linking approach, where two fragments that bind at distinct protein pockets are linked together to obtain a new compound. While this approach theoretically represents a very attractive methodology to rapidly gain in binding affinity from the initial fragments, it faces two major challenges. First, the observation of two fragments binding simultaneously at two distinct binding pockets of a protein is not systematic. This is related to the non-equivalence of the corresponding hot-spots in the binding pockets.

As reported in Table 2, a second screening to identify fragments capable to bind in the second pocket may be necessary. In addition, the fragments do not necessarily bind to the protein in suitable positions for linking. The second challenge remains the design of a linker that will maintain the protein-fragment interactions. Fesik and co-workers [START_REF] Shuker | Discovering high-affinity ligands for proteins : SAR by NMR[END_REF][START_REF] Hajduk | Discovering high-affinity ligands for proteins[END_REF] first successfully used the fragment linking methodology through their Structure-Activity Relationships (SAR) by NMR approach (Figure 1), with the generation of nanomolar compounds for the FK506-binding protein (FKBP), a prolyl isomerase belonging to the immunophilin family. Due to its binding properties to the immunosuppressant molecule FK506, FKBP is a target for neurodegenerative disorders.

In 2011, Whittaker and colleagues [START_REF] Ichihara | Compound design by fragment-linking[END_REF] reviewed examples where the fragment linking approach was used and focused their discussion on the super-additivity concept. The concept of super-additivity (also called positive cooperativity) was defined by Jencks. [START_REF] Jencks | On the attribution and additivity of binding energies[END_REF] It stipulates that the affinity of the linked compound should be greater that the affinity resulting from the addition of the binding energies of the two fragments. The super-additivity energetic term includes changes in translational and rotational entropy, loss of binding energy due to the linker and change in binding orientation of the two fragments. When the orientation of the fragments is not disturbed, changes in translational and rotational entropy dominate. Whittaker and colleagues [START_REF] Ichihara | Compound design by fragment-linking[END_REF] proposed that one key criterion to achieve super-additivity is to maintain the binding modes of the initial fragments. To be successful, linking should therefore involve one fragment that interacts with the protein through polar interactions and a second fragment that interacts via van der Waals interactions, as the latter will be more tolerant to changes in binding mode. This statement will be further discussed later in the perspective.

Here we have reviewed, to the best of our knowledge, all cases where the fragment linking approach has been used, through 2018. To our point of view, the first step that consists in the identification of two fragment hits is not straightforward. As we report below, two fragments bound in two distinct protein pockets were directly identified in 22 cases out of 39, while 17 cases required the conduction of two successive screens. Once two fragments have been identified, the main challenge is the design of the linker, in particular when the distance between the two fragments is large (> 3Å). One possibility consists in reducing the distance between the two fragments. To do so, one of the two fragments is selected and a growing approach is applied until the distance is close to 3Å. Regarding the chemical structure of the linker, simple linkers such as alkyl of different lengths are widely used. More sophisticated linkers include functional groups, which may be utilized to fit particular geometries such as 90° bends required to conserve the positions and orientations of the fragments. Such functionalized linkers also enable the addition of interactions between the designed compound and the protein.

The article is organized as follows. First, we summarize the methods used for the identification of the two fragments that will be further linked. This includes successive identification as well as simultaneous identification of the two fragment hits. We also review the particular case of fragment dimerization.

Then we discuss the second challenge of the linking process and analyze different types of linkers that have been designed to generate the new linked compounds.

Figure 1.

Fragment linking approach with venetoclax. The initial fragments discovered through "SAR by NMR" were linked with an alkene moiety. Navitoclax, exhibiting high affinity for both BCL-XL and BCL-2, was generated and finally, medicinal chemistry led to venetoclax, a selective BCL-2 inhibitor.

The drug was first approved for use in patients with chronic lymphocytic leukemia and 17p deletion.

SECOND-SITE SCREENING FOR IDENTIFICATION OF A SECOND FRAGMENT

As widely reported in the literature, it can be necessary to perform a second screening to identify a fragment bound in another binding site (Table 2). To do so, it may be worth occupying the first site to block its entry. This is partly due to the fact that the fragments typically bind a preferred hot-spot on the protein surface. Moreover, the detection of second-site ligands is generally more difficult because these fragments tend to have a weaker affinity for the target than the first-site ligands. We present below six different strategies that have been used in the literature to identify second-site fragments. The strategies are illustrated in Figure 2.

Screening using protein-observed NMR or "SAR by NMR". NMR is an established technique

for fragment screening and is particularly attractive for linking strategies. In protein-detected experiments, the protein is typically [START_REF] Hajduk | Discovery of potent nonpeptide inhibitors of stromelysin using SAR by NMR[END_REF] N or [START_REF] Ichihara | Compound design by fragment-linking[END_REF] C labeled and two-dimensional heteronuclear NMR experiments are used to observe the protein NMR peak modifications upon ligand addition. Most of the published cases deal with small to medium-sized proteins, such as protein domains, for which the NMR assignment is not a difficult task. Fesik and collaborators [START_REF] Shuker | Discovering high-affinity ligands for proteins : SAR by NMR[END_REF][START_REF] Hajduk | Discovering high-affinity ligands for proteins[END_REF] at Abbott established the "SAR by NMR" approach to design a potent inhibitor of the FKBP binding domain. Two fragments bound in two pockets were identified from the NMR experiments and a linker was elaborated using structural data from 15 N- [START_REF] Ichihara | Compound design by fragment-linking[END_REF] C-filtered protein-ligand Nuclear Overhauser Effect (NOE) (Table 2, entry 1). With the same approach, a linked compound was designed for the stromelysin protein (Table 2, entry 2). [START_REF] Hajduk | Discovery of potent nonpeptide inhibitors of stromelysin using SAR by NMR[END_REF] Navitoclax, the orally active anti-cancer drug, [START_REF] Oltersdorf | An inhibitor of BCL-2 family proteins induces regression of solid tumours[END_REF][START_REF] Petros | Discovery of a potent inhibitor of the antiapoptotic protein Bcl-XL from NMR and parallel synthesis[END_REF][START_REF] Tse | ABT-263: A potent and orally bioavailable Bcl-2 family inhibitor[END_REF] which led to venetoclax, [START_REF] Souers | ABT-199, a potent and selective BCL-2 inhibitor, achieves antitumor activity while sparing platelets[END_REF] was discovered using the SAR by NMR strategy (Figure 1). In another study, the group of Hadjuk 19 designed a highly potent B-cell lymphoma 2 (BCL-2) inhibitor using a library of 17,000 compounds for a first screen and a small library of 70 compounds for a second screen (Table 2, entry 3). Another specific inhibitor of B-cell lymphoma-extralarge (BCL-XL) was designed by Souers and colleagues. [START_REF] Tao | Discovery of a potent and selective Bcl-XL inhibitor with in vivo activity[END_REF] In this case, the first site was blocked using a compound derived from High-Throughput Screening (HTS) and medicinal chemistry. 875 fragments were screened to identify a second-site ligand that could be linked to the first-site compound (Table 2, entry 4). The fragment linking approach was also successfully applied to the heat shock protein 90 (HSP90) (Table 2, entry 5) [START_REF] Huth | Discovery and Design of Novel HSP90 inhibitors using multiple fragment-based design strategies[END_REF] and to the protein tyrosine phosphatase-1B (PTP1B) (Table 2, entries 6, 7). [START_REF] Szczepankiewicz | Discovery of a potent, selective protein tyrosine phosphatase 1B inhibitor using a linked-fragment strategy[END_REF][START_REF] Liu | Fragment screening and assembly: a highly efficient approach to a selective and cell active protein tyrosine phosphatase 1B inhibitor[END_REF] 2.2. Screening using ligand-observed 1 H NMR. Ligand-observed NMR offers different opportunities for identifying second-site ligands that bind at the vicinity of a first-site ligand. By contrast to the "SAR by NMR" approach, ligand-observed experiments are performed for medium-size to very large proteins.

One strategy is to use 2D Nuclear Overhauser Effect Spectroscopy (NOESY) experiments that allow the identification of compounds that bind near (~5Å) a first-site ligand through the observation of Inter-Ligand NOEs (ILOEs). [START_REF] Krimm | INPHARMA-based identification of ligand binding site in fragment-based drug design[END_REF][START_REF] Li | The inter-ligand Overhauser effect: a powerful new NMR approach for mapping structural relationships of macromolecular ligands[END_REF][START_REF] Leone | The Nuclear Overhauser Effect in the lead identification process[END_REF] It is possible to modify the first-site ligand through the addition of methyl group(s), allowing the generation of a suitable NOESY probe for the observation of strong NOESY cross peaks in a spectral region with little overlap. Such experiments, referred as "SAR by ILOE", have been used for the design of protein-protein interaction inhibitors for the targets BCL-XL and MCL1

(Table 2, entry 8). [START_REF] Becattini | Structure-activity relationships by interligand NOE-based design and synthesis of antiapoptotic compounds targeting Bid[END_REF][START_REF] Rega | SAR by interligand nuclear overhauser effects (ILOEs) based discovery of acylsulfonamide compounds active against Bcl-XL and Mcl-1[END_REF] In addition to the identification of second-site ligands, the ILOE experiment can be used to assess the orientation of the second-site ligand relative to the first-site ligand, as shown by the group of Abell for the Mycobacterium turberculosis pantothenate synthetase (PS) (Table 2, entry 9) [START_REF] Sledz | Optimization of the interligand Overhauser effect for fragment linking: application to inhibitor discovery against Mycobacterium tuberculosis pantothenate synthetase[END_REF] and confirm previously results obtained by X-Ray crystallography. [START_REF] Hung | Application of fragment growing and fragment linking to the discovery of inhibitors of Mycobacterium tuberculosis pantothenate synthetase[END_REF] Another strategy is to use paramagnetic labeling, [START_REF] Jahnke | Spin labels as a tool to identify and characterize protein-ligand interactions by NMR spectroscopy[END_REF][START_REF] Jahnke | Second-site NMR screening and linker design[END_REF] taking advantage of the paramagnetic enhancement (PRE) obtained thanks to the paramagnetic labeling of a first-site ligand. Typically, a 2,2,6,6tetramethylpiperidin-1-oxyl (TEMPO) moiety is used. The transverse relaxation rate of the protons located within a radius of ∼10Å of the first-site ligand is drastically enhanced, which significantly reduces the NMR signal intensities. [START_REF] Vazquez | Development of molecular probes for second-site screening and design of protein tyrosine phosphatase inhibitors[END_REF][START_REF] Vazquez | Development of paramagnetic probes for molecular recognition studies in protein kinases[END_REF] This approach has been successfully applied for the development of nanomolar E-selectin antagonists by Ernst and co-workers [START_REF] Egger | Nanomolar E-selectin antagonists with prolonged half-lives by a fragment-based approach[END_REF] (Table 2, entry 10). [START_REF] Petros | Discovery of a potent and selective Bcl-2 inhibitor using SAR by NMR[END_REF] F NMR. [START_REF] Petros | Discovery of a potent and selective Bcl-2 inhibitor using SAR by NMR[END_REF] F NMR spectroscopy is another tool for fragment screening, with the possibility to screen large mixtures of more than 20 fragments, taken advantage of the chemical shift dispersion of the [START_REF] Petros | Discovery of a potent and selective Bcl-2 inhibitor using SAR by NMR[END_REF] F nucleus. [START_REF] Petros | Discovery of a potent and selective Bcl-2 inhibitor using SAR by NMR[END_REF] F NMR is also used to search for second-site ligands, as exemplified for inhibitors of the Alzheimer target beta-secretase 1 (BACE1) (Table 2, entry 11). [START_REF] Cheng | From fragment screening to in vivo efficacy: optimization of a series of 2-aminoquinolines as potent inhibitors of beta-site amyloid precursor protein cleaving enzyme 1 (BACE1)[END_REF][START_REF] Jordan | A fragmentlinking approach using 19F NMR spectroscopy to obtain highly potent and selective inhibitors of β-secretase[END_REF] FAXS-NMR screening (fluorine chemical shift anisotropy and exchange for screening) was used to identify a novel class of HSP90 inhibitors. The principle is to use a fluorinated molecule as a spy to recognize fragments that competitively bind with the spy molecule. A fragment hit selected from a 1,200 fragments library was further characterized by X-ray crystallography and linked to the known HSP90 ligand resorcinol, leading to a low nanomolar compound (Table 2, entry 12). [START_REF] Casale | Fragment-based hit discovery and structure-based optimization of aminotriazoloquinazolines as novel HSP90 inhibitors[END_REF] 2.4. Second-site ligand resulting from elaboration of a first-site ligand. Fragment growing may also generate a new ligand that bind in a pocket that differs from the one of the initial fragment hit. This is exemplified by the group of Dalgarno 39 who screened 735 fragments against the lactate deshydrogenase A (LDHA) using Saturation Transfer Difference (STD) NMR. A fragment bound in the substrate pocket and part of the cofactor site was identified by X-ray crystallography. Fragment growing led to a new compound that do not bind in the cofactor binding site. Therefore, the initial fragment and the optimized compound were linked to generate the final structure (Table 2, entry 13).

Screening using ligand-observed

Second-site ligand following tethering-based first-site ligand.

Wells and colleagues [START_REF] Erlanson | Site-directed ligand discovery[END_REF] have developed an alternative strategy to identify fragments that bind to proteins, using a cysteine residue that can form a disulfide bond with disulfide containing fragments. The disulfide bond of the fragments that display affinity for the protein are entropically stabilized while the other disulfide bonds can be easily reduced. Tethered compounds were then recognized by Mass Spectrometry (MS). The approach was used to generate a novel non-peptidic inhibitor of caspase 3 (Table 2, entry 14). [START_REF] Erlanson | In situ assembly of enzyme inhibitors using extended tethering[END_REF] The methodology was also used by Raimundo and co-workers. [START_REF] Braisted | Discovery of a potent small molecule IL-2 inhibitor through fragment assembly[END_REF] They developed a potent small-molecule inhibitor of interleukin 2 (IL2) starting from a library of 7,000 disulphide-containing fragments (Table 2, entry 15).

Second-site ligand screening with focused libraries.

To identify second-site ligands, another strategy is to screen a fragment library that is chemically oriented to bind a particular protein-binding pocket. For example, the group of Hudson 43 screened a focused library of 450 fragments containing acids to find hits able to bind to the negatively charged region of the LDHA protein. The hit fragment was then linked to another fragment bound in the adenine region of the cofactor nicotinamide adenine dinucleotide (NADH) (Table 2, entry 16). Another example was published by Gaul and co-workers [START_REF] Möbitz | The discovery of potent, selective and structurally novel DOT1L inhibitors by a fragment linking approach[END_REF] regarding disruptor of telomeric silencing 1-like (DOT1L), a potential therapeutic target involved in leukemias. The first site was blocked with a DOT1L inhibitor to search for fragments that bind in the cofactor S-adenosylmethionine (SAM) pocket using NMR (Table 2, entry 17). In parallel, a focused virtual screening followed by a biochemical screen was performed for the cofactor pocket. The NMR and biochemical hits were further studied by X-ray crystallography. Modeling studies suggested to link a fragment and a second-site binder, which led to a new structure with low nanomolar affinity. with focused libraries. When the nature of a second pocket is known, a focused library can be used to identify a second-site binder.

IC 50 = 91 nM K i < 10 pM K i = 42 pM 2 2 N N N N Optimization K d = 150 μM K d = 20 μM K i = 1.9 μM Closed state O O N H H 2 N CH 3 F 3 C N N O O N H S N H N N CH 3 H 2 N O O O O N H N N H 2 N CH 3 K i =
K d = 1.9 μM K d = 30 nM O O CH 3 CO 2 CH 3 OH OH HO H 3 C O O OBz O COOH HO HO O 2 N HN O O CH 3 O H N OH OH HO H 3 C O O OBz O COOH HO HO N N N N O 2 N K d not reported Linking K d = 114 μM N N H N O O NH 2 N H CH 3 H 3 C CH 3 F N CH 3 N H N O O NH 2 N H CH 3 H 3 C CH 3 F Cl K d = 140 nM K d =
K d = 770 μM IC 50 > 500 μM K d = 1.1 mM IC 50 > 500 μM S N H 3 CHN H N CH 3 O Br O OH O OH O S N H 3 C H N O H 3 CO O OH O OH O H N H N H N O O N S H 3 C O H N H N O CH 3 OH OH O O H N H N O O N S S H 3 C OH O OH O K d =

DIRECT IDENTIFICATION OF FRAGMENTS BOUND TO TWO DISTINCT BINDING

SITES

When techniques such as X-ray crystallography, NMR or virtual screening are used, the identification of two fragments that bind in two distinct sites can be a direct outcome of the screening process. As discussed below, additional methods have also been used to identify two fragments that simultaneously bind the protein. The different strategies are summarized in Figure 3.

Screening using X-ray crystallography. X-ray crystallography is a particularly attractive

technique in FBDD as it directly gives the 3D structure of the protein-fragment complex. Using a focused library of 80 fragments on the protein thrombin, [START_REF] Howard | Application of fragment screening and fragment linking to the discovery of novel thrombin inhibitors[END_REF] Astex Therapeutics observed fragments bound to the S1 pocket of the enzyme and one compound that bound to the S2-S4 region. The X-ray structures then helped the design of a linked compound (Table 3, entry 18). The group of Yarnold [START_REF] Barker | Discovery of a novel HSP90 inhibitor by fragment linking[END_REF] identified, using biochemical screening followed by X-ray crystallography, fragments that bound to separate sub-sites within the ATPase pocket of HSP90 (Table 3, entry 19). In another example, Guichou and colleagues 47 used computational screening of 34,409 fragments to identify fragments of the cyclophilin D (CYPD) active site as well as the gatekeeper pocket. Fourteen X-ray structures were obtained, showing four fragments bound to the catalytic site of the protein while five fragments were observed in the gatekeeper pocket (Table 3, entry 20). Using a thermal shift assay followed by X-ray crystallography, Barral and co-workers [START_REF] Benmansour | Discovery of novel dengue virus NS5 methyltransferase non-nucleoside inhibitors by fragment-based drug design[END_REF][START_REF] Hernandez | Optimization of a fragment linking hit toward Dengue and Zika virus NS5 methyltransferases inhibitors[END_REF] identified 3 compounds that bind near the S-Adenosylmethionine (AdoMet) binding pocket of the NS5 AdoMet-dependent mRNA methyltransferase (MTase) domain, highlighting a new targetable site (Table 3, entry 21). More recently, Spring et al. [START_REF] Brear | Specific inhibition of CK2α from an anchor outside the active site[END_REF][START_REF] De Fusco | A fragment-based approach leading to the discovery of a novel binding site and the selective CK2 inhibitor CAM4066[END_REF][START_REF] Iegre | Second-generation CK2α inhibitors targeting the αD pocket[END_REF] found a fragment bound at multiple sites on the protein casein kinase 2 (CK2), including a previously unreported site adjacent to the adenosine triphosphate (ATP) site. The fragment was modified to generate a compound that specifically binds the newly identified pocket and linked with ATP-site ligands (Table 3, entry 22).

Screening using protein-observed NMR.

Protein-observed NMR experiments can also be used to identify two different fragments that bind simultaneously two distinct protein pockets. The group of Fesik 53 examined 14,976 fragments of replication protein A (RPA) by NMR and found compounds that bind to two distinct sites in the basic cleft of RPA70N. X-ray structures revealed the binding modes of the fragments, suggesting a strategy for fragment optimization and linking (Table 3, entry 23).

Screening using MS.

Similarly to the "SAR by NMR", a "SAR by MS" approach has been proposed. Ibis Therapeutics 54 used MS to screen fragments against the 1061 region of bacterial 23S ribosomal ribonucleic acid (rRNA). This process includes a first step where compounds are screened against the RNA by MS. SAR is obtained by chemical modification of the hits and MS experiments.

Data obtained for structurally modified hits, for which competition or absence of competition in the MS experiments was observed, led to distant restraints between initial fragments that were then linked (Table 3, entry 24).

Screening using biochemical assays. Biochemical assays constitute straightforward screening

methods that require knowledge of the biochemical protein functions. A proof of concept of the linking strategy was reported by Green 55 with biotin analogues binding avidin, illustrating the super-additivity that can be achieved with the fragment linking approach (Table 3, entry 25). Another report [START_REF] Maly | Combinatorial target-guided ligand assembly: identification of potent subtype-selective c-SRC inhibitors[END_REF] showed that the knowledge of the 3D structure of the target was not crucial. Ellman and co-workers screened at high concentration a library of oximes and a small library of di-hydroxylamine linkers against the tyrosine kinase c-SRC. They could demonstrate the utility of the method with the identification of a potent and subtype-selective inhibitor (Table 3, entry 26).

Fragment self-assembly.

We have also considered fragment self-assembly as examples of fragment linking, even if no linker is designed to obtain the linked compound. The process called dynamic combinatorial chemistry (DCC) is observed in situ in the presence of a target protein. As a first example, Huc and Lehn 57 reported the generation of carbonic anhydrase (CA) inhibitors by combination of aldehyde and amine moieties to in situ form imines (Table 3, entry 27). Other applications were published for a neuraminidase (NA) inhibitor (Table 3, entry 28) [START_REF] Hochgürtel | Target-induced formation of neuraminidase inhibitors from in vitro virtual combinatorial libraries[END_REF][START_REF] Hochgürtel | Ketones as building blocks for dynamic combinatorial libraries: highly active neuraminidase inhibitors generated via selection pressure of the biological target[END_REF] and an aspartic protease endothiapepsin (Table 3, entry 29). [START_REF] Mondal | Structure-based design of inhibitors of the aspartic protease endothiapepsin by exploiting dynamic combinatorial chemistry[END_REF] Dynamic combinatorial X-ray crystallography, when DCC takes place inside protein crystals, was demonstrated for the cyclin-dependent kinase 2 (CDK2), using a mixture of hydrazines and isatins (Table 3, entry 30). [START_REF] Congreve | Detection of ligands from a dynamic combinatorial library by X-ray crystallography[END_REF] Another methodology called dynamic ligation screening (DLS) was reported by Rademann and co-workers [START_REF] Schmidt | Selective identification of cooperatively binding fragments in a high-throughput ligation assay enables development of a picomolar caspase-3 inhibitor[END_REF] for the protease caspase 3 (Table 3, entry 31). Using a fluorescent probe attached to a fragment, competitors or fragments that bind cooperatively to the fluorescent probe are detected with the fluorescence polarization assay. The authors also extended the approach to form amide bonds (Table 3, entry 32). [START_REF] Jaegle | Protein-templated formation of an inhibitor of the blood coagulation factor Xa through a background-free amidation reaction[END_REF] Protein-templated click chemistry (PTCC) that relies on the 1,3-dipolar cycloaddition of azides and alkynes to generate a triazole moiety was also reported by the team of Sharpless 64 with the generation of a femtomolar inhibitor of acetylcholinesterase (Table 3, entry 33). Additional examples concern inhibitors developed for histone deacetylases (Table 3, entry 34), [START_REF] Suzuki | An unexpected example of protein-templated click chemistry[END_REF] chitinase B (Table 3, entry 35) [START_REF] Hirose | Observation of the controlled assembly of preclick components in the in situ click chemistry generation of a chitinase inhibitor[END_REF] and the aspartic protease endothiapepsin (Table 3, entry 36). [START_REF] Mondal | Fragment-based drug design facilitated by protein-templated click chemistry: fragment linking and optimization of inhibitors of the aspartic protease endothiapepsin[END_REF] The approach can also lead to protein-protein interaction inhibitors, as demonstrated for BCL-XL (Table 3, entry 37). [START_REF] Hu | Bcl-XL-templated assembly of its own protein-protein interaction modulator from fragments decorated with thio acids and sulfonyl azides[END_REF][START_REF] Kulkarni | Screening of protein-protein interaction modulators via sulfo-click kinetic target-guided synthesis[END_REF] 3.6. Inhibitor deconstruction and reconstruction. Deconstruction of known ligands can provide a useful strategy for the reconstruction of a new compound. [START_REF] Chen | Evolutions in fragment-based drug design : the deconstruction-reconstruction approach[END_REF] Using HTS, two compounds were identified to bind the human N-myristoyltransferase 1 (NMT1) protein and X-ray crystallography indicated that the molecules bind in two different pockets. [START_REF] Mousnier | Fragment-derived inhibitors of human Nmyristoyltransferase block capsid assembly and replication of the common cold virus[END_REF] To preclude a steric clash upon compound linking, one of the hits was deconstructed to remove the quinoline moiety (Table 3, entry 38).

The linking approach can be particularly useful to replace a part of a compound that is not suitable. For example, fragments were searched to substitute a chymase inhibitor benzothiophene moiety that produced reactive metabolites. [START_REF] Taylor | Discovery of potent, selective chymase inhibitors via fragment linking strategies[END_REF] Takahashi and co-workers screened one thousand fragments and identified a fragment that binds in the same pocket as the benzothiophene core. The deconstruction and reconstruction of the initial inhibitor led to a compound with improved metabolic properties (Table 3, entry 39).

NH 2 NH 2 F 3 C H 2 N COOH CH 3 H N H N O NH 2 F 3 C S H N COOH O O K d > 500 μM K d = 250 μM K d =
K i not reported CHO S H 2 N O O S H 2 N O O N H 2 H 2 N

Self-assembly

Self-assembly

K i not reported K i = 85 nM O COOH H 2 N NHAc NH 2 COOH N H NHAc NH 2 H 3 C H 3 C H 3 C H 3 C IC 50 =

Self-assembly

K i = 120 μM K i = 25 nM H 3 C N H O H N COOH O N H O H N COOH O O O H 3 C CH 3 COOH H 3 C N H O H N COOH O N H O H N COOH O H N O H 3 C CH 3 COOH O N H 2 N O N K i = 80 pM N CH 3 CH 3 N CH 3 CH 3 Self-assembly K i = 5.5 mM K i = 29 nM S N H H N O OH O O O O S N H H N O N H O O O O NH 2 NH H 2 N NH 2 NH K i = 680 μM
Self-assembly Self-assembly Two fragments that bind simultaneously two distinct protein pockets can be identified on 2D NMR protein spectra. C) Screening using MS. Fragment hits identified by MS are modified and competition experiments are performed to derive structural restraints. D) Screening using biochemical assays. The example shows the use of the oxime function together with a small library of di-hydroxylamine linkers.

IC 50 > 100 μM IC 50 < 1 nM IC 50 = 20 μM F CH 3 N H N N CH 3 CH 3 F O N N N CH 3 CH 3 CH 3 F N N H 3 C CH 3 H 3 C H 3 C O O H 3 C

E)

Fragment self-assembly in the presence of a target protein: dynamic combinatorial chemistry (DCC).

The example is illustrated with aldehyde and amine moieties that in situ form imines.

DIMERIZATION OF A FRAGMENT HIT

A special application of fragment linking is dimerization. This approach allows one to link together two copies of the same fragment binding to either adjacent sites or in the same pocket of a protein.

4.1.

The fragment binds twice in the protein binding pocket. The first example was reported by Brimijoin and co-workers [START_REF] Pang | Highly potent, selective, and low cost bistetrahydroaminacrine inhibitors of acetylcholinesterase. Steps toward novel drugs for treating Alzheimer's disease[END_REF] for the acetylcholine esterase. The observation that two copies of an acridine analogue could be modelled in the active site led to the design of dimeric compounds with different linker lengths. An inhibitor that was 1,000-fold more potent than the original molecule was synthesized (Table 4, entry 40). Another case was reported by Blundell and co-workers [START_REF] Surade | A structure-guided fragment-based approach for the discovery of allosteric inhibitors targeting the lipophilic binding site of transcription factor EthR[END_REF] for the transcription factor from Mycobacterium tuberculosis (EthR), involved in antibiotic resistance. Thermal shift assay followed by crystallography identified two copies of a fragment hit in the allosteric pocket of the protein, starting from 1,250 fragments (Table 4, entry 41). Similarly, biochemical assay (enzyme inhibition by monitoring NADH absorbance) followed by crystallography identified two copies of a fragment hit in the active site of the protein inosine-5'-monophosphate dehydrogenase (IMPDH) (Table 4, entry 42). [START_REF] Trapero | A fragment-based approach to targeting inosine-5'-monophosphate dehydrogenase (IMPDH) from Mycobacterium tuberculosis[END_REF] They synthesized an inhibitor that was 2,500-fold more potent than the original molecule.

4.2.

The fragment binds in the same pocket in two protein sub-units. Human mast cell tryptase, a trypsin-like serine protease, exists as a tetramer. The ligand benzamidine was modified to contain two benzamidine moieties with a flexible linker region spanning the space between two monomers (Table 4, entry 43). [START_REF] Burgess | Potent selective nonpeptidic inhibitors of human lung tryptase[END_REF] With a similar approach, an anti-cancer pyruvate kinase M2 (PKM2) activator was designed (Table 4, entry 44) [START_REF] Matsui | Discovery and structure-guided fragment-linking of 4-(2,3-dichlorobenzoyl)-1-methyl-pyrrole-2carboxamide as a pyruvate kinase M2 activator[END_REF] as well as an inhibitor of the α-amino-3-hydroxy-5-methyl-4isoxazolepropionic acid (AMPA) receptors (Table 4, entry 45). 79

LINKERS

Once two fragments have been identified, a linker is designed, which can be done using various strategies. From a chemical point of view, we have considered two types of linkers, the alkyl-derived linkers, and the functionalized linkers. We also report two promising approaches, a first one that we have called "linkers inspired by known ligands" and the other one "growing before linking".

Alkyl linkers and derivatives.

Alkyl linkers of variable lengths are largely used in the literature to link two fragments bound in two pockets (Table 5). In some studies, the linkers also include more rigid structures such as phenyl core, alkyne function ( 5 entry 24). Linking strategy with an alkyl chain has also successfully generated compounds in the absence of structural information, using biochemical data (Table 5 entries 25 & 43).

In a majority of cases, a small set of compounds with varying linker lengths and attachment points are synthesized to explore the hypotheses. In addition, the linking can start from an ether or an amine moiety, making chemical synthesis more straightforward.

Linkers with functional groups.

Alkyl functions are more problematic when the linking of the two molecules requires a non-linear geometry. For example, the team of Barral used a urea function as suggested by in silico studies (Table 5 entry 21). Hadjuk and co-workers reported a case where a sulfonamide was chosen as it could bend by 180°. In the same study, in order to position correctly a furanone moiety included in the fragment with a 90° twist, an acetylene linker was used (Table 5 entry 5). Sulfonamide and acyl sulfonamide were also used by the Abell group to replace ester and amide moieties in the case of the pantothenate synthase (Table 5 entry 9).

In some cases, the linker has been carefully optimized in order to add molecular interactions between the atoms of the linker and the protein residues. For example, Fesik's team chose to add rigidity and to introduce interactions between the linker and the protein by introducing a thioamide linker for a BCL-2 inhibitor (Table 5 entry 23). In the Dalgarno group, the initial ether-based linker was modified with the addition of functional groups including ester, amide, amine and hydroxyl functions. The eight-atom linker containing four hydroxyl groups interacting with protein atoms provided the most active compound (Table 5 entry 13). A further strategy reported in the literature consisted in selecting the linkers by in silico studies. For example, for the design of a BCL-XL inhibitor (Table 5 entry 8), several types of linkers were proposed and led to more active compounds.

Structural data for the linker design.

As illustrated in Table 5, in the large majority of the cases (26 out of 31 publications), structural information is used to design the linker. X-ray crystallography appears as the method of choice, alone or in combination with modeling, while NMR is much rarely utilized. Structural data can also result from the merging strategy, where a known ligand offers an opportunity to design the linker (see below, 5.4). Also, the growing strategy may be necessary to first reduce the distance between the fragments before linking, which necessitates structural characterization of the protein-fragment complexes (see 5.5).

Linkers inspired by known ligands (mixing merging and linking strategies). Another strategy

for the linker design is to take advantage of the knowledge of natural substrates or other known inhibitors. For example, Guichou et al. chose to use a urea moiety, after superimposition of fragments with the known structures of sangliferin A and cyclosporine A, two cyclophilin inhibitors. Two molecules with two different linkers were obtained and one of them directly showed micromolar activity (Table 5 entry 20). Abell group similarly merged a previously known inhibitor with two copies of a fragment hit (Table 5 entry 42), which has led to a successful linker.

5.5.

Growing before linking. We want to particularly emphasize one strategy that consists in growing one of the fragments before linking, when the distance between the two fragments is particularly large.

This strategy has been used with both alkyl and functionalized linkers. In the report by AstraZeneca (Table 5 entry 16) on the LDHA protein, growing of one of the fragments was performed until the distance between the two fragments was reduced to 3Å. Then, a molecular study was used to select suitable linkers. In the case of the protein kinase CK2, due to the long distance between the initial fragments, Spring and colleagues choose to modify one of the fragments by addition of chemical groups with different lengths. The synthesized molecules were then evaluated and X-ray data were solved to measure the resulting distance between the two fragments ( X-ray 20 [47] 23 [53] 42 [76] S 22 [50-52] 41 [75] • Ranking by calculating the internal-strain energies •• Molecular dynamics simulation

FUTURE PERSPECTIVES AND CONCLUSION

FBDD is a powerful method for the generation of new drug candidates, which so far has led to three FDA-approved drugs and to clinical trials for nearly fifty molecules. While strategies for developing an active molecule from a fragment are typically growing, linking and merging approaches, some examples reported here show that these strategies may be used one after another to increase the chances of success.

S NH O O O S NH O O N H O N H N H O Optimization N H O N H O N H N H O O N H O S S
Nevertheless, the literature clearly shows that the growing strategy is by far the most widely used strategy, even if the linking strategy raised great expectations due to theoretical considerations. [START_REF] Jencks | On the attribution and additivity of binding energies[END_REF] Are there some experimental, technical difficulties that might explain the moderate success of the linking strategy? The first step in this process is the identification of two fragments that bind the protein in two distinct sub-sites. Screening a fragment library with a structural method offers a clear advantage in this context. While both NMR and X-Ray crystallography techniques theoretically offer the opportunity to directly identify two compounds that simultaneously bind the protein target at two different sites, published examples highlight X-Ray crystallography as the most successful (or used) method. By contrast, NMR is a method of choice for the iterative identification of a second-site binder. [START_REF] Sun | A method for the second-site screening of K-Ras in the presence of a covalently attached first-site ligand[END_REF] However, when using NMR as a screening technique, two screens are generally essential. Second-site screening also offers the opportunity to identify new binding pockets, which may open the route for the design of highly selective compound such as allosteric molecules. The optimization of the second-site ligands might be necessary to increase the affinity of those ligands that are typically weaker binders than the first-site ligands. Once the screening has been performed, the most appropriate method to guide the linking process will depend on the distance between the two identified binders. NMR can be an interesting approach for short distances, typically when the two fragments bind in adjacent pockets, using ILOE 2D experiments that do not require protein labeling. We believe this strategy is particularly suitable for catalytic sites of enzymes (Table 2, entries 8 & 9). For larger distances, which might be observed for protein-protein interaction inhibitors or for bivalent compounds targeting two sites (for example active site and allosteric site), modelisation and X-Ray crystallography are more efficient techniques than NMR, as the linker design will necessitate the resolution of the complex 3D structure.

NMR would require labeled protein samples.

For the majority of the fragment-linking published examples, super-additivity is not observed. This might explain why the growing approach remains the preferred strategy. Out of the forty-five examples reported here, nine only fulfill the super-additivity criterium ( Entries 2, 4, 18, 20, 25, 30, 32, 33 & 43), including the well-known avidin case, one dimerization case and three self-assembly examples. This shows that achieving super-additivity is challenging. In particular for non-catalytic sites such as proteinprotein interaction sites, protein conformational rearrangement upon compound binding might induce changes in the fragment binding modes. Translational and rotational entropy changes due to the linker also need to be counteracted. It is not straightforward to understand what criteria (regarding the fragment or the linker) lead to super-additivity. Previously, Whittaker and colleagues [START_REF] Ichihara | Compound design by fragment-linking[END_REF] suggested that successful linking could be achieved by carefully selecting the pair of fragments to be linked, with one forming polar interactions with the protein while the second fragment should interact through van der Waals interactions. The idea is that the binding mode of the fragment that does bind through nonpolar interactions can be changed without penalty upon linking. There are examples in the literature that contradict this assumption and successful cases here showed polar interactions for both fragments. To our point of view, the linking strategy in FBDD essentially depends on the optimization of the two initial fragments, the geometry required to link the fragments and the length of the linker. As observed for the compounds for which the super-additivity was reached, the linkers are rather short and flexible, mainly alkyl-derived linkers. Therefore, these findings tend to suggest that mixing the growing and linking approaches could be a promising method to achieve the super-additivity. Growing one fragment helps reduce the length of the linker required. Also, optimization of the fragments before linking may help adapt the linker in case of protein conformational changes. Another promising approach consists in mixing merging and linking strategies, where the linker is designed thanks to the imitation of chemical groups observed in known ligands.

Jean-François

  BCL-X L ) = 86 nM IC 50 (MCL1) = 140 nM IC 50 (BCL-X L ) = 15.08 μM IC 50 (MCL1) = 25.38 μM K d = 691.8 μM 500 μM K d = 1.8 μM

Figure 2 .

 2 Figure 2. Schematic representation of methods for second-site screening for identification of a

  IC 50 (DENV 2'O-MTase) = 91 μM IC 50 (DENV N7-MTase) = 1.1 mM IC 50 (WNV 2'O-MTase) = 51 μM

Figure 3 .

 3 Figure 3. Schematic representation of methods for direct identification of fragment bounds to two

  F) Inhibitor deconstruction and reconstruction. A known ligand can be improved by replacing a moiety by a fragment hit identified in the presence of the deconstructed fragment. G) Cooperative binding. A first ligand induces a new pocket for a second fragment hit.

  50 = 176 μM EC 50 = 13 nM Linking

  Guichou received his Master's degree in Chemistry in 1996 from Montpellier University, France. He received the diploma of ENSCM (Ecole Nationale Suppérieure de Chimie de Montpellier) in 1997 from Montpellier, France. He received his Doctoral degree in Organic chemistry in 2002 under the supervision of Prof. Manfred Mutter at the Lausanne University, Switzerland. He was assistant professor from 2005 to 2016 in Montpellier University. In 2009, he won a "Chercheur d'Avenir" grant for 2 years, France. From 2016 to date, he is full professor in Montpellier University. He has been working on various therapeutic targets, using SBDD or FBDD approaches. Isabelle Krimm received her Ph.D. from the Chemistry Department in 2000 in the University of Lyon, France. She served as a postdoctoral fellow at the CEA in Paris for 2 years in order to resolve the 3D structure of the N-terminal part of the lamin protein, using NMR. She then joined a Chemistry laboratory at the University of Lyon, and started a new group dedicated to fragment-based approaches for drug design in 2013. In 2019, she joined as Principal investigator both the The Very High field NMR Center in Lyon and the Center for Research in Cancer in Lyon.

Table 1 . Fragment-based compounds in clinical trials or approved (From clinicaltrials.gov, 2020 January)

 1

	Drug • Biological target	Company	Status	Initial fragment strategy
	Erdafitinib FGFR	Johnson & Johnson / Astex	Approved	Growing
	Vemurafenib BRAF-V600E	Plexxikon	Approved	Growing
	Venetoclax BCL-2	AbbVie / Genentech, Inc.	Approved	Linking
	Asciminib BCR-ABL	Novartis	Phase 3	Growing
	PLX3397 CSF1R, KIT, FLT3	Plexxikon	Phase 3	Growing
	Verubecestat BACE1	Merck	Phase 3	Growing
	AZD3293 BACE1	Astex / AstraZeneca / Lilly	Phase 2/3	Growing
	ABT-737 BCL-2 & BCL-XL	AbbVie	Phase 2	Linking
	AT7519
	CDK1, CDK2, CDK4, CDK5	Astex	Phase 2	Growing
	& CDK9
	AT9283 Aurora & JAK2	Astex	Phase 2	Growing
	AUY922 HSP90	Novartis, Vernalis	Phase 2	Growing
	AZD5363 AKT	AstraZeneca / Astex	Phase 2	Growing
	CPI-0610 BET	Constellation	Phase 2	Growing
	eFT508 MNK1 & MNK2	eFFECTOR	Phase 2	Growing
	Indeglitazar PPAR agonist	Plexxikon	Phase 2	Growing
	LY3202626	Lilly	Phase 2	Growing

• Drugs are listed alphabetically within each phase.

Table 2 . Fragment linking by second-site screen.

 2

	Entry [Ref.]
	Target (Pathologies)		Fragments & leads
	Method
	1 [11,12] FKBP (Neuro-degenerative NMR diseases)	N K d = 2 μM O O H 3 CO H 3 CO OCH 3	K d = 100 μM	Linking	K d = 49 nM
	2 [15]
	MMP3 (Cardiovascular diseases)
	NMR
	3 [19]
	BCL-2 (Cancer & autoimmune diseases)
	NMR
	4 [20]
	BCL-XL (Cancer & autoimmune diseases)
	NMR
	5 [21]
	HSP90 (Cancer & neuro-degenerative diseases)
	NMR

Table 3 . Fragment linking by direct identification of two fragments.

 3

	Entry [Ref.]
	Target (Pathologies)		Fragments & leads
	Method
	18 [45] diseases) degenerative (Neuro-Thrombin		H 3 CO	S HN CH 3
	X-Ray	IC 50 = 330 μM	IC 50 = 12 μM	IC 50 = 1.4 nM
	19 [46]
	HSP90 (Cancer & neuro-degenerative diseases)
	Bioassay & X-
	Ray
	20 [47]
	CYPD (Viral & neuro-degenerative diseases)
	X-Ray
	21 [48,49]
	DENV-MTase (Viral diseases)
	X-Ray
	22 [50-52]
	CK2a (Cancer)
	X-Ray

Table 4 . Fragment linking by fragment dimerization

 4

	Entry [Ref.]
	Target (Pathologies)		Fragments & leads
	Method
	40 [74]
	ACHE (Neuro-degenerative diseases) Bioassay	2 K i = 600 nM	Linking	K i = 400 pM
	41 [75]
	EthR (Infectious diseases)
	Bioassay &
	X-Ray
	42 [76]
	IMPDH (Infectious diseases)
	Bioassay &
	X-Ray
	43 [77]
	Tryptase (Asthma)
	Bioassay
	44 [78]
	PKM2 (Cancer)
	HTS
	45 [79]
	AMPA
	receptor (Neuro-degenerative diseases)
	X-Ray

  Table 5 entry 11) and cyclohexane (Table 5 entry 26). Typically, the likely binding mode and structural constraints for the lengths are obtained from NMR experiments (Table 5 entries 1, 2 & 3), crystal structure of fragments (Table 5 entries 4, 6, 18, 38 & 39) or a combination of crystallography and molecular modelling (Table 5 entries 7, 12, 16, 17, 19, 40, 44 & 45). One publication showed that mass spectroscopy competition experiments can also generate distance restraints (Table

  Table 5 entry 22), before linking.

	Journal of Medicinal Chemistry	Page 32 of 48
	ACS Paragon Plus Environment

Table 5 . Linker chemical structures and structural methods.

 5

	Entry
	from tables 2,	Structural methods	Linker structure
	3, 4 [Ref.]
			Alkyl linkers and derivatives
	1 [11,12] 2 [15] 3 [19]	NMR
	4 [20] 6 [22] 18 [45] 38 [71] 39 [72] 17 [44] 19 • [46] 40 [74] 45 [79] 44 •• [78] 16 [43] 12 [38]	X-Ray modeling molecular X-ray &	Variable lenghts n
	25 [55] 43 [77]	-
	7 [23]	X-ray & modeling molecular	O
	11 [36, 37]	NMR, X-ray & molecular modeling	N	N
	24 [54]	MS
	26 [56]	-	n
			Variable lenghts

Journal of Medicinal Chemistry

ACKNOWLEDGMENTS

We thank the CNRS, the Ligue contre le cancer and ANR (Convergence PLAScan) for financial support.

ABBREVIATIONS USED

AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; ACHE, acetylcholinesterase; AdoMet, adenosylmethionine; AKT, protein kinase B; ATP, adenosine triphosphate; BACE1, betasecretase 1; BCL-2, B-cell lymphoma 2; BCL-XL, B-cell lymphoma-extra-large; BET, bromo-and extra-terminal domain family; BRAF, v-raf murine sarcoma viral oncogene homolog B; BRD2, bromodomain-containing protein 2; c-SRC, proto-oncogene tyrosine-protein kinase SRC; CA, carbonic anhydrase; CASP3, caspase 3; CDK1/2/4/5/9, cyclin-dependent kinases 1/2/4/5/9; cIAP1, cellular inhibitor of apoptosis protein 1; CK2, casein kinase 2; CSF1R, colony stimulating factor 1 receptor; CYPD, cyclophilin D; DCC, dynamic combinatorial chemistry; DENV, dengue virus; DLS, dynamic

FXa Lilly / Protherics Phase 2 Growing Navitoclax BCL-2 & BCL

AUTHOR INFORMATION

Corresponding Author *E-mail: isabelle.krimm@univ-lyon1.fr. Phone: +33 4 37 42


Dubiel Yohan Pillon
email: yohan.pillon@ird.fr

Alfonso González

Herizo Randriambanona

Porter P Lowry Ii

Tanguy Jaffré

Sylvain Merlot

Dubiel Alfonso González

Porter P Lowry

Parallel ecological filtering of ultramafic soils in three distant island floras

Keywords: Cuba, Madagascar, New Caledonia ecological filtering, disharmony, exaptation, island, niche conservatism, nickel hyperaccumulator, serpentine

come   L'archive ouverte pluridisciplinaire

 Pillon Y., González D. A., Randriambanona H., Lowry II P. P., Jaffré T., Merlot S. 2019. Parallel ecological filtering of ultramafic soils in three distant island floras. Journal of Biogeography,46 (11): 2457-2465.

| INTRODUCTION

" Quand la nature n'offre pas les mêmes espèces, elle se plaît à répéter les mêmes genres » [When Nature does not offer the same species, she likes to repeat the same genera] A. von Humboldt (1814, p. 601) During his many travels, Alexander von Humboldt was able to observe the floras of many regions of the world with contrasting climates. He noticed that the same grass species could be found in Switzerland or along the Magellan strait, and the same violet occurred on top of the volcano on Tenerife and in the Pyrenees Mountains (Humboldt & Bonpland, 1814, p. 601). He was puzzled by the possible migration of plants across oceans or areas with an inhospitable climate. He acknowledged that the number of species shared by the Old and New Continents may not be as great as previously thought, but speculated that the highest mountains of tropical America probably have plantains, valerians, sandworts, buttercups, medlars, oaks, and pines that look like the European ones but belong to different species (Humboldt & Bonpland, 1814, p. 602). Thus, the same genera or closely-related genera belonging to the same tribe (Humboldt & Bonpland, 1814, p. 602) are to be expected in regions with the same climatic conditions.

Long distance dispersal is now acknowledged as a major force in biogeography [START_REF] De Queiroz | The resurrection of oceanic dispersal in historical biogeography[END_REF], and even improbable dispersal between islands in different oceans has been shown [START_REF] Le Roux | Relatedness defies biogeography: the tale of two island endemics (Acacia heterophylla and A. koa)[END_REF]. When plants disperse over long distances from continents to islands, they will often occupy areas with similar ecological conditions (Carlquist, 1974 p. 81). Lineages have a tendency to retain the same ecological traits or niche characteristics through time and speciation events, which has been termed phylogenetic niche conservatism [START_REF] Ackerly | Community assembly, niche conservatism, and adaptive evolution in changing environments[END_REF][START_REF] Crisp | Phylogenetic niche conservatism: what are the underlying evolutionary and ecological causes?[END_REF][START_REF] Wiens | Niche conservatism as an emerging principle in ecology and conservation biology[END_REF]. It has been shown, for example, Pillon Y., González D. A., Randriambanona H., Lowry II P. P., Jaffré T., Merlot S. 2019. Parallel ecological filtering of ultramafic soils in three distant island floras. Journal of Biogeography, 46 (11): 2457Biogeography, 46 (11): -2465. . that plants tend to occupy similar biomes after transoceanic dispersal across southern continents [START_REF] Crisp | Phylogenetic biome conservatism on a global scale[END_REF]. The assemblage of a community such as the Mediterraneantype woody vegetation of California (chaparral) involved not only adaptation but also niche conservatism [START_REF] Ackerly | Adaptation, niche conservatism and convergence: comparative studies of leaf evolution in the California chaparral[END_REF]. Indeed, the small leaves and low specific leaf area of several plant lineages, characteristic of Mediterranean vegetation, are features that were often already present in their ancestors before dispersal from their ancestral subtropical biome. The greater success of lineages that already possess a suitable combination of functional traits for their novel niche has been termed ecological sorting [START_REF] Ackerly | Community assembly, niche conservatism, and adaptive evolution in changing environments[END_REF][START_REF] Ackerly | Adaptation, niche conservatism and convergence: comparative studies of leaf evolution in the California chaparral[END_REF].

Evidence for niche conservatism and ecological sorting in plants comes primarily from studies on biome preferences (e.g. [START_REF] Ackerly | Adaptation, niche conservatism and convergence: comparative studies of leaf evolution in the California chaparral[END_REF][START_REF] Crisp | Phylogenetic biome conservatism on a global scale[END_REF]. These mechanisms are likely to operate in plant responses to edaphic conditions. For example, Stebbins (1974, p. 43) noted a "selective inertia" leading to a preference of Ericaceae for acid soils, Caryophyllaceae for mineral-rich substrates, and Chenopodiaceae for saline or sub-saline soils. Habitat filtering can be expected to be more obvious in extreme environments such as on ultramafic substrates. Soils derived from ultramafic rocks (including serpentine) offer challenging conditions for plant development because of their low content in essential nutrients (N, P, K), a strong imbalance in their Mg:Ca ratio, high concentrations of certain metals (Ni, Co, Cr, etc.), and low water availability leading to stress, as well as their fire-prone vegetation [START_REF] Kazakou | Hypotheses, mechanisms and trade-offs of tolerance and adaptation to serpentine soils: from species to ecosystem level[END_REF][START_REF] Proctor | Vegetation and soil and plant chemistry on ultramafic rocks in the tropical Far East[END_REF]. Outcrops of ultramafic rocks are often covered with a distinctive vegetation that includes narrowly endemic species, e.g. in California [START_REF] Safford | Serpentine endemism in the California flora: a database of serpentine affinity[END_REF], Cuba [START_REF] Borhidi | Phytogeography and vegetation ecology of Cuba[END_REF], and New Caledonia [START_REF] Wulff | Conservation priorities in a biodiversity hotspot: analysis of narrow endemic plant species in New Caledonia[END_REF]. Previous work has suggested that ultramafic substrates may have acted as a filter that could explain the over-or under-representations of certain plant families (i.e. disharmony) on the island of New Caledonia [START_REF] Goodman | Ultramafic soils and species sorting in the flora of New Caledonia[END_REF]. Pillon Y., González D. A., Randriambanona H., Lowry II P. P., Jaffré T., Merlot S. 2019. Parallel ecological filtering of ultramafic soils in three distant island floras. Journal of Biogeography,46 (11): 2457-2465.

To test the ecological filtering effects of ultramafic substrates, Cuba, Madagascar, and New Caledonia represent an ideal comparative system. Ultramafic outcrops are found on all three islands, which occur in three different oceans and belong to distinct floristic kingdoms or subkingdoms [START_REF] Takhtajan | Floristic regions of the world[END_REF]. They share very few species and are thus almost totally independent systems, but share more higher rank taxa and are sufficiently diverse to permit a broad sampling of the tree of life. In this insular context, the total pool (i.e. the entire island) from which the ultramafic flora is most likely derived is easily defined and characterized. We have intentionally limited our study to tropical islands, thus excluding temperate floras, to limit possible effects of climate as a filtering factor. In order to see if Humboldt's observation can be expanded to edaphic conditions, we tested whether the same families tended to be selected for or against on the ultramafic substrates of all three islands, and thereby demonstrate whether soil can be a driver of ecological sorting. Assuming that ultramafic floras should be a random subset of the total flora for each island, we measured how each family deviates from this null-model and assessed if their behaviour was consistent between islands.

| MATERIAL AND METHODS

| Model systems

The southwest Pacific island of New Caledonia has a continental basement that emerged above sea level ca. 37 My ago, when it was covered with ultramafic substrates that now occupy one third (5,470 km 2 ) of the surface of Grande Terre, the main island [START_REF] Cluzel | Upper Eocene unconformity and preobduction events in New Caledonia[END_REF][START_REF] Pelletier | Geology of the New Caledonia region and its implications for the study of the New Caledonian biodiversity[END_REF]. New Caledonia's biota is considered to have reached the island via long-distance dispersal from neighbouring areas, particularly Australasia [START_REF] Grandcolas | New Caledonia: a very old Darwinian island?[END_REF]. Mount Humboldt (1,617 m), the second highest peak of Pillon Y., González D. A., Randriambanona H., Lowry II P. P., Jaffré T., Merlot S. 2019. Parallel ecological filtering of ultramafic soils in three distant island floras. Journal of Biogeography,46 (11): 2457-2465.

New Caledonia, entirely comprised of ultramafic rocks, is a nature reserve and a major centre of micro-endemism [START_REF] Bradford | Plant species microendemism and conservation of montane maquis in New Caledonia: two new species of Pancheria (Cunoniaceae) from the Roche Ouaïème[END_REF][START_REF] Munzinger | A second species in the endemic New Caledonian genus Gastrolepis (Stemonuraceae) and its implications for the conservation status of high-altitude maquis vegetation: coherent application of the[END_REF].

The island of Cuba emerged 30-35 My ago [START_REF] Iturralde-Vinent | Paleogeography of the Caribbean region: implications for Cenozoic biogeography[END_REF][START_REF] Iturralde-Vinent | Meso-cenozoic Caribbean paleogeography: implications for the historical biogeography of the region[END_REF], and according to the GAARlandia hypothesis, ancestors of a portion of the terrestrial biota may have colonized the island through a land bridge with South America [START_REF] Ali | Colonizing the Caribbean: is the GAARlandia land-bridge hypothesis gaining a foothold[END_REF][START_REF] Iturralde-Vinent | Paleogeography of the Caribbean region: implications for Cenozoic biogeography[END_REF]. Nevertheless, long-distance dispersal seems to have been the dominant source of colonizers among seed plants [START_REF] Nieto-Blázquez | Historical biogeography of endemic seed plant genera in the Caribbean: Did GAARlandia play a role?[END_REF]. Ultramafic substrates, recorded on Cuba by Humboldt (1826), cover c. 7% (2,700 km 2 ) of the island, including areas of ancient origin (10-30 My) in the Western and Eastern regions [START_REF] Finko | Sobre la edad de la cortea del intemperismo y las lateritas de Cuba[END_REF], while Cuba's central ultramafic area was exposed more recently (< 1My, Zonn, 1968). Alejandro de Humboldt National Park is a UNESCO World Heritage Site in eastern Cuba with great biological and geological diversity, including ultramafic-adapted ecosystems.

Madagascar is a portion of Gondwana that was located between the Tanzania-Mozambique coast, India and Antarctica prior to its detachment from the African continent starting at the end of the Jurassic (about <140 My), followed by the separation of India in the late Cretaceous (<83 My, [START_REF] Fröhlich | La position de Madagascar dans le cadre de l'évolution géodynamique et de l'environnement de l'océan Indien[END_REF]. While a small portion of Madagascar's rich flora may thus have an ancient continental (vicariant) origin, recent reviews indicate that subsequent dispersal has played an important role [START_REF] Buerki | Spatiotemporal history of the endemic genera of Madagascar[END_REF][START_REF] Yoder | Has vicariance or dispersal been the predominant biogeographic force in Madagascar? Only time will tell[END_REF]. Ultramafic soils are rare in Madagascar, but a lateritic nickel deposit occurs at Ambatovy, which covers an area of 12 × 7 km at an elevation of approximately 1,000 m above sea level on Madagascar's eastern escarpment [START_REF] Delbos | Travaux du bureau géologique N°160[END_REF] and has been dated to c. 89-91 My [START_REF] Melluso | Geochronology and petrogenesis of the Cretaceous Antampombato-Ambatovy complex and associated dyke swarm, Madagascar[END_REF]. Botanical inventories of the area were recently conducted and several micro-endemic species have been reported [START_REF] Phillipson | Floristic inventory of the Ambatovy-Analamay mine site and comparison to other sites in Madagascar[END_REF], most of which have now been also found in surrounding areas (Madagascar Catalogue, 2019).

| Data sets

For the flora of New Caledonia, we used the list of native species occurring on ultramafic substrates from [START_REF] Isnard | How did the ultramafic soils shape the flora of the New Caledonian hotspot?[END_REF] and the complete checklist of the indigenous flora from FLORICAL (Morat et al., 2012, updated ;[START_REF] Munzinger | FLORICAL: checklist of the vascular indigeneous flora of New Caledonia vers[END_REF]. For the flora of Madagascar, we used the plant checklist from the Ambatovy-Analamay region [START_REF] Phillipson | Floristic inventory of the Ambatovy-Analamay mine site and comparison to other sites in Madagascar[END_REF], the main ultramafic outcrop of the island, and the complete checklist from the Madagascar Catalogue [START_REF] Callmander | The endemic and non-endemic vascular flora of Madagascar updated[END_REF]Madagascar Catalogue, 2019 accessed 15/09/2016) taking into account only native species. In Cuba, we developed a partial checklist of plants (appendix S1 in supporting information) occurring on ultramafic substrate by compiling lists from various regions and sources: Guamuhaya (Glaría, González-Torres, Iturralde, & Granados, 2006) Lomas de Galindo [START_REF] Lara | Recuperación natural de la flora serpentinícola en "Lomas de Galindo": una alternativa de conservación[END_REF][START_REF] Robledo | Flora i vegetación de "Lomas de Galindo" Canasí, La Habana[END_REF], Yamanigüey [START_REF] Valdés | Notas sobre la flora y vegetación de la zona de Yamanigüey, Moa, Provincia de Holguín[END_REF], Central Cuba [START_REF] Franco | Motembo, a serpentine center of plant evolution in Cuba and the West Indies[END_REF], and the Oriental Region of Cuba [START_REF] Quesada | Riqueza de especies y endemismo de la erspermatófitas en las plurisilvas de la Región Oriental de Cuba[END_REF]. We removed synonyms and non-native species using [START_REF] Greuter | The spermatophyta of Cuba: a preliminary checklist[END_REF], which also served as a checklist for the entire Cuban flora. Our analyses were restricted to flowering plants.

| Representation indices

To quantify the under-or over-representation of plant families, we defined a representation index. For each plant family and each island, we first calculated the expected number of species (Nexp,i) that should be observed on ultramafic substrates if they represented a random subset of the entire flora. ,

where NT,i is the total number of species of family i on the island, Mum is the total number of species across all families on ultramafic substrates, and MT is the total number of species across all families on the island. A representation index (Ri) was calculated for each family, as follows (neglog transformation):

, , . ln 1 , ,
where Nobs,i is the observed number of species in family i on ultramafic substrate. Ri is positive for over-represented families and negative for under-represented families. The more the number of species observed in a family deviates from expectation, the larger the absolute value of the index is. Representation indices were calculated in the same way for each angiosperm order.

We also identified the families that were significantly under-or over-represented by comparing their number of species on ultramafic substrates compared to that expected under a null hypothesis where the flora on this substrate was a random subset of the entire island flora. We tested deviation from a normally distributed sampling, applying a Bonferroni correction for multiple tests.

| Phylogenetic patterns

Since closely related families are expected to be similar in many ways, spurious correlations between characters can be inferred if families are treated as independent replicates. To circumvent this bias, [START_REF] Felsenstein | Phylogenies and the comparative method[END_REF] introduced a method called pairwise independent contrasts. It involves the comparison of the variables (X and Y) between two lineages (1 and 2) descending from the same node and the calculation of contrasts (X2-X1; Y2-Y1). Contrasts are calculated for pairs of lineages that descend from the same node, but are phylogenetically independent. A positive correlation between contrasts indicates a positive correlation between X and Y, free of phylogenetic bias. Such an approach requires knowledge of phylogenetic relationships between families. We used the family-level phylogenetic tree of the angiosperms from [START_REF] Magallón | A metacalibrated time-tree documents the early rise of flowering plant phylogenetic diversity[END_REF], which includes 366 of 416 currently recognized plant families, representing over 99% of global angiosperm species diversity and over 97% of each of the three floras investigated. This tree was also simplified to build a tree of angiosperm orders.

Pairwise independent contrasts of the representation indices between the three possible island pairs were calculated using PHYLOCOM 4.2 [START_REF] Webb | Phylocom: software for the analysis of phylogenetic community structure and trait evolution[END_REF].

| Concordance between all three islands

Independent contrasts cannot be used to compare three variables. To test concordance between representation of families on the three islands considered, we classified the families naturally present on all of them in four categories, those that have more species than expected on all three islands, on two islands, on a single island, or on none (i.e. under-represented on all islands). We compared this to a null-model distribution, where under-and overrepresentation is completely independent between islands. For example, if the proportions of under-represented families in Cuba, Madagascar, and New Caledonia are 0.3, 0.5 and 0.46, the expected proportion of families under-represented on all three islands should be 0.3 × 0.5 × 0.46 = 0.1403. We compared the observed distribution and the distribution under the nullmodel using a  2 test. Note that this approach is not free of phylogenetic effects, contrary to independent contrasts. Between 115 and 136 plant families included in the phylogenetic analysis of [START_REF] Magallón | A metacalibrated time-tree documents the early rise of flowering plant phylogenetic diversity[END_REF] were shared between pairs of the three islands examined, viz. Cuba, Madagascar and New Caledonia. The relationships between contrasts of representation indices of the floras for each island pair are plotted in Figure 1. Within sister family pairs, the better represented family (based on number of species) on ultramafic substrates on a given island was generally also better represented on ultramafics on the other island, a situation observed in a significant proportion of family pairs (table 1). When we compared orders, we also found a significant relationship between representation indices on each island pair. When we compared the 120 families that were represented on all three islands, more often than expected by chance, these families were over-or under-represented on ultramafic substrates on all three islands ( 2 = 9.017, p = 0.029, Figure 2). Fabaceae, Malvaceae, and Poaceae were under-represented on all three islands, and significantly so on Madagascar and New

Caledonia (Appendix S2 & S3

). Clusiaceae and Erythroxylaceae were over-represented on all three islands, and significantly so in Madagascar and Cuba. The same pattern was observed among the 46 orders represented on Cuba, Madagascar and New Caledonia, which were overor under-represented on ultramafic substrate on all three islands more often than expected by chance ( 2 = 11.777, p = 0.008, Figure 3).

A group of interest is the COM clade, which comprises the orders Celastrales, Oxalidales, and Malpighiales (figure 3). These three orders are over-represented on all three islands and the clade contains an important number of families that are over-represented on ultramafic substrates on some or all three islands. As a whole, this group has an excess of species on ultramafic substrate, compared to random expectation, of 56% in Madagascar, 50% in Cuba, and 10% in New Caledonia.

| DISCUSSION

This study indicates that the same plant families tend to be over-represented (e.g.

Clusiaceae, Phyllanthaceae, Salicaceae) or under-represented (e.g. Acanthaceae, Fabaceae, Malvaceae, Acanthaceae) on the ultramafic substrates of Cuba, Madagascar, and New Caledonia. This pattern was also observed at higher levels, as the same orders tended to be over-or under-represented on ultramafic substrates of the three islands. The phylogenetic signal observed here is rather deep considering that it can be detected at the family and ordinal levels. Indeed, molecular clocks [START_REF] Magallón | A metacalibrated time-tree documents the early rise of flowering plant phylogenetic diversity[END_REF] indicate that most angiosperm families are older than the biota of Cuba or New Caledonia and most of that of Madagascar.

Our comparison involves three islands with a high rate of endemism, each located in a different biogeographical setting. It seems unlikely that direct dispersal from ultramafic substrate on one island to another, could account for more than a small portion of the taxonomic similarities between these communities. Recruitment from the local pool of species appears more likely, which suggests that ecological sorting processes [START_REF] Ackerly | Community assembly, niche conservatism, and adaptive evolution in changing environments[END_REF] were at play. Some ancestral characteristics of plant families (e.g. exaptation, [START_REF] Ackerly | Adaptation, niche conservatism and convergence: comparative studies of leaf evolution in the California chaparral[END_REF][START_REF] Gould | Exaptation-a missing term in the science of form[END_REF] probably contributed to their greater success on ultramafic substrates on all three islands.

Plants capable of adapting to ultramafic substrates may have descended in most cases from an evolutionary precursor [START_REF] Marazzi | Locating evolutionary precursors on a phylogenetic tree[END_REF] that belongs to a group in which this adaptation is more likely to evolve. The traits, or exaptation [START_REF] Gould | Exaptation-a missing term in the science of form[END_REF], that would confer on a plant a greater success on ultramafic substrates remain to be identified. The constrains of such environment are multiple, including low nutrients, high Mg:Ca ratio, high concentrations of metals, etc. [START_REF] Kazakou | Hypotheses, mechanisms and trade-offs of tolerance and adaptation to serpentine soils: from species to ecosystem level[END_REF][START_REF] Proctor | Vegetation and soil and plant chemistry on ultramafic rocks in the tropical Far East[END_REF]. Sclerophylly has been associated with drought tolerance and soil nutrient deficiency [START_REF] Salleo | Sclerophylly: Evolutionary advantage or mere epiphenomenon?[END_REF], and is indeed a common feature in New Caledonia's scrubland (maquis) on ultramafic substrate

Pillon Y., González D. A., Randriambanona H., Lowry II P. P., Jaffré T., Merlot S. 2019. Parallel ecological filtering of ultramafic soils in three distant island floras. Journal of Biogeography,46 (11): 2457-2465. [START_REF] Read | Sclerophylly in two contrasting tropical environments: low nutrients vs. low rainfall[END_REF]. Recent genetic and genomic studies have revealed in Brassicaceae species that several genes coding for proteins involved in the transport of nutrients (e.g. Ca, Mg, K) and transition metals (e.g. Fe, Ni) are linked to adaptation to serpentine [START_REF] Arnold | Borrowed alleles and convergence in serpentine adaptation[END_REF][START_REF] Bradshaw | Mutations in CAX1 produce phenotypes characteristics of plants tolerant to serpentine soils[END_REF][START_REF] Sobczyk | Evolution of nickel hyperaccumulation and serpentine adaptation in the Alyssum serpyllifolium species complex[END_REF][START_REF] Turner | Population resequencing reveals local adaptation of Arabidopsis lyrata to serpentine soils[END_REF]. Among these candidate genes, transporters of the Ferroportin/IREG family were shown to transport Ni in Brassicaceae and some other plant families [START_REF] Halimaa | Gene expression differences between Noccaea caerulescens ecotypes help to identify candidate genes for metal phytoremediation[END_REF][START_REF] Meier | Comparative RNA-seq analysis of nickel hyperaccumulating and nonaccumulating populations of Senecio coronatus (Asteraceae)[END_REF][START_REF] Merlot | The metal transporter PgIREG1 from the hyperaccumulator Psychotria gabriellae is a candidate gene for nickel tolerance and accumulation[END_REF][START_REF] Morrissey | The ferroportin metal efflux proteins function in iron and cobalt homeostasis in Arabidopsis[END_REF][START_REF] Schaaf | AtIREG2 encodes a tonoplast transport protein involved in iron-dependent nickel detoxification in Arabidopsis thaliana roots[END_REF], and could therefore constitute a link between adaptation to ultramafic soils and Ni hyperaccumulation. The latter qualifies plants that accumulate particular metals in their leaves to levels that may be hundreds or thousands of times greater than is normal for most plants [START_REF] Reeves | A global database for plants that hyperaccumulate metal and metalloid trace elements[END_REF]. In a majority of cases, this trait has been recorded on ultramafic substrate and could be seen as an extreme case of specialization to these edaphic conditions. Genes involved in metal transport may thus characterise the evolutionary precursors of plants adapted to ultramafic substrates. Most of our knowledge on adaptation to ultramafic substrates and metal hyperaccumulation is derived from the mostly temperate and herbaceous Brassicaceae. Although this family favours ultramafic substrates elsewhere (Europe, California), a preference was not observed on the tropical islands studies here, where the family is poorly represented (less than 10 species on each island). Further studies on the genes involved in metal transport on a phylogenetically broad range of tropical plants would be necessary to draw any broad conclusions.

The COM clade stands out for its over-representation on ultramafic substrates on all three islands. Members of this clade also seem to have a predisposition to become nickel hyperaccumulators [START_REF] Goodman | Ultramafic soils and species sorting in the flora of New Caledonia[END_REF], a syndrome strongly associated with ultramafic substrates. The COM clade represents just 7.1% of angiosperm species but 51% of the known nickel hyperaccumulators [START_REF] Reeves | A global database for plants that hyperaccumulate metal and metalloid trace elements[END_REF], Global hyperaccumulator database, 2018). Interestingly, this group is sister to the Cucurbitales-Fabales-Fagales-Rosales clade, also known as the nitrogen-fixing clade [START_REF] Griesmann | Phylogenomics reveals multiple losses of nitrogen-fixing root nodule symbiosis[END_REF][START_REF] Soltis | Parallel ecological filtering of ultramafic soils in three distant island floras[END_REF][START_REF] Werner | A single evolutionary innovation drives the deep evolution of symbiotic N2-fixation in angiosperms[END_REF], generally under-represented on ultramafic substrates (Figure 3). Their exact relationship is nevertheless not fully resolved (Angiosperm Phylogeny Group IV, 2016; [START_REF] Sun | Deep phylogenetic incongruence in the angiosperm clade Rosidae[END_REF]. The ancient divergence between these two large clades could be associated with the rise of contrasting nutritional strategies. Similarly the order Caryophyllales is characterized by a prevalence of sodium hyperaccumulators and the high frequency of halophytes [START_REF] White | Evolutionary origins of abnormally large shoot sodium accumulation in nonsaline environments within the Caryophyllales[END_REF]. This is in line with a certain evolutionary conservatism of nutritional traits, particularly on nutrient-poor soils, suggested by [START_REF] Verboom | Specialization to extremely lownutrient soils limits the nutritional adaptability of plant lineages[END_REF]. Future studies may identify further physiological features that characterise the large clades within angiosperms, which have mostly been defined based on molecular phylogenetic evidence (Angiosperm Phylogeny Group IV, 2016).

Because of the lack of a robust phylogeny of all angiosperm genera, we could not test whether ecological sorting on ultramafic substrates holds true at the generic level.

  Pillon Y., González D. A., Randriambanona H., Lowry II P. P., Jaffré T., Merlot S. 2019. Parallel ecological filtering of ultramafic soils in three distant island floras. Journal ofBiogeography, 46 (11): 2457Biogeography, 46 (11): -2465. .

  Pillon Y., González D. A., Randriambanona H., Lowry II P. P., Jaffré T., Merlot S. 2019. Parallel ecological filtering of ultramafic soils in three distant island floras. Journal ofBiogeography, 46 (11): 2457-2465. , , .

  Pillon Y., González D. A., Randriambanona H., Lowry II P. P., Jaffré T., Merlot S. 2019. Parallel ecological filtering of ultramafic soils in three distant island floras. Journal of Biogeography, 46 (11): 2457-2465.

Figure 1 .

 1 Figure 1. Relationships between contrasts (differences between sister families) of representation indices on ultramafic substrates between each island pair. Points above the x axis represent a positive association.

Figure 2 .

 2 Figure 2. Representation on ultramafic substrates of 120 plant families present on all three islands (Cuba, Madagascar, and New Caledonia). Families tended to respond accordingly more often than expected by chance.

Figure 3 .

 3 Figure 3. Phylogenetic patterns in under-and over-representation on the ultramafic substrates of Cuba, Madagascar, and New Caledonia. Excesses or deficits in the number of species for each order compared to a null model are mapped on the phylogenetic tree of Magallón et al. (2015). The orders Acorales, Petrosaviales, Trochodendrales, Berberidopsidales, Bruniales (absent on all three islands) and Metteniusales (not included in Magallón et al., 2015) are not shown. The shaded branch corresponds to the COM clade (Celastrales, Oxalidales, Malpighiales). The photograph shows Phyllanthus peltatus (Phyllanthaceae, Malpighiales) on ultramafic substrate in New Caledonia.

  Pillon Y., González D. A., Randriambanona H., Lowry II P. P., Jaffré T., Merlot S. 2019. Parallel ecological filtering of ultramafic soils in three distant island floras. Journal ofBiogeography, 46 (11): 2457Biogeography, 46 (11): -2465. .

  Pillon Y., González D. A., Randriambanona H., Lowry II P. P., Jaffré T., Merlot S. 2019. Parallel ecological filtering of ultramafic soils in three distant island floras. Journal ofBiogeography, 46 (11): 2457Biogeography, 46 (11): -2465. .

  Pillon Y., González D. A., Randriambanona H., Lowry II P. P., Jaffré T., Merlot S. 2019. Parallel ecological filtering of ultramafic soils in three distant island floras. Journal ofBiogeography, 46 (11): 2457Biogeography, 46 (11): -2465. .

  Pillon Y., González D. A., Randriambanona H., Lowry II P. P., Jaffré T., Merlot S. 2019. Parallel ecological filtering of ultramafic soils in three distant island floras. Journal ofBiogeography, 46 (11): 2457Biogeography, 46 (11): -2465. .

Table 1 :

 1 Pillon Y., González D. A., Randriambanona H., Lowry II P. P., Jaffré T., Merlot S. 2019. Parallel ecological filtering of ultramafic soils in three distant island floras. Journal ofBiogeography, 46 (11): 2457-2465. Results of independent comparisons between representation indices (Ri) on Cuba, Madagascar, and New Caledonia.

	Phyllanthus on both Cuba and New Caledonia (Reeves, 2003), but has not yet been
	investigated on Madagascar. Our results thus corroborate the observation made by Humboldt
	(1814) over two centuries ago that the same genera may be found on different continents
	under similar ecological conditions. Although Humboldt's comment was mostly inspired by
	climatic considerations, our study shows that it can be extended to edaphic preferences.

Nevertheless, it can be noted that Garcinia (Clusiaceae), Phyllanthus (Phyllanthaceae), Psychotria (Rubiaceae), and Weinmannia (Cunoniaceae) are present on ultramafic substrates in Cuba, Madagascar, and New Caledonia, but represented in each case by distinct islandendemic species. Remarkably, nickel hyperaccumulation has been recorded in Psychotria and Modern comparative approaches enable statistical tests of Humboldt's statement, and future

ACKNOWLEDGMENTS

We thank L. Barrabé for help with phylogenetic analyses and R. B. Iturralde for assistance with compiling the bibliography. Peter Linder, Félix Forest, and three anonymous reviewers made helpful comments on previous versions of the manuscript. S.M. and D.A.G. were supported by funding from the CNRS (Defi Enviromics Gene-4-Chem) and from the Cooperation and Cultural Action Service of the French Embassy in Cuba.

The authors have broad interest in the ecology, systematics and evolution of the flora of Cuba, Madagascar, and New Caledonia, including the ecosystems on ultramafic soils, their restoration, and metal accumulating plants.

DATA AVAILABILITY STATEMENT

The partial checklist of plants occurring on ultramafic substrate in Cuba is provided in Appendix 1.

Authors' contributions. Y.P. conceived the study and conducted the analyses; D.A.B., H.R., P.P.L. and T.J. contributed data; D.A.B., H.R., P.P.L., S.M., T.J. and Y.P. drafted the manuscript.

SUPPORTING INFORMATION

Additional supporting information may be found online in the Supporting Information section at the end of the article.

BIOSKETCH

Pillon Y., González D. A., Randriambanona H., Lowry II P. P., Jaffré T., Merlot S. 2019. Parallel ecological filtering of ultramafic soils in three distant island floras. Journal of Biogeography, 46 (11): 2457Biogeography, 46 (11): -2465. .