R Blossey

H Schiessel

Biophysical Perspective: The Latest Twists in Chromatin Remodeling

In its most restrictive interpretation, the notion of chromatin remodeling refers to the action of chromatin remodeling enzymes on nucleosomes with the aim to displace and remove them from the chromatin fiber (the effective polymer formed by a DNA molecule and proteins). This local modification of the fiber structure can have consequences for the initiation and repression of the transcription process and, when the remodeling processes spreads along the fiber, also results in long-range effects essential for fiber condensation. There are three regulatory levels of relevance that can be distinguished for this process: the first is the intrinsic sequence preference of the histone octamer which rules the positioning of the nucleosome along the DNA, notably in relation to the genetic information coded in DNA, the second is the recognition or selection of nucleosomal substrates by remodeling complexes, and the final one the motor action on the nucleosome exerted by the chromatin remodeler. On each of these three levels recent work has been able to provide crucial insights which add new twists to this exciting and unfinished story, which we highlight in this perspective.

I. INTRODUCTION

The nucleosome, the basic structural unit of the chromatin fiber, remains a key subject of interest for chromatin biologists and physicists. A key basic insight of recent years was the (indirect) sequence-dependence of its positioning along DNA [START_REF] Segal | A genomic code for nucleosome positioning[END_REF]. Already much earlier it was noticed that the nucleosome structure is a flexible, dynamic structure with an intrinsic, thermally driven mobility [START_REF] Eslami-Mossallam | Nucleosome dynamics: Sequence matters[END_REF]. In the context of many chromatin-related processes, the nucleosome needs to be actively displaced or removed. The molecular machines that organize this process are called chromatin remodelers [START_REF] Smith | ATP-dependent chromatin remodeling[END_REF], commonly grouped in four distinct families, see Figure 1 [START_REF] Clapier | The biology of chromatin remodeling complexes[END_REF][START_REF] Blossey | Chromatin: Structure, Dynamics, Regulation[END_REF]. Chromatin remodelers are increasingly understood as key factors also in the development of diseases due to the deregulation of gene expression such as in cancer, see, e.g. [START_REF] Kumar | Epigenomic regulation of oncogenesis by chromatin remodeling[END_REF]. The definition of the chromatin remodeler families relies on the basic motor unit, the ATPase, which derives from helicases, and the positioning of additional regulatory subunits around the ATPase.

How can we imagine a chromatin remodeler to work on a nucleosome? Being researchers in the low countries (The Netherlands and French Flanders), we allow ourselves to resort to an analogy drawn from a favorite transportation means in our regions, the bicycle. Manpowered bikes may still be the most frequently used type of bikes, but additional power is built in in the ever more popular eBikes. For those who want to have the best of both worlds, there are the 'clip-on' motors: if one wants to go faster, one fixes a small add-on motor to power the bike. An example is shown in Figure 2. Chromatin remodelers, within this analogy, are nothing but 'clip-on' FIG. 1. Definition of the remodeler families according to [START_REF] Clapier | The biology of chromatin remodeling complexes[END_REF]. DExx and HELICc (shown in red) are ATPase domains, Bromo, SANT and SLIDE, and Tandem chromo (shown in blue) are accessory domains that recognize specific histone tail modifications, or DNA. Adapted from [START_REF] Clapier | The biology of chromatin remodeling complexes[END_REF].

motors to nucleosomes. However, they do not propel the nucleosome in a linear fashion, as many molecular motors do that simply transport cargo. Chromatin remodeling motors displace the DNA wrapped around the histone octamer, and this obviously is a much more complex task than linear transport. Our simplistic analogy, however, also makes us ask simple questions such as: where are these motors actually fixed on the nucleosome? How do they pull on the DNA? How is the DNA transported around the nucleosome? Recent research has given at Before turning to the topic of the motor action, we will address the two additional levels of chromatin regulation concerning the nucleosome we mentioned at the beginning. The first and most fundamental one is the positioning of the nucleosome along the fiber, which is dictated in a complex way by the DNA sequence -or rather, the physical properties associated with the stacking of the base pairs. The second level is the problem of how the machinery recognizes the right nucleosome at the right time. Some time ago we have postulated a 'kinetic proofreading' scenario for this process, for which some experimental justification has become available shortly afterwards. New high-throughput data that have been published only very recently lend new experimental support to our idea. Finally, on level three, recent progress in the understanding of the action of remodeling motors has become available through new experimental techniques that allow to trace the dynamics of the remodelernucleosome complexes, which we highlight as we go through some new results. Key in this field have been the substantial advances in cryo-electron microscopy, freshly honoured with the Nobel prize in Chemistry in 2017 for the pioneers of this technique. In the following, we will step through the recent insights on these three levels.

II. NUCLEOSOME POSITIONING

In a nucleosome about one persistence length of DNA is wrapped one and three quarter turns around an octamer of histone proteins, which amounts to a substantial amount of bending energy (about 60 k B T [START_REF] Langowski | Chromatin simulations[END_REF]). On the other hand, the elasticity and geometry of the DNA double helix depends on the underlying sequence of base pairs [START_REF] Olson | DNA sequence-dependent deformability deduced from protein-DNA crystal complexes[END_REF][START_REF] Lankaš | Sequencedependent elastic properties of DNA[END_REF]. It is thus to be expected that the affinity of a given stretch of DNA to be complexed in a nucleosome varies strongly with sequence. Moreover, the binding of the DNA to the octamer occurs mainly at 14 patches where the minor groove of the DNA touches the surface of the octamer [START_REF] Davey | Solvent Mediated Interactions in the Structure of the Nucleosome Core Particle at 1.9 Å Resolution[END_REF]. As the sugar-phosphate backbones do not change with the underlying base pair sequence, the pure binding energy (mainly based on charge-charge interactions between the DNA phosphates and histones [START_REF] Fenley | Charge state of the globular histone core controls stability of the nucleosome[END_REF]) is expected to be only weakly sequence dependent. Taken together this suggests that the nucleosome performs an indirect readout of the basepair sequence, with the affinity reflecting the overall ability of 147 base pairs to wrap around the octamer.

Nucleosomes show indeed characteristic sequence preferences [START_REF] Segal | A genomic code for nucleosome positioning[END_REF][START_REF] Satchwell | Sequence periodicities in chicken nucleosome core DNA[END_REF], e.g. high affinity sequences feature an increased occurrence of GC dinucleotides (a G followed by a C) at locations where the minor groove faces outwards, whereas AA, TT and TA dinucleotides peak in between, where the minor groove faces inwards towards the histone octamer (i.e. at the binding sites), see Figure 3(a). These preferences have been proposed to constitute a "genomic code for nucleosome positioning" [START_REF] Segal | A genomic code for nucleosome positioning[END_REF] (for earlier versions of this idea see e.g. Refs. [START_REF] Satchwell | Sequence periodicities in chicken nucleosome core DNA[END_REF][START_REF] Trifonov | The pitch of chromatin DNA is reflected in its nucleotide sequence[END_REF]). In fact, the sequence preferences of the nucleosome give rise to two types of positional preferences: rotational and translational positioning. Rotational positioning results from the fact that DNA is attached at its minor groove to the octamer. Therefore when a nucleosome is repositioned one base pair step at a time the DNA needs to perform a corkscrew-like motion. As a given wrapped stretch of DNA has typically a preferred bending direction (even for a completely random base pair sequence), there is every 10 base pairs a minimum in the bending energy, causing the rotational positioning of the nucleosome. The second type of preference, translational positioning, reflects an overall preference of nucleosomes to sit on DNA stretches with high GC content and to avoid certain motifs like e.g. poly(dA:dT) tracts.

Nucleosomes have been mapped genome-wide in vivo and in vitro (in the latter case nucleosomes are reconstituted on DNA from their pure components) using a variety of methods, e.g. through digestion by nuclease [START_REF] Segal | A genomic code for nucleosome positioning[END_REF] and by a chemical cleavage technique [START_REF] Brogaard | A map of nucleosome positions in yeast at base-pair resolution[END_REF]. Such nucleosome maps have taught us that indeed non-random mechanical cues can be found written along genomes. This becomes especially clear by focusing on particular locations, e.g. at transcription start sites (TSSs), and then by averaging over all such locations (e.g. over the TSSs of all genes of an organism). Such studies revealed that yeast shows on average a depletion of nucleosomes just in front of genes [START_REF] Segal | A genomic code for nucleosome positioning[END_REF][START_REF] Brogaard | A map of nucleosome positions in yeast at base-pair resolution[END_REF]. But is this depletion reflecting nucleosome sequence preferences or is it caused by other mechanisms, e.g. the action of chromatin remodellers [START_REF] Struhl | Determinants of nucleosome positioning[END_REF]? In general this is a complex question and the answer clearly depends on the organism at hand.

To answer this question it is necessary to develop methods that allow to estimate nucleosome sequence preferences along DNA molecules. Bioinformatics models trained on experimental nucleosome maps provide a common approach [START_REF] Segal | A genomic code for nucleosome positioning[END_REF]. Deeper insight into the relation between DNA mechanics and nucleosome positioning can be gained from coarse-grained computer models of nucleosomes that give fairly reliable estimates of sequence preferences [START_REF] Morozov | Using DNA mechanics to predict in vitro nucleosome positions and formation energies[END_REF][START_REF] Freeman | DNA Shape Dominates Sequence Affinity in Nucleosome Formation[END_REF][START_REF] Eslami-Mossallam | Multiplexing genetic and nucleosome positioning codes: a computational approach[END_REF], see e.g. Figure 3(b). These models, however, typically tend to be too slow to perform e.g. genome wide averages of the DNA mechanics around TSSs. The recently developed Mutation Monte Carlo method [START_REF] Eslami-Mossallam | Multiplexing genetic and nucleosome positioning codes: a computational approach[END_REF] overcomes this problem as it allows to determine the sequence preferences of a given model nucleosome that in turn can be used to build probabilistic models [START_REF] Tompitak | Benchmarking and refining probability-based models for nucleosome-DNA interaction[END_REF][START_REF] Wondergem | Performing SELEX experiments in silico[END_REF]. Such models are similar to the above-mentioned bioinformatics approaches but ultimately are based on specific microscopic models with well-known ingredients. Starting from an already fairly efficient coarse-grained nucleosome model, this approach leads to a speedup of about 10 5 [START_REF] Tompitak | Benchmarking and refining probability-based models for nucleosome-DNA interaction[END_REF]. Applied to TSSs of yeast [START_REF] Tompitak | Genomes of multicellular organisms have evolved to attract nucleosomes to promoter regions[END_REF] we found excellent agreement with experimental data. As the approach is based on a purely mechanical model, this suggests that nucleosome depletion at TSSs in yeast is indeed caused by sequence-dependent DNA mechanics.

An exciting question to ask is to what extent genomes position nucleosomes. This turns out to be a tricky question. For instance, in Ref. [START_REF] Segal | A genomic code for nucleosome positioning[END_REF] it was claimed that about 50% of the nucleosomes on the yeast genomes can be "explained solely by sequence preferences." But this statement has to be taken with a grain of salt as it does not automatically mean that there are dedicated locations for 50% of the nucleosomes directly encoded into the DNA sequence. When one looks at the in vivo nucle-osome maps of yeast it seems that nucleosomes are wellpositioned along the beginning of each gene such that one can count them, one after the other (one speaks indeed of the +1 nucleosome, the +2 nucleosome and so on [START_REF] Brogaard | A map of nucleosome positions in yeast at base-pair resolution[END_REF]). But when one calculates the average positional preferences of the nucleosomes around the TSSs [START_REF] Tompitak | Genomes of multicellular organisms have evolved to attract nucleosomes to promoter regions[END_REF], one finds mainly an "anti-positioning" element just in front of genes, an AT-rich piece of DNA that repels nucleosomes. The appearance of clearly distinguishable "positioned" nucleosomes along the beginning of genes in yeast is then in fact just a statistical effect, caused by the relatively large density of nucleosomes and the presence of a boundary constraint as suggested in the classical work of Kornberg and Stryer [START_REF] Kornberg | Statistical distributions of nucleosomes: nonrandom locations by a stochastic mechanism[END_REF]. For the much lower density of nucleosomes obtained via in vitro reconstitution, these seemingly well-defined peaks vanish completely [START_REF] Kaplan | The DNAencoded nucleosome organization of a eukaryotic genome[END_REF] (the situation is in fact a bit more complex; for details we refer the reader to the excellent review by Struhl and Segal [START_REF] Struhl | Determinants of nucleosome positioning[END_REF]).

Even though nucleosomes seem not to be positioned individually in yeast, the statistical ordering of nucleosomes caused by the presence of a boundary can still be said to be "explained" by nucleosomal sequence preferences. But the more exciting question remains whether there are also examples of nucleosomes that are specifically positioned by sequence at certain genomic locations. There are indeed numerous examples. In fact, the genomes of many organisms attract nucleosomes to TSSs. A rule that seems to be generally true (at least for the 35 eukaryotic genomes we analysed [START_REF] Tompitak | Genomes of multicellular organisms have evolved to attract nucleosomes to promoter regions[END_REF]) is that genomes of unicellular organisms repel nucleosomes from TSSs whereas multicellular life forms show a region that attracts nucleosomes. For instance, nematodes like C. elegans have (on average) a strongly positioned nucleosome just at the entrance to its genes [START_REF] Tompitak | Genomes of multicellular organisms have evolved to attract nucleosomes to promoter regions[END_REF]. Mammals (e.g. mouse, chimpanzee, human) show a broad region with high overall nucleosome affinity [START_REF] Tompitak | Genomes of multicellular organisms have evolved to attract nucleosomes to promoter regions[END_REF].

What is the biological function of those various motifs? At this point one can only speculate. For yeast the encoded nucleosome depletion "may indicate that eukaryotic genomes direct the transcriptional machinery to functional sites by encoding unstable nucleosomes over these elements, thereby enhancing their accessibility" [START_REF] Segal | A genomic code for nucleosome positioning[END_REF]; the same would be true for all unicellular lifeforms [START_REF] Tompitak | Genomes of multicellular organisms have evolved to attract nucleosomes to promoter regions[END_REF].

In C. elegans one finds a single well-positioned nucleosome at the beginning of many genes in vitro and in vivo, as predicted by our mechanical model [START_REF] Tompitak | Genomes of multicellular organisms have evolved to attract nucleosomes to promoter regions[END_REF], and this might function as a mechanism for TSS selection for RNA polymerase [START_REF] Dreos | Influence of rotational nucleosome positioning on transcription start site selection in animal promoters[END_REF]. In humans the situation is complex. The high nucleosome occupancy encoded by the sequence at promoters [START_REF] Tompitak | Genomes of multicellular organisms have evolved to attract nucleosomes to promoter regions[END_REF] has been speculated "to restrict access to regulatory information that will ultimately be utilized in only a subset of differentiated cells" [START_REF] Tillo | High nucleosome occupancy is encoded at human regulatory sequences[END_REF]. However, the in vivo nucleosome occupancy does not correlate well with that intrinsic nucleosome affinity, possibly reflecting transcription related processes [START_REF] Vavouri | Chromatin organization in sperm may be the major functional consequence of base composition variation in the human genome[END_REF]. A possible explanation is that the mechanical cues serve here a different purpose, namely to determine which nu-cleosomes are retained in sperm cells (where most nucleosomes are replaced by protamines), allowing the transmission of paternal epigenetic information. In fact, the mechanical cues correlate well with the nucleosome retention score in sperm cells [START_REF] Tompitak | Genomes of multicellular organisms have evolved to attract nucleosomes to promoter regions[END_REF][START_REF] Vavouri | Chromatin organization in sperm may be the major functional consequence of base composition variation in the human genome[END_REF]. This does, however, not mean that there are no mechanical cues for nucleosomes in somatic human cells. A spectacular example are 6 × 10 6 nucleosomes mechanically positioned around nucleosome inhibiting barriers, but the biological function has yet to be determined [START_REF] Drillon | Evidence of selection for an accessible nucleosomal array in human[END_REF].

We finally mention two more important points that are not well understood yet. The first one is that the nucleosome density varies with cell type or developmental state even though all cells of an organism have the same DNA sequences. This is not inconsistent with the idea of mechanical cues on DNA molecules as regions of nucleosomes might act as "nucleosomal switches" [START_REF] Schwab | Nucleosome switches[END_REF]. Another complication is the presence of higher order chromatin structures that might influence nucleosome positioning, a subject that to the best of our knowledge has not yet been addressed.

Sequence-dependent DNA mechanics is thus one of several players that determine nucleosome positions along genomes. We turn now to another key mechanism: the action of chromatin remodelers.

III. NUCLEOSOME RECOGNITION BY REMODELERS

Chromatin remodelers have a very high affinity for DNA so that they will 'remodel' even a naked DNA molecule, and this even without ATP [START_REF] Lia | Direct observation of DNA distortion by the RSC chromatin remodelling complex[END_REF]. This basic background mechanism therefore needs to be tuned such that the remodelers act on the right objects (the nucleosomes) at the right place (e.g. near a TSS), and at the right time.

A mechanism by which the remodelers identify their targets has been proposed by us in 2008 [START_REF] Blossey | Kinetic proofreading of gene activation by chromatin remodeling[END_REF]. It is a variant of the famous kinetic proofreading mechanism originally proposed by John Hopfield in the context of mRNA translation [START_REF] Hopfield | Kinetic proofreading: a new mechanism for reducing errors in biosynthetic processes requiring high specificity[END_REF]. In the context of chromatin remodeling, histone tail modifications placed by 'histone writers' can be read by accessory subunits of the chromatin remodelers which are sensitive to these states. The essentially same idea was put forth shortly after by Geeta Narlikar for the ISWI/ACF system, based on her very detailed experiments [START_REF] Narlikar | A proposal for kinetic proof reading by ISWI family chromatin remodeling motors[END_REF][START_REF] Blossey | Kinetic proofreading in chromatin remodeling: the case of ISWI/ACF Biophys[END_REF].

Very recently, a high-throughput analysis confronting different remodelers with the different possible histone tail modifications lends very strong support for the correlation between histone tails states and the outcomes of remodeler actions [START_REF] Dann | ISWI chromatin remodellers sense nucleosome modifications to determine substrate preference[END_REF]. While the results have not been interpreted within the proofreading scenario, the observations themselves are clearly in support of it. Apart from the recognition of histone modifications, a key element of the kinetic proofreading scenario, there are also additional levels of recognition encoded which provide alternative means of regulation. Since ISWI recognizes the H4-tail only without modifications, there is an additional level embedded in this system as a 'basic patch' on the tail enters in competition with a like patch on the remodeler itself: the remodeling motor is inhibited by some of its own domains (called AutoN and NegC), an inhibition that is only relieved in the presence of the histone tail [START_REF] Clapier | Regulation of ISWI involves inhibitory modules antagonized by nucleosomal epitopes[END_REF]. Recently, the multivalency of bromodomains of the BAF chromatin remodeling complex has been elucidated, as they bind to both DNA and the histone tail modification H3K14ac [START_REF] Morrison | DNA binding drives the association of BRG1/hBRM bromodomains with nucleosomes[END_REF]. These insights show that even within the interaction of a specific remodeler type with a nucleosome a rich network of recognition mechanisms has evolved for the fine tuning of remodeler action. These developments are clearly important for more general predictive models of remodeler recruitment on similar large-scale levels as for sequence-directed nucleosome positioning. Statistical physics-based models in this direction were already put forward in [START_REF] Teif | Predicting nucleosome positions on the DNA: combining intrinsic sequence preferences and remodeler activities[END_REF][START_REF] Padintheeri | Nucleosome positioning in a model of active remodeling enzymes[END_REF].

IV. CHROMATIN REMODELER ACTION

A. Motor engagement

Three recent studies have provided new key insights into the engagement of chromatin remodeling motors on the nucleosome; as they consider remodelers from different families, the insights they yield nicely complement each other.

In a study by Liu et al. [START_REF] Liu | Mechanism of chromatin remodelling revealed by the Snf2-nucleosome structure[END_REF], summarized by Wigley and Bowman [START_REF] Wigley | A glimpse into chromatin remodeling[END_REF], the interaction between the ATPase unit of Snf2 (also called SWI-SNF) and the nucleosome was studied by cryo-EM. The study reveals that the AT-Pase is bound to an internal site on superhelical location 2 (SHL2). As the structure is nucleotide-free, the ATP-binding cleft is in an open conformation, which by comparison with closed conformations allows to conclude that the closure of the cleft would push 1 bp of DNA towards the nucleosome dyad. An important insight from this study is that in addition to the binding site of the ATPase at SHL2, an acidic patch on the first lobe of the ATPase contacts the DNA 90 bp away. Upon ATP binding and closure of the cleft, the second lobe can move the DNA duplex toward the dyad axis across the histone octamer. After ATP-hydrolysis, lobe 2 would need to reset without sliding the DNA back with it. This study therefore provides an answer to the question we asked in the introduction. On the eBike, the 'clip-on' motor on the nucleosome needs to be fixed on a position at the frame, and then contacts the rolling wheel of the bike to transmit the power. In the case of the ATPase, the remodeler remains attached with its lobe 1 at SHL2, while lobe 2 dynamically resets upon translocation, see the schematic illustration in Figure 4. Very similar findings on the workings of the remodeler ATPase were obtained in work by Nodelman et al. on the Chd1 remodeler [START_REF] Nodelman | Interdomain communication of the Chd1 chromatin remodeler across the DNA gyres of the nucleosome[END_REF], which has also been studied by the Bowman group in much detail [START_REF] Mcknight | Sequence-targeted nucleosome sliding in vivo by a hybrid Chd1 chromatin remodeler[END_REF][START_REF] Winger | The sequence of nucleosomal DNA modulates sliding by the Chd1 remodeler[END_REF]. Chd1 is a (smaller) remodeling complex that plays a key role in nucleosome positioning over coding regions [START_REF] Gkikopoulos | A role for Snf2-related nucleosome-spacing enzymes in genome-wide nucleosome organization[END_REF].

The third study we refer to is the work by Sundaramoorthy et al. [START_REF] Sundaramoorthy | Structural reorganization of the chromatin remodeling enzyme Chd1 upon engagement with nucleosomes[END_REF], which also considers Chd1. Like other remodelers, it belongs to the Snf2-family and contains two helicase-like domains for which previous structural information was available for the ATPase domain associated to N-terminal chromodomains, which serve to recognize methylation states of the histone tails. Chd1 resembles ISWI in one important aspect, as it also contains a C-terminal DNA-binding domain composed of the SANT and SLIDE domains. The structural information available on all these structures, however, was limited to these subdomains, not reaching up to the level of the full structure. In the experiments described in [START_REF] Sundaramoorthy | Structural reorganization of the chromatin remodeling enzyme Chd1 upon engagement with nucleosomes[END_REF], SAXS data obtained on full Chd1 complexes (without the nucleosome) showed that Chd1 is a monomeric complex. To obtain higher resolution data, the complex was studied with pulsed EPR after cysteine-serine replacements. The mutant protein showed unmodified remodeler activity. Reintroduction of specifically chosen labelled cysteine pairs allowed to determine distances in the complex, which can be related to the crystal structure. The resulting distance distributions reveal the possible dynamics of the structure through their difference to residue distances in the crystal structure. Together with docking poses these data thus allow to develop an idea of the dynamic contacts the complex can undergo. These results also corroborated by a very recent structural study on the Chd1-nucleosome complex published by the Cramer group [START_REF] Farnung | Nucleosome-Chd1 structure and implications for chromatin remodeling[END_REF].

Further insights into the role of the N-terminal tail in the regulation of remodeler activity was studied by performing mutations in several regions, and assessing their effect on the repositioning of remodelers. Finally, also the engagement of the remodeler to the nucleosome was studied by cryo-EM. These highly detailed studies allow substantial insights into the different contacts the remodeler domains make with nucleosomes during remodeling.

B. A dynamical role for the histone octamer

In most studies of chromatin remodeling, the histone octamer is considered as a rather static bystander of remodeling events: it is essentially the spool around which the DNA is wrapped and hence unspooled or transported around this protein structure. A recent study by Sinha, Gross and Narlikar [START_REF] Sinha | Distortion of histone octamer core promotes nucleosome mobilization by a chromatin remodeler[END_REF] challenges this purely 'passive' role of the histone octamer. The findings by Sinha et al. have been summarized by Flaus and Owen-Hughes [START_REF] Flaus | Unlocking the nucleosome[END_REF].

The key idea of this work has been to first introduce artificial modifications in the histone octamer structure, and to correlate the direct observations of the effect of these modifications on NMR spectra of nucleosomes bound to a remodeler with positioning assays of nucleosomes under the action of remodelers.

Specifically, the authors studied 13 C-isotope modifications in the methyl groups of buried H4 side chains, and detected the structural changes these modifications induce via nuclear magnetic resonance spectroscopy. The nucleosomes were bound to an Snf2-remodeler. The observed changes appeared prominently in the H3-H4 dimer near the superhelical location 0 (SHL0), close to the center of the nucleosome, which is distant from the location SHL2 where the remodeler is expected to bind the nucleosome. In a separate set of experiments, the authors engineered disulfide linkages between H3 and H4 to restrict their flexibility. In accord with the NMR data, such crosslinks in the vicinity of SHL0 led to a statistically significant number of disrupted nucleosomes, while crosslinks near SHL2 showed no significant effect. By studying remodelers from different families, ISWI, INO80 and RSC, differences between the remodelers became apparent: the crosslinking near SHL2 affects both ISWI and RSC-remodeled nucleosomes, but shows no relevant effect on INO80.

These results clearly show that the full structure of a nucleosome-remodeler complex features dynamical behaviour in all of its subunits. Remodeling has become yet more dynamic than previously thought.

C. Nucleosomes and CRISPR-Cas

As a final point of this perspective article we address the relevance of chromatin remodeling for the currently hottest topic in DNA biology, CRISPR-Cas9. In a recent study involving one of the co-inventors of the CRISPR technology, the role of nucleosomes on the chromatin fiber undergoing the genome editing process has been investigated, under conditions of the presence or absence of chromatin remodelers [START_REF] Isaac | Nucleosome breathing and remodeling constrain CRISPR-Cas9 function[END_REF]. In biochemical assays in which the preferred positioning of nucleosomes on the 601-sequence was used in specifically constructed sequences, the authors could establish that the efficiency of the Cas9 enzyme to cut the DNA is consistent with the well-known nucleosome breathing behaviour [START_REF] Polach | Mechanism of protein access to specific DNA sequences in chromatin: a dynamic equilibrium model for gene regulation[END_REF]. According to this mechanism nucleosomal DNA unpeels temporarily from the ends favouring the interaction of enzymes with sites that are close to the nucleosomal flanks. This has been shown first for restriction enzymes [START_REF] Polach | Mechanism of protein access to specific DNA sequences in chromatin: a dynamic equilibrium model for gene regulation[END_REF][START_REF] Anderson | Sequence and position-dependence of the equilibrium accessibility of nucleosomal DNA target sites[END_REF] and can now be observed more directly via fluorescence resonance energy transfer [START_REF] Buning | Single-pair FRET experiments on nucleosome conformational dynamics[END_REF][START_REF] Gansen | Closing the gap between single molecule and bulk FRET analysis of nucleosomes[END_REF]. In addition, Cas9 efficiency is promoted by the presence of chromatin remodelers which displace the nucleosomes from the cut sites. Therefore, due to these two mechanisms, the CRISPR-Cas9 system can reach efficiencies in eukaryotes that equal those in bacterial systems, in which the DNA is more readily accessible.

The authors of Ref. [START_REF] Isaac | Nucleosome breathing and remodeling constrain CRISPR-Cas9 function[END_REF] also point out that the design of CRISPR-Cas9 systems can be optimized by taking into account the intrinsic nucleosome positions. But to what extent can such a prediction be made? If nucleosome positions are known, coarse-grained models can predict the sequence-dependent accessibility for proteins to DNA target sites inside nucleosomes [START_REF] Polach | Mechanism of protein access to specific DNA sequences in chromatin: a dynamic equilibrium model for gene regulation[END_REF][START_REF] Anderson | Sequence and position-dependence of the equilibrium accessibility of nucleosomal DNA target sites[END_REF] as recently demonstrated [START_REF] Culkin | The role of DNA sequence in nucleosome breathing[END_REF]. However, as mentioned earlier, not all nucleosomes are positioned by mechanical signals alone (see e.g. our discussion of nucleosome positioning at human TSSs). We therefore expect that all three levels of chromatin regulation concerning the nucleosome need to be taken into account to arrive at reliable predictions of Cas9 efficiency.

V. CONCLUSIONS

We have provided an overview about recent developments in understanding how nucleosomes give limited access to their DNA. One important aspect is the sequence-dependent DNA elasticity which can be tuned over evolutionary time scales to create regions of high or low nucleosome stability. We discussed how in the context of transcription start sites this might give access to regulatory information or, depending on the organisms, might determine nucleosome retention in sperm cells. We then turned to chromatin remodellers and first discussed new detailed experimental evidence how remodellers might recognise the "right" nucleosomes for their action. Finally, we discussed how very recent work begins to provide detailed insight how the motor activity of remodellers translates to nucleosome repositioning. The high level of understanding of the microscopic details of these three mechanisms that influence nucleosome positioning and dynamics makes us hopeful that a comprehensive picture of how nucleosomes influence gene expression might be within reach in the near future. In this perspective we have focused on the very recent developments which in our view significantly advance the field. Obviously, we have not been able to cover all aspects here in which progress has been made in the last years. For an extended description covering earlier work we refer the reader to the recent review by Lai and Pugh [START_REF] Lai | Understanding nucleosome dynamics and their links to gene expression and DNA replication[END_REF].

FIG. 2 .

 2 FIG. 2. Top: a 'clip-on' motor that turns a bike into an eBike; the bottom figure shows how the motor is fixed on the bike. Courtesy: add-e

FIG. 3 .

 3 FIG. 3. (a) Nucleosome positioning rules [1]: Key dinucleotides are shown relative to one-half of the symmetric nucleosome. (b) A coarse grained computational model of the nucleosome as employed in Refs. [18-20].

FIG. 4 .

 4 FIG.[START_REF] Clapier | The biology of chromatin remodeling complexes[END_REF]. Schematic drawing of the remodeler engagement for the Snf2-construct, following Figure3 a) from[START_REF] Liu | Mechanism of chromatin remodelling revealed by the Snf2-nucleosome structure[END_REF] and Figure2from[START_REF] Wigley | A glimpse into chromatin remodeling[END_REF]. Grey: histone octamer; black: nucleosomal DNA; red: ATPase units; blue: accessory remodeler domains.

Acknowledgements. This perspective article is dedicated to the memory of Jörg Langowski, a longtime friend and collaborator. As many colleagues could witness, Jörg was a unique individual with a limitless source of friendliness, support and criticism. We deeply miss him.


Thomas F Bristow
email: thomas.f.bristow@nasa.gov

Elizabeth B Rampe
email: elizabeth.b.rampe@nasa.gov

Cherie N Achilles

David F Blake

Steve J Chipera

Patricia Craig

Joy A Crisp

Marais David Des

Robert T Downs

Ralf Gellert

D J Des Marais

J P Grotzinger

S Gupta

R M Hazen

B Horgan

J V Hogancamp

N Mangold

P R Mahaffy

A C Mcadam

D W Ming

J M Morookian

R V Morris

S M Morrison

A H Treiman

D T Vaniman

A R Vasavada

A S Yen

Affiliations

Clay mineral diversity and abundance in sedimentary rocks of Gale crater, Mars

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

Introduction

The Mars Science Laboratory (MSL) rover Curiosity has documented sedimentary rocks on the floor of Gale crater and lower slopes of the crater's central mound, Aeolis Mons (informally known as Mt. Sharp), since landing in August 2012 (1,2). Early in the mission, at Yellowknife Bay (YKB) (1), lacustrine mudstones of the Sheepbed member were shown to contain ~20 wt.% clay mineral, which was identified as Fe-rich saponite. Saponite was proposed as forming close to the time of sediment deposition by isochemical aqueous alteration of detrital olivine under anoxic to poorly oxidizing conditions (3-5). By providing constraints on pH and possible substrates for chemolithoautotrophs during deposition, the clay minerals are key indicators of an ancient habitable lake (1,3,4). D/H ratios of clay minerals combined with their mode of formation also constrain the global inventory of martian water ~3.5 Ga (6).

YKB gave an early glimpse of part of a spatially and temporally extensive Early Hesperian (~3.5 Ga) fluvial-lacustrine system that likely occupied much of Gale crater, which MSL continues to explore as part of its now >18 km traverse of the crater floor and lower slopes of Mt. Sharp (2). Mineralogical and geochemical investigations of these sediments have revealed the dynamic natures of lake water chemistry and early diagenetic conditions, with evidence of redox stratification in the lake, and/or variations in pH and Eh during subsequent diagenesis (7,8).

Clay minerals were found in sandstones and mudstone samples stratigraphically above YKB at Windjana in the Kimberley formation, and various samples of the Murray formation outcrops in the Pahrump Hills (7,9,10) (Fig. 1,2). The identified clay minerals largely belong to the 2:1 group (7,9), a family of phyllosilicates with a diverse range of physical, chemical, and crystallographic properties (11-13). However, their low abundance (~10 wt.% or less), combined with XRD peak overlap from co-occurring pyroxene minerals (7,9,10), prevent detailed crystallography and chemical characterization. As a result, key constraints on the origin and genesis of clay minerals are not accessible. Here we provide these details for clay minerals in the four most recent drill samples of Murray formation mudstones, stratigraphically above the basal Pahrump Hills member (Fig. 1).

Results

The four Murray formation samples we investigated are drill powders, collected from a depth of 5 to 6 cm in the bedrock, by Curiosity's Sample Acquisition, Processing, and Handling (SA/SPaH). The samples in stratigraphic order, shown in Fig. 1, are: Oudam, from a ~25 m-thick unit of cross-stratified siltstones and very-fine grained sandstones of the Hartmann's Valley member, likely aeolian in origin, although a fluvial interpretation has been discussed; Marimba and Quela, which come from a ~30 m-thick package of finely laminated mudstones of the Karasburg member, representing a return to subaqueous deposition; and Sebina, from the Sutton Island member that consists of heterolithic mudstone-sandstones containing desiccation cracks and other sedimentary structures suggesting episodic drying and subaerial exposure of the Gale lake (14-16). By examining the clay mineralogy of samples in their geological context, we constrain the timing, locus and mechanisms of clay mineral formation. Our findings are relevant to debates concerning surficial vs. crustal origins of clay minerals detected from orbit, which have implications for the planetary hydrology and climate of early Mars (17-19).

CheMin X-ray diffraction (XRD) analysis (Materials and Methods) show that clay minerals make up ~15 to 28 wt.% of the bulk rock with similar contributions to XRD patterns in Marimba, Quela and Sebina (Table 1). As observed in nearly every clay mineral bearing sample collected by MSL (Oudam is the exception, as described below), broad basal reflections at ~10 2 CoK, (~10 Å), indicate the presence of 2:1 group clay minerals (Fig. 3A). The 02l clay mineral band, which is sensitive to the occupancy and species of cations within the octahedral sheets of clay minerals [START_REF] Moore | X-ray Diffraction and the Identification and Analysis of Clay Minerals[END_REF], peaks at ~22.9 2 CoK (4.50 Å; Fig. 3B). This band position is characteristic of dioctahedral 2:1 clay minerals (13,[START_REF] Moore | X-ray Diffraction and the Identification and Analysis of Clay Minerals[END_REF]. Trioctahedral smectites in YKB samples (John Klein and Cumberland) have a distinctly different 02l band position (~22.7 2, 4.58 Å) (Fig. 3B) (3,4).

The CheMin sample cells maintain near-constant, very low humidity, which would promote loss of interlayer H2O and collapse of smectite interlayers, making them difficult to distinguish from illite based on basal reflection position alone (3,4). However, illite, which typically contains fixed K in the interlayer (4,11-13), does not appear to be a significant component of these samples based on the lack of correlation between clay content of Murray formation mudstones and K content of the bulk samples (Fig. S1). Smectitic clay minerals appear to be most abundant, with the K contents of samples accounted for by sanidine, jarosite and Xray amorphous material (10, Table S1). SAM evolved gas analyses (EGA, see Materials and Methods) give additional information on the nature of the octahedral sheets of the Murray clay minerals. The temperature of H2O loss during heat-driven dehydroxylation of clay minerals is sensitive to cation content, occupancy, and the position of the vacant octahedral sites in dioctahedral clay minerals [START_REF] Heller-Kallai | Dehydroxylation of dioctahedral phyllosilicates[END_REF][START_REF] Derkowski | Nature of rehydroxylation in dioctahedral 2:1layer clay minerals[END_REF][START_REF] Cuadros | Crystal-chemistry of interstratified Mg/Fe-clay minerals from seafloor hydrothermal sites[END_REF].

Peak H2O release of the Marimba sample occurred at 610C and 780C, indicating the presence of both dioctahedral and trioctahedral components, respectively (Fig. 4) [START_REF] Heller-Kallai | Dehydroxylation of dioctahedral phyllosilicates[END_REF][START_REF] Derkowski | Nature of rehydroxylation in dioctahedral 2:1layer clay minerals[END_REF][START_REF] Cuadros | Crystal-chemistry of interstratified Mg/Fe-clay minerals from seafloor hydrothermal sites[END_REF][START_REF] Ming | Volatile and Organic Compositions of Sedimentary Rocks in Yellowknife Bay, Gale Crater, Mars[END_REF]. EGA data are inconsistent with the most Fe(III)-rich dioctahedral smectites such as nontronite, which have diagnostic dehydroxylation temperatures of <550C [START_REF] Heller-Kallai | Dehydroxylation of dioctahedral phyllosilicates[END_REF][START_REF] Derkowski | Nature of rehydroxylation in dioctahedral 2:1layer clay minerals[END_REF][START_REF] Cuadros | Crystal-chemistry of interstratified Mg/Fe-clay minerals from seafloor hydrothermal sites[END_REF][START_REF] Ming | Volatile and Organic Compositions of Sedimentary Rocks in Yellowknife Bay, Gale Crater, Mars[END_REF]. Comparison of the dioctahedralassigned EGA peak at 610C with laboratory studies of dehydroxylation temperature systematics as a function of Fe content suggest that the dioctahedral smectite likely contains ~5 wt.% Fe2O3, requiring at least half of octahedral sites be occupied by Al [START_REF] Brigatti | Relationships between composition and structure in in Fe-rich smectites[END_REF]. The peak water release at 780C, assigned to Mg-rich trioctahedral smectite, is higher than the 725C peak observed for YKB samples [START_REF] Ming | Volatile and Organic Compositions of Sedimentary Rocks in Yellowknife Bay, Gale Crater, Mars[END_REF] The formation of Al-bearing dioctahedral smectites from basaltic precursors requires greater element mobility and more oxidizing conditions than the suboxic, isochemical aqueous alteration environments proposed for the Fe-saponite of YKB (3,4). For example, in terrestrial weathering profiles of basaltic rocks, the initial alteration products of olivine typically contain trioctahedral smectite species with compositions (e.g., Mg/Fe) related to those of the primary minerals (13).

As alteration progresses, trioctahedral smectites are replaced by dioctahedral clay minerals via removal of Fe 2+ and Mg 2+ and/or oxidation of Fe 2+ and passive enrichment of Si and Al (11-13).

Oxidizing conditions also tend to lower pH, which is less favorable for formation of Mgtrioctahedral clay minerals [START_REF] Bristow | Terrestrial perspective on authigenic clay mineral production in ancient martian lakes[END_REF]. As a consequence, trioctahedral smectites are rarely found in basaltic soils. Formation of dioctahedral smectite formation is not restricted to surface weathering profiles; on a basaltic planet like Mars dioctahedral smectites could form in a variety of surface and subsurface aqueous environments (17-19,28). Several observations indicate that the smectitic clay minerals in Marimba, Quela and Sebina were subject to modification or formed close to the time of deposition within the Gale sedimentary system. There is no evidence from orbital VNIR spectra for Al-bearing clay minerals in the rim and walls of Gale crater, suggesting that clay minerals we describe were not sourced there [START_REF] Buz | Mineralogy and stratigraphy of the Gale crater rim, wall, and floor units[END_REF]. The accompanying trends in sedimentary facies, as well as bulk mineralogy and geochemistry of mudstones, indicate that the occurrence of Al-bearing, dioctahedral smectite corresponds with a shift in environmental conditions and the degree of chemical alteration in Gale crater lake sediments. Fig. 2 shows the changes in abundances of environmentally sensitive mineral components along MSL's traverse, including: 1) a transition from magnetite to hematite as the main Fe-oxide; 2) an increase in the abundances of Ca-sulfates, which appear to be matrix components above the Pahrump Hills member of the Murray formation and thus indicators of near-surface evaporative processes; and 3) an overall reduction in the quantity of reactive mafic mineralspyroxene and olivine. These mineralogical trends broadly correspond with observed sedimentary indicators of shallowing and episodic lake desiccation (14,16). The observed increase in the degree of aqueous alteration of mafic detritus in the upper part of the Murray formation is also reflected in chemical indices of alteration derived from bulk geochemical analyses [START_REF] Mangold | Open-System Weathering at Gale Crater from the Chemistry of Mudstones Analyzed by the Curiosity Rover[END_REF].

Our preferred mechanism for the production of Murray dioctahedral smectites involves open-system aqueous alteration of basaltic detritus in the lake, near the time of deposition, with elemental mobilization and oxidation driven by periodic desiccation and migration of the water table. Trioctahedral smectites may be the remnants of early-stage alteration of olivine or pyroxene, as proposed for YKB (2-5), or the product of high Mg 2+ activities in lake water and sedimentary pore water caused by evaporation [START_REF] Hover | Mg-smectite authigenesis in a marine evaporative environment, salina Ometepec, Baja California[END_REF]. Higher dehydroxylation temperatures indicate elevated Mg content in Murray saponite compared with YKB -a finding that is consistent with salinity-driven clay mineral formation mechanisms [START_REF] Bristow | Terrestrial perspective on authigenic clay mineral production in ancient martian lakes[END_REF].

Although Oudam contains just ~3 wt.% phyllosilicates, their basal (001) diffraction at ~9.6 Å is distinct from those of every other phyllosilicate-bearing sample measured by CheMin to date. Interlayer collapse to <10 Å, potentially induced by the dry air inside CheMin (4), is observed in certain smectites with small, monovalent cations, such as Na + , in the interlayer [START_REF] Sato | Effects of layer charge, charge location, and energy change on expansion properties of dioctahedral smectites[END_REF]. However, we do not think the ~9.6 Å peak in Oudam stems from collapsed smectite. Other smectite-bearing samples maintain basal spacings of ~10 Å inside CheMin, indicating the prevalence of bivalent interlayer cations (4). No evidence of a process leading to the preferential Na-exchange of Oudam clays is apparent from rover observations. Instead, Oudam siltstones appear to have been bathed in Ca 2+ -rich fluids as shown by Ca-sulfate minerals in the matrix (Table 1).

A basal diffraction of ~9.6 Å is also characteristic of high-charge or zero-layer-charge phyllosilicates that lack interlayer H2O, which include certain micas (e.g., paragonite, margarite) and species in the pyrophyllite-talc series (e.g. ferripyrophyllite). The EGA trace for Oudam shows a single H2O release at 470C, which is reasonably attributable to dehydroxylation of a Fe(III)-rich phyllosilicate [START_REF] Heller-Kallai | Dehydroxylation of dioctahedral phyllosilicates[END_REF]. Ferripyrophyllite is consistent with both XRD and EGA data [START_REF] Chukhrov | The ferric analogue of pyrophyllite and related phases[END_REF].

All of the candidate 9.6 Å phyllosilicate analogs recognized are the products of high-temperature hydrothermal and metamorphic processes. The absence of textural or mineralogical indicators of these processes at Oudam, and the presence of sedimentary structures indicative of an aeolian origin ( 14), leads us hypothesize that Oudam phyllosilicates did not form in situ, but are windblown detritus sourced elsewhere [START_REF] Buz | Mineralogy and stratigraphy of the Gale crater rim, wall, and floor units[END_REF].

Discussion

The contrast between the clay mineralogy of the Marimba, Quela, and Sebina mudstones with that of the 9.6 Å phyllosilicate in Oudam provides additional support linking smectite abundance and speciation with syn-depositional neoformation and transformation processes rather than later period of genesis during burial diagenesis. We note that the Oudam sample has the same basic mineralogy as other Murray formation mudstones (Table 1), much of which could serve as precursors to smectite formation in an invasive diagenetic fluid model (e.g., 26). In terrestrial sedimentary basins, the high porosity and permeability typical of sandstones promotes more extensive production of burial diagenetic clay minerals compared with mudstones. Based on sedimentological observations it appears that Oudam sediments spent less time in a dynamic aqueous environment close to the water table.

The discovery of a broad spectrum of mineralogical facies at Gale crater provides a window into the history of near-surface aqueous alteration processes on ancient Mars. On Earth, aqueous reactions at the juncture of the atmosphere, hydrosphere and lithosphere (termed the critical zone) are central to biogeochemical cycles that regulate climate and biological productivity of the planet through the draw-down of greenhouse gases and release of nutrients [START_REF] Amundson | Coupling between biota and Earth materials in the Critical Zone[END_REF]. The unexpected abundance and diversity of clay minerals in sedimentary rocks at Gale crater and longevity of this sedimentary system (2) indicate near-surface aqueous alteration continued into the Early Hesperian on Mars. When integrated with sedimentological observations, clay mineral diversity provides additional insight into how environmental conditions evolved during and after the deposition of the strata of lower Aeolis Mons. Through the continued survey of sedimentary rocks at Gale, which are predicted to encompass a range of conditions, MSL has the opportunity to develop an understanding of how the martian critical zone operated and influenced planetary evolution. geometry (see ref. [START_REF] Blake | Characterization and calibration of the CheMin mineralogical instrument on Mars Science Laboratory[END_REF] for more details). Curiosity's Sample Acquisition, Processing, and Handling (SA/SPaH) system was used to drill Oudam, Marimba, Quela, and Sebina from the Murray fm. bedrock (the majority of sample powder delivered to CheMin comes from a depth of 5 to 6 cm), sieve the resulting powder to <150 μm, and deliver ~50 mm 3 of material to CheMin analysis cells, which have either Mylar or Kapton windows. Oudam and Marimba were delivered to Mylar cells, and Quela and Sebina were delivered to Kapton cells. Sample cells are reusable and located on a rotating sample wheel. The cells are shaken piezoelectrically during analysis to randomize grain orientations, presenting all lattice orientations to the incident Co X-rays. A CCD detector is used to determine the energy and position of photons striking the CCD; fluoresced photons provide XRF data and the two-dimensional (2D) position of each diffracted Co Kα photon is used to construct the diffraction pattern; circumferential integration of Debye diffraction rings, corrected for arc length, produces a conventional 1D XRD pattern with 2θ resolution of ~0.3° (e.g. Fig. 3). Positions of detected photons are summed over repeated 10-sec measurements for several hours during each night of analysis. Samples are generally analyzed for four or more nights, spaced at time intervals determined by rover energy budget, allowance for operating other instruments, and other operational considerations. Plagioclase is a common phase in almost all samples, and the 1D diffraction patterns are corrected for minor variations in sample-to-detector distance using the best fit to plagioclase c and γ unit cell parameters (10).

Abundances of crystalline phases in the Oudam, Marimba, Quela, and Sebina samples were determined by Rietveld analysis using Jade software. The mineral quantities presented in this paper (Table 1) differ slightly from results available in the Planetary Data System (PDS) (https://pds.nasa.gov). These new results are products of Jade-based Rietveld refinements that incorporate clay mineral standard patterns in addition to structural models of crystalline phases.

We find this method improves model fits of measured XRD patterns.

The clay mineral standards used in these hybrid refinements were measured on CheMin 4 a prototype instrument that is similar to the CheMin flight instrument. We found that including both dioctahedral and trioctahedral smectite clay mineral standards led to the best replication of measured XRD patterns in Jade, consistent with our conclusion that Marimba, Quela, and Sebina contain both di-and trioctahedral smectite phases. Smectite clay mineral standards were heated to 200ºC for 10 hours before analysis to remove interlayer H2O and collapse basal spacing to ~10 Å, to more closely replicate the state of clay minerals within MSL. These approaches produce accurate identification and detection for virtually all crystalline phases at abundances greater than ~1 wt.%. The abundances of amorphous components and poorly crystalline clay minerals are determined using the program FULLPAT [START_REF] Chipera | FULLPAT: A full-pattern quantitative analysis program for Xray powder diffraction using measured and calculated patterns[END_REF] and remain as reported in the PDS.

The phases contributing to the pattern in the vicinity of the clay mineral 02l band were examined in more detail using BGMN, a Rietveld refinement program that can generate XRD patterns of partially disordered clay minerals and simultaneously consider contributions from crystalline phases [START_REF] Bergmann | BGMN a new fundamental parameter-based Rietveld program for laboratory X-ray sources, its use in quantitative analysis and structure investigations[END_REF][START_REF] Ufer | Description of X-ray powder pattern of turbostratically disordered layer structures with a Rietveld compatible approach[END_REF]. BGMN uses instrument profiles as part of the XRD pattern modeling procedure. BGMN instrument profiles are generated based on description of the XRD instruments' geometry using raytracing simulations. BGMNs' ray tracer does not support simulation of the flat CCD detector that collects CheMin patterns; therefore, we approximate profiles using a point detector, making adjustments to instrument geometry parameters until the profile function reproduces unit cell parameters and peak shapes of the beryl standard that resides inside CheMin and was measured earlier in the mission. BGMN refinements were used to estimate the relative proportions of dioctahedral and trioctahedral smectites in Marimba, Quela and Sebina. These refinements include structural models of dehydrated dioctahedral smectite (based on montmorillonite) and a trioctahedral smectite (saponite). The b-unit cell parameter of the dioctahedral smectite phase was constrained to <9 angstroms during refinements, based on the relation between dehydroxylation temperature and Fe content of dioctahedral smectites in ref. [START_REF] Brigatti | Relationships between composition and structure in in Fe-rich smectites[END_REF]. Note that we report the ratios of dioctahedral:trioctahedral smectites from BGMN refinements because we do not have a reliable way to quantify the amount X-ray amorphous material in our samples with BGMN.

To distinguish between illitic and smectitic clay minerals, we use potassium abundance data obtained by the APXS onboard the Curiosity rover for Murray formation samples analyzed between sols 782 and 1496. These data are reproduced in Table S2 with their reported analytical uncertainties. A complete description of the instrument, as well as the methods used for calibration and quantification of APXS data, can be found in Gellert et al. [START_REF] Gellert | The Alpha Particle X-Ray Spectrometer (APXS): Results from Gusev Crater and calibration report[END_REF] and Campbell et al. [START_REF] Campbell | Calibration of the Mars Science Laboratory Alpha Particle X-ray Spectrometer[END_REF]. In summary, APXS is a contact instrument with Curium-244 sources that induce Particle-Induced X-ray Emission (PIXE) and X-ray Fluorescence (XRF) to determine the

  , indicating that trioctahedral smectites in Marimba have a comparatively lower Fe content. These observations are consistent with the CheMin XRD analyses. The intermediate position of the 02l clay mineral band is best modeled when measured clay mineral standards or structural models of both trioctahedral and Al-rich dioctahedral smectites are used in Rietveld refinements of the XRD patterns (Fig. 3C, Materials and Methods). Based on the combination of XRD, EGA, and bulk chemical data measured by the Alpha Particle X-ray Spectrometer (APXS) we find that a mixture of Al-rich dioctahedral and Mg-rich trioctahedral smectite is present in Marimba, Quela and Sebina. This is the first in situ detection of dioctahedral smectite in Gale crater. There have been no direct orbital detections of phyllosilicate in strata traversed (so far) by Curiosity. Recent orbital VNIR spectra have documented signatures of Al/Fe-smectites in laterally (and presumably time equivalent) units of the Karasburg and Sutton Island members of the Murray formation (26, 27, Fig. S2). Our rover-based results presented here are the first crystallographic ground truth of the thousands of orbital phyllosilicate detections, which are essential inputs for Mars science and mission planning (17-19). Our results confirm the presence of Al-rich dioctahedral smectite (albeit in conjunction with additional trioctahedral smectite) and other mineralogical information that helps constrain the origin and environmental implications of these clay minerals. By incorporating structural models of clay minerals, with the b-unit cell parameter constrained by dehydroxylation temperatures observed in Marimba, Rietveld refinements of XRD patterns provide an estimate of the ratio of dioctahedral to trioctahedral smectites (Materials and Methods). The proportion of dioctahedral smectite increases up-section, with dioctahedral:trioctahedral ratios of 1:2, 1:1 and 5:3 for Marimba, Quela, and Sebina, respectively.

Figure 3 .

 3 Figure 3. X-ray diffraction patterns of clay mineral bearing sample from Gale Crater. (A) Comparison of XRD patterns from Oudam, Marimba, Quela, and Sebina, with peaks assigned to clays minerals and other component minerals (A = anhydrite, B = bassanite, H = hematite, P = plagioclase). (B) Close up comparison of Marimba, Quela, Sebina, and YKB XRD patterns with trioctahedral and dioctahedral smectite standards (SapCa-1 saponite and SAz-1 montmorillonite), showing the difference in 02l band position corresponding to a difference in octahedral occupancy. (C) BGMN model of the 02l region of Marimba showing contributions from trioctahedral and dioctahedral smectites.

table S1 .

 S1 Data used to infer the K content of clay minerals. fig. S1. Comparison of the clay mineral and potassium content of Murray formation samples. fig. S2. Comparisons of CRISM smectite signatures in the Murray Formation vs. the phyllosilicate trough.

	mineral	Oudam	Marimba	Quela	Sebina
	Andesine	27.8 ± 0.5	14.0 ± 0.9	13.5 ± 0.7	10.7 ± 0.4
	Hematite	13.9 ± 0.4	6.4 ± 0.4	7.1 ± 0.4	6.9 ± 0.2
	Ca-sulfate	6.3 ± 0.3	7.0 ± 0.6	5.5 ± 0.4	7.4 ± 0.6
	Sanidine	-	2.4 ± 0.6	2.3 ± 0.5	1.4 ± 0.4
	Pyroxene	5.3 ± 0.9	0.7 ± 0.6	2.7 ± 0.7	2.8 ± 0.4
	Jarosite	-	At detection	At detection	0.9 ± 0.2
	Quartz	0.7 ± 0.1	At detection	At detection	At detection
	Clay Minerals	3 ± 1	28 ± 5	16 ± 3	19 ± 4
	Amorphous	43 ± 20	40 ± 20	51.7 ± 25	51.1 ± 25

abundance of major, minor, and trace elements from sodium to bromine in soil and rock targets.

Low atomic number (Z) element X-rays stem from the topmost 5 microns of the sample, higher Z elements like Fe are detected from the upper ~50 microns. The APXS is mounted on a turret at the end of the Curiosity rover's arm and is deployed on selected targets along the rover traverse to determine their elemental composition. The sampled area is about 1.7 cm in diameter when the instrument is in contact with the sample, and APXS spectra represent the average composition over the sampled area.

The Sample Analysis at Mars (SAM) instrument suite consists of a quadrupole mass spectrometer (QMS), a six-column gas chromatograph (GC), and a tunable laser spectrometer (TLS) connected to a gas processing system that includes two pyrolysis ovens [START_REF] Ming | Volatile and Organic Compositions of Sedimentary Rocks in Yellowknife Bay, Gale Crater, Mars[END_REF][START_REF] Mahaffy | The sample analysis at Mars investigation and instrument suite[END_REF][START_REF] Leshin | Volatile, Isotope, and Organic Analysis of Martian Fines with the Mars Curiosity Rover[END_REF]. Gases evolved during pyrolysis of samples can be sent to several of these instruments; here we focus on data from the direct QMS detection of gases during sample heating, referred to as evolved gas analysis mass spectrometry (EGA-MS). Volatiles evolved during pyrolysis and their evolution temperatures can be used to inform the mineralogy or organic chemistry of samples. For further information about the SAM instrument and its operation see refs. 24,41,42. Splits of the <150 μm portion of the Oudam and Marimba sample powders were delivered by SA/SPaH into cleaned (by heating to >800°C) quartz sample cups. The mass of portions delivered to SAM cups is not measured in situ but is estimated to be 45 ± 18 mg (2σ) based on sample volume delivered during experiments with the Collection and Handling for Interior Martian Rock Analysis (CHIMRA) system testbed on Earth and analytical models [START_REF] Mahaffy | The sample analysis at Mars investigation and instrument suite[END_REF][START_REF] Leshin | Volatile, Isotope, and Organic Analysis of Martian Fines with the Mars Curiosity Rover[END_REF]. Sample fines were then heated from ~30°C (ambient SAM temperature) to ~860°C at 35°C /min under a helium carrier gas flow of ~0.8 standard cubic centimeters per minute (sccm) and ~25 mb of gas pressure in the sample pyrolysis ovens. A split of gases evolved from the sample during heating was swept into the QMS inlet and detected by the mass-to-charge ratio (m/z) of the molecules. If the main mass of a molecule saturated the MS detector, as was the case for m/z 18 from H2O, then a signal for an isotopolog (e.g., H2 18 O at m/z 20 for H2O) or QMS fragment of the molecule (e.g., m/z 17 for H2O) was used to study the evolution of a molecule with temperature.

Supplementary Materials


Alain Burr

François Hild

Frederick A Leckie

On the mechanical behaviour under cyclic loading of ceramic matrix composites


Alain Burr

François Hild

Frederick A Leckie

Isochronous analysis of the behaviour of ceramic-matrix composites under thermomechanical cyclic loading conditions

teaching and research institutions in France or abroad, or from public or private research centers.


Dr Benjamin A Parris
email: bparris@bournemouth.ac.uk

Brendan Stuart

Hackett Weekes

Mohammad Momenian

Maria Augustinova

Ludovic Ferrand

Phonological processing in the Stroop task

Keywords: Stroop, selective attention, response mode, phonological, facilitation

A long-standing debate in the Stroop literature concerns whether the way we respond to the colour dimension determines how we process the irrelevant dimension or whether word processing is purely stimulus-driven. Models and findings in the Stroop literature differ in their predictions about how response modes (e.g., responding manually versus vocally) affect how the irrelevant word is processed (i.e. phonologically, semantically) and the interference and facilitation that results, with some predicting qualitatively different Stroop effects. Here we investigated whether response mode modifies phonological facilitation produced by the irrelevant word. In a fully within-subjects design, we sought evidence for the use of a serial print-to-speech prelexical phonological processing route when using manual and vocal responses by testing for facilitating effects of phonological overlap between the irrelevant word and the colour name at the initial and final phoneme positions. The results showed phoneme overlap leads to facilitation with both response modes, a result that is inconsistent with qualitative differences between the two response modes.

The Stroop task [START_REF] Stroop | Studies of interference in serial verbal reactions[END_REF] is a test of the mechanisms of selective attention. It is also used to test predictions about the relative automaticity of written word processing (see [START_REF] Augustinova | Automaticity of Word Reading: Evidence from the Semantic Stroop Paradigm[END_REF]MacLeod, 1991, for reviews). The task yields a Stroop effect which refers to slower naming of the print color of the word when the word refers to another colour (e.g., the word red is printed in blue; called an incongruent trial) compared to when the word is the same as the print colour name (e.g., the word red is printed in red; called a congruent trial). Furthermore, when trials are compared to neutral non-colour related trials (e.g., top printed in blue; called a neutral trial), incongruent trials produce interference whilst congruent trials produce facilitation.

One finding to emerge from Stroop experiments is that the mode of response used to classify the colour of the printed word (e.g., responding manually via a keypress vs. responding vocally by saying the colour name aloud) determines the magnitude and type of facilitation and interference that results [START_REF] Fennell | Does response modality influence conflict? Modelling vocal and manual response Stroop interference[END_REF][START_REF] Glaser | Context Effects in Stroop-like Word and Picture Processing[END_REF][START_REF] Kinoshita | The magic of words reconsidered: Investigating the automaticity of reading colour-neutral words in the Stroop task[END_REF][START_REF] Mcclain | Effects of response type and set size on Stroop color-word performance[END_REF][START_REF] Redding | Stroop effects: Interference and facilitation with verbal and manual responses[END_REF][START_REF] Sharma | Differential components of the manual and vocal Stroop tasks[END_REF][START_REF] Turken | Response selection in the human anterior cingulate cortex[END_REF]Zahedi, Abdel Rahman, Stürmer & Sommer, 2019; see also [START_REF] Macleod | Half a Century of Research on the Stroop Effect: An Integrative Review[END_REF]Parris, Hasshim, Wadsley, Augustinova, & Ferrand, submitted). For example, [START_REF] Sharma | Differential components of the manual and vocal Stroop tasks[END_REF] reported evidence they argued showed no semantic Stroop effects with the manual response Stroop task (although see [START_REF] Brown | On a variant of Stroop's paradigm: Which cognitions press your buttons?[END_REF].

Accounts of differences between these two response modes propose differential access to the systems (i.e., phonological, lexico-semantic, or response level processing) that are assumed to produce interference and facilitation [START_REF] Glaser | Context Effects in Stroop-like Word and Picture Processing[END_REF][START_REF] Kinoshita | The magic of words reconsidered: Investigating the automaticity of reading colour-neutral words in the Stroop task[END_REF][START_REF] Sharma | Differential components of the manual and vocal Stroop tasks[END_REF][START_REF] Sugg | Time course of inhibition in color-response and word-response versions of the Stroop task[END_REF][START_REF] Turken | Response selection in the human anterior cingulate cortex[END_REF][START_REF] Virzi | Toward a translational model of Stroop interference[END_REF]; see also [START_REF] Fennell | Does response modality influence conflict? Modelling vocal and manual response Stroop interference[END_REF]Zahedi et al., 2019). Consistent with this, it has recently been argued that vocal and manual responding involve different tasks (naming vs classification, respectively) and as such the type of evidence that is accumulated during Phonological processing in the Stroop task 4 Stroop task performance is different, leading to qualitatively different Stroop effects [START_REF] Kinoshita | The magic of words reconsidered: Investigating the automaticity of reading colour-neutral words in the Stroop task[END_REF]. This account predicts that reading and the semantic access that this entails is not an automatic invariant process, as argued by others [START_REF] Augustinova | Automaticity of Word Reading: Evidence from the Semantic Stroop Paradigm[END_REF]Neely & Kahan, 2001).

In contrast to the differential access position, [START_REF] Roelofs | Goal-referenced selection of verbal action: modeling attentional control in the Stroop task[END_REF] argued for similar effects with manual and vocal response modes based on his WEAVER++ model of Stroop task performance positing mandatory verbal mediation of manual responses; under this account participants subvocally produce the colour name when responding manually and thus any effect found with vocal responses should be observed with manual responses. In the present study we investigated whether phonological processing of the irrelevant word differs between manual and vocal responses.

Previous investigations have provided evidence for phonological processing with a vocal response. To seek evidence for a serial grapheme-to-phoneme processing route in word reading, [START_REF] Coltheart | A position-sensitive Stroop effect: Further evidence for a left-to-right component in print-to-speech conversion[END_REF] developed a novel Stroop paradigm by creating stimuli that either shared the initial phoneme or end phoneme of a colour name.

For example, if the colour to be named was red, the to-be-ignored word would be rat (sharing initial phoneme) or pod (sharing the end phoneme) or a word that shares no phoneme at all e.g. fit. Words that share a phoneme with a colour name have been shown to produce a naming latency advantage when naming the colour not the printed word [START_REF] Regan | Involuntary automatic processing in colour-naming tasks[END_REF].

Coltheart and colleagues reasoned that if, as assumed by the Dual Route Cascaded (DRC) model (see [START_REF] Coltheart | DRC: A dual route cascaded model of visual word recognition and reading aloud[END_REF], there is a serial order component in processing of print-to-speech, there will be an advantage for colour names that share an initial phoneme with the to-be-ignored written word compared to items that share an end phoneme because the computation of phonology from print is left to. Consistent with their predictions, Coltheart and colleagues reported that phonemic overlap was significant at both positions. However, facilitation was greater for items with overlap in the initial phoneme which they argued is incompatible with models that do not assume a serial grapheme-tophoneme processing route in reading (see also [START_REF] Marmurek | Stroop-like serial position effects in colour naming of words and nonwords[END_REF], for a partial replication of this effect).

Evidence for automatic phonological processing in the Stroop task was also sought by [START_REF] Besner | Unintentional reading: Can phonological computation be controlled?[END_REF] who employed a manual response. [START_REF] Besner | Unintentional reading: Can phonological computation be controlled?[END_REF] employed nonwords that sound like colour words (e.g. bloo), known as pseudohomophones, as the irrelevant stimuli, and found substantial Stroop effects when compared to a neutral baseline (see also [START_REF] Tzelgov | Unintentional word reading via the phonological route: The Stroop effect with cross-script homophones[END_REF]. They argued that phonological computation cannot be controlled, even with a manual response. However, a limitation of their design given present purposes is the fact that whilst phonological processing must be activated during responding to produce a pseudohomophone effect, there are alternative sources of interference and facilitation (orthographic/semantic) that cannot be ruled out, which could have led to any subsequent Stroop effects. As such, on the basis of this study alone it is not possible to determine whether phonological processing of the irrelevant word contributed to the Stroop effects with the manual response.

Recent work has presented evidence suggesting that the manual response Stroop task does not produce phonologically-based Stroop effects [START_REF] Kinoshita | The magic of words reconsidered: Investigating the automaticity of reading colour-neutral words in the Stroop task[END_REF]. [START_REF] Kinoshita | The magic of words reconsidered: Investigating the automaticity of reading colour-neutral words in the Stroop task[END_REF] asked what aspects of the reading process is triggered by the irrelevant word stimulus to produce interference in the colour-word Stroop task. They compared performance on five types of colour-neutral letter strings to incongruent words. They included real words (e.g., HAT), pronounceable pseudowords (e.g., HIX), consonant strings (e.g., HDK), nonalphabetic symbol strings (e.g., &@£), and a row of Xs. They reported that there was a wordlikeness or pronounceability gradient where real words and pseudowords showed an equal amount of interference (with interference increasing with string length) and more than that produced by the consonant strings which in turn produced more interference than the symbol strings and the row of Xs (which did not differ from each other). Under their account, the phonological encoding (the segment-to-frame association processes in articulation planning) of the colour name was slowed by the computation of phonology that occurs independent of lexical status and as long as the word is pronounceable. Importantly for present purposes, the authors reported this effect when participants responded vocally, but did not observe it when participants responded manually. [START_REF] Kinoshita | The magic of words reconsidered: Investigating the automaticity of reading colour-neutral words in the Stroop task[END_REF] suggested that it is the process of generating a speech code from the irrelevant letter string that produced the pronounceability in the vocal task. They argued that because the task in the manual Stroop task is to categorize and not name the relevant colour, generation of the speech code of the irrelevant word does not cause interference. This task-dependent effect is consistent with the Bayesian reader model [START_REF] Norris | The Bayesian reader: Explaining word recognition as an optimal Bayesian decision process[END_REF][START_REF] Norris | Perception as evidence accumulation and Bayesian inference: Insights from masked priming[END_REF]) that stipulates that it is the task goal and not some invariant automatic reading processing that determines the outcome of processing. And the task goal in vocal and manual responding is different (naming vs. classification, respectively). Such a finding is inconsistent with Roelofs' (2003) lexical mediation account of manual responses.

The present study was designed to test the prediction that manual and vocal Stroop tasks lead to qualitatively different phonological Stroop effects. We employed [START_REF] Coltheart | A position-sensitive Stroop effect: Further evidence for a left-to-right component in print-to-speech conversion[END_REF] paradigm to investigate whether phonological facilitation arises with a manual response when the initial or final phoneme is shared by the irrelevant word and colour name.

In a fully within-subjects design, participants were asked to complete the Coltheart et al. paradigm using both manual and vocal responses (in counterbalanced order). A finding of phonological facilitation with a vocal response but not with a manual response would be supportive of a qualitative difference between response modes perhaps resulting from the different task goals and resulting differential access associated with responding vocally (naming) and manually (classification). In contrast, a finding of phonological facilitation with manual and vocal responses would be consistent with similar mechanisms underpinning both response modes [START_REF] Roelofs | Goal-referenced selection of verbal action: modeling attentional control in the Stroop task[END_REF].

Method

Participants. 44 participants (30 females) were recruited from Universities of Bournemouth and Sussex (UK). Based on the effect size reported by Coltheart et al.,44 participants were considered sufficient to achieve a power estimate of 0.95. Materials. The 184 stimuli used in the present study were taken directly from the Coltheart et al. study. The full list of items, with a full description, is reported in [START_REF] Coltheart | A position-sensitive Stroop effect: Further evidence for a left-to-right component in print-to-speech conversion[END_REF]. There were 92 stimuli in the Phoneme overlap condition, half of which possessed phonemic overlap with the initial phoneme of one of the three colours names (red, blue, green; e.g. rack, bait, gasp) the other half overlap with the final phoneme (e.g. cud, skin, few). The stimuli were selected on the bases of having meeting the following criteria: 1) Being between 3 and 5 letters in length; 2) Having the same number of phonemes as the colour name; 3) Having an absence of phonemic overlap with any other phoneme in the colour name; 4) Lack any obvious colour association. The remaining 92 stimuli were match to the critical stimuli on number of letters and phonemes and contained no phonemic overlap with the colour names. Each control stimulus was presented only in the same colour as its matched critical stimulus.

All stimuli were presented in lowercase via Superlab Pro Version 2.0 software running on a PC desktop computer. The ink colours chosen were red (RGB: 255, 0, 0), green (RGB: 0, 255, 0), and blue (RGB: 0, 0, 255). Three-letter stimuli were approximately 15mm long x 11mm high, four-letter stimuli were approximately 22mm long x 10mm high, and five-letter stimuli were approximately 29mm long x 10mm high. During a practice session, the stimuli consisted of a string of five repeated X symbols presented on a gateway solo P2-550 laptop pc using a TFT screen with a refresh rate of 60 Hz. For the manual response condition the response keys were labelled green, blue and red using colour patches placed over the 'z', 'v', and 'm' keys, respectively. Half of the participants used two fingers of their right hand to respond and one finger from their left hand, whilst the other half used two fingers from their left hand and one finger from their right hand. For the vocal response condition, a voice key connected to the laptop using a microphone with a voice activated trigger was used to record the responses and responses were also taped to enable the experimenter to check for errors.

Procedure. Each participant sat approximately 60 cm away from a screen. They were asked to identify the colour of the stimulus print, which would be red, green, or blue, quickly and as accurately. Each stimulus was preceded by a fixation cross appearing at the centre of the screen for 497 msec. The stimulus then appeared and was replaced by a blank screen as soon as a participant initiated their response. The blank screen was subsequently replaced after 1000msec by a fixation cross. Participants completed 184 trials of both manual and vocal responses giving a total of 368 trials. The same stimuli were presented in each condition i.e. for both response modes. Half of the participants performed the manual task first and half the vocal task first. Data analysis. Following Coltheart et al. (1999) the data were analysed using a repeated measures ANOVA and for which the dependent variable was reaction time.

However, in response to reviews of a previous version of this manuscript we also carried out Phonological processing in the Stroop task 9 Linear Mixed Effects (LME) Modelling of the data. The benefit of LME over ANOVA is that is models random effects, reducing the Type 1 error rate. A full description of the steps taken during the analysis are available in the Supplementary Materials and on request. We used the lme4 package (https://cran.r-project.org/web/packages/lme4/) with R software (R Development Core Team, 2008) to analyze RT data. We followed the practice recommended by [START_REF] Bates | Parsimonious mixed models[END_REF]. In contrast to the ANOVA analysis, our dependent variable was log RT following the mainstream tradition in psycholinguistics [START_REF] Baayen | Analyzing Linguistic Data: A Practical Introduction to Statistics[END_REF][START_REF] Baayen | Mixed-effects modeling with crossed random effects for subjects and items[END_REF]. Finally, we calculated p values for this model (see Table 2) with the lmerTest package [START_REF] Kuznetsova | lmerTest Package: Tests in Linear Mixed Effects Models[END_REF].

Results

Following [START_REF] Coltheart | A position-sensitive Stroop effect: Further evidence for a left-to-right component in print-to-speech conversion[END_REF] analysis was conducted on correct reaction times (RTs) only. 6.3% and 2.6% of the vocal and manual data, respectively, were classed as errors.

Following [START_REF] Coltheart | A position-sensitive Stroop effect: Further evidence for a left-to-right component in print-to-speech conversion[END_REF] reaction times 2 standard deviations from the mean were defined as outliers and removed before analysis. This outlier removal procedure was done independently for the manual and vocal responses. For the manual responses, 3.9% of the data were classed as outliers. For the vocal response, 7.4% of the data were classed as outliers. See Table 1 for reaction times and standard errors in each condition. There was a Condition x Response type interaction, F(1, 43) = 8.023, p = .007, partial  2 = .157, indicating that the vocal response led to greater overall phonological facilitation (13ms; irrespective of position of overlap) than the manual response (3ms). The two-way Condition x Overlap position interaction was significant, F(1, 43) = 41.290, p<.001, partial  2 = .490, indicating that there was Stroop facilitation at the initial position (15ms), but not at the end position (-4ms) when collapsed across response modality. The Overlap position x Response type interaction was not significant, F(1, 43) = 2.020, p > .1, partial  2 = .045.

Analysis of latencies

There was a trend toward a three-way interaction where F(1, 43) = 3.795, p = .058, partial  2 = .081.

Since the main aim of the present work was to establish whether phonological processing contributes to Stroop effects with manual responses we decomposed the threeway interaction into two 2 (Condition: Phoneme overlap, No phoneme overlap) x 2 (Overlap position: initial-letter, end-letter) repeated measures ANOVAs. For the manual response, there was no main effect of Condition F(1, 43) = 1.049, p = .311, partial  2 = .024, and no main effect of Overlap position F(1, 43) = 1.264, p = .267, partial  2 = .029, but the interaction was significant (F(1, 43) = 7.733, p = .008,  2 = .152. The interaction was the result of significant phonological facilitation at the initial letter position (9ms; t(43) = 2.653, p = .011), but not at the end position (-4ms; t(43) = -.989, p = .328). The significant facilitation at the initial letter position supports the notion that phonological processing of the irrelevant word occurs when participants respond manually in the Stroop task.

For the vocal response, there was a main effect of Condition F(1, 43) = 30.967, p < .001, partial  2 = .419, and a main effect of Overlap position F(1, 43) = 10.780, p = .002, partial  2 = .2. The interaction was also significant (F(1, 43) = 32.299, p < .001,  2 = .429.

As with the manual response, the interaction was the result of significant phonological facilitation at the initial letter position (27ms; t(43) = 7.831, p < .001), but not at the end position (1ms; t(43) = .201, p = .841).

Linear Mixed Effects Modelling

As in other analyses using LME we first started with a maximal model [START_REF] Barr | Random effects structure for confirmatory hypothesis testing: Keeping it maximal[END_REF] entering all the fixed variables including Word Type, Overlap Position and Response Type and the control variable (colour), together with random intercepts for the items and the subjects, plus by-subject and by-item random slopes for all the fixed effects. The interaction between the fixed variables was also included in the models following [START_REF] Barr | Random effects structure for confirmatory hypothesis testing: Keeping it maximal[END_REF].

Next, we calculated a Singular Value Decomposition (SVD) on the covariance matrix of the maximal model using the PCA [START_REF] Bates | Parsimonious mixed models[END_REF]. SVD analysis reveals which slopes in the random effects structure do not contribute to the model. This removes unnecessary complications such as over-specification from the model, elimination of redundant analyses, and makes the model easier to converge overall. SVD analysis is accompanied by a Likelihood Ratio Test (LRT) during evaluation. After the SVD specifies which slopes have a variance close to zero, they are then removed from the model one at a time. Using a series of LRTs, the new models are compared with previous models to determine whether the removal of a slope(s) makes any significant changes to the model fit.

SVD analysis and then LRTs suggested a random effects structure which consisted of both random intercepts for subjects and items, plus by-subject and a by-item random slopes for Response Type. This means that the effect of Response Type was variable across subjects and items. However, because of a singularity problem and a ceiling correlation between the item intercept and its slope, we needed to remove the by-item slope from the model. This did not have a significant impact on the model estimates because the removed variance was contributing to less than 5% of the whole random effects variance. The resulting structure is then assumed to explain our data using the best fit from among most other alternatives.

After specifying the best random effects structure for our data, we then started looking into the main effects and their interactions. Determining which main effects and interactions are significant is a controversial issue in LME research. We followed the practice suggested by [START_REF] Baayen | Mixed-effects modeling with crossed random effects for subjects and items[END_REF]. LRT showed that the main effects of colour (x 2 (2)=146.85, p <0.001), Word Type (x 2 (1)=12.75, p <0.001), Overlap Position (x 2 (2)=8.94, p <0.01), and Response Type (x 2 (1)=41.79, p <0.001) were significant. When it comes to interactions, Word Type* Overlap Position (x 2 (1)=24.89, p <0.001), Response Type* Overlap Position (x 2 (1)=9.45, p <0.01), and Word Type*Response Type (x 2 (1)=7.56, p <0.01) were significant. The three-way interaction was not significant (x 2 (1)=1.57, p =0.20). We used Akaike Information Criterion (AIC) as the criterion to select the best model which explained

Summary of the analyses

The results from the ANOVA and LME modelling were consistent for all key effects including the non-significant three-way interaction. Whilst the three-way interaction trended towards significance with the ANOVA, this was not the case for the LME modelling which has better control over Type 1 error rate.

Discussion

The aim of the present work was to investigate whether the manual response colourword Stroop task leads to phonological Stroop effects. Using a fully-within subjects design, we replicated the serial position Stroop facilitation effect reported by [START_REF] Coltheart | A position-sensitive Stroop effect: Further evidence for a left-to-right component in print-to-speech conversion[END_REF] Phonological processing in the Stroop task 13 using a vocal response with both manual and vocal responses,. The results support the notion that manual responding in the Stroop task does produce phonological Stroop facilitation when the irrelevant word shares phonemes with the colour name despite the fact that participants are not required to produce the name of the relevant colour. The results are consistent with models that predict verbal mediation of manual responses and thus similar components comprising Stroop interference in both response modes [START_REF] Roelofs | Goal-referenced selection of verbal action: modeling attentional control in the Stroop task[END_REF]. Our data suggest that both manual and vocal responding lead to phonological processing of the irrelevant word and that this processing facilitates the process of phonologically encoding the colour name.

Our data are inconsistent with the notion that manual and vocal responding involve different task goals that determine the nature of the evidence to be accumulated [START_REF] Kinoshita | The magic of words reconsidered: Investigating the automaticity of reading colour-neutral words in the Stroop task[END_REF] and that manual and vocal responding have differential access to the systems that produce Stroop effects [START_REF] Fennell | Does response modality influence conflict? Modelling vocal and manual response Stroop interference[END_REF][START_REF] Glaser | Context Effects in Stroop-like Word and Picture Processing[END_REF][START_REF] Kinoshita | The magic of words reconsidered: Investigating the automaticity of reading colour-neutral words in the Stroop task[END_REF][START_REF] Mcclain | Effects of response type and set size on Stroop color-word performance[END_REF][START_REF] Redding | Stroop effects: Interference and facilitation with verbal and manual responses[END_REF][START_REF] Sharma | Differential components of the manual and vocal Stroop tasks[END_REF]Zahedi, Abdel Rahman, Stürmer & Sommer, 2019). However, one clear and obvious difference between the manual and vocal response modes is the need to actually utter the colour name with a vocal response. This requires the full repertoire of speech production including syllabification and articulation. The engagement of speech production processes might modify the way the irrelevant word is processed [START_REF] Burt | Why do non-colour words interfere with colour naming?[END_REF]VanVoorhis & Dark, 1995). Thus, whilst our data and those from others (e.g., [START_REF] Brown | On a variant of Stroop's paradigm: Which cognitions press your buttons?[END_REF] support the notion that both manual and vocal responding might involve access to phonology and semantics, leading to phonologically and semantically based Stroop effects, a key difference might be the increased access to these systems produced by feedback from the phonological encoding of the relevant colour name. Whilst we do not report a significant difference in the amount of phonological processing between the two response modes our data are somewhat consistent with a potential quantitative difference.

In contrast to Coltheart et al.'s results, facilitation at the end letter position in the present study was small (vocal) and even negative (manual; see Figure 1). Closer examination of [START_REF] Marmurek | Stroop-like serial position effects in colour naming of words and nonwords[END_REF]'s data also reveals they observed a similar lack of facilitation from final position overlap (with a vocal response). We used a Bayes Factor (Dienes, 2011) to determine if each response type showed no evidence of an effect of phoneme overlap at the end position. Here, B H(0, x) refers to a Bayes factor in which the predictions of H1 were modelled as a half-normal distribution with an SD of x (see Dienes, 2014Dienes, , 2016)). To calculate the Bayes Factors we used a half normal of 12ms which was the raw effect size reported by [START_REF] Coltheart | A position-sensitive Stroop effect: Further evidence for a left-to-right component in print-to-speech conversion[END_REF]. For the manual response a Bayes Factor of B H (0,12) = .15 was returned confirming no facilitation. For the vocal response a Bayes Factor of B H (0,12) = .31 was returned again confirming no facilitation. Our data are therefore somewhat inconsistent with the findings from [START_REF] Coltheart | A position-sensitive Stroop effect: Further evidence for a left-to-right component in print-to-speech conversion[END_REF] who reported a final position overlap effect. Our data do not enable us to determine why this difference has been observed but this could be an avenue for future research.

In conclusion, the present results are not consistent with a qualitative difference between manual and vocal responding in the Stroop task with regards to phonological processing. When there is an overlap between the phonemes of the irrelevant word and the colour name at the initial letter position, phonological facilitation is observed with both response modes. We interpret our data as being consistent with Roelofs' ( 2003

  Ages ranged from 18 to 35 years (mean = 23.5, SD=3.2). Participants were right-handed, undergraduate psychology students. Design. The experiment used a 2 (Condition: Phoneme overlap, No phoneme overlap) by 2 (Overlap Position: initial, end) by 2 (Response type: manual, vocal) within-subjects design.

.

  The data were entered into a 2 (Condition: Phoneme overlap, No phoneme overlap) x 2 (Response type: manual, vocal) x 2 (Overlap position: initial-letter, end-letter) repeated measures ANOVA. There was a main effect of Condition F(1, 43) = 23.225, p < .001, partial  2 = .351, indicating that the words with phoneme overlap were responded to faster than the words with no phoneme overlap, and a main effect of Response type F(1, 43) = 106.390, p < .001, partial  2 = .712, indicating that the vocal responses were longer than the manual responses, and a main effect of Overlap position F(1, 43) = 10.275, p = .003, partial  2 = .193.

  our data best. The following model was the best model since it had the lowest AIC: RTlog ~ Colour + Word Type + Overlap Position + Response Type + Word Type * Overlap Position + Overlap Position*Response Type + Word Type*Response Type + (1 +Response Type|Subject) + (1|Item).

  ) model of the Stroop task which argues for verbal mediation of the manual Stroop task. Zahedi, A., Abdel Rahman, R., Stürmer, B., & Sommer, W. (2019). Common and specific loci of Stroop effects in vocal and manual asks, revealed by event-related brain potentials and posthypnotic suggestions. Journal of Experimental Psychology: General. Advance online publication. http://dx.doi.org/10.1037/xge0000574 Phonological processing in the Stroop task 21

Table 1 :

 1 Mean reaction times (ms), standard errors (in parentheses), and magnitudes of Stroop facilitation when identifying colour as a function of the position of phonological overlap (Initial-or End-letter) between the irrelevant word and the colour name.

			VOCAL		MANUAL
			Position of overlap	Position of overlap
	Condition		Initial-	End-	Initial-	End-
			letter	letter	letter	letter
	Control	RT	614	609	533	529
	(no	SE	(9.1)	(8.1)	(9.5)	(10.3)
	overlap)
	Critical	RT	587	608	524	533
	(overlap)	SE	(8.2)	(3.7)	(9.5)	(9.5)
	Stroop		27	1	9	-4
	Facilitation

Table 2 .

 2 Summary of the mixed and random effects models

		Estimate	Std. error	t value	P*
	Fixed effects
	Position (Initial)	0.007	0.002	2.74	0.006
	Word Type (Critical)	0.006	0.002	2.48	0.013
	Response Type (Vocal)	0.065	0.007	9.22	0.001
	Colour (Green)	0.013	0.002	6.33	0.001
	Colour (Red)	-0.019	0.001	-10.08	0.001
	Position*Word Type	-0.016	0.003	-5.16	0.001
	Word Type*Response Type	-0.008	0.002	-2.75	0.001
	Position*Response Type	-0.009	0.002	-3.08	0.001
	Random effects	Variance	SD	Correlation
	Item (Intercept)	0.00001	0.003

Open Practices Statement

The minimal dataset underlying the findings in the manuscript (i.e. the values behind statistics, graphs, figures, and tables) and a description of the Linear Mixed Effects analysis steps are freely available to other researchers on the Open Science Framework (https://osf.io/2xzqd/?view_only=c9fae46897fa49e9823c91767e78e8d2). There are no ethical or legal restrictions. Requests for data may be sent to the corresponding author.

The authors declare no conflict of interest. This experiment was not preregistered. Any original materials used to conduct the research (including analysis code) will be made available to other researchers for purposes of replicating the procedure or reproducing the results.