Paul Fabry

Adrien Barton

Jean-François Ethier

Jean-Francois Ethier

Version control for interdependent ontologies: challenges and first propositions

Keywords: OWL ontologies, interoperability, version control

published or not. The documents may come

Introduction

Advances in modern healthcare have significantly increased the variety and quantity of health data generated. Ontologies are well positioned to serve as mediation models to support interoperability between diverse clinical data sources as they provide formal, source-independent representations and do not rely on specific data models that might be tied to specific technologies or formats. One such example is PARS3 [START_REF]PARS3 • Solutions • GRIIS[END_REF], a distributed data access platform that is currently being deployed to support activities of the Health Data Research Network Canada [START_REF] Mcgrail | Introducing Health Data Research Network Canada (HDRN Canada): A New Organization to Advance Canadian And International Population Data Science[END_REF] and to enable the implementation of a mortality prediction tool in hospitals in Quebec. Implementing these projects implies of series of tasks from ontology development, to pivot relational model generation, to mappings with data sources. While this approach has been demonstrated and validated to enable the support of various tools like ReflexD [START_REF]ReflexD • Primary health care and follow-up for people with diabetes • Solutions • GRIIS[END_REF], an audit-feedback tool for diabetes treatment in primary care, the next step is to support the evolution of users' needs. Science evolves, new requirements emerge, mistakes are identified, ideas are refined, etc. This will often imply changes in the knowledge representation generated at the inception of the project. This ontological evolution has a significant effect on the activities downstream of ontology development. It is critical to be able to identify what changes constitute a modification that can affect a user's understanding of what is represented or what should be instantiated in a table.

To follow on the questions above, if an ontology changes, how can one identify which of its parts might need to be reviewed for potential adjustments? If the answer is always "all of them", scalability is obviously at risk. What could be the consequences of this change on the applications which depend on it? In the context of PARS3, what are the impacts of a change in the ontology on the mappings between a data source and the pivot relational model? If the answer here again is to ask every source to review or redo every mapping between their assets and the pivot model, maintenance, scalability and as a result, acceptability and sustainability will be markedly reduced.

Background

This paper focuses on ontologies expressed in OWL2, as it is the most used Description-Logic language for biomedical ontologies. Good engineering principles have been proposed to mitigate the impact of ontology change. The Open Biomedical Ontologies Foundry [START_REF] Smith | The OBO Foundry: coordinated evolution of ontologies to support biomedical data integration[END_REF] has proposed guidelines [START_REF]OBO Foundry -Principles overview[END_REF] including an emphasis on reusing the existing classes, a clearly defined scope for ontologies to ensure orthogonality of ontologies representing different domains and unique textual definitions following an Aristotelian form [START_REF] Seppälä | The Functions of Definitions in Ontologies[END_REF]. These principles were followed for the development of the ontologies supporting PARS3. A modular approach was chosen for the creation of the ontology model with several domain ontologies addressing specific topics such as drug prescriptions [START_REF] Ethier | The prescription of drug ontology 2.0 (PDRO): More than the sum of its parts[END_REF], laboratory results [START_REF] Barton | LABO: An ontology for laboratory test prescription and reporting[END_REF] or clinical forms [START_REF] Fabry | QUESTO -An ontology for questionnaire[END_REF]. All these ontologies are built upon the upper-level ontology Basic Formal Ontology (BFO) [START_REF] Arp | Building ontologies with Basic Formal Ontology[END_REF] and reuse classes and properties from reference ontologies in the biomedical domain like the Ontology of Biomedical Investigations (OBI) [START_REF] Bandrowski | The Ontology for Biomedical Investigations[END_REF]. Finally, these ontologies are linked together via mid-level ontologies describing relevant parent categories pertaining to e.g. health procedures [START_REF] Fabry | An Ontological Analysis of Health Procedure Information[END_REF] or services [START_REF] Fabry | An informational perspective on the ontology of services[END_REF].

Consequently, the clinical model structure consists of a network of ontologies or part of ontologies, which are either imported or created within our group, and are strongly interdependent. On the one hand it adds great flexibility and allows us to add representations of various domains as needed. In addition, ontology reuse enables us to leverage the domain expertise of other groups. On the other hand this structure is very sensitive and any change in its parts requires more and more maintenance efforts as the model grows and evolves, which may compromise its sustainability in the long run. Interestingly, the challenge of tracking change, evaluating its significance, and identifying the cascading effects is also an issue in computer science and is often addressed through version control.

Version control has already been discussed in the context of ontologies and is even part of the OBO Foundry principles [14] as well as FAIR [START_REF] Wilkinson | The FAIR Guiding Principles for scientific data management and stewardship[END_REF]. However, in this context it consists in adding a version date in the metadata of the ontology. The change of the version date is at the discretion of the authors. Yet, different types of modifications in the ontology, such as the addition or removal of a class or the correction of an obvious typo in annotations such as a comment, can have very different impacts and it would be useful to differentiate between them. This is especially true since a version date refers to the whole ontology and does not in itself reveal which classes have been modified, which is all the more challenging in the case where a user imports only a few from a source ontology that may have several thousands.

The objective of this article is to lay the foundation for a versioning methodology that would allow the safe and efficient identification of what is impacted (or not) by a change in a context of highly interconnected ontologies. The core principles concerning both ontologies and versioning upon which this work is based and the issues at stake will be outlined, and some recommendations will be proposed and discussed. The resulting methodology and its implementation will be addressed in subsequent works.

Versioning challenges for ontologies

Version control as it pertains to computer science encompasses all the tools and methods for tracking and providing control over changes to a given project [START_REF] Plaice | A New Approach to Version Control[END_REF]. This control is performed at a granularity level deemed pertinent to manage the project most efficiently without creating an undue maintenance burden. To achieve this, artefacts of interest that are relevant to the project will be tracked individually. In many software projects, these artefacts are text files parts of the project's source code or documentation for example.

To ensure the tracking of an artefact, it is necessary to endorse a diachronic identity criterion on artefacts that allow us to identify some of them as evolutions of the same entity. An artefact like a computer file is identified (that is, uniquely denoted in a given context) by a resource identifier composed by the location in the file tree and the name of the file. This resource identifier provides a criterion of diachronic identity of the file it refers to, i.e. two files are considered as two states (versions) of the same informational artefact if they are identified by the same resource identifier. From a versioning perspective, even if the content of a file completely changes over time, the different versions will be considered as the same file at different states over time as long as they are identified by the same resource identifier. When trying to apply this methodology to ontologies, challenges emerge.

Ontology as the artefact of interest for versioning

One option could be to choose the ontology as the artefact of interest for the versioning. This calls for the characterization of what an ontology is. The word "ontology" can refer, depending on the context, to artefacts of various kinds, belonging to the fields of philosophy and computer science, which have as a common goal the representation of entities of the world or a particular domain of it, the categories to which they belong, their properties and the relations that hold between them.

In knowledge engineering, ontologies have been classically defined [START_REF] Gruber | A translation approach to portable ontology specifications[END_REF][START_REF] Guarino | What Is an Ontology?[END_REF] as follows :

An ontology is a formal explicit specification of a shared conceptualization.

A definition grounded in a realist interpretation of the world is proposed by Smith et al. [START_REF] Arp | Building ontologies with Basic Formal Ontology[END_REF]:

Ontology = def. "A representational artefact, comprising a taxonomy as proper part, whose representations are intended to designate some combination of universals, defined classes, and certain relations between them."

The definition from [START_REF] Arp | Building ontologies with Basic Formal Ontology[END_REF] above assumes the existence of universals independently of the conceptualizations that we can make of the reality. In addition, an important element of Gruber and Guarino's definitions is the notion of shared conceptualization, that is an ontology is meant to be shared, usually between its authors and a community that is using it [START_REF] Neuhaus | Ontology development is consensus creation, not (merely) representation[END_REF].

Many formalisms have been proposed for ontologies; among them, OWL is a widely used and recognized standard [START_REF]OWL 2 Web Ontology Language Primer (Second Edition)[END_REF]. An OWL ontology is uniquely denoted with an internationalized resource identifier (IRI), which can be used for versioning purposes. For example, OBI is associated with the IRI: "http://purl.obolibrary.org/obo/obi.owl" that is specific to this ontology. While the ontology will undergo modifications over time, e.g. by adding and deleting classes or properties, it will be considered as the same ontology as long as its IRI remains the same. However, using the ontology as the versioning artefact may not be the best suited method.

Firstly, this approach considers an ontology as a monolithic artefact, one that is therefore used as a whole. This is a questionable assumption, especially in a context of interconnected ontologies where sometimes only parts of an ontology might be reused in another. Let's consider for example an ontology A that imports some part of an ontology B. If B changes, should a new version of A be declared even if not a single class declared directly in ontology A has been modified? If a new version of B is proposed, how to know if the classes imported in A are affected? If they are not, should this entail the creation of a new version of A?

Secondly, according to the W3C OWL2 reference documents [START_REF]OWL 2 Web Ontology Language Primer (Second Edition)[END_REF], an ontology is defined as: "[…] a certain kind of computational artefacti.e., something akin to a program, an XML schema, or a web pagegenerally presented as a document." While this allows a document-based approach to versioning, facilitated by tools such as Git [START_REF] Chacon | Pro Git[END_REF], this might lead to counterintuitive consequences. For example, two classes with different namespaces located in the same computational artefact would be part of the same ontology according to the W3C definition above; however, in a different conception of ontology that would emphasize namespaces, they would belong to different ontologies.

Thirdly, the size of an ontology may represent a significant challenge for its versioning. The Gene Ontology [START_REF] Carbon | The Gene Ontology resource: Enriching a GOld mine[END_REF] for example has more than 40 000 classes. If the modification of one of them leads to a new version of the whole ontology, this implies the tedious task for all its users of checking for possible changes in all the classes at each new version. Therefore, using the whole ontology as the most fine-grained artefact of interest for versioning is not an ideal solution.

Parts of ontology as the artefact of interest for versioning

An ontology proposes a representation of a domain by one or several authors. Such a representation takes place initially in the form of a collection of cognitive representations1 of its author on the basis of which they create publicly available informational content entities (ICE) [START_REF] Ceusters | Aboutness: Towards Foundations for the Information Artifact Ontology[END_REF].

In the context of OWL2 ontologies, such ICEs will be called "OWL component" (OC). At least some of those OCs are associated with an identifier, such as an International Resource Identifier (IRI) that consists of a permanent location, a namespace and a unique string (for example: "http://purl.obolibrary.org/obo/OBI_0000011" is an IRI from OBI), which is of particular interest for versioning. Also at least2 some of those OCs refer to a portion of reality that is part of the domain, in a way that can be understood by other users. These can be analyzed using a three level framework inspired by Ceusters and Smith [START_REF] Ceusters | Foundations for a realist ontology of mental disease[END_REF]:

• Level L1: a portion of reality;

• Level L2: the cognitive representation of this portion of reality;

• Level L3: a publicly accessible ICE, the OWL component, that emanates3 from the L2 cognitive representation and is also about the same portion of reality.

What is named in the literature as a 'class' or 'term' is an important type of such OCs. OCs can also include annotations such as definitions, or elucidations. Neuhaus [START_REF] Neuhaus | What is an Ontology?[END_REF] characterizes some of the annotations as "assertive annotations", i.e. "…the kind of annotations that are intended to assert a true proposition about the domain of the ontology." Neuhaus also regroup assertive annotations and logical axioms under the term of "assertive statements". A definition would specify in natural language a way to group certain particulars, while logical axioms can be automatically processed by a reasoner to classify the OWL components.

An OC can be associated with some logical axioms. For example, a class might be mentioned by some logical axioms.

For example, the OC with the IRI "http://purl.obolibrary.org/obo/OBI_0000011" (that has for label "planned process") is associated to the following assertive statements (among others):

• Definition: "A process that realizes a plan which is the concretization of a plan specification." • planned process SubclassOf process Two kinds of relations between a class and a portion of reality are proposed. Firstly, a relation of aboutness that is determined by the cognitive acts of its author: in the IAO framework, inspired by Chisholm [START_REF] Chisholm | The primacy of the intentional[END_REF], the "aboutness of those of our representations formulated in speech or writing […] is to be understood by reference to the cognitive acts with which they are or can in principle be associated" [START_REF] Ceusters | Aboutness: Towards Foundations for the Information Artifact Ontology[END_REF]. Secondly, a relation that is determined by the interpretation of the assertive statements by another competent reader who will make their own cognitive representation of a portion of reality on the basis of this interpretation. In this case, the assertive statements can be considered as describing that portion of reality (we will presume here that any competent external user will have the same interpretation). The goal when creating a class is that its assertive statements describe a portion of reality identical to the one pointed to by its aboutness.

Identity criteria for ontological components

One may propose two identity criteria of OC based on cognitive representations and assertive statements, depending on whether one relies on what we will call (inspired by theories in philosophy of language [START_REF] Michaelson | Reference. The Stanford Encyclopedia of Philosophy[END_REF]) the "intentionalist" or "descriptivist" conceptions of the identity of an OC:

• R1: In the intentionalist conception, the identity is determined by its author's related cognitive representation. • R2: In the descriptivist conception, the identity is determined by its associated assertive statements.

Hence, in the case of R1, even though the assertive statements might change significantly, as long as the OC emanates from the same cognitive representation of the author, the identity of the OC does not change and so the IRI should remain the same. Meanwhile in R2, even though the author's cognitive representation associated with an OC might change, as long as the assertive statements are not significantly modified 4 , the identity of the OC does not change and so the IRI should remain the same.

The distinction between these conceptions is very rarely made explicit when creating and sharing an ontology, and this may negatively affect its evolution or its external reuse. The cognitive representation of the author (level 2) and the assertive statements are therefore two aspects that need to be managed appropriately to evaluate an OC as a potential artefact of interest for versioning, and as described above, two possible approaches, namely R1 and R2, need to be considered.

Let's consider the following example about a hypothetical ontology about fruits, the Fruit Ontology (FO). At time t1 the author of this ontology adds an OWL component that refers to its own cognitive representation of the class of oranges (Citrus sinensis) in the reality. This OC (let's call it "OC1") is associated with the IRI: "FO_004" and is associated with the following assertive statements:

• Definition D1: "A citrus fruit which is the edible fruit of the orange tree and that is orange in color." • orange subClassOf citrus fruit At time t2, the author is made aware that the definition D1 does not explicitly mention unripe oranges, which are green, and therefore that some of the ontology's users assume OC1 is about only ripe oranges (Figure 1).

The author is now faced with two options: either A) he considers that his initial cognitive representation is still the relevant one and so the assertive statements need to be updated accordingly, or B) he considers that what needs to be represented is instead ripe oranges and so he is now working with a new cognitive representation but as it turns out, the assertive statements derived from this new representation are similar to those initially derived.

Each of these options may have an impact on the identity of OC1 that must be evaluated according to the R1 and R2 conceptions discussed above, we thus have four distinct scenarios:

• Scenario R1A: The cognitive representation of the class of oranges is not modified between t1 and t2 and a new definition D2 ("A citrus fruit which is the edible fruit of the orange tree and that is orange in color when ripe, and green when unripe.") is proposed at t2 for clarification. In this scenario, OC1 is still conformant to the same • Scenario R2A: R2A describes the same reality as R1A but endorses the R2 conception rather than the R1 conception. A new definition D2 has been formulated at t2, and consequently the assertive statements have changed. In this case, a new OC (OC3), and thus a new IRI, is created along the new assertive statements (Figure 3). • Scenario R2B: Here also, R2B describes the same reality as R1B, but endorses the R2 conception rather than the R1 conception. The assertive statements have not been modified and thus the identity of OC1 has not changed even though OC1 is now related to another cognitive representation (Figure 3). Let's consider that OC1 is imported at time t1 in two fictive distinct ontologies:

• The Plant Parasites ontology (PPO), created by the author of OC1, imported it in the axiom: medfly subClassOf (is_parasite_of some orange). As a matter of fact, the author of PPO considered that FO_004 was about oranges at any stage of development.

• The Juice Ontology (JO) imported it in the axiom: orange juice subClassOf (is_made_of some orange). The authors of JO have initially imported "FO_004" as they understood the assertive statements as describing ripe oranges.

For these two ontologies, the previously mentioned scenarios will have various implication. For example, in the scenario R1A, as OC1 is now about oranges at any stage of development, JO needs to modify its axiom and include another OC while PPO doesn't need to. On the contrary, in the scenario R2B, as OC1 is now about ripe orange, JO could keep the axiom as is while PPO need to modify it. In context of a direct import, this will lead to incompatible OCs that might be difficult to identify, and the "live" change might affect users before the problem can be identified and fixed.

These scenarios are found in practice without being explicitly identified as such, which leads to many ambiguities when reusing ontologies. However, each conception has its advantages and disadvantages depending on whether one favors the author's cognitive representation importance in contributing to the coherence of the OC over the users' perspective which is based on their understanding of the assertive statements.

The examples above illustrate that OCs can be good candidates as artefacts of interest for versioning, provided that we are able to track both the cognitive representation of the author (via the operationalization of R1) and the assertive statements (via the operationalization of R2).

Discussion

Despite emerging tools that provide an invaluable help [START_REF] Matentzoglu | Ontology Development Kit: a toolkit for building, maintaining and standardizing biomedical ontologies[END_REF], managing the evolution of interconnected ontologies is a tedious and error-prone task. Versioning processes are a way forward to support the management of ontology evolution. In this work, two issues at the heart of this question are addressed.

Artefact of interest for ontology versioning

The first issue concerns the artefacts of interest, whose evolution needs to be tracked individually. As mentioned above, the current processes imply a versioning at the level of the ontology, but this presents some important problems, if only because it is not simple to outline the target of versioning in a context of interconnected modular ontologies where sometimes only parts of an ontology might be reused in another. Therefore, ontologies should no longer be considered as standalone and monolithic but rather as highly interconnected informational constructs, and a focus on a more fine-grained artefact is required, leading to the introduction of so-called "OWL components" as the versioned artefacts.

In the current work, we only discussed one kind of OC, namely classes. However, other components of an ontology such as object properties, data properties or even ontological instances are possible candidates as OCs of interest for versioning. Indeed, all these elements can be associated with assertive statements that can change over time. Defining OCs in the least ambiguous and most comprehensive way possible is of the utmost importance and will be the subject of subsequent work. In addition, not all parts of an ontology are of interest for versioning and therefore it will be important to determine whether certain OCs are not relevant for versioning.

It seems also relevant to consider what an ontology is in relation to OCs. On the one hand, ontologies are conceptually envisioned through high-level definitions such as the one previously mentioned [START_REF] Arp | Building ontologies with Basic Formal Ontology[END_REF][START_REF] Guarino | What Is an Ontology?[END_REF]. On the other hand, ontologies as defined by W3C are described as computerized constructs and manipulated via files or namespaces. As it has been discussed in this work, the relationship between the two is not so clear cut and this calls for approaching OWL ontologies according to principles more in line with those of software engineering.

Identity of the artefact of interest

The next issue is the determination of the identity of the artefact of interest. Even tough the OCs relevant for versioning have only been characterized through their role for versioning, two possibly relevant aspects for their identity have been identified: their cognitive representations and their associated assertive statements (logical axioms and assertive annotations).

While the formal aspect of an ontology allows the use of reasoners and query tools to automatically process an ontology, an ontology is not limited to its formal elements. An ontology is also a way to share knowledge between communities of humans and for this goal, assertive annotations such as natural language definitions may have a role as significant as the logical axioms [START_REF] Neuhaus | Ontology development is consensus creation, not (merely) representation[END_REF].

However, not all annotations are assertive and one needs to distinguish assertive annotations from annotations concerning the OC itself, such as its author or the date of creation. An annotation that will require additional exploration to arbitrate its inclusion or not as an assertive statement is the label of an OC. Given the uncertainty around its status, it is not included in the examples above. A natural language label in itself may be too ambiguous for playing an assertive role: experience shows that a given label could be associated with several distinct OC from distinct ontologies. However, labels are not randomly chosen either and the eventual import that the semantics of natural language labels should have into ontologies will need to be analyzed.

In an ideal world, one could envision a one-to-one relation between cognitive representations and assertive statements, as the cognitive representation of the author of an OC would be exactly translated in its assertive statements, which in turn would be exactly interpreted in a similar cognitive representation by the users that have only access to its assertive statements. However, OCs may retain the same IRI while their assertive statements undergo significant changes through time. Despite everyone's best effort, discrepancies exist and will likely remain and therefore the chosen versioning process must be able to handle these situations.

However, regardless of the quality of the assertive statements, an evolution of knowledge may imply a modification of the cognitive representation associated with an OC. Since OCs are developed in interlinked coherent groups (ontologies), the cognitive representation of the author is likely to play an important role in keeping this group coherent. Being able to track the cognitive representation of the author would allow the user community to know when something fundamental changed in the way the author structures the knowledge representation (e.g. because of a shift in science) versus when they attempt to improve the assertive statements to better match their cognitive representation.

To this end, being able to individually track the changes of both the cognitive representations and assertive statements would be a significant advantage. As it stands though only one IRI is associated with an OC and it is sometimes used to track the author's cognitive representation (R1) and sometimes the assertive statements (R2). However, it cannot do both in parallel. Associating two IRIs, one that tracks OC identity according to conception R1, and another one that tracks OC identity according to conception R2, would allow the tracking of both the cognitive representation and the assertive statements. Further work should elaborate the details of such a proposition.

Conclusion and Future Work

The work presented here aims at laying the foundation for a safe handling of changes to OWL components when they are reused. Being able to track changes in the cognitive representations of the authors or the assertive statements is the first step to further scalability and sustainability while diminishing the incentive to build "yet another model".

Other challenges lie ahead. It will likely be desirable to identify various types of changes without semantic implications including:

1. Changes to the computational representations e.g. changes of OWL syntax or in the order of elements; 2. Changes in non-assertive annotations e.g. additions of contact information annotations; 3. Changes in natural language assertive statements e.g. correcting a typographical error or replacing a word by a perfect synonym; 4. Changes in logical axioms producing a logically equivalent result e.g. replacing "OCA OR OCB" by "OCB OR OCA".

Automatically detecting changes of type 3 or 4 is not trivial in all cases. Finally, when the changes include the cognitive representation of the author or assertive statements, it will be greatly beneficial to be able to have an approach to navigate through OCs of interest and identify those not impacted by the changes, leaving only a subset for manual review and adjustments as needed.

Overall, an approach based on the principles presented here will ensure not only a better change management through a cohesive, complete, and fined-grained version control, but also a better import process while contributing to supporting ontology engineering methods.

Figure 1 :

 1 Figure 1: Relations between the OWL component (OC1) from the Fruit Ontology, the portion of reality it is about, and the associated author's and users' cognitive representations at time t1.

  cognitive representation, but one of the associated assertive statements has been modified. As the identity of OC1 is determined by the cognitive representation in R1 and this representation didn't change, what comes out of this scenario is a new version of OC1, which is thus associated with the same IRI "FO_004". (Figure2). • Scenario R1B: A new cognitive representation of interest (about ripe oranges) has been identified by the author of OC1. Therefore, a new OWL component (OC2) and thus a new IRI, is created by reusing assertive statements of OC1 (in this case the definition).(Figure2).

Figure 21 :

 21 Figure 21: Scenarios R1A and R1B at t2. In R1A the cognitive representation of the class of oranges remains unchanged between t1 and t2 and a new definition is proposed at t2 for clarification. In R1B a new cognitive representation of interest has been identified by the author and therefore, a new OWL component (OC2) is created.

Figure 3 :

 3 Figure 3: Scenarios R2A and R2B at t2. In R2A, the assertive statements have changed, thus a new OC and a new IRI are created along them. In R2B, the assertive statements have not changed. OC's identity has not changed even though it is associated with a new cognitive representation of the author.

Cognitive representations are also concretizations of ICEs according to IAO[START_REF] Ceusters | Aboutness: Towards Foundations for the Information Artifact Ontology[END_REF].

"at least" because it remains a complex open question whether all OCs are about a portion of reality (e.g. object properties).

The precise nature of the relation of emanation is outside the scope of this work.

We are talking about changes that might affect the understanding of the OC's meaning, not merely a typo fix or a logically equivalent reformulation of a statement.


Mathieu Raingeval

Basile Leduque

Pierre Baduel

Alejandro Edera

Fabrice Roux

Vincent Colot

Leandro Quadrana
email: leandro.quadrana@universite-paris-saclay.fr

Basile

Retrotransposon-driven environmental regulation of FLC leads to adaptive response to herbicide

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

Abstract:

The mobilization of retrotransposons yields major-effect mutations. Here, we report an adaptive retrotransposon insertion within the first intron of the Arabidopsis floral-repressor locus FLOWERING LOCUS C (FLC). The insertion-mutation augments the environmental sensitivity of FLC by affecting the balance between coding and non-coding transcript isoforms in response to environmental threads. We show that this balance is modulated epigenetically by DNA methylation and orchestrated by IBM2, a factor involved in the processing of intronic heterochromatin. The stress-sensitive allele of FLC has recently spread across populations subjected to recurrent chemical weeding, and we demonstrate that retrotransposon-driven acceleration of life cycle represents a rapid response to herbicide.

Our findings illustrate how retrotransposition can create environmentally-sensitive alleles that facilitate adaptation to anthropogenic disturbances of the environment.

2

INTRODUCTION

Environmental changes due to direct human activities have impacted biodiversity at unprecedented pace, putting a countless number of natural populations at evolutionary risk.

Cropland expansion, intensified chemical input, and urbanization of natural habitats have favored the replacement of native populations by colonizing ruderal species. Identifying the type, mode of action, and history of mutations enabling adaptation to ruderal habitats is crucial to forecast the impact of ongoing and future anthropogenic activity on biodiversity.

Among the different types of mutations, those that occur spontaneously following DNA replication errors or damage are arguably the best characterized [START_REF] Mackay | The Genetics of Quantitative Traits: Challenges and Prospects[END_REF][START_REF] Weigel | Population Genomics for Understanding Adaptation in Wild Plant Species[END_REF]. However, structural variants, notably those produced by the mobilization of transposable elements (TEs), which are DNA sequences that have the ability to self-propagate within and across genomes, account for the largest fraction of varying base pairs among individuals [START_REF] Michael | High Contiguity Arabidopsis Thaliana Genome Assembly with a Single Nanopore Flow Cell[END_REF][START_REF] Sudmant | An Integrated Map of Structural Variation in 2,504 Human Genomes[END_REF][START_REF] Weischenfeldt | Phenotypic Impact of Genomic Structural Variation: Insights from and for Human Disease[END_REF]. TE mobilization has been proposed to have a greater potential than single nucleotide polymorphisms to generate large effect mutations [START_REF] Schrader | The Impact of Transposable Elements in Adaptive Evolution[END_REF], which may be further accentuated by the exquisite sensitivity of some TEs to environmental signals [START_REF] Casacuberta | The Impact of Transposable Elements in Environmental Adaptation[END_REF]. Despite these considerations, the actual contribution of new TE insertions to adaptive walks remains controversial.

Previous analyses of TE insertions polymorphisms based on population genomics data for hundreds of accessions of the model species Arabidopsis thaliana revealed that the first intron of the Flowering Locus C (FLC) gene is the most frequent target of TE insertions in nature, with up to 16 independent TE-containing alleles [START_REF] Quadrana | The Arabidopsis Thaliana Mobilome and Its Impact at the Species Level[END_REF][START_REF] Baduel | Genetic and Environmental Modulation of Transposition Shapes the Evolutionary Potential of Arabidopsis Thaliana[END_REF]. FLC encodes a MAD-box transcription factor that negatively regulates floral transition by impairing the expression of flowering signals. Vernalization (i.e. overwintering or long cold exposure at the seedling stage) represses FLC and this repression is maintained epigenetically upon the return to warmth, allowing plants to flower in spring [START_REF] Bastow | Vernalization Requires Epigenetic Silencing of FLC by Histone Methylation[END_REF]). However, with few exceptions [START_REF] Qüesta | Arabidopsis Transcriptional Repressor VAL1 Triggers Polycomb Silencing at FLC during Vernalization[END_REF], we currently have limited knowledge about how mutations at FLC modulate flowering time variation in nature.

RESULTS

3

A recent retrotransposon insertion within FLC modulates flowering response

Using long-read sequencing data, we assembled a highly contiguous, telomere-to-telomere, genome assembly of the Arabidopsis accession Ag-0 (Supplementary Figure 1), which carries a retrotransposon insertion within FLC [START_REF] Quadrana | Transposition Favors the Generation of Large Effect Mutations That May Facilitate Rapid Adaption[END_REF]. Our assembly confirmed the localization and identity of a full-length ATCOPIA78/ONSEN retrotransposon insertion within FLC's first intron (Figure 1a). Based on the nucleotide divergence between retrotransposon's LTRs [START_REF] Sanmiguel | The Paleontology of Intergene Retrotransposons of Maize[END_REF] we estimated that this insertion is less than 0.16 million years old (MYO). The Ag-0 genome has two additional full-length ONSEN insertions, located in chromosomes 1 and 3. LTRs of these copies were significantly divergent (2.27% and 0.66%, respectively), suggesting they inserted ~2.27 and ~0.66 MYO, respectively. Consistently, these insertions are respectively fixed or at intermediate frequency (14%) among 1069 natural Arabidopsis accessions previously characterized [START_REF] Baduel | Genetic and Environmental Modulation of Transposition Shapes the Evolutionary Potential of Arabidopsis Thaliana[END_REF]. The insertion within FLC is more closely related to ONSEN copies carried by the reference accession Col-0 than the ones on chromosome 1 and 3 of Ag-0 (Figure 1b), suggesting that none of these two served as donors for the insertion within FLC. To characterize the origin of that insertion, we set out to identify accessions carrying the same FLC haplotype as Ag-0 using SNP data available for the 1069 natural accessions (1001 Genomes Consortium 2016). Twenty-seven accessions, mostly from northern latitudes, carry the Ag-0 haplotype (Hap1) but lack the retrotransposon insertion (Figure 1c). Based on the number of SNPs accumulated in the haplotype block we estimated that the transposon insertion within FLC occurred less than 246 years ago. Considering that Ag-0 was collected by Denis Ratcliffe almost 60 years ago from a railway ballast [START_REF] Kranz | Genetic resources in Arabidopsis : computerized listing of seed bank material[END_REF], it is most likely that the insertion within FLC took place during the XIX century.

Numerous TE insertions within FLC have been identified in Brassicaceae species, most of which produce loss-of-function alleles [START_REF] Quadrana | The Contribution of Transposable Elements to Transcriptional Novelty in Plants: The FLC Affair[END_REF]. To test if the TE-containing FLC allele in Ag-0, which has one of the longest (8435nt-long) introns described in Arabidopsis (Supplementary Figure 2), is functional and subjected to its canonical regulation, we grew Ag-0 plants under standard long-days conditions or eight weeks of vernalization. Vernalized plants flowered soon after the treatment (28±1.39 days 4 after vernalization), while control plants flowered more than 120 days after germination (Figure 1d). Consistently, FLC expression was initially high and strongly reduced following vernalization (Supplementary Figure 3). Vernalization-induced down-regulation of FLC was maintained at least four weeks after returning plants to warm temperatures, similarly to the Northern Sweden ecotype Lov-1 [START_REF] Coustham | Quantitative Modulation of Polycomb Silencing Underlies Natural Variation in Vernalization[END_REF]) that requires long cold exposure to saturate the vernalization requirement (Supplementary Figure 3). These results confirm that the FLC allele carried by Ag-0 is functional and characterized by a canonical vernalization response.

Because ONSEN retrotransposons are induced by heat shock (HS) (Tittel-Elmer et al. 2010), we tested the flowering response of Ag-0 to single or multiple HS (Figure 1d).

Compared to the late flowering response observed when plants are grown under control conditions, plants subjected to heat stresses flowered much earlier (40-70 days after germination). This acceleration in flowering correlates with the severity and number of heat-shocks. For instance, while only 30% of plants subjected to a single day of heat shock flower less than 50 days after germination, all plants subjected to three heat shocks flowered ~40 days after germination.

To determine the genetic architecture of the flowering response to HS, we generated an F2 population by crossing Ag-0 with two accessions with different degree of vernalization response (Ws-2 and CIBC17). Bulk segregant analysis of F2 plants flowering early in response to HS identified a single peak of association spanning the Ag-0 allele of FLC in both crosses (Figure 1e), further suggesting that the TE-containing allele of this gene is causal for the HS-induced flowering response.

Heat-induced retrotransposon activation impairs FLC activity

To investigate the molecular mechanisms underpinning the flowering response of FLC to HS, we performed RNA-seq experiments on Ag-0 seedlings grown under control or HS conditions. FLC was highly expressed in control plants (Figure 2a), in agreement with the strong vernalization requirement observed for this accession. ONSEN was strongly induced in response to HS (Figure 2a), consistent with previous work reporting activation of this TE in several Arabidopsis accessions [START_REF] Masuda | Characterization of a Heat-Activated Retrotransposon in Natural Accessions of Arabidopsis Thaliana[END_REF]. Alongside ONSEN reactivation, the first intron of FLC is significantly retained and overall FLC expression was 5 markedly reduced in response to stress. RT-qPCR confirmed these observations (Figure 2b) [START_REF] Quadrana | Transposition Favors the Generation of Large Effect Mutations That May Facilitate Rapid Adaption[END_REF]. We did not find, however, significant readthrough transcription from the ONSEN insertion towards the first exon of FLC, suggesting that nonsense-mediated degradation, rather than retrotransposon-derived antisense transcripts likely cause the observed downregulation of FLC. Moreover, the canonical FLC antisense transcript COOLAIR, which mediates the epigenetic silencing of FLC [START_REF] Liu | Targeted 3' Processing of Antisense Transcripts Triggers Arabidopsis FLC Chromatin Silencing[END_REF], is also downregulated in response to heat stress (Supplementary Figure 4), excluding its participation in the reduction of FLC expression. In addition, expression of FLM, another negative regulator of flowering time known to be downregulated in response to warm temperatures [START_REF] Posé | Temperature-Dependent Regulation of Flowering by Antagonistic FLM Variants[END_REF], is not altered in response to HS (Supplementary Figure 4).

Crucially, HS-induced downregulation of FLC was accompanied by a strong induction of direct targets of FLC [START_REF] Deng | FLOWERING LOCUS C (FLC) Regulates Development Pathways throughout the Life Cycle of Arabidopsis[END_REF], such as the major flowering integrators

FLOWERING LOCUS T (FT), SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1

(SOC1), and AGAMOUS LIKE 24 (AGL24) (Figure 2b and Supplementary Figure 4). In combination, these results indicate that environmental reactivation of ONSEN impairs FLC expression, inducing the expression of flowering integrators that trigger flowering transition.

Experimental loss of heterochromatin modifications, including DNA methylation, can affect the expression of TE-containing genes [START_REF] Saze | Mechanism for Full-Length RNA Processing of Arabidopsis Genes Containing Intragenic Heterochromatin[END_REF][START_REF] Berthelier | Long-Read Direct RNA Sequencing Reveals Epigenetic Regulation of Chimeric Gene-Transposon Transcripts in Arabidopsis Thaliana[END_REF].

However, whether HS-induced reactivation of ONSEN is associated with its hypomethylation is still unclear [START_REF] Tittel-Elmer | Stress-Induced Activation of Heterochromatic Transcription[END_REF][START_REF] Sun | Heat Stress-Induced Transposon Activation Correlates with 3D Chromatin Organization Rearrangement in Arabidopsis[END_REF], likely due to the difficulty to investigate DNA methylation of such repetitive sequences. In contrast to the reference Arabidopsis genome Col-0, which contains at least four nearly identical full-length ONSEN copies, Ag-0 has only three, and significantly divergent, copies (Figure 1b). Indeed, bisulfite-sequencing (Bi-seq) data obtained from control Ag-0 plants provide robust DNA methylation information across the complete ONSEN annotation and revealed extensive non-CG methylation of the LTR sequences (Figure 2c). In response to HS, the 5' LTR of ONSEN, which contains heat-shock response elements and acts as a promoter of transcription [START_REF] Pietzenuk | Recurrent Evolution of Heat-Responsiveness in Brassicaceae COPIA Elements[END_REF], becomes strongly and rapidly hypomethylated, indicating that transcriptional reactivation of ONSEN is accompanied by DNA methylation loss.

DNA methylation-dependent modulation of FLC

To test directly the role of DNA methylation in the modulation of flowering response in Ag-0, we generated CRISPR-Cas9-mediated mutants of the SWI2/SNF2 chromatin remodeler DDM1, which is required to maintain DNA methylation in Arabidopsis [START_REF] Jeddeloh | Maintenance of Genomic Methylation Requires a SWI2/SNF2-like Protein[END_REF]. After two generations with the CRISPR-Cas9 cassette, transgene-free offspring were propagated and genotyped to identify plants carrying mutations between the end of exon 6 and the beginning of exon 7 of DDM1, where guide

RNAs were targeted (Supplementary Figure 5a). Two lines (ddm1-5 and ddm1-6) were obtained in this way (Figure 3a). Target sequencing revealed that both mutants carry a similar frameshift mutation in exon 6 that generates multiple premature stops within the ATPase domain of DDM1 (Supplementary Figure 5b). DNA methylation levels of ONSEN were strongly reduced in the CRISPR-mutant compared to wild-type Ag-0 plants (Figure 3b), confirming the loss of DDM1 activity in these plants. Strikingly, compared to the late flowering phenotype of control Ag-0 plants, ddm1 plants flower 25±6 days after germination (Figure 3a). We then analyzed the expression of canonical as well as intron-retention (IR) transcript isoforms of FLC. ddm1-induced hypomethylation of ONSEN was accompanied by a dramatic downregulation of FLC expression and production of IR-FLC transcript isoforms (Figure 3c and Supplementary Figure 5b). Notably, ONSEN expression was not altered in ddm1 compared to wild-type Ag-0 plants (Supplementary Figure 5c), In combination, these results established that it is the hypomethylation of ONSEN, rather than its transcriptional activation, that modulates FLC expression and floral transition in Ag-0 (Figure 3d).

Processing of gene transcripts produced from intronic heterochromatin is mediated by a protein complex comprising the RNA binding protein [START_REF] Saze | Mechanism for Full-Length RNA Processing of Arabidopsis Genes Containing Intragenic Heterochromatin[END_REF]). To test the involvement of IBM2 in the expression of FLC in Ag-0, we introduced the ibm2-4 mutant allele [START_REF] Deremetz | Antagonistic Actions of FPA and IBM2 Regulate Transcript Processing from Genes Containing Heterochromatin[END_REF] into Ag-0 plants. We selected F3 plants carrying the Ag-0 alleles of FLC in combination with either the WT or the mutant IBM2 alleles (Figure 3e). These plants (Ag-0 ibm2 -/-and Ag-0 ibm2 +/+ , respectively) were grown under control conditions to measure flowering time.

INCREASE IN BONSAI METHYLATION 2 (IBM2)
7

Ag-0 ibm2 -/-mutant plants flowered 22 +/-10 days after germination, while all Ag-0 ibm2 +/+ control plants flowered more than 120 days after germination (Figure 3e).

Expression analysis showed that acceleration of flowering in Ag-0 ibm2 -/-plants was accompanied by a significant reduction in FLC expression and induction of IR-FLC transcripts isoforms (Figure 3f). As expected, misregulation of FLC in ibm2 plants leads to higher FT expression levels (Supplementary Figure 6), further supporting that failed processing of the ONSEN-containing intron of FLC transcripts, such as in response to HS or ddm1-induced hypomethylation, underlie the early flowering of Ag-0. Importantly, DNA methylation of the ONSEN insertion was not affected in Ag-0 ibm2 -/-plants (Figure 3g), indicating that IBM2 functions downstream of DNA methylation. Altogether, these results demonstrate that the environmental modulation of the TE-containing allele of FLC is mediated by IBM2's methylation-dependent transcript processing (Figure 3h).

Retrotransposon-mediated acceleration of life cycle in response to herbicide

Given the key adaptive role of FLC in aligning flowering time with seasons, the retrotransposon-driven HS impairment of FLC expression may reflect a more general ability of Ag-0 plants to flower early in response to specific environmental conditions [START_REF] Quadrana | Transposition Favors the Generation of Large Effect Mutations That May Facilitate Rapid Adaption[END_REF]). To investigate whether the TE-containing allele of FLC has spread across populations experiencing similar climates as that of Ag-0, we collected in spring 2019 and 2021, 74 Arabidopsis accessions across a 160km2 area near the city of Argentat in central France (Figure 4a and table S1), where Ag-0 was initially collected in the 50s [START_REF] Kranz | Genetic resources in Arabidopsis : computerized listing of seed bank material[END_REF]. The newly collected accessions were growing in diverse habitats, including brownfields, sidewalks, railways, and field margins. Genotyping showed that 23% of the accessions carry the retrotransposon insertion in FLC, demonstrating that this allele has been maintained in the area for the past 60 years. Intriguingly, none of the populations from Argentat, or nearby cities, carry the insertion (Figure 4a), as the railway where Denis Ratcliffe initially collected Ag-0 was dismantled in 1970 and the zone became heavily urbanized since then. Conversely, the insertion is frequent (~40%) in Arabidopsis populations nearby the city of Archignac and Turenne, yet carrier and non-carrier populations share the same climate niche (Figure 4b). Therefore, the retrotransposon-containing FLC allele more likely plays a role in adaptation to local 8 environments, rather than providing an adaptive response to warmer climates as previously proposed [START_REF] Quadrana | Transposition Favors the Generation of Large Effect Mutations That May Facilitate Rapid Adaption[END_REF]. Consistent with this possibility, carriers of the TE-containing allele of FLC were found most often along railways and field margin strips (Figure 4c), two environments exposed to intense chemical weeding [START_REF] Marshall | Field Margins in Northern Europe: Their Functions and Interactions with Agriculture[END_REF][START_REF] Antuniassi | Soil and Weed Survey for Spatially Variable Herbicide Application on Railways[END_REF]. Selection of weeds in such environments has been linked to mutations in several flowering pathways, notably FLC [START_REF] Baduel | Genetic Basis and Evolution of Rapid Cycling in Railway Populations of Tetraploid Arabidopsis Arenosa[END_REF], enabling plants to cycle faster and complete a full life-cycle in high-stress and high-disturbance environments [START_REF] Kreiner | Rapid Weed Adaptation and Range Expansion in Response to Agriculture over the Past Two Centuries[END_REF]). To test this hypothesis, we investigated the presence of the retrotransposon-containing allele in 165 natural populations of Arabidopsis collected across south-west of France in 2015 [START_REF] Frachon | A Genomic Map of Climate Adaptation in at a Micro-Geographic Scale[END_REF]. Remarkably, and despite its recent origin, the retrotransposon-containing allele of FLC is present in 56% of these populations (Figure 4a, table S2). Carrier populations were significantly enriched in railways, and, to a lesser extent, field margins, backyards, and silo sites (Figure 4d), supporting a role for this allele in the response to chemical weeding. To determine if this allele is under positive selection, we quantified haplotype-lengths using the Extended Haplotype Homozygosity (EHH) test [START_REF] Sabeti | Detecting Recent Positive Selection in the Human Genome from Haplotype Structure[END_REF], which detects recent selective sweeps associated with de novo mutations. This analysis showed that haplotypes carrying retrotransposon insertions are more frequent and much longer than ancestral haplotypes (Figure 4e), indicating recent or ongoing positive selection of the insertion. In combination, these results demonstrate that the de novo retrotransposon insertion within FLC is likely involved in adaptation to herbicide-heavy environments.

To test directly this possibility, we sprayed Ag-0 plants with sublethal doses of the broad-spectrum herbicide Glufosinate. Strikingly, at least half of the Ag-0 plants treated with herbicide flowered before 60 days, compared to none of the control plants (Figure 4f).

RT-qPCR experiments show that under these conditions ONSEN becomes strongly upregulated in response to herbicide (Figure 4g), supporting the notion that this retrotransposon is sensitive to severe stresses [START_REF] Matsunaga | A Small RNA Mediated Regulation of a Stress-Activated Retrotransposon and the Tissue Specific Transposition during the Reproductive Period in Arabidopsis[END_REF], rather than being induced by specific environmental signals such as HS. Together with retrotransposon activation, the balance between full-length and IR FLC transcripts drastically shifts, leading to high expression of the non-functional FLC isoform, FT induction, and early flowering (Figure 4g). In conclusion, the retrotransposon insertion within FLC enables plants to cycle 9 faster in response to chemical spraying, providing a rapid adaptive response of Arabidopsis to modern weed control practices (Supplementary Figure 7).

DISCUSSION

Agricultural intensification and extensive urbanization, aided by the massification of agrochemicals, has transformed natural habitats at unprecedented pace. How populations react to such drastic environmental changes, and whether it relies on standing variation or de novo mutations, is still a matter of debate. We demonstrate that a gain-of-function retrotransposon insertion within the flowering gene FLC enables Arabidopsis plants to cycle faster in response to herbicide-exposure, leading to an escape response to chemical weeding. This escape mechanism may apply to a wide array of stresses, setting them apart from conventional herbicide resistance responses, which entail the accumulation of mutations at genes encoding specific herbicide target enzymes or related metabolic pathways [START_REF] Kreiner | Rapid Weed Adaptation and Range Expansion in Response to Agriculture over the Past Two Centuries[END_REF][START_REF] Kersten | Standing Genetic Variation Fuels Rapid Evolution of Herbicide Resistance in Blackgrass[END_REF]. Additionally, while target-site herbicide resistances are typically caused by fixed-effect mutations, the effect of the retrotransposon insertion in FLC allele is stress-dependent. Such a type of mutation should reduce fitness costs in the absence of stress, and therefore increase its chance to persist in the population as segregating alleles [START_REF] Merenciano | The Interplay Between Developmental Stage and Environment Underlies the Adaptive Effect of a Natural Transposable Element Insertion[END_REF][START_REF] Baduel | Genetic and Environmental Modulation of Transposition Shapes the Evolutionary Potential of Arabidopsis Thaliana[END_REF]). Indeed, this insertion occurred before the discovery of herbicides in the late 1800s, and likely maintained in the population . In line with this scenario, the TE-containing allele of FLC is found only at intermediate frequencies across recently collected populations (Figure 4a).

Thus, environmentally-dependent fitness effects may facilitate the spreading of TE insertions through balancing selection at the regional scale, providing an alternative explanation for the excess of rare TE insertion alleles observed across natural populations at the worldwide scale [START_REF] Merenciano | The Interplay Between Developmental Stage and Environment Underlies the Adaptive Effect of a Natural Transposable Element Insertion[END_REF][START_REF] Baduel | Genetic and Environmental Modulation of Transposition Shapes the Evolutionary Potential of Arabidopsis Thaliana[END_REF][START_REF] Baduel | Relaxed Purifying Selection in Autopolyploids Drives Transposable Element over-Accumulation Which Provides Variants for Local Adaptation[END_REF][START_REF] Niu | Transposable Elements Drive Rapid Phenotypic Variation in[END_REF].

Our findings provide a compelling example of a recent retrotransposon insertion enabling a rapid evolutionary response to anthropogenic activities. Adaptive TE insertions were previously reported [START_REF] Casacuberta | The Impact of Transposable Elements in Environmental Adaptation[END_REF]. For instance, the industrial melanism of the British peppered moths was caused by a de novo TE insertion within the gene cortex involved, through still unclear mechanisms, in pigmentation of Lepidoptera's 10 wings ( [START_REF] Van't Hof | The Industrial Melanism Mutation in British Peppered Moths Is a Transposable Element[END_REF]. Similarly, natural pesticide resistance in Drosophila melanogaster was found associated with the presence of a TE insertion within the gene CHKov1, likely affecting a target of organophosphate pesticides [START_REF] Aminetzach | Pesticide Resistance via Transposition-Mediated Adaptive Gene Truncation in Drosophila[END_REF]. Unlike these adaptive TE insertions mutations, however, the response reported here relies on the ability of the retrotransposon ONSEN to respond to environmental threats. The ONSEN-containing allele of FLC is reminiscent of the retrotransposon KARMA within the gene MANTLED, the hypomethylation of which leads to splicing alterations and somaclonal variations in oil palm [START_REF] Ong-Abdullah | Loss of Karma Transposon Methylation Underlies the Mantled Somaclonal Variant of Oil Palm[END_REF].

Hypomethylation of KARMA occurs in response to repeated in vitro culture. Hence, environmentally-and artificially-induced hypomethylation of intronic TEs can be an important source of phenotypic changes in plants, with implications for adaptation and crop production.

FLC, and notably its first intron, is a common target of TE insertions in Arabidopsis and other Brassicaceae species [START_REF] Quadrana | The Contribution of Transposable Elements to Transcriptional Novelty in Plants: The FLC Affair[END_REF][START_REF] Li | Common Evolutionary Trajectory of Short Life-Cycle in Brassicaceae Ruderal Weeds[END_REF], which adds to the remarkable regulatory complexity of this gene. The large number of TE containing alleles of FLC likely reflects the major adaptive role of this gene, which we show can be catalyzed by complex mutations such as the insertion of environmentally-sensitive retrotransposon sequences. The insertion-mutation carried by Ag-0 lies close to the H3K27me3 nucleation region, a key sequence for vernalization response that plays a pivotal role in determining FLC's epigenetic state [START_REF] Yang | Antagonistic Roles for H3K36me3 and H3K27me3 in the Cold-Induced Epigenetic Switch at Arabidopsis FLC[END_REF]. If and how such TE insertion modulates the chromatin-mediated vernalization response of FLC warrants further investigation. Similarly, the large number of TE insertions within FLC offers a unique opportunity to explore how transposition can contribute to novel gene regulation mechanisms. Our findings reveal how retrotransposition can enable rapid response to environmental threads, with implications for our understanding of the genetic basis of past and future adaptations to anthropogenic activities.

mutant seeds and Fabien Nogué for sharing the pDE-Cas9-dsRed plasmid as well as advice on guide RNA design.

Materials and Methods

Samples and experimental models. The following A. thaliana plants were used: wild-type Ag-0, Ws-2, CIBC-17, and Lov-1 obtained from the NASC collection. The Col-0 ibm2-4 mutant was obtained from Deremetz et al 2019. Ibm2 Ag-0 plants were obtained by crossing Col-0 ibm2-4 with Ag-0. F2 plants were genotyped and ibm2-4 -/-, FLC ag-0/ag-0 , and FRI ag-0/ag-0 , as Col-0 has a week FRI allele that impairs FLC expression, were selected and AGCTGCCACCAGTGTTAACA) and candidate mutants validated by sanger sequencing. Two ddm1 mutant lines were identified in this way. Heat shock stresses were performed as previously described [START_REF] Quadrana | Transposition Favors the Generation of Large Effect Mutations That May Facilitate Rapid Adaption[END_REF]. Herbicide treatments were performed on two weeks old plants by spraying 10mg/ml Glufosinate once per week, during three weeks.

Unless stated otherwise, all plants were grown in long-days (16h:8h light:dark) at 23°C.

13

Vernalization treatments were performed by growing ten days old plants in short days at 6C for eight weeks.

De novo assembly and annotation of Ag-0. High-molecular weight genomic DNA of two weeks old Ag-0 plants was extracted as previously reported [START_REF] Mayjonade | Extraction of High-Molecular-Weight Genomic DNA for Long-Read Sequencing of Single Molecules[END_REF]) with minor modifications. Briefly, plant material was grinded under liquid nitrogen and 100mg of powder was incubated in 1 ml of lysis buffer (PVP40 1%, NaCl 500mM, Tris ph8 100mM, EDTA 50mM, SDS1.25%, Sodium bisulfite 1%, DRR5mM) during 30 minutes at 50 degrees.

DNA was cleaned using KOAc precipitations (1.3M, pH7.5) and DNA purified using 0.05X of seraMAG beads. ONT library (LSK1010) was prepared using 1 ug of HMW DNA and loaded in a R9.4 Minion flow-cell. Sequencing was performed in a Mk1C device during 72hs. Basecalling was performed using Guppy (V) with the HAC model. Long-read fastq files were used for de novo genome assembly using CANU [START_REF] Koren | Canu: Scalable and Accurate Long-Read Assembly via Adaptive -Mer Weighting and Repeat Separation[END_REF]. low error rate Illumina short reads from Ag-0 (1001 genomes project) were used for sequence polishing using pilon software [START_REF] Walker | Pilon: An Integrated Tool for Comprehensive Microbial Variant Detection and Genome Assembly Improvement[END_REF]. Chromosome scaffolding was achieved by running RagTag [START_REF] Walker | Pilon: An Integrated Tool for Comprehensive Microbial Variant Detection and Genome Assembly Improvement[END_REF][START_REF] Alonge | RaGOO: Fast and Accurate Reference-Guided Scaffolding of Draft Genomes[END_REF]) with default parameters. TEs and repetitive sequences were annotated using REPET [START_REF] Flutre | Considering Transposable Element Diversification in de Novo Annotation Approaches[END_REF]. Quantification of expression by RT-qPCR. RNA was extracted using the RNeasy plant mini kit (Qiagen) from plants. Contaminating DNA was removed using Turbo DNase (Invitrogen) following manufacturer instructions. 1ug of RNA was used for RT using SuperScriptIV (Invitrogen). RT-qPCR was performed using LightCycler 480 SYBR Green I Master mix (Roche) and run in a LightCycler 480 System (Roche). Primer sequences are provided in table S3. RT-qPCR expression levels relative to the PP2A housekeeping gene were calculated using the 2 -∆∆Ct method. DNA methylation of ONSEN was performed by

Mapping by sequencing of HS

McrBC-digestion followed by qPCR as described before and using the primers listed in table S3.

Methylome analysis. DNA from Ag-0 plants was extracted using a standard CTAB protocol. Bisulfite conversion, BS-seq libraries and sequencing (paired-end 100nt reads)

were performed by BGI Tech Solutions (Hong Kong). Bioinformatic analysis was performed as previously described [START_REF] Sasaki | Fast Co-Evolution of Anti-Silencing Systems Shapes the Invasiveness of Mu-like DNA Transposons in Eudicots[END_REF] knowing the presence/absence information. To investigate the presence of selective sweeps, the same re-sequencing data [START_REF] Frachon | A Genomic Map of Climate Adaptation in at a Micro-Geographic Scale[END_REF]) was aligned to the Arabidopsis reference genome TAIR10 using Bowtie2 with default parameters. Reads mapping to multiple genomic locations or duplicates were removed using samtools with parameter -q 5 and Picard MarkDuplicates with default parameters (REMOVE_ DUPLICATES = true), respectively. SNPs were called using bcftools mpileup and bcftools call commands. Variant calls with quality lower than 30 or covered by less than 10 reads were removed. Biallelic SNPs around the FLC locus were extracted using bcftools view -v snps -M 2 and phased using beagle (v5.2). Extended haplotype homozygosity (EHH) around the ONSEN insertion within FLC was calculated using selscan [START_REF] Szpiech | Selscan: An Efficient Multithreaded Program to Perform EHH-Based Scans for Positive Selection[END_REF] with the -ehh option.

16

  propagated till F3 for phenotypic and molecular characterization. Collections of seeds from Arabidopsis plants around Argentat were carried out in May 2019, August 2019, and April 2021. In order to break potential seed dormancy, seeds were kept at least four months at 15 degrees previous to germination. CRISPR-CAS9 ddm1 mutants of Ag-0 were generated by floral-dip transformation using the binary vector pDE-Cas9-dsRed following reported protocol[START_REF] Morineau | Selective Gene Dosage by CRISPR-Cas9 Genome Editing in Hexaploid Camelina Sativa[END_REF]. Briefly, two guide RNAs targeting the end of exon 6 //crispor.tefor.net/crispor.cgi) and synthesized by Invitrogen GeneArt (ThermoFisher) in tandem repeat, each preceded by a U6 promoter and separated by a tracrRNA (GTTTAAGAGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCC GTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT), and flanked by attB sites within a pDONR221 plasmid. The guide RNA construct was then ligated into the pDE-Cas9-dsRed vector by LR Gateway (ThermoFisher) by selection against ccdB. Transgenic T1 plants were identified by seed fluorescence followed by PCR genotyping of the transgenic cassette (primers: sgU6-F CGGTGCTTTTTTTGGATCC and DsRed-proR TGCTTCTTGCTGAGCTCACA), and then propagated in the heterozygous state (characterized by the production of both fluorescent and non-fluorescent seeds) for a second generation. Non-fluorescent offspring (G1) plants of the T2s were genotyped

  -induced flowering. Ag-0 plants were crossed with Ws-2 or CIBC-17, which show different degrees of vernalization response. Derived F1 plants were backcrossed. Approximately 100 F2 plants were germinated in solid medium (½ MS without sugar) and subjected to a 48h HS one week after germination. Plants were transferred to soil after one week of recovery. Flowering plants were sampled 56 days after germination (n=6 and n=20 for Ag-0 x Ws-2 and Ag-0 x CIBC17, respectively) and polled for DNA extraction by CTAB. DNA was also extracted from pooled leaves of F2 plants as control. One μg of total DNA was used to construct libraries and sequencing (paired-end 100nt reads) by BGI Tech Solutions (Hong Kong). Approximately 20M pair-end reads were obtained for each sample and aligned to the Ag-0 genome assembly using Bowtie2 with default parameters. Reads mapping to multiple locations or duplicates were removed using samtools with parameter -q 5 and Picard MarkDuplicates with default parameters (REMOVE_ DUPLICATES = true), respectively. SNPs were called using bcftools mpileup and 14 bcftools call commands. Variant calls with quality lower than 30 or covered by less than 10 reads were removed. Allele frequency of non-reference alleles were calculated and averaged through windows of 200Kbp, with 100Kbp steps. The difference between allele frequencies of HS-induced flowering F2 plants and control F2 plants were calculated and plotted genome wide. Windows with the highest 1% Δ(SNP index) were considered as significant.Transcriptomic analysis. RNA from Ag-0 plants was isolated using Rneasy Plant Minikit (Qiagen) according to the supplier's instructions. One μg of total RNA was used to construct directional libraries and sequencing (paired-end 100nt reads) by BGI Tech Solutions (Hong Kong). About 20M 100nt-long pair-end reads were obtained per sample. Expression level was calculated by mapping reads using STAR v2.5.3a63 on the Ag-0 reference genome with the following arguments --outFilterMultimapNmax 50 --outFilterMatchNmin 30 --alignSJoverhangMin 3 --alignIntronMax 10000. Duplicated pairs were removed using picard MarkDuplicates.

Figure 1 .

 1 Figure 1. A recent retrotransposon insertion within FLC modulates flowering response. a. Circos plot of Ag-0 genome assembly with full-length (violet) and truncated (yellow) ATCOPIA78/ONSEN insertions. The localization, minor allele frequency (MARF) across 1069 natural accessions, and estimated age of full-length insertions are indicated. b. Phylogenetic tree of full-length ONSEN insertions in Ag-0 and the reference genome Col-0. Bootstrap probabilities are shown beside the branches. c. Polymorphisms along the FLC haplotype block between accessions with related haplotypes. Ag-0 accession and location of the ONSEN insertion are indicated. Geographic distribution of accessions sharing the Ag-0 haplotype of FLC (Hap1) is shown. d. Boxplots of days to flowering of Ag-0 plants grown under control conditions (Ctl.), subjected to eight weeks of vernalization (8W Vern.), or subjected to a single, or repeated heat shock (HS) at the seedling stage. Timing of the HSs are indicated by red bars. Statistical significance for differences were obtained using a two-sided MWU test. e. Genome-wide Δ(SNP index) plot of F2 (Ag-0 x Ws-2 and Ag-0 x CIBC-17) bulk segregant analysis of flowering responses to heat-shock. Red line indicates the genome-wide 1% threshold. Localization of FLC locus is indicated.

Figure 2 .Figure 3 .Figure 4 .

 234 Figure 2. Heat-induced retrotransposon activation impairs FLC activity. a. Genome browser view of normalized RNA-seq coverage at FLC locus in Ag-0 plants seven days after a heat-shock (HS t7) or control treatment (Ctl. t7). Light blue and orange arrowheads represent the location of primers used to quantify FLC and ONSEN expression, respectively. b. Relative expression levels of ONSEN, FLC, SOC1, and FT in Ag-0 plants 14 days after a heat-shock (HS t14) or control treatment (Ctl. t14). Data are mean ± s.d. (n>10 samples, two biological experiments) and statistical significance for differences was obtained using the MWU test. c. Genome browser tracks showing local CHH hypomethylation of the 5' end of the ONSEN insertion (gray box) in response to heat-shock (HS t7).

Natural Arabidopsis populations from south-west of France.

  ). Paired-end reads were trimmed using To call presence/absence of the ONSEN insertion within FLC, Illumina re-sequencing data from[START_REF] Frachon | A Genomic Map of Climate Adaptation in at a Micro-Geographic Scale[END_REF] was aligned to 150bp sequence spanning each insertion extremity of the ONSEN insertion within FLC from the Ag-0 assembly, as well as to the empty insertion site of the reference Col-o genome (TAIR10). Reads mapping uniquely (qual greater than 24) and entirely (CIGAR string lacking any S) to each extremity and the empty insertion site were counted and compared to estimate the TE insertion allele frequency. Local environments at each

	"ILLUMINACLIP:TruSeq3-PE.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15
	MINLEN:36" (Bolger, Lohse, and Usadel 2014). Mapping of trimmed sequences to Ag-0
	assembled genome, removal of identical reads, and counting of methylated and
	unmethylated cytosines were performed by Bismark ver. 0.15.0 (Krueger and Andrews
	2011).
	Trimmomatic	program	(version	0.33)	with	following	parameters
				15

collection site were retrieved by inspecting satellite images and aerial pictures available at GEOportail (https://www.geoportail.gouv.fr/carte). This process was performed without

Acknowledgements:

We thank members of the Colot and Quadrana group for discussions and critical reading of the manuscript. We thank members of the Colot group, Mercedes Tkach, Hugo Vergne, and Claire Delamare Deboutteville for helping with the collection of Arabidopsis plants nearby Argentat. We also thank Nicolas Bouché for sharing ibm2-4

Funding: This work was supported by grants from the Centre National de la Recherche Scientifique (MOMENTUM program, to L.Q.) and the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (grant agreement No. 948674 to LQ). Pierre Baduel was supported in part by a postdoctoral fellowship from the Fondation pour La recherche Médicale.

Data and material availability: Original Scripts are available on GitHub. WGBS and RNA-seq of Ag-0 plants are available at the European Nucleotide Archive (ENA) under project XXXX.

Authors contributions: LQ conceived the project. MR performed most HS and herbicide experiments, RT-qPCRs, and generated Ag-0 ibm2 mutants, with additional help from LQ.

PB and VC generated Ag-0 ddm1 mutant lines. BL genotyped Arabidopsis accessions nearby Argentat, generated ONT sequencing data, assembled and annotated Ag-0 genome, with additional help from AE. FR contributed to the analysis of south-west French populations. LQ performed BS-seq, RNA-seq, QTL mapping, genomic and population genomic analyses, interpreted the data, and wrote the manuscript. All the authors read and approved the paper.

Competing interests:

The authors declare that they have no competing interests.