AIMS AND STRATEGIES RESULTS PART I: TRANSCRIPTOMIC DATA FOR CARPEL EVOLUTIONARY STUDIES Aims and strategies of part 1

Keywords: Amborella trichopoda, Arabidopsis thaliana, WGCNA, gene-clustering, orthogroup, angiosperm, flower, carpel SPATULA, ALCATRAZ, PHYTOCHROME INTERACTING FACTOR5, PHYTOCHROME INTERACTING FACTOR7, basic Helix-Loop-Helix, carpel, gynoecium, carpel margin meristem, flower, angiosperm, evolution, Arabidopsis, Amborella, Nymphaea, Petunia, Picea, Selaginella

A proof-of-concept using novel transcriptomic data from the early-diverging angiosperm Amborella trichopoda to identify genetic modules conserved over deep evolutionary time.

RESUME

La base moléculaire de l'évolution du carpelle.

Le carpelle est l'organe reproducteur femelle des plantes à fleurs. Nous présentons une analyse transcriptomique comparative, focalisant particulièrement sur le développement des tissus femelles reproducteurs, entre la plante modèle établie Arabidopsis thaliana et la soeur probable du restant des plantes à fleurs vivantes Amborella trichopoda. Des modules de co-expression de gènes ont été d'abord définis et ensuite comparés statistiquement entre espèces sur la base de relations d'orthologie entre tous les gènes dans les deux génomes sous comparaison. Cette étude a révélé des modules génétiques entiers, plutôt que seulement des gènes individuels, qui auraient conservé leurs patrons d'expression depuis le dernier ancêtre commun des plantes à fleurs. D'autres modules d'origine plus récente ont été également mis en évidence, modules qui ont peut-être contribué à une complexification dans la morphologie des carpelles qu'a eue lieu plus tardivement pendant l'évolution des plantes à fleurs. Le deuxième thème majeur de cette thèse concerne l'un des régulateurs de ce deuxième groupe, le facteur de transcription SPATULA (SPT) de la famille bHLH. Nous présentons des données suggérant que SPT aurait acquis son rôle actuel dans les tissus à l'apex du carpelle chez un ancêtre commun des Brassicacées. Nos données suggèrent également que l'acquisition de ce nouveau rôle dépendait de changements au niveau des séquences cis-régulatrices de SPT. Nous décrivons des expériences en cours qui ont pour objectif d'identifier les séquences d'ADN précises responsables pour ce changement évolutive.

ABSTRACT

The molecular basis of carpel evolution.

The carpel is the female reproductive organ of flowering plants. We present a comparative transcriptomic analysis, focusing in particular on the development of female reproductive tissues, between the established model plant Arabidopsis thaliana and the probable sister of all remaining living flowering plants Amborella trichopoda. Gene co-expression modules were first defined and then compared statistically between species on the basis of orthology relationships between all genes in the two genomes under comparison. This study revealed entire genetic modules, rather than just individual genes, that appear to have retained their expression patterns since the last common ancestor of living flowering plants. Other modules of more recent origin have also been brought to light, which may have contributed to a complexification in the morphology of carpels that occurred later during the evolution of flowering plants. The second major theme of this thesis concerns one of the regulators of this second group, the transcription factor SPATULA (SPT) of the bHLH family. We present data suggesting that SPT acquired its present role in the tissues at the apex of the carpel in a common ancestor of Brassicaceae. Our data also suggest that the acquisition of this new role depended on changes in the cis-regulatory sequences of SPT. We describe ongoing experiments that aim to identify the precise DNA sequences responsible for this evolutionary change.

ACKNOWLEDGMENTS

If you ever ask me what are my favorite colors, my answer will be: the blue, the green and the flowers. When I was a little girl, I used to collect flowers in my way home back from the school, they were a specific species, the Ruellia flowers. Once at home, I used to run to a large flower pot in the shape of a swan that my mom has at the front garden. There I inserted my collected flowers in a different plant that had no flower of its own. In my own way, I was doing plant cross, and got (to my eyes) a successful hybrid.

As I grew, I was always curious of what happened inside the plants, inside the trunks of the trees, and I used to imagine small cities inside them, with tiny little people that work at the leaves and branches, they were highways connecting one city to another, people were always busy, working to make the plant grow. It was my nine-yearold picture of plant regulatory development. And now, that I think about it, it wasn't that far from reality. Only, instead of people, there are genes and transcription factors, always busy, working in these little towns, called cellules, with plasmodesmata that connect one cell to another.

I fell completely and irreversibly in love with plants, in my first practical lecture of plant histology, in college. I could finally see what I had imagined for so long. Over microscopy observation my fascination with plants grew, and there I was combining histology, morphology, taxonomy, anatomy, physiology, cytology and genetics to understand my favorite living beings on earth.

What more could I ask for?

Well, what about me crossing the ocean, landing in France for full four years of a passionate plant biology PhD work? Let's live the dream! I still remember the first time I saw the RDP web site from Paraguay, there were sparkles on my eyes, I imagine myself working there! And now, here I am, at the end of this magical adventure in the Evo-Devo team, in which I had discovered a new dimension of third favorite color: the flowers. As the end approach, it comes the time to say thank you… The magic start with BECAL who put at my disposal the pot of gold coins to fly over the Atlantic and to SFERE for the guidance in my path. Also thank you to the ENS of Lyon for helping me to complete the gold needed to live my dreams.

My dreams were fueled by the thoughts and prayers of my family and friends. Mom and Dad, thank you, your crazy daughter is about to be a Doctor and I have not forgotten to eat healthy during these years. To my sisters Lily and Victoria, thank you for your support.

My path was made easier by the hand of my greatest loves, Hector and Cynthia. I am grateful to life for having given me their existence and their company. Magical times require powerful and enlightened beings like you to complete all the journeys. Gracias por su amor.

In this fairy tale, I got to this magical chapter called the PhD thesis, and I want to thank to all the other charmed characters that helped me in this incredible adventure. I shall start by thanking My Magic Goblin: Charlie Scutt, with his Irish flute he got me dancing around carpel origin and evolution, for the last four years. Thank you for your scientific passion, your patience, and thank you for showing me the path of the Abominable mystery of Charles Darwin. My First Master, Amelie Andres Robin, she transferred me my first bench-powers, I will always remember her lectures of how to hold a micropipette, how a PCR is made and how to do a miniprep. Thank you Amelie.

My Science Elf, Aurelie Vialette, I have an image engraved in my memory, of she asking me to sit at her desk and kindly explaining me how to organize mi scientific work. She was there for me at the beginning and at the end, but even when she moved toward new scientific horizons she was there enlightening me with her advices. Gracias.

My Fairy godmother, Pierre Chambrier, instead of a magic wand he has a P10 micropipette, with an unmatched power for molecular biology. Whenever I had a question, he had the answer or the way to find it. Thanks for your magic on the bench and on the dance floor. Merci.

The Wizard of the command lines, Jérémy Just, thanks for your time and your patience. Thank you for helping me understand new languages and guiding me in using them.

Many thanks to the rest of the enchanted beings that are part of my team, especially Michiel and Patrice who have closely contributed to this fairy tale. But I must also thank Suzanne, Marie, Lucie, Nelu and Mathilde, who throughout these years have accompanied this process in one way or another.

To my magical beings, GéGé, Judit, Isa and Hervé: Thank you for those chats at the paillasse, corridor and coffee, thank you for enchanting my working days with your kindness. With the most special mention for Isa and Hervé whose work is and was indispensable for my magical experiments.

To the Elves who have cared for my plants with the greatest of jealousy, Alexis, Patrice and Justin, THANK YOU.

To my Elves from the 017 of M3, Priscilla and Antoine, thank you for your company, for our talks, cups of tea and your constant encouragement.

Virginie, the Fairy of the most ordered protocols of RDP. Thanks for the talks, help and advice during the experiments and for your magical protocols. Merci du fond de mon Coeur! To the companions and friends of the enchanted village of the Afterwork and the stairs, Sana, Raquel, Yannik, Jack, Adrien, Julie, Juan, Julie and Justine. Thank you, it has been a pleasure to share with you our dishes, joys and sorrows.

Raquel, thank you for being a person of light and giving me your support and your friendship. Voy a extrañar las patatas entre clonaciones, las risas y los bailes al son de los Decadentes en la poyata. ¡GRACIAS por todo guapa! Finally, thanks to all the RDP members, who make the lab a pleasant place to work. MERCI tout le monde! It is time for me, to undertake new adventures, to search for other treasures, to explore other horizons. I will keep in mind all the people who have surrounded me at this point, but today I want to say: thank you, merci, gracias, aguije.

TABLE OF CONTENTS

GENERAL INTRODUCTION

The origin of angiosperms: when and where did they appear?

What is a flower? -The modern flower and its components.

What did the first flower look like, and what were the possible ancestors of angiosperms?

A closer look at female reproductive tissues.

The ancestral carpel and its origin.

Molecular mechanism that control the floral development in extant angiosperms

The genetic regulation of female reproductive tissues in Arabidopsis thaliana

The Gene Regulatory Network of the ancestral carpel Non-model species for studies of the origin of the flowers Techniques and methods that could help to explain the origin of the flower Unsolved questions of flower origin and evolution

Introduction

Results

Discussion

Materials and Methods

GENERAL INTRODUCTION

Introduction

The origin of angiosperms: when and where did they appear?

Angiosperms, or flowering plants, are the main component of most terrestrial ecosystems. They represent more than 95% of the vascular plants existing today [START_REF] Mandel | A Jurassic leap for flowering plants[END_REF]. Their morphological, functional and ecological diversity have made angiosperms the dominant group of living plants, with more than 290,000 species described [START_REF] Ramírez-Barahona | The delayed and geographically heterogeneous diversification of flowering plant families[END_REF]. Flowering plants are critical components of aquatic and terrestrial habitats. This clear dominance as a group was established in the Lower Cretaceous, in which the fossil record clearly documents the rapid expansion and diversification of angiosperms.

Nevertheless, there are controversies regarding the dating of the origin of flowering plants. Research based on molecular clock analyses place the origin of angiosperms between 256 and 139 million years ago (MYA) (Figure 1), in which the earlier spectrum of dates corresponds to the Jurassic or even the Triassic [START_REF] Li | Origin of angiosperms and the puzzle of the Jurassic gap[END_REF][START_REF] Salomo | The Emergence of Earliest Angiosperms may be Earlier than Fossil Evidence Indicates[END_REF]. However, the earliest known fossils with certain affinities to angiosperms date from the early Cretaceous, between 135 and 125 MYA. These fossils are fossilized pollen grains which have been found in or around regions corresponding to the northern parts of the ancient supercontinent of Gondwana (Figure 2). However, as mentioned above, the dates of these fossils are not very consistent with that of the most-recent common ancestor (MRCA) of living angiosperms, as determined by molecular-clock based studies, the most recent of which gives a date of 209 MYA [START_REF] Li | Origin of angiosperms and the puzzle of the Jurassic gap[END_REF].

There is therefore a gap of perhaps about 70 million years (MY) between the earliest known fossil angiosperms and dates obtained using molecular analyses, and this has been called the "Jurassic Gap" [START_REF] Li | Origin of angiosperms and the puzzle of the Jurassic gap[END_REF][START_REF] Mandel | A Jurassic leap for flowering plants[END_REF]. The age of the flowering plant group has been widely discussed over the years, from early studies in the 1960s to the present, but no real consensus has been reached [START_REF] Coiro | How deep is the conflict between molecular and fossil evidence on the age of angiosperms?[END_REF][START_REF] Herendeen | Palaeobotanical redux: Revisiting the age of the angiosperms[END_REF]. For some authors, the sudden rapid diversification of angiosperms in the lower Cretaceous (Figure 1) is an indication of their origin and radiation in earlier periods [START_REF] Herendeen | Palaeobotanical redux: Revisiting the age of the angiosperms[END_REF][START_REF] Ramírez-Barahona | The delayed and geographically heterogeneous diversification of flowering plant families[END_REF][START_REF] Smith | An uncorrelated relaxed-clock analysis suggests an earlier origin for flowering plants[END_REF].

Pollen-wall morphology comprises one of the most useful sets of characters to reconstruct the early evolution of flowering plants. The pollen-fossil record of angiosperms during the Cretaceous period is continuous, while reports of angiosperm pollen prior to this period are very sporadic and highly contested in their possible relationship to angiosperms [START_REF] Herendeen | Palaeobotanical redux: Revisiting the age of the angiosperms[END_REF]. Recent studies support the hypothesis of an early origin of angiosperms, followed by a long period of persistence, before their rapid diversification [START_REF] Li | Origin of angiosperms and the puzzle of the Jurassic gap[END_REF][START_REF] Ramírez-Barahona | The delayed and geographically heterogeneous diversification of flowering plant families[END_REF]. This greater diversification is located between the middle of the Cretaceous and the end of this period, giving rise to most of the existing lineages [START_REF] Vea | Putting scales into evolutionary time: The divergence of major scale insect lineages (Hemiptera) predates the radiation of modern angiosperm hosts[END_REF]. The correspondence of this major expansion of angiosperms with the diversification of insects and other pollinators has been extensively studied, but in addition to this factor, favorable ecological conditions and substantial morphological changes in the first species of flowering plants can be added [START_REF] De Boer | A critical transition in leaf evolution facilitated the Cretaceous angiosperm revolution[END_REF][START_REF] Ramírez-Barahona | The delayed and geographically heterogeneous diversification of flowering plant families[END_REF].

Three hypotheses have been proposed by [START_REF] Mandel | A Jurassic leap for flowering plants[END_REF], to try to explain the "Jurassic Gap" in the fossil record. The first of these suggests that early angiosperms were rare, and that their fossils are therefore difficult to find and identify. The second hypothesis postulates an environment for early angiosperms that was not very conducive to the preservation of plant fossils. The third hypothesis suggested by [START_REF] Mandel | A Jurassic leap for flowering plants[END_REF] points to the anatomy of the first angiosperms, which might have had very small flowers and fruits, complicating their identification as angiosperms. The closest outgroup to the flowering plants is the remaining seed plants, or living gymnosperms. The separation of the living angiosperm and gymnosperm clades has been dated to around 310 MYA (Figure 1), which places a logical upper limit on the date of the MRCA of living angiosperms.

Another point of discussion concerns the geographical origin of angiosperms. The earliest known angiosperm fossils are monoaperturate pollen grains from strata dating from 139-130 MYA, and have been found at locations corresponding to present-day parts of North and Central Africa, the Middle-East, Western Europe and South America [START_REF] Coiro | How deep is the conflict between molecular and fossil evidence on the age of angiosperms?[END_REF]. Many of these pollen types can be ascribed to particular groups of angiosperms within the living angiosperm clade, and must therefore have originated later than the MRCA of all living angiosperms. The earliest known fossil pollen grains may, therefore, represent an early expansion of angiosperms from their original habitat, rather than the first populations of angiosperms to have existed. Later strata at these same locations, and strata at many other locations extending out of the palaeotropics and into temperate regions, contain the tricolpate pollen (containing three elongated apertures) characteristic of the more recent eudicot group of angiosperms, which seems to have appeared 10-15 MY after the earliest known angiosperm pollen types.

Much of northern Gondwana in the early Cretaceous had a hot and dry climate, and early angiosperm pollen from Congo, Gabon and Brazil have typically been found in association with pollen of xerophytic groups including Cheirolepidiaceae (extinct conifers) and Gnetales (James A. Doyle et al., n.d.)(J. A. [START_REF] Doyle | The rise of angiosperms as seen in the African Cretaceous pollen record[END_REF]. However, a band of more humid paleohabitats is known, running from present-day Israel and Egypt to Colombia and Peru [START_REF] Carvalho | Late Aptian (Cretaceous) climate changes in northeastern Brazil: A reconstruction based on indicator species analysis (IndVal)[END_REF]. These regions show evidence of early angiosperm pollen together with pollen and spores of plants typical of humid habitats, including Matoniaceae (ferns) and Araucariaceae (conifers). Therefore, the angiosperms may have originally expanded from humid habitats of equatorial northern Gondwana, first into drier habitats in the surrounding territories, and then within the next 10-15 MY out of northern Gondwana and into temperate zones of both the northern and southern hemispheres. This migration was accompanied by a series of innovations of floral structures in the emerging clades of angiosperms, increasing interactions with pollinating and seed-dispersing agents, and favorable ecological conditions [START_REF] Endress | Evolutionary diversification of the flowers in angiosperms[END_REF][START_REF] Li | Origin of angiosperms and the puzzle of the Jurassic gap[END_REF]. The angiosperms finally came to dominate terrestrial ecosystems in the Paleocene period, which began 66 MYA [START_REF] Ramírez-Barahona | The delayed and geographically heterogeneous diversification of flowering plant families[END_REF].

From the evolutionary point of view, some of the synapomorphic characters that define angiosperms as a group are: the presence of the carpel (and of the fruit, which is derived from the carpel), ovules with two integuments (except in taxon-specific cases), bisexuality of the reproductive axis, the presence of 4-loculate stamens and double fertilization. In addition to reproductive characters, ecophysiological and vegetative characters particular to flowering plants can also be listed, such as the presence of vessels in the xylem, net-veined leaves and the physiological adaptation to the atmosphere with decreasing concentrations of carbon dioxide, as reviewed by Charles P. Scutt, (2018).

Two large groups of flowering plants can be recognized, the so-called ANA-grade and the mesangiosperms (Figure 1) [START_REF] Group | An update of the Angiosperm Phylogeny Group classification for the orders and families of flowering plants: APG III[END_REF][START_REF] Group | An update of the Angiosperm Phylogeny Group classification for the orders and families of flowering plants: APG IV[END_REF]. Recognized as the most basal group of flowering plants, the ANA-grade is made up of the three orders Amborellales, Nymphaeales and Austrobaileyales, among which Amborella trichopoda, the only living species of the order Amborellales, is considered the single sister species of all other living angiosperms [START_REF] Project | The Amborella Genome and the Evolution of Flowering Plants[END_REF].

Mesangiosperms, by contrast, include more than 99.95% of flowering plants, and are divided into four main clades: magnoliids (including Chloranthaceae), monocots, Ceratophyllales and eudicots (Figure 1) [START_REF] Frohlich | After a dozen years of progress the origin of angiosperms is still a great mystery[END_REF][START_REF] Group | An update of the Angiosperm Phylogeny Group classification for the orders and families of flowering plants: APG III[END_REF][START_REF] Group | An update of the Angiosperm Phylogeny Group classification for the orders and families of flowering plants: APG IV[END_REF][START_REF] Zeng | Resolution of deep angiosperm phylogeny using conserved nuclear genes and estimates of early divergence times[END_REF].

What is a flower? -The modern flower and its components.

As mentioned above, most of the synapomorphic characters of angiosperms are reproductive characters, which are grouped in the structure called the flower. The flower can be described as a compact reproductive axis, with lateral organs arising in close juxtaposition. Floral organs can be arranged in a spiral phyllotaxy leading to a variable number of organs. However, most flowering plants present relatively fixed numbers and positions of floral organs arranged in whorls. Typically, a

bisexual flower has a central gynoecium formed by carpels, surrounded by the androecium of stamens that produce the pollen grains (Figure 3). However, there are also angiosperm species bearing unisexual flowers, in which only the gynoecium or androecium develops.

Enclosing the reproductive organs in a typical flower is the perianth, composed of sterile bract-like organs. The perianth is frequently divided into an inner whorl of petals, which may participate in interactions with pollinators, and an outer whorl of sepals, which may function mainly to protect the floral bud (Figure 3). Also, in some cases, the perianth may be undifferentiated, in which case its organs are called tepals, and these can be either petals-like or sepal-like.

What did the first flower look like, and what were the possible ancestors of angiosperms?

As the main character that differentiated angiosperms from their precursors, the gymnosperms, a large part of the research effort in plant evolution has been devoted to the reconstruction of the ancestral flower. Over the last few decades, a series of techniques and methods have been developed and combined in order to attempt to reconstruct this structure. These methods include the comparative study of fossils and the morphological and molecular phylogeny of living angiosperms. Sophisticated statistical models can now be applied to the data obtained from comparative studies to reconstruct the flower of the MRCA of flowering plants [START_REF] De-Paula | Unbuttoning the Ancestral Flower of Angiosperms[END_REF][START_REF] Rümpler | Reconstructing the ancestral flower of extant angiosperms: The 'war of the whorls' is heating up[END_REF][START_REF] Sauquet | The ancestral flower of angiosperms and its early diversification[END_REF][START_REF] Sokoloff | Was the ancestral angiosperm flower whorled throughout?[END_REF].

Since the beginning of the 21st century, botanical experts have tested proposals for the possible structure of the ancestral flower. At the end of the 1990's, the identification of the ANA-grade group greatly facilitated the reconstruction of the MRCA of angiosperms [START_REF] Rümpler | Reconstructing the ancestral flower of extant angiosperms: The 'war of the whorls' is heating up[END_REF]. In the following years, several attempts were made, in most of the cases using phylogenetic analyses based on the principle of parsimony (James A. [START_REF] Doyle | Integrating Molecular Phylogenetic and Paleobotanical Evidence on Origin of the Flower[END_REF][START_REF] Doyle | Molecular and Fossil Evidence on the Origin of Angiosperms[END_REF][START_REF] Endress | Floral phyllotaxis in basal angiosperms: Development and evolution[END_REF][START_REF] Group | An update of the Angiosperm Phylogeny Group classification for the orders and families of flowering plants: APG III[END_REF], 2015). The development of new methods, and the availability of new fossil and molecular data, has further facilitated these reconstructions.

In the reconstruction presented by [START_REF] Sauquet | The ancestral flower of angiosperms and its early diversification[END_REF] (Figure 4. A and B), the ancestral flower is presented as a bisexual and actinomorphic flower, with a superior gynoecium composed of five free carpels with spiral insertion. Six or more stamens are present, arranged in two whorls, and surrounded by a whorled perianth of tepals, arranged in trimers. This model has, however, been criticized because of a switch it proposes in floral organ phyllotaxy between the androecium and gynoecium: such a change in is not found in extant angiosperms [START_REF] Sokoloff | Was the ancestral angiosperm flower whorled throughout?[END_REF]. Also, the reconstructions of [START_REF] Sauquet | The ancestral flower of angiosperms and its early diversification[END_REF] assessed character states independently from each other, and so may have produced a list of characters that were mutually incompatible [START_REF] De-Paula | Unbuttoning the Ancestral Flower of Angiosperms[END_REF]. [START_REF] Reyes | Testing the impact of morphological rate heterogeneity on ancestral state reconstruction of five floral traits in angiosperms[END_REF], postulated that the high heterogeneity of extant flowers may have an impact in the reconstruction of ancestral states of characters, and that this effect is mostly observed in perianth organs. Despite these various caviats, in may be concluded, from a synthesis of studies, that flowers in the MRCA living angiosperms were probably small, protogynous and bisexual. Their gynoecium was most likely superior and composed of five or more carpels. These flowers probably had an undifferentiated perianth of tepals, it is possible that the androecium and the perianth were arranged in two or more trimerous whorls. The first flowering plant were probably insect-pollinated, and provided pollen as a reward.

Another aspect of the origin of angiosperms to be addressed concerns the gymnosperm group from which the angiosperms emerged. The patchy nature of the relevant fossil record, and the absence of close living relatives of the angiosperms, have been major aspects contributing to this problem. In the "anthophyte hypothesis", the results of morphological cladistic analyses had suggested the gymnosperm groups closest to angiosperms were living Gnetales and mesozoic (extinct) Bennettitales.

However, with the inclusion of molecular analyses from all major groups of living seed plants, this hypothesis (at least regarding Gnetales) was largely refuted (James A. [START_REF] Doyle | Molecular and Fossil Evidence on the Origin of Angiosperms[END_REF].

Morphological data from both living and extant group have, however, permitted the tentative placement of extinct gymnosperms on a molecular phylogenetic backbone of living seed plants (Figure 5) (James A. [START_REF] Doyle | Integrating Molecular Phylogenetic and Paleobotanical Evidence on Origin of the Flower[END_REF][START_REF] Doyle | Molecular and Fossil Evidence on the Origin of Angiosperms[END_REF]. From these studies, the two extinct taxa shown to be the closest possible angiosperm stem-lineage relatives are Caytoniales and Bennettitales (Figure 5).

Caytonia was the first member of Caytoniales to be discovered. Caytonia fossils represents female reproductive structures, while the pollen-producing structures of Caytonanthus and the leaves of Sagenopteris may correspond to the same species.

Caytonia consists of an axis bearing laminate cupules, each of which contains multiple ovules. These cupules are folded from the tip to the base and could be compared to the ovule-containing outer integument in angiosperms. However, homology between structures in Caytonia and the angiosperm carpel is more difficult to define (Figure 5) (James A. [START_REF] Doyle | Integrating Molecular Phylogenetic and Paleobotanical Evidence on Origin of the Flower[END_REF]. Some interpretations propose that the angiosperm carpel derives from the female rachis in a Caytonia-like ancestor. However, Bennettitales, which have no outer integument or carpel-like organs are also considered strong candidates for a close relationship to angiosperms. Bennettitales present the angiosperm-like features of net-veined leaves, a bisexual reproductive axis (in some species), nonsaccate pollen grains, and a perianth-like, which gives the reproductive axis a flower-like appearance (Figure 5). Another shared character with angiosperms is the capacity of Bennettitales to synthesize oleananes, which are terpenoid compounds, which are so stable they can persist in fossils [START_REF] Taylor | Biogeochemical evidence for the presence of the angiosperm molecular fossil oleanane in Paleozoic and Mesozoic non-angiospermous fossils[END_REF].

A closer look at female reproductive tissues.

Moving on from the origin, appearance and ancestors of the first flowering plants, I will focus in this work mainly on the evolution of the female part of the flower. Situated at the center of the typical flower, the gynoecium is composed of one or several carpels. The carpel has probably conferred on angiosperms several major advantages in the process of sexual reproduction. These functions include the mechanical and biochemical protection of the ovules, the reception of pollen grains, and pollentube guidance. Carpel tissues also act as a selective barrier to fertilization. After fertilization, the wall of the ovary within the carpel develops further to form the fruit, which protects the seeds and then facilitates their dispersion.

The ancestral carpel and its origin.

The gynoecium of the flower in the MRCA of living angiosperms was most probably composed of separate, ascidiate (bottle-shaped) carpels. The carpel apex was probably not completely fused by cellular structures, but instead had a restricted entrance in the form of an aperture or canal. From this canal, substances would have been secreted to close the apex of the carpel and facilitate the entry of pollen tubes [START_REF] Endress | Ancestral traits and specializations in the flowers of the basal grade of living angiosperms[END_REF].

The stigmatic surfaces of the ancestral carpel were probably covered by multicellular striations that may have been physically in contact among adjacent carpels, forming an intragynoecial compitum to allow the exchange of pollen tubes [START_REF] Endress | Ancestral traits and specializations in the flowers of the basal grade of living angiosperms[END_REF]. The ancestral carpel probably enclosed a single pendant ovule, or a small number of such ovules. These ovules were likely bitegmic and contained, in addition to the female gametophyte, a large nucellus, or female-derived storage tissue. The fruits of the MRCA of living angiosperms were probably indehiscent [START_REF] Becker | A molecular update on the origin of the carpel[END_REF][START_REF] Endress | Ancestral traits and specializations in the flowers of the basal grade of living angiosperms[END_REF].

To get an idea of the appearance of the ancestral carpel, it may be useful to look at ANA-grade carpels, such as those of Trimenia (Austrobaileyales), which conserve most of the inferred features of the ancestral carpel (Figure 4. C).

The carpel is considered as a modified leaf that has closed around the ovules, but this structure is probably more directly related to a sporophyll: a specialized leaf bearing one or more sporangia. If the carpel is considered as related to the (female) megasporophyll (though this is not the case in at least one hypothesis by M. W. [START_REF] Frohlich | An evolutionary scenario for the origin of flowers[END_REF], then the female cone scales of conifers, for example, would be considered homologous to the carpel. Depending on which fossil candidate is taken as the possible ancestor of flowering plants, different possible anatomical precursors to the carpel can be recognized. [START_REF] Doyle | Integrating Molecular Phylogenetic and Paleobotanical Evidence on Origin of the Flower[END_REF](Figure 6) identified three groups as possible close stem-relatives of angiosperms, and these are related to three different evolutionary scenarios for the origin of the carpel and outer integument of angiosperms. In the first of these scenarios, Caytonia-like gymnosperms are considered as potential ancestors of angiosperms, and in this case the outer integument would have evolved from the cupule by a reduction in ovule-number to one, leaving a single ovule now enclosed in two tissue layers, corresponding to the two integuments of angiosperms. If Caytonia-like gymnosperms were ancestral to angiosperms, it has been hypothesized that the female rachis then expanded and folded around the bitegmic ovules to form the carpel.

In a second evolutionary scenario, a glossopterid-type of female reproductive axis is considered as a possible progenitor to the outer integument and carpel of angiosperms. In this scenario, the cupule became the outer integument, as in the first scenario above, relating to Caytonia. However, in the case of glossopterids, the cupules emerge from a subtending bract, and it this bract that is considered to have possibly closed around the cupule/bitegmic ovule to form the carpel. One difficulty for this scenario relates to the date of the extinction of glossopterids, which may have preceded the origin of flowering plants (James A. [START_REF] Doyle | Integrating Molecular Phylogenetic and Paleobotanical Evidence on Origin of the Flower[END_REF]. Indeed, glossopterids have also been suggested as possible ancestors of Caytoniales (including Caytonia). However, as there is still much uncertainly in the date of origin of angiosperms (as discussed above), glossopterids remain contenders as possible angiosperm ancestors. The third alternative scenario, based on the analyses of [START_REF] Doyle | Integrating Molecular Phylogenetic and Paleobotanical Evidence on Origin of the Flower[END_REF], postulates that Bennettitales were the ancestors of angiosperms. These plants have megasporophylls attached to a central rachis, which might be homologous to the carpel, though they have no cupule-like organs of possible homology to the angiosperm outer integument (Figure 6).

Molecular mechanism that control the floral development in extant angiosperms

Despite its great morphological diversity, the flower is, in general, composed of four types of organs arranged in whorls. Floral-organ identity is specified by homeotic genes which function in a genetic model termed the ABC model [START_REF] Coen | THE WAR OF THE WHORLS -GENETIC INTERACTIONS CONTROLLING FLOWER DEVELOPMENT[END_REF]. This model was extended to the ABCDE model, which notably incorporates a further class of genetic component, the E-class, which was found to act together with the other floral homeotic genes (Figure 7) [START_REF] Pelaz | B and C floral organ identity functions require SEPALLATA MADS-box genes[END_REF][START_REF] Theissen | Plant biology-Floral quartets[END_REF]. In the ABC model, A-class genes are required to specify sepal identity, A and B-class genes together establish petal identity, B and C-class genes together establish stamen identity, and C-class genes specify carpel identity. The E-class are genes necessary to support the activities of the A, B and C class genes (Figure 7), while D-class genes were originally identified as necessary for specifying ovule development [START_REF] Colombo | THE PETUNIA MADS BOX GENE FBP11 DETERMINES OVULE IDENTITY[END_REF]. However, D-class genes are closely related to C-class genes and may have overlapping activities with these, depending on the species analyzed. [START_REF] Soltis | The ABC Model and its Applicability to Basal Angiosperms[END_REF][START_REF] Thomson | Chapter Eight-Molecular regulation of flower development[END_REF].

Almost all homeotic genes of the ABCDE model are MIKC-type MADS-domain transcription factors, which contain four conserved domains, among which the MADS (M) domain is the most highly conserved region and is associated with DNA-binding activity, dimerization and nuclear localization [START_REF] Theißen | MADS-domain transcription factors and the floral quartet model of flower development: Linking plant development and evolution[END_REF][START_REF] Thomson | Chapter Eight-Molecular regulation of flower development[END_REF]. In Arabidopsis thaliana, the A-class genes are APETALA1 (AP1) and APETALA2 (AP2), the B-class genes are APETALA3 (AP3) and PISTILLATA (PI), the C-class gene is AGAMOUS (AG), the D-class genes are SEEDSTICK (STK), SHATTERPROOF1 and 2 (SHP1, SHP2), and finally SPETALLATA 1-4 (SEP1-4) encode E-class genes [START_REF] Becker | Arabidopsis flower development-Of protein complexes, targets, and transport[END_REF]. Of the above-listed genes, only AP2 is not a member of the MADSbox family, but is instead the founder member of the AP2-domain family of transcription factors [START_REF] Okamuro | The AP2 domain of APETALA2 defines a large new family of DNA binding proteins in Arabidopsis[END_REF].

The floral quartet model explains how the homeotic genes of the ABCDE model interact with downstream target genes to control the formation of different floral organs (Figure 7), as reviewed by [START_REF] Thomson | Chapter Eight-Molecular regulation of flower development[END_REF]. In addition to regulating diverse downstream targets, genes of the ABDCE model also establish an extensive genetic cross talk and regulate their own expression [START_REF] Becker | Arabidopsis flower development-Of protein complexes, targets, and transport[END_REF].

The ABCDE model has only been directly investigated in a few model angiosperms [START_REF] Becker | Arabidopsis flower development-Of protein complexes, targets, and transport[END_REF][START_REF] Litt | The ABC model and the diversification of floral organ identity[END_REF]. However, in-situ hybridization assays, protein-DNA interaction data, and functional analyses suggest conserved mechanism of class B and C genes between extant gymnosperm species and flowering plants [START_REF] Moyroud | A link between LEAFY and B-gene homologues in Welwitschia mirabilis sheds light on ancestral mechanisms prefiguring floral development[END_REF][START_REF] Wang | Molecular interactions of orthologues of floral homeotic proteins from the gymnosperm Gnetum gnemon provide a clue to the evolutionary origin of 'floral quartets[END_REF][START_REF] Winter | On the origin of class B floral homeotic genes: Functional substitution and dominant inhibition in Arabidopsis by expression of an orthologue from the gymnosperm Gnetum[END_REF][START_REF] Zeng | Resolution of deep angiosperm phylogeny using conserved nuclear genes and estimates of early divergence times[END_REF].

The genetic regulation of female reproductive tissues in Arabidopsis thaliana

Evolutionary changes within the clades of MADS-box genes of the ABCDE model may have been a

key step for the origin of the flower, and of the carpels within it [START_REF] Litt | The ABC model and the diversification of floral organ identity[END_REF] AG interacts with SEPALLATA1-4 proteins (SEP1-4) to specify carpel identity (Pfannebecker et al., 2017a). After carpel identity is determined by AG expression, several genes of several classes, most of which encode transcription factors, act to regulates the developmental program of the A thaliana gynoecium. A meristematic zone appears at the margins of the carpel tissues to produce new tissues in the medial region of the gynoecium. This meristem is termed the Carpel Margin Meristem (CMM) [START_REF] Reyes-Olalde | Inside the gynoecium: At the carpel margin[END_REF]. Tissues that are very important for sexual reproduction derive from this meristem [START_REF] Reyes-Olalde | Synergistic relationship between auxin and cytokinin in the ovary and the participation of the transcription factor SPATULA[END_REF].

Besides the genes involved in gynoecium development, phytohormones also play an essential role.

These include auxin and cytokinin, which together promote the development medial domain of the gynoecium [START_REF] Reyes-Olalde | Synergistic relationship between auxin and cytokinin in the ovary and the participation of the transcription factor SPATULA[END_REF]. Mainly following the recent review of [START_REF] Zúñiga-Mayo | Gynoecium development: Networks in Arabidopsis and beyond[END_REF], I present below a summary of the regulation of gynoecium development in A. thaliana.

From Stages 3-5, AG expression is necessary to determine gynoecium/carpel identity and the termination of the FM. AG coordinates both of these processes through the regulation of crosstalk between cytokinin and auxin, repressing WUS both directly and indirectly through KNUCKLES (KNU). At Stage 5, there is a peak of cytokinin at the apex of the gynoecium, while auxin is concentrated in two lateral foci in the apical cells. These patterns of hormone distribution are coordinated by the action of the flavin monooxigenases YUCCA1 and 4 (YUC1/4), and of the auxin efflux carriers PINOID1, 3 and 7 (PIN1, 3 and 7). Active repression of cytokinin also occurs through ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN6 (AHP6) (Figure 8). 8). This is a very quick and general review of early gynoecium development, as there are many other genes that interact in the control of A thaliana carpel development For example, more than 80 genes are believed to regulate the development of the CMM [START_REF] Reyes-Olalde | Inside the gynoecium: At the carpel margin[END_REF]. It is important to highlight that Arabidopsis does not necessarily typify the gynoecium regulatory network of all angiosperms. Indeed, the carpel regulatory networks of other core eudicots, such as Solanaceae, shows important differences to that of A thaliana [START_REF] Ortiz-Ramírez | Evolution of genes associated with gynoecium patterning and fruit development in Solanaceae[END_REF].

The Gene Regulatory Network of the ancestral carpel

The A thaliana gynoecium is highly complex and contains two fused carpels, a dry stigma, a false septum and transmitting tract. ANA-grade species, representing the basal-most living angiosperms, mostly have unfused ascidiate carpels, and no transmitting tract or false septum. The ancestral carpel described above, similar to those of present-day basal angiosperms, may have had a limited GRN when compared with present-day A thaliana, partly because of the absence in the former of several specialized tissues described in the latter (Pfannebecker et al., 2017b).

Through the phylogenetic reconstruction of transcription factors (Pfannebecker et al., 2017a(Pfannebecker et al., , 2017b) ) have estimated the age of gene families involved in carpel development. According to these authors, lineages including AG and SEP were already present in the MRCA of seed plants, whereas other regulators were already present in the MRCA of all land plants. The lineages encoding the bHLH transcription factors SPT and HEC, present also in living gymnosperms, were probably therefore present in the MRCA of living seed plants.

Becker A (2020) reviewed carpel development regulators, and presented a list of genes that were most probably active in the carpel regulatory network in the MRCA of flowering plants (Figure 9). The above description represents a simplified regulatory network of the ancestral carpel. To improve our knowledge in this domain, it will be necessary to include data from more species that are strategically placed across seed plant evolution. The next section of this introductory chapter, therefore, concerns species which occupy key phylogenetic positions for the reconstruction of carpel development mechanisms from the earliest stages of angiosperm evolution.

Non-model species for studies of the origin of the flowers

In addition to the work carried out on model species, taxa from strategically-placed groups should be included in studies aimed at elucidating the origin of the carpel. In particular, gymnosperms and basal angiosperms could help to elucidate the genetic regulation of reproductive tissues in the MRCA of flowering plants by comparing the molecular networks that control reproductive tissue development in these two groups.

Scutt, CP. 2019, has made a summary of the requirements for potential new model species to study early angiosperm evolution. Such models should be amenable to standard molecular biology procedures and functional genetic approaches, and have a genome size that facilitates sequencing and assembly tasks. Further requirements include easy access to biological material at all developmental stages for laboratory work, small size at maturity, self-fertility, a short generation time, and the production of copious amounts of seed. Below, I summarize the features of groups of model and nonmodel species that are strong candidates in the light of the above-listed requirements.

Gymnosperms. As the closest living relatives of the angiosperms, the gymnosperms form an However, most of the data currently available from gymnosperms are from a few species of high economic interest such as the conifers Picea abies and Pinus taeda, and do not cover the whole range of living gymnosperms. Another problem in working with gymnosperm species concerns sampling, including the slow growth of many gymnosperm reproductive tissues, and difficulties accessing cones that develop at tens of meters from the ground among dense and fragile branches. Also gymnosperms tend to have very large genomes, which complicates genome sequencing and assembly. However, some of these problems may be solved, notably with the emergence of new methods and decreasing costs of sequencing, and the use of gymnosperms that have either shorter developmental cycles like

Welwitschia mirabilis and Ephedra spp, or which produce cones at accessible levels, like the acrocona mutant of P. abies.

ANA-grade angiosperms:

The orders Amborellales, Nymphaeales and Austrobaileyales form the three most basal lineages of flowering plants, termed the ANA grade. Amborellales contains the single living species Amborella trichopoda, considered as probable sister to all other extant angiosperms. The second earliest diverging ANA-grade order Nymphaeales regroups three families: Nymphaeaceae (water lilies, with a cosmopolitan distribution), Cabombaceae and Hydatellaceae (aquatic and semiaquatic herbs with more restricted distributions). The Austrobaileyales order is the probable third earliest-diverging order of the ANA grade, and includes approximately 100 woody plant species in three families.

Amborella trichopoda (Figure 10. A and B) is a key taxon for reconstructions of the GRN in early flowers, due to both its unique phylogenetic position and the absence of any whole-genome duplications in its lineage since the MRCA of living angiosperms. The genome of Amborella is therefore also an important reference to study genome evolution among angiosperms. A trichopoda is an understorey shrub, endemic to sub-tropical cloud forests of New Caledonia. It is a dioecious species, whose flowers have an undifferentiated perianth of tepals. Amborella female flowers have free, ascidiate carpels, each of which produces a secretion that fills an apical canal to provide a route for pollen-tube growth. Each carpel also has a large stigmatic surface for pollen contact. Inside each Amborella carpel is a single, pendent, bitegmic ovule. The Amborella ovule possesses a unique eightcelled embryo sac that, after double fertilization, generates a diploid zygote and triploid endosperm.

Morphological analyses of Amborella have helped establish a list of plesiomorphic features in angiosperms. Interestingly, some differences in Amborella compared to other ANA-grade angiosperms, such as its near orthotropous ovule symmetry, lead to ambiguities in reconstructions of the MRCA of extant flowering plants. Molecular analyses such as in situ hybridization can be performed in Amborella, and this technique has been extensively used to study the conservation of expression of flower development genes. However, its dioecious breeding and woody habit make Amborella a poorly adapted species for functional-genetic studies.

Several species of Nymphaeales have been used as evolutionary developmental models, including cultivars of Nuphar and Nymphaea. However, the most promising Nymphaeales model for functionalgenetic studies is perhaps Nymphaea thermarum. This species was endemic to Rwanda, but is now considered extinct in the wild. N thermarum (Figure 10. C and D), is the smallest water lily, the diameter of adult plants being 10-20cm, and that of mature, open flowers being up to 2 cm. It is a perennial herb whose life cycle from seed to flowering takes 2-3 months. It can be easily cultivated in a warm, humid and well-lit environment, and grown in pots of wet compost, rather than completely immerse in water.

The N. thermarum flower has whorled phyllotaxy. Its perianth has two sets of tepals: four greenish outer tepals and six to eight whitish inner tepals. The gynoecium of N. thermarum is comprised of seven to nine basally-fused carpels with post-genital fusion at the carpel margins. Within each carpel, a high number of bitegmic, anatropous ovules develop. These ovules have four-celled embryos sacs which, after double fertilization, produce a diploid embryo and a diploid endosperm. N. thermarum is self-compatible, and each fruit typically produces around 150 seeds.

Currently, the scientific literature on N thermarum is limited. However, this species possesses several advantages as a potential model ANA-grade angiosperm: the small size of its flowers makes it practical for in situ hybridization and other microscopic procedures, its diploid genome is one of the smallest within basal angiosperms, and its seeds can easily be sterilized for in-vitro culture. The development of tissue culture and protocols for stable genetic transformation might be possible in this species, and it might also be possible also to generate seed-based mutant collections, depending on the (currently unknown) suitability of N. thermarum seed for long-term storage.

The Austrobaileyales order includes plants with various breeding systems, such as a mixture of monoecy and dioecy in Schisandra chinensis, protogyny in Austrobaieleya scandens and selfincompatibility in Illicium floridanum and Trimenia mooreii [START_REF] Bernhardt | The pollination of Trimenia moorei (Trimeniaceae): Floral volatiles, insect/wind pollen vectors and stigmatic self-incompatibility in a basal angiosperm[END_REF][START_REF] Thien | THE REPRODUCTIVE-BIOLOGY OF A RELICT ILLICIUM-FLORIDANUM ELLIS[END_REF]. Flowers of Austrobaileyales mostly possess a spiral phyllotaxy with an undifferentiated perianth of tepals. The typical Austrobaileyales gynoecium consists of unfused carpels (except partially fused in Illicium), each of which has a secretion-filled apical canal for pollen tube growth.

Inside each carpel a single or a low number of bitegmic ovules develop.

Species of Austrobaileyales have been extensively incorporated in analyses for morphological

reconstructions of the MRCA of extant angiosperm. However, their woody habit makes them poorly adapted as potential molecular-genetic models, and in consequence this group has not been extensively used for molecular studies of flower evolution.

Magnoliids. The magnoliids are a large clade of basal angiosperms which emerged shortly after the ANA-grade lineages. There are five orders of magnoliids regrouping around 11000 species, including both herbaceous and woody plants. Herbaceous magnoliids may make more practical models for the study of the origin of angiosperms than the woody species of Amborellales and Austrobaileyales, or the aquatic herbs of Nymphaeales, within the ANA grade. The phylogenetic position of magnoliids make this clade also an important external reference point for the origin of later-emerging clades of angiosperms, such as the eudicots and monocots.

Aristilochia fimbriata is a promising model magnoliid. Plants of this species are small, have a rapid life cycle, are self-compatible and possess a small genome. A. fimbriata is a vine that attract its pollinators (flies) into a chamber formed by a bilaterally symmetrical perianth of fused sepals (Figure 10. E and F). Bilateral symmetry has arisen several times independently in angiosperms, but this may have occurred first inside the magnoliid clade. The presence of an inferior ovary, and the stamens fused to the style to form a gynostemium, are further distinct floral features of A. fimbriata. A. fimbriata has important advantages for laboratory work as it can be transformed using Agrobacterium tumefaciens, and is readily amenable to standard molecular procedures such as in situ hybridization.

Techniques and methods that could help to explain the origin of the flower

Several approaches are needed to elucidate the origin of the flower and the angiosperms. Recent developments offer the possibility of better understanding this major event in plant evolution. Here, I describe the techniques that are of particular importance for these studies:

1. Next Generation Sequencing (NGS): The increasing efficiency and decreasing costs of NGS facilitate whole genome and transcriptome sequencing. The problem of genome assembly in the absence of physical linkage maps and genetic markers etc, is compensated now by certain recent NGS methods that provide reads extended to tens of kilobases. These methods will be very useful for the sequencing of basal angiosperms and gymnosperms, which in general have large genomes.

2. Bioinformatics and statistical analyses: the development of new bioinformatics and statistical packages could help comparisons of the expression profiles of genes in model and non-model species. Also, such methods could facilitate the analysis of genetic modules involved in flower development across the flowering plants.

Synteny and phylogeny:

The reconstruction of the structure of ancestral genomes from those of their living descendants [START_REF] Murat | Reconstructing the genome of the most recent common ancestor of flowering plants[END_REF]) is now possible due to progress in methods based on the combined analysis of synteny and phylogeny. It may soon possible to reconstruct, in considerable detail, the genome of the ancestor of living angiosperms, and even that of their more distant ancestor from before the epsilon whole genome duplication event (Jiao et al., 2011).

Functional studies:

Recent advances in gene editing should significantly facilitate functional studies, allowing the direct study of the function of developmental regulators in basal angiosperms and gymnosperms. Well-adapted model ANA-grade angiosperms could be adopted to study the function of genes that were crucial genes to the origin of the angiosperms.

Strong candidates for such models are the recently discovered Nymphaea thermarum, and possibly also certain Trithuria spp. (both from Nymphaeales), if transformation methods can be developed for these species. 7. Paleobotany: it would be very useful to discover further fossils of potential stem lineagerelatives of the angiosperms, and also of early flowers. The latter may have been preserved as mesofossils (fossils of a few millimeters in diameter) which can be generated by events such as forest fires which generate "coalified" specimens [START_REF] Schonenberger | Rise from the ashes-The reconstruction of charcoal fossil flowers[END_REF]. Recently developed tomographic methods could help examine anatomical details at the cellular and even sub-cellular level in such fossils [START_REF] Von Balthazar | Potomacanthus lobatus gen. Et sp nov., a new flower of probable Lauraceae from the Early Cretaceous (Early to Middle Albian) of eastern North America[END_REF].

Unsolved questions of flower origin and evolution

We are still far from a complete knowledge of the GRN that was present in the ancestral flower or The carpel is one of the main characters that differentiates angiosperms from its closest sister clade, the gymnosperms. This thesis is centered on the study on the evolution of the carpel and other female reproductive tissues. Its main aim is to help elucidate the Gene Regulatory Network (GRN) that controlled female reproductive development in the MRCA of living flowering plants.

In this work, I have attempted to answer the following two specific questions:

1. Is it possible to identify groups of co expressed genes involved in the development of the carpel and ovule, which have been conserved from the MRCA of living flowering plants?

Aims and strategies of Part 1

Basal angiosperms of the ANA-grade provide key morphological, ecological and molecular data for the evolutionary study of angiosperms. The comparative analysis of distantly related species such as the eudicots and the ANA-grade provides enables us to infer the ancestral states of different characters in the first flowering plants.

This chapter explores the application of interspecific transcriptomic analysis, in order to answer the following question: Is it possible to identify groups of co-expression of genes involved in the development of the carpel and ovule that have been conserved from the most recent common ancestor of flowering plants?

As mentioned above, the genome of Amborella trichopida is an important reference for carpel regulatory networks in early angiosperms, and can help us understand the evolution of the molecular mechanisms that lay behind the appearance of new structures, such as the carpel, in early flowering plants. In this work, novel transcriptomic data of female reproductive tissues from A. trichopoda, produced through laser microdissection and RNA-seq technology, were analyzed.

A proof-of-concept using novel transcriptomic data from the early-diverging angiosperm Amborella trichopoda to identify genetic modules conserved over deep evolutionary time.

Rivarola Sena AC 1 *, Andres-Robin A 1 *, Vialette AC1, Just J 1 , Alexandra Launay-Avon 2 , Dubreucq B 3 and Scutt CP 1 #

Introduction

A major branch of evolutionary-developmental biology (evo-devo) uses inter-species comparisons within an established phylogenetic framework to reconstruct the evolution of the gene regulatory networks (GRNs) that control organismal development. In parallel, gene clustering methods have been widely used to define GRNs within individual species, and in some cases to make comparisons of networks between closely related species [START_REF] Morandin | Comparative transcriptomics reveals the conserved building blocks involved in parallel evolution of diverse phenotypic traits in ants[END_REF][START_REF] Muntane | Interhemispheric gene expression differences in the cerebral cortex of humans and macaque monkeys[END_REF][START_REF] Yu | Divergent gene expression networks underlie morphological diversity of abscission zones in grasses[END_REF].

However, few studies to date have used clustering methods to compare gene expression between highly diverged species, as is often necessary in evo-devo studies that aim to answer macro-evolutionary questions.

The origin of the angiosperm flower, and of its numerous unique organ systems and processes, is one of the major unresolved questions of macro-evolutionary biology. To address this question from a molecular angle, numerous studies have compared flower-development regulators from model angiosperms such as Arabidopsis thaliana with their orthologs from members of the three earliestdiverging "ANA-grade" angiosperm orders: Amborellales, Nymphaeales and Austrobaileyales (reviewed by Scutt, 2018). Indeed, many of these studies have focused on Amborella trichopoda, the only living representative of Amborellales and probable sister to all other living flowering plants [START_REF] Stevens | Angiosperm Phylgeny Website[END_REF]. Amborella is a scrambling, dioecious shrub, endemic to the sub-tropical rainforests of New Caledonia. Flowers of female Amborella individuals contain spirally arranged organs that make up a perianth of typically 7-8 tepals, surrounding a gynoecium of 5(-6) unfused carpels (Endress & Igersheim, 2000b). One or two staminodes (sterile, stamen-like organs) may also be present in Amborella female flowers, inserted between the tepals and carpels. The undifferentiated perianth of the Amborella flower is a probable pleisiomorphic feature of angiosperms, while the carpels of its gynoecium also contain a number of likely pleisiomorphies, including the presence of a compitum, generated through physical contact between the stigmatic surfaces of adjacent carpels, which facilitates the exchange of pollen tubes and thus increases the efficiency of fertilization. Amborella carpels also show the probable pleisiomorphies of an ascidiate (vase-like) shape; ridged, multicellular protrusions on the stigmatic surface; a secretion-filled apical canal for pollen tube growth; and a single, pendent, bitegmic ovule.

The Amborella nuclear genome measures approximately 810 Mb [START_REF] Albert | The Amborella Genome and the Evolution of Flowering Plants[END_REF]. Its detailed analysis reveals clear traces of the At-ε whole genome duplication (WGD), which is believed to have preceded the radiation of living flowering plants [START_REF] Jiao | Ancestral polyploidy in seed plants and angiosperms[END_REF], but provides no evidence of any subsequent WGD events along the Amborella lineage. The availability of flower tissue-specific transcriptomic resources in Amborella has until now been limited, though a detailed study of gene expression in the Amborella egg apparatus was recently published [START_REF] Flores-Tornero | Transcriptomics of manually isolated Amborella trichopoda egg apparatus cells[END_REF].

Here, we contribute the first detailed transcriptomic study of Amborella female flower development, performed using Laser Capture Microdissection (LCM). As a pilot study for the application of geneexpression clustering methods to early flower evolution, we have applied weighted gene co-expression network analysis (WGCNA; [START_REF] Langfelder | WGCNA: an R package for weighted correlation network analysis[END_REF] to our Amborella datasets, and in parallel to reasonably comparable published datasets from the molecular-genetic model angiosperm Arabidopsis thaliana. We have developed a simple work-flow and computer codes to compare the results of gene-expression clustering between distantly related species on the basis of gene-orthology relationships at the genomic scale. Using these procedures, we have succeeded in identifying 13 pairs of genetic modules that show both a highly significant intersection in orthogroup-content and congruent expression dynamics between Amborella and Arabidopsis flowers, strongly suggesting their conservation since the most recent common ancestor (MRCA) of living angiosperms.

Materials and methods

Plant material

Seedlings of Amborella trichopoda (Bail.) were generously provided by Gildas Gâteblé and Bruno Fogliani (IAC, St Michel, New Caledonia) and grown to maturity in Lyon in a greenhouse under conditions of ~70% relative humidity, 18-30°C, and natural daylight attenuated by artificial shadecovering. Flower buds for LCM procedures were harvested from five-year-old plants during peak flowering season in the autumn. These materials were used to initiate the workflow indicated in Fig. 1.

Laser Capture Microdissection

Amborella female flower buds were fixed, embedded, sectioned and subjected to LCM procedures on a PALM Micro-Beam system (Zeiss) as described by [START_REF] Sakai | Combining laser-assisted microdissection (LAM) and RNA-seq allows to perform a comprehensive transcriptomic analysis of epidermal cells of Arabidopsis embryo[END_REF]. Approximately 10 tissue sections were combined for each sample. RNA was extracted from these using an Arcturus Pico RNA Extraction Kit (Thermo Fisher Scientific). RNA integrity was assessed using a 2100 BioAnalyzer (Agilent Technologies) and samples showing RNA Integrity Numbers (RINs) of >7.0 were processed for library construction.

Library construction, sequencing, read mapping and data normalization Amborella libraries were constructed using a cDNA SMARTer Ultra-Low Kit (Illumina) and pooled in groups of five using a barcoding system to be processed in three runs of Hi-seq2000 (Illumina) highthroughput sequencing. Read quality was assessed using FastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/ ) and reads were then mapped to the Amborella V1 complete genome sequence [START_REF] Albert | The Amborella Genome and the Evolution of Flowering Plants[END_REF] using HISAT2 [START_REF] Sirén | Indexing graphs for path queries with applications in genome research[END_REF]).

Read-counts were performed using FeatureCounts [START_REF] Liao | featureCounts: An efficient general purpose program for assigning sequence reads to genomic features[END_REF] and the resulting count data normalized using DEseq2 [START_REF] Love | Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2[END_REF].

Gene expression clustering

Gene clustering was performed in parallel using the WGCNA package [START_REF] Langfelder | WGCNA: an R package for weighted correlation network analysis[END_REF] on Amborella female flower datasets generated in the present study and on published Arabidopsis flower datasets (Yuling [START_REF] Jiao | Cell-type specific analysis of translating RNAs in developing flowers reveals new levels of control[END_REF]. Clustering was performed using the WGCNA automated method, and default settings including the selection of an unsigned network topology. After scrutiny of plots of Scale Free Model Topology Fit again Scale Independence, a Soft Thresholding Power of 7 was chosen for clustering procedures in both species, and genetic modules were generated by cutting dendrograms at a cut-height of 0.5. Eigengene values for these modules, generated in WGCNA, were averaged over biological replicates and converted to a green-red color scale in Microsoft Excel for use in schematic representations.

Inter-species comparison of gene-expression modules and statistical analysis

A global comparison of gene orthology between the Amborella and Arabidopsis genomes (SI Tab 1)

was generated using BIOMART in PHYTOZOME (https://phytozome.jgi.doe.gov/pz/portal.html).

This comparison classes genes from each species into orthogroups, each of which has a unique identifier and is putatively derived from a single ancestral gene that was present in the MRCA of Amborella and Arabidopsis (i.e. in the probable MRCA of all living angiosperms). Orthogroups are classed in this analysis as "one-to-one", "one-to-many", "many-to-one" or "many-to-many", depending on whether gene or genome duplication events in one or both plant lineage have, since their separation, expanded the orthogroup in question.

The Amborella and Arabidopsis modules generated using WGCNA were processed into suitable lists using Perl-Code-1 (SI Methods) , and converted into their respective orthogroup identifiers using Perl-Code-2 (SI Methods), which also takes as input a mapping file in .csv format, derived from the geneorthology relationships obtained using Phytozome. These mapping files each contained a list of Amborella or Arabidopsis (as appropriate) gene identifiers in the first column, and the corresponding orthogroup identifiers in the second. The lists of orthogroups in each module, generated using Perl-Code-2, were compared pairwise between species using Perl-Code-3 (SI Methods). Perl-Code-3 is set up such that the Amborella module is the second argument used in each pairwise comparison, which is significant as this argument represents the trials used to find matches in the list provided by the first (Arabidopsis) argument. Accordingly, in Perl-Code-3, an Amborella module containing N members of a given orthogroup will find N corresponding elements in an Arabidopsis module that contains one or more members of that same orthogroup. Output from Perl-Code-3 comprises lists of the orthogroups present in all pairwise module-intersections between the species analyzed. This output was used as input for Perl-Code-4 (SI Methods), which returns the number of orthogroups in each list.

The statistical significance of the intersection between each pair of modules was calculated using the hypergeometric test. The number of sample successes (q) in this test is represented by the number of orthogroup members in the intersection between the pair of modules under consideration (from the output of Perl-Code-4). Population successes (m) are represented by the number of unique orthogroups (i.e. counting two or more identical entries as one) in the entire Arabidopsis transcriptome that are also present in the Amborella module under consideration. Population failures (n) thus correspond to the total number of unique orthogroups in the Arabidopsis transcriptome, minus the number of population successes (m). The number of trials (k) corresponds to the total number of orthogroups (i.e. counting N identical entries as N) in the Amborella module under consideration. The upper-tail, cumulative pvalue was calculated in this way for each pairwise module comparison between Amborella and Arabidopsis using the R code:

>phyper(q-1, m, n, k, lower-tail=FALSE);

The Amborella and Arabidopsis orthologs occupying the intersections between highly conserved modules of interest from the two species were then complied in lists, together with their orthogroup IDs, using Perl-Code-5 (SI Methods), and Arabidopsis gene annotations for one gene from each orthogroup (obtained from Yuling Jiao & Meyerowitz, 2010) added using Perl-Code-2 on appropriately formatted input files.

Results

Laser Capture Microdissection proves an efficient method to define the floral transcriptome of the early-diverging angiosperm Amborella trichopoda.

We have used LCM and RNA-seq procedures to generate transcriptomic datasets from two stages of Amborella female flower development as defined by [START_REF] Buzgo | Floral developmental morphology of Amborella trichopoda (Amborellaceae)[END_REF]. The stages samples correspond to (1) Stage 5-6, and (2) a stage between Stage 7 and that of the mature female flower (female anthesis), referred to here as "Late Stage 7". At Stage 5-6, the young tepals are expanding laterally and the carpels are developing from a solid primordium into a hollow structure that will later enclose the ovule. At this stage, young carpels are clearly distinguishable from any staminodes that may also be present. Ovule initiation occurs at Stage 7, and by Late Stage 7, ovule development is well underway, while distinct regions of the carpel corresponding to the ovary wall and carpel apex are also clearly apparent.

We sampled two tissues from Stage 5-6, corresponding to young tepals and carpels (Figs 2A andB).

From Late Stage 7, we sampled three tissues corresponding to the developing ovule, ovary wall and carpel apex (Figs 2C and D). All samples were obtained in triplicate, making a total of 15 samples for library construction and paired-end read sequencing. Between 89% and 96% of reads generated from the 15 samples could be mapped to the Amborella genome sequence, giving an average of 2.62 x 10 7 mapped reads per sample. The datasets obtained (SI Tab 2, normalized counts) constitute the first detailed transcriptomics resource from flower tissues of an ANA-grade angiosperm.

Orthogroup-based comparisons identify 24 highly significant intersections between genetic modules from Amborella and Arabidopsis.

We performed clustering analyses in WGCNA on the Amborella female flower datasets generated in and AGAMOUS (AG, expressed in whorls 3-4).

Our clustering analyses, using identical parameters for each species, yielded totals of 14 and 13 modules in Amborella and Arabidopsis flowers, respectively, not including a "grey" module of unassigned genes in each case (SI Tab 3). The remaining modules are labelled in WGCNA using a fixed series of RGB colour-names (see key in SI Tab 4) in descending order of gene-content (so modules bearing the same colour-name may be of similar size, but are not necessarily closely related between species). Module eigengenes [START_REF] Langfelder | Eigengene networks for studying the relationships between coexpression modules[END_REF], calculated in WGCNA, were averaged between biological replicates (SI Tab 4) and plotted on a red/green color-scale for the schematic diagrams in Fig 3 . Each eigengene is the theoretical gene which typifies its module, the value for each tissue/sample being the first principal component of the variance in gene expression.

High positive eigengene values thus represent high expression, while high negative values represent low expression. As we used an unsigned clustering procedure, modules may contain some genes that show inverted expression dynamics compared to their module eigengene (as well as many others whose expression dynamics correlate positively with those of the module eigengene). Such unsigned networks may be particularly useful to identify negative as well as positive regulators in GRNs of interest.

We used a custom procedure to compare the contents of the genetic modules identified in WGCNA (SI Tab 3) by first converting their Amborella or Arabidopsis gene-identifiers to orthogroup-identifiers generated in PHYTOZOME (SI Tab 1). We then compared all modules pairwise between species and indicates that Atr_green is also related to Ath_magenta with the moderately low p-value of p=0.092.

Indeed, it appears that, in most cases of multiple modules in one species that show very similar expression dynamics, the main distinction between these modules resides in subtle differences between biological replicates, rather than more pronounced differences between the tissues or stages analyzed.

In the case of Atr_tan and Atr_green, for example, the former shows a particularly high eigengene value for the first biological replicate of the ovary wall tissue, while the latter shows a similarly high eigengene value for the second ovary wall replicate (SI Tab 4).

Several of the modules that show highly significant (p<0.01) positive intersections in our study do so with more than one module from the other species under comparison. This is the case, for example, for the Atr_turquoise module, which shares a close relationship with five different Arabidopsis modules (Fig. 2, SI Tab 6).

Thirteen highly significant module intersections show congruent expression dynamics, suggesting their conservation since the MRCA of living angiosperms.

It is not trivial to compare gene-expression dynamics between such widely diverged angiosperm species as Amborella and Arabidopsis. Such comparisons are affected by problems of defining organ and tissue homology [START_REF] Roux | What to compare and how: Comparative transcriptomics for Evo-Devo[END_REF]. For example, Amborella contains an undifferentiated perianth of tepals, while Arabidopsis contains a differentiated perianth containing both a calyx of sepals and a corolla of petals. Furthermore, the petals of core eudicots such as Arabidopsis may be "andropetals", derived from sterilized former stamens, whereas petaloid perianth organs in more basally-diverging angiosperm taxa such as Amborella are usually interpreted as "bracteopetals", derived from leaf-like organs [START_REF] De Craene | Origin and evolution of petals in angiosperms[END_REF]. Furthermore, it is not clear that all tissue-types in the complex syncarpous gynoecium of Arabidopsis possess a counterpart in the much simpler, unfused carpels of Amborella (Endress & Igersheim, 2000a). Flower development between Amborella and Arabidopsis also exhibits heterochrony, making it very difficult to designate equivalent stages of flower bud development between these two species. Most notably, Amborella floral organ primordia are produced along a continuous spiral, whereas the primordia of each whorl of the Arabidopsis flower are initiated simultaneously.

Despite these various differences, there are clear indications that several of the highly significant module-intersections identified in the current work derive from the conservation of gene expression.

We accordingly observe that, in each module identified in the present study from either Amborella or Arabidopsis, one or two tissues/stages show relatively high eigengene values of >0.25, and this information can be used to broadly characterize the expression dynamics of the module. Using this information, 13 of the 24 highly significant intersections identified through statistical testing appear to show congruent expression patterns between Amborella and Arabidopsis, and these link together all but six of the modules (three from each species) for which highly significant intersections (p< 0.01)

were found (Tab 1). These 13 intersections contain a total of 1809 orthogroups, which appear to have conserved important elements of their expression patterns, as typified by their module eigengenes, since the MRCA of living angiosperms. Full data on these 13 sets of conserved orthologs are presented in SI Tab. 7, including annotation information for the first-listed Arabidopsis gene from each orthogroup.

Orthologs from conserved modules show several different types of relationship with each other and with their module eigengenes.

We cannot summarize here the expression characteristics of all the genes of the 1809 orthogroups from

Discussion

What is modularity and why is it so important?

Modularity is a key concept of evolutionary-developmental biology (evo-devo). However, the definition given of a "module" may not always correspond perfectly between evolutionary morphologists and developmental biologists [START_REF] Mabee | Integrating evolution and development: The need for bioinformatics in evo-devo[END_REF]. Evolutionary morphologists tend to think of modules as elements of an organism in which internal connectivity is greater than external connectivity. Such modules, which might for example correspond to a particular organ system or tissue, may be able to undergo evolutionary change without substantial change to the rest of the organism. Developmental biologists, by contrast, think increasingly in terms of Gene Regulatory Networks in which a module might represent a particular set of genes that can be regulated together to produce a given developmental effect, perhaps analogous to a computer subroutine that can be invoked to perform a precise task from within any part of the overall program.

Clearly, profound links must exist between morphological and molecular modules, and it is perhaps surprising that relatively few clear examples can be cited to link evolutionary change to modular processes at both the genetic and morphological levels. One possible reason for this, particularly in plant biology, is that large-scale gene expression datasets from a wide phylogenetic range of species have only become available over the last few years. Indeed, even though many plant transcriptomes have now been assembled [START_REF] Leebens-Mack | One thousand plant transcriptomes and the phylogenomics of green plants[END_REF], the availability of large-scale gene-expression datasets that cover different organs, tissues and stages continues to lag considerably behind in all but a few model species. It is likely, however, with techniques such as the LCM method employed in the present work, and also the very promising single-cell RNA-sequencing technologies [START_REF] Frank | Cell-specific transcriptomic analyses of three-dimensional shoot development in the moss Physcomitrella patens[END_REF][START_REF] Ryu | Single-Cell RNA Sequencing Resolves Molecular Relationships Among Individual Plant Cells[END_REF]) that detailed large-scale expression data will become increasingly available from a much wider range of plant species. It will consequently become increasingly important to devise methods to compare global expression data between species, including those separated by great evolutionary distances, which is one of the reasons we decided to perform the present pilot study.

This study shows that conserved gene-expression modules can be identified over large phylogenetic distances.

In this work, we show that it is possible to meaningfully compare the results of independentlyperformed gene-expression clustering analyses between plant species that are believed to have diverged at least 149 MYA [START_REF] Barba-Montoya | Constraining uncertainty in the timescale of angiosperm evolution and the veracity of a Cretaceous Terrestrial Revolution[END_REF]. By comparing gene-expression modules on the basis of gene orthology, we have been able to identify 13 highly significant intersections between modules whose eigengenes show congruent expression patterns between the species under comparison. Accordingly, the members of the 1809 orthogroups contained in these 13 intersections appear to have conserved their expression profiles in both the Amborella and Arabidopsis lineages since the MRCA of living angiosperms. These results represent an initial proof-of-concept, answering the first of the questions we asked at the outset of this work concerning the feasibility of comparing gene-expression clustering data over a vast phylogenetic distance.

Analyses of genome structure indicate the Amborella genome to have undergone no whole genome duplication since the At-ε event, believed to have occurred before the radiation of living angiosperms (Yuannian Jiao et al., 2011). In the Arabidopsis genome, by contrast, there is evidence of one genome triplication (At-γ) and two genome duplications (At-α and At-β) that have taken place since the At-ε event (Yuannian Jiao et al., 2011). These multiple, large-scale duplication events in the Arabidopsis lineage inevitably render gene-orthology relationships with Amborella more complex than with, for example, a recently diverged member of Brassicaceae. Nonetheless, the use of a genome-wide analysis of orthology relationships has permitted, in the present study, a meaningful comparison of global transcriptome data between species whose lineages diverged at the dawn of the angiosperm clade.

The majority of the conserved orthogroup members identified in the present work show congruent expression patterns between the two species under comparison. However, inversions of expression dynamics appear to have occurred between orthologs in a few cases (e.g. Fig. 4F), which may point to a switch in an upstream regulatory component in one or other of the plant lineages concerned. These examples of converse expression patterns between orthologs may merit further attention, as some of them might be indicative of important evolutionary changes to upstream developmental regulators.

How useful are datasets derived from independent transcriptomic analyses?

Clearly, it is very useful for practitioners of evo-devo to be able to use published datasets from other workers, in addition to their own novel data. Accordingly, the second question we asked at the outset of this study concerned the usefulness of comparing datasets from different sources, which by definition were not generated as part of a unified experimental design. The answer to this question, from the results presented here, seems to be "both yes and no". We used partially similar flower RNAseq datasets from Amborella (this study) and Arabidopsis (Yuling Jiao & Meyerowitz, 2010), which differed in the exact tissues and stages sampled and in the experimental approach used (i.e. LCMdissection of discrete tissues/organs in Amborella, compared to sampling of overlapping tissue zones in Arabidopsis). As stated above, this approach was nevertheless successful in identifying 13 cases of apparent large-scale conservation between genetic modules. However, a further 11 pairs of highly significant module intersections were identified for which the corresponding module eigengenes did not show congruent expression patterns between the species analyzed. In addition, many of the module-intersections identified in our study showed deviations from a 1:1 relationship between species. It is likely that two major factors contributed to the above two effects: (1) we used datasets produced in independent studies, in which the tissues and developmental stages sampled corresponded only partially between species. Secondly, real differences between the species under study, resulting from evolutionary processes since the separation of their lineages, may also have contributed to changes in gene expression patterns and/or to a departure from a 1:1 correspondence between genetic modules.

It seems clear that the best way to identify, with reasonable confidence, real, intrinsic differences between gene-expression clustering data from different species will be to minimize variation between datasets that are due to different sampling strategies and/or experimental methods. This caveat means that, to reach solid conclusions about evolutionary change as well as evolutionary conservation, it will be very important to compare datasets that are as closely matched as possible. This stringent requirement could, however, still permit the use of data obtained from other studies. In particular, the general applicability of detailed gene expression data might be facilitated by the adoption by the scientific community of data standards that maximize equivalence and comparability between datasets.

Aims and strategies of this part

The following manuscript aims to reconstruct the evolutionary history of the gene SPATULA (SPT), which encodes a bHLH transcription factor that plays important roles in gynoecium development and other developmental processes in the molecular-genetic model angiosperm Arabidopsis thaliana. In the Arabidopsis gynoecium, SPT is known for its role in the development of the replum, which develops from the carpel margin meristem (CMM), and the style and stigma which develop at the apex of the gynoecium.

At the outset of this doctoral work, SPT was one of the best-characterized regulators of gynoecium development in Arabidopsis. It was known to have undergone a structural change before the origin of flowering plants, which may have been a prerequisite for the evolution of its role in the gynoecium.

Coding sequences of SPT orthologs from asterids such as tomato were known to be able to rescue spt mutants in transgenic Arabidopsis, and SPT orthologs were known to be upregulated in gynoecium tissues of widely diverged angiosperms. All the above lines of evidence suggested that SPT was recruited very early to carpel development and that this factor accordingly formed part of the basic Gene Regulatory Network (GRN) of the carpel over 145 MYA in the first angiosperms.

We set out to test the above hypothesis using a large range of studies performed on living species chosen for their informative phylogenetic positions, including several basal angiosperms and the eudicot model Petunia, which is extensively studied in our research group. To study the preangiosperm evolution of the SPT lineage, we also incorporated into our investigation non-flowering vascular plants including gymnosperms and lycophytes.

The above studies have led to a manuscript which is a "work-in-progress" that can hopefully be submitted for publication on the completion of a few more experiments. Its main provisional conclusion is that SPT does not form part of the basic GRN of the carpel in all angiosperms, but was recruited to the development of CMM tissues much later, in a common ancestor of Brassicaceae. In Brassicaceae, SPT appears to contribute to a very specific type of tissue organization, involving the growth from the CMM of a false septum which divides the syncarpous gynoecium into two chambers.

However, we have also obtained data relating to an apparent spt double-mutant phenotype associated specifically with the stigma in Petunia (asterids). The exact list of mutant genes that contribute to this phenotype, however, remains to be clarified, and this awaited clarification could lead to a reappraisal of our current view that SPT was recruited to the development of CMM tissues in an ancestor of Brassicaceae. The alternative possibility is that SPT may have acquired a role the formation of tissues at the carpel apex or margins much earlier in the evolution of angiosperms, but this role became more pronounced and specific during the evolution of the novel and atypical gynoecium structure in Brassicaceae.

We show in our manuscript that SPT orthologs are very highly expressed in the abaxial domain of the carpel, and in the placenta and ovules, in a very wide spectrum of angiosperms, including some of the most angiosperm basal lineages. However, no mutant phenotypes are known in model angiosperms to be associated with these peaks of SPT expression, which may indicate the present of a complex set of genetic redundancies involving genes from outside the SPT gene-clade.

In addition to the above questions of SPT function and evolution, in which further clarification would be useful, our manuscript provides some clearer insights in a number of areas. These more successfully answered questions include: (1) the phylogenetic origin of the SPT lineage in early land plants, (2) evolution of the domain structure of SPT proteins in land plants, (3) conservation of biochemical properties and downstream pathways associated with widely-diverged SPT orthologs, (4) evolutionary changes to the SPT-promoter which may have led to its current role in Brassicaceae-specific gynoecium morphology and ( 5), the origin of lineages labelled as "ALCATRAZ" (ALC) in Solanaceae and Brassicaeae.

We envisage submitting a revised version of this manuscript after the completion of our ongoing analyses of spt mutants of Petunia, which we expect to clear up some of the remaining important questions left partially unanswered in the current manuscript version.

Evolution of the transcription factor SPATULA and its role in Arabidopsis gynoecium development.

Ana C. Rivarola-Sena

Introduction

In the syncarpous gynoecium of the model angiosperm Arabidopsis thaliana (referred to below as Arabidopsis), the margins of the two fused carpels form a Carpel Margin Meristem (CMM) which undergo cell division to generate the placenta, ovules and vertical septum or adaxial replum [START_REF] Ferrandiz | Carpel Development[END_REF]. The carpel margins then later differentiate to generate a dehiscence zone and separation layer, both of which contribute to fruit dehiscence.

Many genes are known to function in tissues that develop from the CMM, including the basic Helix-Loop-Helix (bHLH) transcription factor SPATULA (SPT; Heisler et al., 2001). In spt loss-of-function mutants, the gynoecium remains unfused at the apex and the septum fails to develop properly (Alvarez & Smyth, 1999). Fertility is reduced is spt mutants due to a reduction in the size and extent of the pollen transmitting tissue within the style and septum, in which cells fails to properly elongate. Outside the flower, SPT also functions in seed dormancy [START_REF] Penfield | Cold and light control seed germination through the bHLH transcription factor SPATULA[END_REF] and in leaf and cotyledon expansion [START_REF] Josse | A DELLA in Disguise: SPATULA Restrains the Growth of the Developing Arabidopsis Seedling[END_REF]. In gynoecium and fruit tissues, SPT acts partially redundantly with its close paralog ALCATRAZ (ALC), though the latter plays the major role in the development of fruit dehiscence zones and shows no single mutant phenotype at earlier stages in the developing gynoecium (Groszmann et al., 2011).

SPT is capable of homodimerization, and of heterodimerization with both ALC (Groszmann et al., 2011) and with the bHLH factors HECATE1-3 (HEC1-3; [START_REF] Gremski | The HECATE genes regulate female reproductive tract development in Arabidopsis thaliana[END_REF], which are redundantly necessary for normal style and septum development. SPT can also form heterodimers with INDEHISCENT (IND), a close relative of HEC1-3 [START_REF] Girin | INDEHISCENT and SPATULA Interact to Specify Carpel and Valve Margin Tissue and Thus Promote Seed Dispersal in Arabidopsis[END_REF]. [START_REF] Reymond | A Light-Regulated Genetic Module Was Recruited to Carpel Development in Arabidopsis following a Structural Change to SPATULA[END_REF] identified 24 putatively direct targets of SPT, including a high proportion of genes that are known to be regulated also by phytochrome-interacting transcription factors (PIFs) which, like SPT and ALC, belong to Group 24 of the very large bHLH family [START_REF] Carretero-Paulet | Genome-Wide Classification and Evolutionary Analysis of the bHLH Family of Transcription Factors in Arabidopsis, Poplar, Rice, Moss, and Algae[END_REF]. These lightregulated target genes are known to act downstream of PIFs to promote cell-elongation, and thereby bring about shade avoidance responses in stem internodes and the hypocotyl. They are hypothesized also to act downstream of SPT to promote cell-elongation in the style and transmitting tissue of the gynoecium. Indeed, [START_REF] Reymond | A Light-Regulated Genetic Module Was Recruited to Carpel Development in Arabidopsis following a Structural Change to SPATULA[END_REF] proposed that SPT evolved from a PIF by the loss of an active phytochrome binding (APB) domain, thereby possibly escaping from light-regulation acting through phytochrome photoreceptors.

In addition to light-regulated targets, SPT has been shown to regulate several genes related to auxin signaling, one of which encodes the serine/threonine protein kinase PINOID (PID), which positively regulates cellular auxin efflux [START_REF] Friml | A PINOIDdependent binary switch in apical-basal PIN polar targeting directs auxin efflux[END_REF]. SPT has been shown to interact with IND to downregulate PID in the style and carpel margins [START_REF] Girin | INDEHISCENT and SPATULA Interact to Specify Carpel and Valve Margin Tissue and Thus Promote Seed Dispersal in Arabidopsis[END_REF]. A further direct target of SPT,

ARABIDOPSIS RESPONSE REGULATOR1 (ARR1), promotes cytokinin-dependent cell division at

the carpel margins (Irepan [START_REF] Reyes-Olalde | Synergistic relationship between auxin and cytokinin in the ovary and the participation of the transcription factor SPATULA[END_REF].

The domain structure of SPT-like proteins has been analyzed in a wide variety of angiosperms, including magnoliids, monocots, basal eudicots, asterids and rosids [START_REF] Groszmann | Functional domains of SPATULA, a bHLH transcription factor involved in carpel and fruit development in Arabidopsis[END_REF][START_REF] Ortiz-Ramirez | Evolution of genes associated with gynoecium patterning and fruit development in Solanaceae[END_REF]. SPT expression patterns have furthermore been studied in the basal eudicot Boccinia fructescens (Papaveraceae; [START_REF] Zumajo-Cardona | Evolution of the SPATULA/ALCATRAZ gene lineage and expression analyses in the basal eudicot, Bocconia frutescens L. (Papaveraceae)[END_REF], and comparison of these data with SPT expression in Arabidopsis (Heisler et al., 2001;Groszmann et al., 2010) suggested the role of SPT in the development of tissues from the carpel margins to be widely conserved in eudicots. However, functional-genetic studies using virus-induced gene silencing (VIGS) in three Solanaceae species recently found no SPT-knockdown phenotypes associated with early gynoecium development [START_REF] Ortiz-Ramirez | Expression and function of the bHLH genes ALCATRAZ and SPATULA in selected Solanaceae species[END_REF]. Instead, these studies concluded SPT-like genes in Solanaceae to control petal expansion, leaf pigmentation and lignification in the fruit. [START_REF] Ortiz-Ramirez | Expression and function of the bHLH genes ALCATRAZ and SPATULA in selected Solanaceae species[END_REF] speculate that other genetic systems may be acting redundantly with SPT-like genes to control gynoecium development in Solanaceae, or else that VIGS methods may not have produced a sufficient reduction in SPT mRNA levels to reveal a loss-of-function phenotype in Solanaceae models.

Here, we attempt to reconstruct the evolutionary history of the SPT lineage and use a series of in-vitro, in-vivo and in-silico approaches to try to determine the stage at which SPT acquired its current role in the development of tissues that arise from the carpel margins of the Arabidopsis gynoecium. We furthermore attempt to identify molecular changes that might have been responsible for this recruitment. We conclude that Arabidopsis SPT probably acquired major aspects of its role in the gynoecium in a common ancestor of Brassicaceae, rather than at a much earlier evolutionary stage within angiosperms. We present evidence to suggest that the acquisition of this role did not involve a major change in the biochemical capacities of SPT with regard to protein dimerization or protein-DNA interactions. We also find no evidence of any major evolutionary change in the direct targets regulated by SPT during angiosperm evolution. Rather, we conclude that SPT's novel role in the Brassicaeae gynoecium resulted from a change to its expression pattern, and we present preliminary data suggesting that major rearrangements in the SPT promoter region may have correlated with this change.

Results

The SPT lineage is conserved throughout euphyllophytes and shows a complex history of gene duplication, gene loss and structural modification.

To provide a framework to understand the evolutionary history of the SPT clade and its role in gynoecium development, we first performed a phylogenetic analysis of SPT-like sequences, together with selected sequences from Group 24 of the bHLH family [START_REF] Carretero-Paulet | Genome-Wide Classification and Evolutionary Analysis of the bHLH Family of Transcription Factors in Arabidopsis, Poplar, Rice, Moss, and Algae[END_REF]. These sequences were sampled from a wide range of vascular plants, some of which have sequenced genomes (SI Tab. 1). The results of the analysis, which used HEC sequences from Group 31 of the bHLH family as a close out-group [START_REF] Carretero-Paulet | Genome-Wide Classification and Evolutionary Analysis of the bHLH Family of Transcription Factors in Arabidopsis, Poplar, Rice, Moss, and Algae[END_REF], are shown in Fig. 1.

Phylogenetic analysis shows both SPT and ALC from Arabidopsis in a clade of SPT-like sequences that lies in sister position to the PIF7 clade of phytochrome-interacting transcription factors. Genes from the monilophyte (fern) species Azolla filiculoides and Salvinia cucullata occupy relatively basal and well-supported positions within the SPT clade, while a gene from the lycophyte Selaginella moellendorffii forms a basal polytomy, together with the PIF7 and SPT clades. Further sampling of basal vascular plant taxa will be necessary to reach firmer conclusions, but these preliminary data suggest that the SPT lineage may have originated in a common ancestor of euphyllophytes (ferns+seed plants), perhaps by duplication of an PIF7-like gene.

Our phylogenetic analysis provides evidence of two important duplication events in the SPT lineage that took place within angiosperms. The first of these was found at the base of the core-eudicot clade, and apparently gave rise to the SPT and ALC lineages present in Solanaceae and other asterids, as identified by [START_REF] Pabon-Mora | Evolution of fruit development genes in flowering plants[END_REF]. This gene duplication corresponds in timing with the welldocumented At-γ genome triplication event [START_REF] Jaillon | The grapevine genome sequence suggests ancestral hexaploidization in major angiosperm phyla[END_REF]. It appears, however, from our data that only one of the two sister lineages generated at this time was retained in rosids.

The second and more recent major duplication observed in the present study occurred somewhere around the base of Brassicaceae and appears to have generated the Brassicaceae SPT and ALC lineages, as defined by SPT and ALC in Arabidopsis. It therefore appears that genes labelled "ALC" in Brassicaceae [START_REF] Rajani | The Arabidopsis myc/bHLH gene ALCATRAZ enables cell separation in fruit dehiscence[END_REF]Groszmann et al., 2011) and Solanaceae [START_REF] Pabon-Mora | Evolution of fruit development genes in flowering plants[END_REF][START_REF] Ortiz-Ramirez | Expression and function of the bHLH genes ALCATRAZ and SPATULA in selected Solanaceae species[END_REF] may not be orthologous between these families. Rather, the Solanaceae SPT lineage may be a pro-ortholog of SPT and ALC in Brassicaceae, while "ALC" genes from Solanaceae and other asterids may have no true orthologs in Brassicaceae, this lineage having been lost early in the evolution of the rosid clade.

We subjected a range of sequences from the SPT+PIF7 clade in vascular plants, and also Arabidopsis PIF5 for comparative purposes, to structural analyses using MEME software (Fig. 2A). PIF5 was chosen as this gene, together with its paralog PIF4, was shown to be redundantly necessary for the rescue of Arabidopsis spt mutants under specific light conditions [START_REF] Reymond | A Light-Regulated Genetic Module Was Recruited to Carpel Development in Arabidopsis following a Structural Change to SPATULA[END_REF]. All the bHLH proteins we analyzed showed a well-conserved bHLH domain, which is known to be responsible for DNA-binding, homodimerisation, and heterodimerisation with other bHLH proteins.

Of the proteins analyzed here, only Arabidopsis PIF5 and the putative PIF7/SPT pro-ortholog from S.

moellendorffii possess an N-terminal APB domain (Figs 2B-C). Our findings suggest, therefore, that the SPT lineage lost an APB domain, presumably through a genetic truncation event, in a common ancestor of euphyllophytes, shortly after its origin through duplication (Fig. 2D). The evolution of SPT through the loss of the APB domain was already proposed by [START_REF] Reymond | A Light-Regulated Genetic Module Was Recruited to Carpel Development in Arabidopsis following a Structural Change to SPATULA[END_REF], though these authors could only conclude this event to have taken place in or before the MRCA of living seed plants.

As noted by [START_REF] Reymond | A Light-Regulated Genetic Module Was Recruited to Carpel Development in Arabidopsis following a Structural Change to SPATULA[END_REF], the SPT lineage clearly lost its APB domain, and potentially therefore escaped from light-regulation acting through phytochrome, long before the evolutionary origin of the carpel in a common ancestor of angiosperms. [START_REF] Groszmann | Functional domains of SPATULA, a bHLH transcription factor involved in carpel and fruit development in Arabidopsis[END_REF] reported SPT orthologs from eudicots to possess two functionally important domains, in addition to the bHLH domain, that were not found to be conserved in monocots. These domains correspond to an amphipathic helix that was determined by deletion experiments to enhance SPT function in the gynoecium, and an acidic domain that proved essential to this same gynoecium function. Multiple alignment of bHLH sequences indeed shows sparse amino-acid similarity, outside eudicots, over the amphipathic domain identified by [START_REF] Groszmann | Functional domains of SPATULA, a bHLH transcription factor involved in carpel and fruit development in Arabidopsis[END_REF], as shown in Fig 2E.

However, it is possible that functionally equivalent regions containing similarly charged residues may be present in SPT orthologs from outside the eudicots, but are difficult to identify by multiple alignment. The acidic domain reported by [START_REF] Groszmann | Functional domains of SPATULA, a bHLH transcription factor involved in carpel and fruit development in Arabidopsis[END_REF] is, by contrast, clearly present in SPT orthologs from basal taxa including Amborella and Picea (Fig 2F). Amino-acid similarities in this domain are also conserved to a significant extent in the putative PIF7/SPT pro-ortholog from S.

moellendorffii and in PIF proteins including Arabidopsis PIF5. The essential acidic domain of eudicot SPT orthologs appears, therefore, to be widely conserved in angiosperms, and in Group 24 bHLH proteins as a whole, even if it has become lost or indistinct in the SPT lineage in monocots.

In-vitro assays suggest SPT proteins to have conserved both important cofactors and target genes, at least since the MRCA of living angiosperms.

The phylogeny and domain structure of SPT-like proteins provide interesting insights into the origin of this lineage, but fail to indicate any molecular changes that may have been responsible for SPT's recruitment to gynoecium development, or suggest when this recruitment occurred. To throw some light on these questions, we attempted to determine whether the interactors and target genes of SPT proteins have changed over evolutionary time, particularly during the evolution of angiosperms.

We first used yeast-2-hybrid analyses to determine whether the SPT protein from the likely basal-most living angiosperm, Amborella trichopoda, was capable of homodimerization, and/or of heterodimerization with the HEC-like genes from the same species, as does its ortholog in Arabidopsis.

Amborella contains two HEC-like genes, one of which, AtrHEC1/2, is the likely pro-ortholog of Arabidopsis HEC1 and HEC2, while the other, Atr_bHLH87, is the likely pro-ortholog of Arabidopsis HEC3 and IND (Pabon-Mora et al., 2014). The results of this analysis (Fig. 3) show that Amborella SPT is able to homodimerize, and to heterodimerize with both Amborella HEC proteins, strongly suggesting that both of these types of physical interactions involving SPT proteins have been conserved in the Arabidopsis and Amborella lineages since the MRCA of living angiosperms.

We then attempted to compare the likely direct targets of SPT-clade transcription factors, this time extending our analysis to cover the gymnosperm Picea abies. SPT coding sequences from potential binding site for SPT, while the equivalent figures were 13/22 for Amborella and 10/13 for Picea (not including genes for which truncated sequences only were available).

To determine whether the above figures represented a significant enrichment in sites predicted to bind SPT with high affinity, we further obtained datasets of 3-kb upstream sequences from the entire sequenced genomes of Arabidopsis and Amborella. It was not, however, possible to obtain equivalent data from the relatively incomplete genome assembly of Picea. We scanned the Arabidopsis and Amborella promoter datasets with the corresponding top-scoring SPT PWM, and then used the hypergeometric test to evaluate the significance of the results obtained, compared to the equivalent scans of Arabidopsis or Amborella putative SPT-target promoters. This procedure identified significantly more promoters containing high-scoring sites among putative targets, at p-values of 6.00x10 -7 and 0.0150 for Arabidopsis and Amborella, respectively (SI Tab. 3, Analysis 1). High scoring sites were over-represented in putative SPT promoters by a factors of 4.83 and 2.66 in Arabidopsis and Amborella, respectively (SI Tab. 3, Analysis 2). As the proportion of putative target promoters containing at least one high-scoring site was higher in Picea than Amborella, we suppose that an equal or lower p-value might have been obtained for Picea, had the necessary sequence data been available to make a full analysis.

These data suggest that direct target genes of SPT have been largely conserved, at least since the MRCA of living angiosperms, and probably since that of living seed plants. These targets include genes identified by [START_REF] Reymond | A Light-Regulated Genetic Module Was Recruited to Carpel Development in Arabidopsis following a Structural Change to SPATULA[END_REF], which encode proteins significantly over-represented in shade-avoidance-and auxin-related processes, among others. Two further SPT targets identified in Arabidopsis, PID [START_REF] Girin | INDEHISCENT and SPATULA Interact to Specify Carpel and Valve Margin Tissue and Thus Promote Seed Dispersal in Arabidopsis[END_REF] and ARR1 (Irepan [START_REF] Reyes-Olalde | Synergistic relationship between auxin and cytokinin in the ovary and the participation of the transcription factor SPATULA[END_REF], were confirmed to be likely conserved as SPT targets in Amborella, though this confirmation was not possible in Picea due to a lack of sequence data from the upstream regions of these genes.

It seems, from the above in-vitro and in-silico studies, that most of the major direct molecular interactions known to involve Arabidopsis SPT, both with its protein cofactors and its direct target genes, have likely been conserved, at least since the MRCA of living angiosperms.

SPT orthologs from a wide range of vascular plants can complement strong loss-of-function spt mutations in transgenic Arabidopsis.

The conservation of the in-vitro biochemical properties of Arabidopsis SPT with its orthologs from basal angiosperms and gymnosperms led us to ask whether the in-vivo biological properties of SPTlike proteins might be similarly conserved between very distant taxa. Indeed, [START_REF] Reymond | A Light-Regulated Genetic Module Was Recruited to Carpel Development in Arabidopsis following a Structural Change to SPATULA[END_REF] had found that PIF4 and 5, two paralogous bHLH proteins from outside the SPT clade, contributed to the rescue of spt loss-of-function mutants grown under low red/far-red light ratios, which suggested that even these evolutionarily distant molecules might be able to replace the biochemical functions of SPT. Furthermore, [START_REF] Groszmann | Functional domains of SPATULA, a bHLH transcription factor involved in carpel and fruit development in Arabidopsis[END_REF] and Groszmann et al. (2011) had already demonstrated the rescue of spt-2 mutants using both Arabidopsis ALC and an SPT ortholog from Solanum lycopersicum (tomato).

To extend the above type of analysis over a greater phylogenetic range, we transformed strong lossof-function Arabidopsis spt-11 mutant plants using the coding sequences from a range of SPT orthologs, pro-orthologs and other bHLH Group 24 members, from taxa as far removed as lycophytes, all driven using the fully functional 6.3-kb promoter sequence of Arabidopsis SPT defined by Groszmann et al. (2010). Two versions each of a putative PIF7/SPT pro-ortholog from S.

moellendorffii and of Arabidopsis PIF5 were included in these studies, one of which had been artificially truncated to remove its N-terminal APB domain while leaving intact its initiation codon and reading frame. The untransformed Arabidopsis spt-11 mutant typically shows reduced carpel fusion over the style and stigma region, fails to generate a septum in the upper portion of the gynoecium, and after fertilization produces shorter fruits, containing fewer seeds, due to poor pollentube transmission prior to fertilization (Fig. 5A-C). At least 16 independent transformants were observed for each transgene construct tested, and untransformed mutants were grown for comparison in each batch of plants observed.

All the constructs tested were able, in at least some of the transgenic lines analyzed, to fully restore fusion of the gynoecium apex (Fig 5D-N, Tab. 2). There was no significant difference (at the p<0.05 level) in the efficiency of the constructs tested, compared to the positive control AthSPT::AthSPT, with the exceptions of: pSPT::PIF5, pSPT::SmoPIF7/SPT and pSPT::SmoΔPIF7/SPT, which proved less efficient than the others. Interestingly, the proportion of lines restored was higher in the N-terminally deleted versions of both AthSPT::Smo PIF7/SPT and AthSPT::AthPIF5. The constructs tested varied more markedly in their capacity to fully restore fruit-size/fertility (Fig. 6, Tab. 2). Accordingly, AthSPT::AthALC, AthSPT::NcaSPT and AthSPT::PaxALC all complemented the fruit-size phenotype with no significant difference in efficiency from the positive control construct (at p<0.05).

AthSPT::AtrSPT, AthSPT::AthPIF5 and AthSPT::ΔAthPIF5 complemented the fruit-size phenotype with intermediate efficiency (p-value between 0.001 and 0.05), while the remaining constructs tested showed very low efficiency (p<0.001), with no complementation in any transformed line generated using AthSPT::PabSPT. Again, the N-terminally deleted versions of AthSPT::AthPIF5 and AthSPT::SmoPIF7/SPT were more efficient at complementing the fruit mutant phenotype than the unmodified versions, though the difference was not significant in the latter case.

These results indicate the in-vivo biological activity of the SPT coding sequence in the Arabidopsis gynoecium to be broadly conserved with widely diverged proteins from Group 24 of the bHLH family.

Though some differences in complementation efficiency were observed between constructs, these did not appear to correlate with any given level of phylogenetic distance from Arabidopsis SPT. The results obtained using AthPIF5 are interesting and support the conclusion of [START_REF] Reymond | A Light-Regulated Genetic Module Was Recruited to Carpel Development in Arabidopsis following a Structural Change to SPATULA[END_REF] that the rescue of Arabidopsis spt mutants grown under low red/far-red light ratios may depend in part on this gene.

In general, the results of these complementation experiments do not indicate a level of phylogenetic distance within bHLH Group 24 genes in angiosperms at which the rescue of Arabidopsis spt mutants is no longer possible, and therefore do not help to establish a stage during land plant evolution at which the SPT lineage might have acquired the biochemical properties needed to play its current developmental role in the Arabidopsis gynoecium. Indeed, these results are consistence with the hypothesis that almost any coding sequence from the PIF7/SPT clade in vascular plants, or even perhaps from the entire Group 24 of bHLH proteins, might be able to replace the function of AthSPT in transgenic Arabidopsis, providing it is appropriately expressed from the AthSPT promoter.

SPATULA orthologs show similarities in gynoecium expression patterns between widely diverged angiosperms, though strong expression in the style and replum appears specific to Brassicaceae.

The results of the above in-vitro and in-vivo analyses indicate that the biochemical properties of SPT proteins have been largely conserved in vascular plants for over >400 MY, and therefore suggest that evolutionary changes to the SPT coding region may not have been involved in the recruitment of SPT to gynoecium development. These considerations led us to ask whether the role of SPT in the gynoecium might have originated from a change in its expression pattern. We therefore tested the expression of SPT genes in the asterid (eudicots) model Petunia axilaris, and in the ANA-grade (basal)

angiosperms Amborella trichopoda and Nymphaea thermarum.

Petunia axillaris contains two SPT-like lineages which we refer to here as PaxSPT (orthologous to previously identified Solanaceae SPT genes; Ines [START_REF] Ortiz-Ramirez | Expression and function of the bHLH genes ALCATRAZ and SPATULA in selected Solanaceae species[END_REF] and PaxALC (orthologous to previously identified Solanaceae ALC genes; Ines [START_REF] Ortiz-Ramirez | Expression and function of the bHLH genes ALCATRAZ and SPATULA in selected Solanaceae species[END_REF]. PaxSPT is expressed in the L1 cell-layer of both the floral meristem and stamen primordia (Fig. 7A-B), with a peak of expression also at the centre of the early gynoecium in the region where the placenta will form (Fig. 7B-C). PaxSPT is also very highly expressed in the L1 cell-layer of the inflorescence meristem and in deeper cell layers in the central zone of this structure (Fig. 7A). At later stages (Fig. 7D-F AtrSPT, the SPT ortholog from the dioecious basal angiosperm Amborella trichopoda, is expressed in the stamens of male flower buds, particularly in the zones that will form the anther loculi (Fig. 8A-B).

In female buds, AtrSPT is expressed very strongly in carpel primordia and moderately in young tepals (Fig. 8C). Strong AtrSPT expression can be observed at later stages (Fig. 8D-E) in the ovule, and in the adaxial tissues of the carpel wall that line the route of pollen-tube growth from the stigmatic crest to the micropyle of the ovule. NthSPT, the SPT ortholog from the basal angiosperm Nymphaea thermarum, is expressed during the formation of the anther loculi (Fig. 8F) and in the L1 cell-layer of all floral organs (Fig. 8F-G). NthSPT is also strongly expressed adaxially in the gynoecium and in the placenta and developing ovules (Fig. 8F-G).

Important aspects of SPT expression are thus conserved between the three widely-diverged angiosperms studied here, and with the well-documented expression of SPT in Arabidopsis (Heisler et al., 2001;Groszmann et al., 2010). In particular, SPT orthologs appear highly expressed in anther loculi, ovules and adaxial tissues of the carpel/gynoecium. Interestingly, the very strong expression of SPT in the transmitting tissue in Arabidopsis is not matched by similar levels of expression of the two SPT-like genes in Petunia axilaris. Both of the basal angiosperms studied in the present work lack specialized transmitting tissue, so no direct comparison with core eudicots is possible on this point.

Promoters of SPT orthologs show diverse activities in transgenic Arabidopsis.

The in-situ hybridization studies described above, combined with published data, revealed both similarities and differences between the expression patterns of SPT orthologs in widely diverged angiosperms. We were curious to know whether apparent differences, such as the much higher expression of SPT in the stylar tissues in Arabidopsis compared with other species, were due to differences between SPT-promoter sequences, or to some other factor. To investigate this question, we decided to test the activity of SPT promoters from a range of species in transgenic Arabidopsis. An important caveat of this work is that observed differences in expression between promoters in heterogeneous systems may be complex to interpret with regard to promoter evolution: the promoter sequences may have diverged between taxa, but so too may trans-acting components that interact with them. By contrast, similar expression patterns generated by orthologous promoters in a heterologous system may provide useful information, pointing in particular to a likely conservation of activity in the promoters under comparison.

Promoter activities of SPT orthologs from three species representing diverse seed plants were tested using GUS reporter-gene expression in transgenic Arabidopsis, together with a positive-control construct containing the 6.3-kb upstream sequence of AthSPT (Groszmann et al., 2010). The control construct generated very high reporter activity in the style and extending down into the replum within the ovary (Fig. 9A-B, Tab. 3), in good agreement with the known expression pattern of AthSPT (Heisler et al., 2001;Groszmann et al., 2010). A reporter construct containing the promoter region of PaxSPT from Petunia axiliaris also generated strong gynoecium expression in transgenic Arabidopsis (Fig.

9C-D, Tab. 3), but the precise pattern generated by this construct was in contrast to that of the control, with generalized expression in the valve tissues and no visible expression in the style. The upstream sequence of PabSPT, the likely SPT ortholog from the gymnosperm Picea abies, generated very high reporter-gene activity in sepals and at the base of pedicels, but no detectable reporter expression in the gynoecium (Fig. 9E-F, Tab. 3). Finally, the orthologous promoter from the NcaSPT gene of the basal angiosperm Nymphaea careulea, a very close relative of N. thermarum from the same subgroup of African waterlilies, generated a low level of reporter expression in anthers, but no detectable expression in the gynoecium (Fig. 9G-H, Tab. 3).

Of the three species tested here in heterogeneous combinations, none showed SPT-promoter activity that was very similar to that of the native Arabidopsis SPT promoter. Arguably, the reporter-gene expression generated by PaxSPT was most similar to that of Arabidopsis, as strong expression in the gynoecium was noted in both species, though the precise tissues-specific patterns observed were clearly distinct. The results of these experiments are not easy to interpret, but seem broadly to indicate that SPT promoters have likely undergone evolutionary changes of significance to gene expression patterns during angiosperm evolution, both before and after the origin of the core eudicots. We considered from this tentative conclusion that it would be interesting to compare SPT-promoter sequences in detail to identify elements that may account for the similarities and differences observed in SPT-expression between taxa.

Comparison of SPT promoters in core eudicots reveals novel motifs and structural rearrangements of potential functional significance.

To attempt to identify conserved, functional elements of SPT promoters, we compared promoters from 20 species of core eudicots, including seven species of Solanaceae, five species of Brassicaceae, and eight species of other rosids. The use of MEME software identified ten motifs shared between two or more of the species under comparison (Fig. 10). The Brassicaceae species analyzed showed a particularly striking similarity in the spatial arrangement of conserved motifs (Fig. 11). We found Solanaceae SPT promoters to share numerous conserved motifs with Brassicaceae. For example, the P. axillaris SPT promoter shares seven of the ten motifs identified in Arabidopsis, lacking only Motifs 2, 7 and 9 (Fig. 11). Groszmann et al. (2010) used a series of deletions of the Arabidopsis SPT promoter to define the functions of conserved promoter regions. Though some additional enhancers and suppressers were found in upstream regions, reporter expression in the transmitting tissue and replum was lost when the SPT promoter was shortened from -260 bp to -180 bp, thus removing a motif containing a G-box (CACGTG) that partially overlapped with an auxin response element (AuxRE, TGTCTC). A similar combined G-box/AuxRE motif present at -68 bp in the Arabidopsis SPT promoter was found by Groszmann et al. (2010) to be necessary for reporter-gene expression in the silique dehiscence zones. These two hybrid motifs identified by Groszmann et al. (2010) were identified in the present study as Motifs 1 and 3 (Fig. 10). These two motifs were found to participate in an ordered cluster of Motifs 1-6-7-3 (from upstream to downstream), situated near the transcriptional start site of almost all Brassicaceae SPT genes (Fig. 11). Interestingly, three of these four motifs, Motifs 1, 3 and 6, are also found in a closely clustered formation in four of the five Solanaceae SPT promoters analyzed. However, this cluster occurs in Solanaceae SPT promoters in the order 6-1-3 (rather than the 1-6-(7)-3 order of Brassicaceae). This Solancaeae cluster is also typically augmented by the presence of Motif 4 near its proximal end. Motif 4 is universally present too in the Brassicaeae promoters analyzed, but in much more variable positions, and in most cases far upstream of the well-conserved cluster of Motifs 1-6-7-3 near the transcriptional start site. Motif 7, present in Brassicaceae but absent in Solanaceae SPT promoters, contains a CAAG box, already identified by Groszmann et al. (2010). It is worth noting, however, that despite the absence of this element, the Pax_SPT promoter from Petunia axillaris is capable of directing strong reporter expression in the gynoecium of transgenic Arabidopsis, albeit with a different tissue-specific pattern to the native Arabidopsis SPT promoter (Fig. 9C-D).

Mutant analyses provide inconclusive data on the biological role of SPT-like genes in Petunia.

To investigate the biological role of SPT-like genes in Petunia hybrida (a hybrid species from a cross reach a definitive answer.

Discussion

The origin of the SPATULA lineage in vascular plants.

We have traced the likely origin of the SPT lineage to a common ancestor of euphyllophytes (ferns+seed plants; Fig 1). We confirm the findings of [START_REF] Reymond | A Light-Regulated Genetic Module Was Recruited to Carpel Development in Arabidopsis following a Structural Change to SPATULA[END_REF] that a defining characteristic of SPT-clade proteins appears to be the loss of an N-terminal active phytochromebinding (APB) domain, present in their close relatives the PIF proteins, which is responsible in the latter for interactions with the active form of phytochrome photoreceptors (Fig. 2A-C). In the more distantly related lycophytes, a putative pro-ortholog of SPT is equally closely related to the PIF7 lineage, and encodes an N-terminal APB-like domain (Fig. 2D). We provide evidence that the biochemical characteristics of Arabidopsis SPT can be replaced by a high proportion of SPT coding sequences from diverse land plant taxa (Figs 5 and6), suggesting that the SPT protein in Arabidopsis has not acquired and retained significant novel biochemical capacities since its distant ancestor in the MRCA of euphyllophytes. This conclusion is supported by the observation that SPT proteins from the basal angiosperm Amborella and the gymnosperm Picea both bind preferentially to G-box-containing motifs (Fig. 4), as does SPT in Arabidopsis. Furthermore, the SPT protein from Amborella is capable of equivalent protein-protein interactions to those shown by Arabidopsis SPT (Fig. 3). The downstream pathways regulated by Arabidopsis SPT, as assessed by the presence of high-affinity SPT-binding sites in putative target promoters, appear also to have been largely conserved, at least since the MRCA of living seed plants (Tab. 1, SI. Tab 2). [START_REF] Groszmann | Functional domains of SPATULA, a bHLH transcription factor involved in carpel and fruit development in Arabidopsis[END_REF] identified two domains outside the bHLH domain that supported SPT functions in transgenic Arabidopsis: an amphipathic helix and an acidic domain. These domains were identified as specific to SPT/ALC-like proteins from eudicots, being absent in SPT orthologs from monocots. The acidic domain identified by [START_REF] Groszmann | Functional domains of SPATULA, a bHLH transcription factor involved in carpel and fruit development in Arabidopsis[END_REF] is, however, encoded in most SPT orthologs/pro-orthologs including those from basal angiosperms, gymnosperms, and the lycophyte Selaginella moellendorffii, as well as in other PIF genes such as AthPIF5 (Fig. 2F). The amphipathic helix of eudicot SPT orthologs is less easy to recognize in other taxa (Fig. 2G), but it is possible that a structurally similar region, or several regions split over different parts of the protein molecule, might functionally replace this amphipathic helix in non-eudicot SPT and related proteins and thus contribute to the in-vivo functional equivalence of these with Arabidopsis SPT, as observed in the present work (Figs 5 and 6).

When and how did the role of SPATULA arise in the development of carpel margin tissues?

In Arabidopsis, SPT controls the development of tissues arising from the carpel margin meristem (CMM), including the pollen-transmitting tissue of the style and adaxial replum. Solanaceae species also contain a specialized transmitting tissue in the style. However, SPT-like genes are much less strongly expressed in these tissues in Solanaceae, compared to Arabidopsis, and studies using VIGS [START_REF] Ortiz-Ramirez | Expression and function of the bHLH genes ALCATRAZ and SPATULA in selected Solanaceae species[END_REF] and mutant approaches (Fig. 12) have failed to show any clear phenotype associated with loss of SPT function in these tissues. It therefore seems likely that the SPT lineage was recruited to the development of CMM tissues in a common ancestor of Brassicaceae, and that it does not share this role in widely diverged angiosperms such as asterids or basal angiosperms.

The replum of Arabidopsis is a false septum that grows from the carpel margins to divide the ovary into two loculi. The ovary of Petunia (Solanaceae) also contains two loculi, but these are separated by congenitally fused ovary-wall tissues, rather than a false septum. Thus, the anatomy of the typical Brassicaceae gynoecium is highly specialized, and it seems highly plausible that modifications to the role of SPT could have been involved in the evolution of this family-specific anatomical arrangement.

Our study suggests Arabidopsis SPT to possess few if any unique biochemical properties, compared to its orthologs from diverse vascular plants, and also that its downstream genetic pathways and protein-protein interactions are largely conserved, at least within angiosperms. We therefore propose that a change to SPT's expression pattern was likely responsible for its recruitment to CMM development in Brassicaceae. Solanaceae SPT promoters in transgenic Arabidopsis fail to induce high levels of reporter-gene expression in the style and replum, though are highly active in other floral tissues, suggesting that changes to the SPT promoter in a common ancestor of Brassicaceae may have been responsible for the evolutionary acquisition of novel functions in the style and replum. This hypothesis might be tested by making subtly modified versions of a Solanaceae SPT promoter to determine which modifications might produce high levels of reporter expression in the style and replum of transgenic Arabidopsis.

We show SPT to be very strongly expressed in the placentae, ovules and abaxial regions of the gynoecium in Petunia and in the basal angiosperms Amborella and Nymphaea. However, the functional significance of this expression is unclear as mutant and VIGS approaches have not identified any clear phenotypes associated with the loss of SPT function in these tissues in Brassicaceae or Solanaceae models (Alvarez & Smyth, 1999;Groszmann et al., 2011;[START_REF] Ortiz-Ramirez | Expression and function of the bHLH genes ALCATRAZ and SPATULA in selected Solanaceae species[END_REF].

Given our findings (Figs 5 and6) and those of [START_REF] Reymond | A Light-Regulated Genetic Module Was Recruited to Carpel Development in Arabidopsis following a Structural Change to SPATULA[END_REF] relating to the biological equivalence between SPT and certain PIF proteins, it could be that SPT-like genes are acting redundantly with PIFs in these tissues of high expression which have so-far revealed no mutant phenotypes.

The origin of ALCATRAZ lineages in Brassicaceae and asterids.

There is currently in the literature a debate over the relationship between genes named "ALCATRAZ" (ALC) in Brassicaceae, compared to those in distantly-related core-eudicot taxa such as Solanaceae.

Groszmann et al. ( 2011) considered the Arabidopsis ALC lineage to have arisen by a duplication of the pre-existing SPT lineage within Brassicales (more precisely in a common ancestor of Brassicaceae, Cleomaceae and Capparaceae), and this conclusion is in agreement with the results of recent Bayesian phylogenetic analyses (Pfannebecker et al., 2017). Considerations of synteny suggested (Groszmann et al., 2011;[START_REF] Bowers | Unravelling angiosperm genome evolution by phylogenetic analysis of chromosomal duplication events[END_REF], in agreement with the above findings, the SPT/ALC duplication to have corresponded to the At-β whole genome duplication [START_REF] Bowers | Unravelling angiosperm genome evolution by phylogenetic analysis of chromosomal duplication events[END_REF] However, the Maximum Likelihood phylogeny presented here (Fig. 1) is in agreement with the conclusions of Groszmann et al. (2011), and with the Bayesian analyses of Pfannebecker et al. (2017).

We do not consider it likely that LBA could have pushed the Brassicaceae ALC lineage to an erroneously terminal position in molecular phylogenies, as LBA tends to have the opposite effect of pushing fast-evolving lineages to erroneously basal positions. Synteny between duplicated blocks of Arabidopsis chromosomes has already been used to support the origin of the AthSPT and AthALC lineages within Brassicales (Groszmann et al., 2011). It may soon be possible to use a more extensive combined phylogeny+synteny approach [START_REF] Murat | Reconstructing the genome of the most recent common ancestor of flowering plants[END_REF] to clear up any remaining questions relating to the origin of SPT/ALC-like genes in diverse core eudicot groups.

Materials and Methods

Database-searching, phylogenetic reconstructions and DNA-sequence analyses.

Sequences of interest were identified by TBLASTn [START_REF] Altschul | Gapped BLAST and PSI-BLAST: a new generation of protein database search programs[END_REF]

Protein Binding Microarray analyses

Coding sequences of SPT orthologs were inserted into the pDEST-TH1 expression vector as translational fusions to a Maltose Binding Protein domain, and the resulting plasmid was transferred to E. coli BL21 cells [START_REF] Franco-Zorrilla | DNA-binding specificities of plant transcription factors and their potential to define target genes[END_REF]. Production of recombinant protein was induced by adding isopropyl β-D-1-thiogalactopyranoside (IPTG, 1 mM) to log-phase cultures and confirmed through SDS-PAGE analysis of cell lysates. The resulting recombinant proteins were analyzed on PBM11 protein binding microarrays (PBMs), as described by [START_REF] Godoy | Improved protein-binding microarrays for the identification of DNA-binding specificities of transcription factors[END_REF] and the resulting data analyzed as described by [START_REF] Berger | Universal protein-binding microarrays for the comprehensive characterization of the DNA-binding specificities of transcription factors[END_REF] to produce position weight matrices (PWMs)

describing DNA-binding preferences, which were converted to diagrams using ENOLOGOS [START_REF] Workman | EnoLOGOS: a versatile web tool for energy normalized sequence logos[END_REF]. PWMs were also used to scan promoter sequences in RSAT MotifScan [START_REF] Nguyen | RSAT 2018: Regulatory sequence analysis tools 20th anniversary[END_REF]. Numbers of promoters containing one or more high-scoring binding sites were compared in R-software between sets of putative targets promoters and full genomic promoter sets using the hypergeometric test, as described in SI Tab. 3.

GUS reporter gene analysis

Promoter sequences of SPT orthologs were PCR-amplified using high-fidelity thermostable polymerases and primers given in SI Tab. 5. The resulting molecules were inserted by recombination in the pENTR/D/TOPO (Invitrogen) vector and sequenced using automated Sanger DNA sequencing reactions to ensure the absence of mutations. Promoters were then inserted by Gateway LR (Invitrogen) recombination reactions into the pKGWFS7.0 GUS-expression vector and the resulting plasmids transferred by electroporation to Agrobacterium tumefaciens C58PMP90 cells for plant transformation, as described above. Flower tissues of T1 transformants were incubated for 20 min on ice in acetone (90% v/v), rinsed for 10 min in sodium phosphate buffer (0.1 M, pH 7.0), and then transferred to staining solutions containing 5-bromo-4-chloro-3-indolyl-β-glucuronique acid (X-Gluc, 1 mM), potassium ferrocyanide (0.5 mM), potassium ferricyanide (0.5 mM), EDTA (5 mM), Triton X100 (0.05% v/v) and sodium phosphate buffer (0.1 M, pH 7.0) at 37°C for 24 to 48h, depending on the extent of staining observed. Samples were then rinsed and stored if necessary at 4°C in sodium phosphate buffer (0.1 M, pH 7.0) prior to examination and imagery using a Keyence VHX-900F digital microscope.

Genetic complementation assays

Coding sequences of interest were amplified by standard RT-PCR procedures employing hi-fidelity, thermo-stable DNA polymerases and primers shown in SI Tab. 6. The resulting DNA molecules were inserted by recombination into the pENTR/D/TOPO vector (Invitrogen) and sequenced in automated Sanger sequencing reactions to verify the absence of mutations. Two different 5'-primers were used to amplify each of AthPIF5 and SmoPIF7/SPT, one of which caused a 5'-truncation of the APB domain. The 6.3 kb AthSPT promoter fragment (Groszmann et al., 2010) was ligated into the pENTR5'TOPO vector (Invitrogen). The AthSPT promoter fragment and each required coding sequence were then transferred to pK7m24GW plasmids [START_REF] Karimi | Building blocks for plant gene assembly[END_REF] using a Multisite

Gateway LR reaction (Invitrogen) to generate the required plant transformation vectors. Homozygous spt-11 mutant Arabidopsis plants were transformed using the resulting plasmids, as described above.

Phenotypes were analyzed in T1 transformants. Numbers of plants in which mutant phenotypes were complemented were analyzed to derive p-values using the two-tailed version of Fisher's Exact Test.

Images of transformed plants were obtained using both a Keyence VHX-900F digital microscope, and a HIROX-3000 environmental scanning electron microscope, the latter using a stage-temperature of -10°C to -20°C and a tube-voltage of 10 Kv.

In-situ hybridization

In-situ hybridizations to Petunia axillaris and Nymphaea thermarum flower buds were performed using the protocol of [START_REF] Morel | The Floral C-Lineage Genes Trigger Nectary Development in Petunia and Arabidopsis[END_REF], while those to Amborella trichopoda flower buds were performed according to [START_REF] Vialette-Guiraud | Cabomba as a model for studies of early angiosperm evolution[END_REF]. For both procedures, dioxygenin-labelled riboprobes were prepared from PCR-amplified, full-length coding sequences, as described by [START_REF] Vialette-Guiraud | Cabomba as a model for studies of early angiosperm evolution[END_REF]. Images were captured using an Imager-M2 fluorescence microscope (Zeiss)

fitted with an AxioCam MRc digital camera (Zeiss).

PERSPECTIVES

Discussion and perspectives

Identification of genetic modules conserved between two divergent species for evo-devo studies of angiosperms.

The main goal of this thesis has been to study the molecular basis of carpel evolution. In the first of the manuscripts included in the thesis, I identified and compared genetic modules from two key species for the study of angiosperm evolution: the basal angiosperm Amborella trichopoda and the model angiosperm Arabidopsis thaliana. We showed that it was possible to compare the contents and expression patterns of genetic modules between these very distant species and identify modules that have been conserved since the evolutionary separation of the two lineages concerned. The methodology that we developed in this pilot study shows promise, and could perhaps be developed further to make a valuable contribution to macroevolutionary studies of the origin of the carpel and other evo-devo topics.

We identified in this study the importance of maximizing the compatibility of datasets to be compared, and we also highlighted difficulties in selecting equivalent samples from very diverse species due both to uncertain homology between organ systems and to heterochrony. Though this type of analysis can be carried out, as in the present work, using a mixture of data obtained from diverse studies, it may frequently be better to use custom data, specifically obtained for the purpose, by applying equivalent sampling and bioinformatics procedures to all species to be compared, as was done in the study of [START_REF] Kivivirta | A protocol for laser microdissection (LMD) followed by transcriptome analysis of plant reproductive tissue in phylogenetically distant angiosperms[END_REF]. To extend our present pilot study further, it may be useful to include in future analyses a wider range of flower developmental stages for each species, and perhaps also to include more species in the analysis, conditionally on the availability of the appropriate sequenced genomes.

Using such an approach, we envisage that it may be possible to track changes in GRNs across angiosperm evolution and determine the composition of the carpel GRN at any node of interest in angiosperm phylogeny. It would also be useful to extend this type of analysis to gymnosperms. Finally, as the molecular regulation of flower development in distantly related species will show differences related to different types of anatomical organization, it will be important to include histological analyses of flowers from the chosen species to gain full insight into the evolution of the genetic modules which regulate floral organ development.

The role of SPATULA in the gynoecium and its evolution in angiosperms SPATULA may have played a role in the female reproductive tissues of the MRCA of living angiosperms, as suggested by the conservation of strong SPT expression in the placentae and ovule primordia of the ANA-grade species Amborella tricopoda and Nymphaea thermarum, as well as in numerous core-eudicot models such as Arabidopsis thaliana and Petunia spp. However, none of the core-eudicot models so far investigated have revealed a function associated with this very clear and highly conserved peak of gene expression in ovules and placentae. At present, we lack functional approaches to perform the equivalent experiments in ANA-grade species. The probability is not high of developing in the near future functional genetics approaches in Amborella. By contrast, N.

thermarum offers many advantages as a potential functional-genetics model. The recent publication of the N thermarum genome [START_REF] Povilus | Water lily (Nymphaea thermarum) genome reveals variable genomic signatures of ancient vascular cambium losses[END_REF] should facilitate the task of genetically modifying this species, maybe using CRISPR-Cas9 technologies (Mao et al., 2019).

The current role of SPT in tissues such as the style, stigma and transmitting tract of A. thaliana seems to be a recent acquisition, probably near the base of Brassicaceae. Based on the results obtained in the present work, we can suggest that the acquisition of this role by SPT occurred through changes to its expression pattern, probably at least in part brought about by changes to the SPT promoter itself. In our promoter analyses, we identified ten motifs grouped in highly conserved regions within Brassicaceae SPT promoters, most of which coincide with regions of high sequence conservation previously reported by Grozsmann et al. (2010) (SI Figure 3). Interestingly, seven of these ten motifs are present also in SPT promoters of Solanaceae species. However, in Solanaceae species, these conserved motifs are located at very variable distances from the start codon, compared to the arrangement in Brassicaeae, and several of them do not occur in the same order or close juxtaposition as they do in Brassicaceae spp. Similar distinctions in SPT-promoter structure are noted when comparing other rosids with Brassicaceae.

To test the functional significance of the above observations, it would be very interesting to make native and rearranged versions of the SPT promoters from a range of angiosperm species such as Nymphaea thermarum (ANA-grade, Nymphaeales, Nymphaeaceae), Petunia axillaris (core-eudicots, asterids, Solanaceae) and Carica papaya (core-eudicots, rosids, Caricaceae), which show varying levels of similarity in SPT-promoter structure with Arabidopsis. Such experiments could identify any precise changes in promoter structure which were linked to the acquisition by Arabidopsis SPT of its current role in style, stigma and replum tissues.

Although VIGS approaches have not identified any clear phenotypes associated with the loss of SPT function in Solanaceae species [START_REF] Ortiz-Ramírez | Expression and function of the bHLH genes ALCATRAZ and SPATULA in selected Solanaceae species[END_REF], it would be interesting to continue with the analysis of mutants in Petunia, begun in the present study, perhaps by adding other genetic backgrounds and testing all allelic combinations in each of them, in order to reliably confirm the absence of a carpel phenotype linked to SPT and ALC functions.

The position of SPATULA orthologs in gene-expression modules identified using WGCNA.

The second manuscript included in this thesis focused on SPT, and so it is logical to ask where SPT orthologs were to be found in the results obtained from the analysis of gene-expression modules performed in the first manuscript of this thesis. For reasons that we do not know, Arabidopsis thaliana SPT, its paralog AthALC, and their mutual pro-ortholog AtrSPT (AM_TR_V1_scaffold00046.26) from Amborella trichopoda, were not placed in an orthogroup in the global analysis of gene orthology performed in PHYTOZOME (SI Tab 1 of Manuscript/Part 1 of this thesis). However, these three genes were present in the Arabidopsis and Amborella transcriptome data analyzed in our WGCNA gene-clustering procedures. According to these analyses, AthSPT (AT4G36390) and AthALC This observation is perhaps unsurprising as the expression data presented in the second manuscript of this thesis (Manuscript/Part 2, Fig. 8) shows that AtrSPT's expression in Amborella flowers differs in many respects from the published expression profiles of both of its orthologs in Arabidopsis (Groszmann et al., 2010(Groszmann et al., , 2011;;Heisler et al., 2001). This result from gene-clustering (Manuscript 1) is thus consistent with the conclusion of Manuscript 2 that the SPT lineage adopted novel expression profiles and functions in a common ancestor of Brassicaeae, long after the divergence of the Arabidopsis and Amborella lineages in the MRCA of living angiosperms.

The future of carpel evolution studies

To continue evolutionary studies of the carpel and floral organs in general, it will be interesting to concentrate efforts in species that occupy key phylogenetic positons such as Nymphaea thermarum, which additionally shows potential as a possible functional-genetic model. If stable transform of N. thermarum becomes possible, an interesting approach to test would be Interspecies Gene Transfer (IGT) [START_REF] Nikolov | Interspecies Gene Transfer as a Method for Understanding the Genetic Basis for Evolutionary Change: Progress, Pitfalls, and Prospects[END_REF]. In this method, entire genetic loci of interest, including non-coding regulatory elements, are transferred between species. In this way, both regulatory and coding sequences can be analyzed to study molecular changes that lead to evolutionary events. Beyond the genetic transformation of new model species, we could also use transcriptomic approaches to study the regulatory network of flower development in a range of both model and non-model species. The laser-capture microdissection method used in Manuscript 1 of the present work, followed by RNAseq, and/or single-cell RNA sequencing techniques, could also be used in N. thermarum and other species occupying key phylogenetic positions.

Studies of individual genes (rather than transcriptomics approaches) will also be very important to further elucidate the molecular basis of floral-organ evolution. In the case of the carpel, there are many further candidate genes to be analyzed, such as the YABBY gene CRABS CLAW (CRC), which has a carpel-specific expression profile in A. thaliana (J. Alvarez & Smyth, 1999;[START_REF] Alvarez | CRABS CLAW and SPATULA Genes Regulate Growth and Pattern Formation during Gynoecium Development in Arabidopsis thaliana[END_REF]. YABBY members are expressed in the abaxial side of lateral organs [START_REF] Siegfried | Members of the YABBY gene family specify abaxial cell fate in Arabidopsis[END_REF]. In A. thaliana, CRC is expressed specifically in the abaxial domain of the carpel wall (John [START_REF] Alvarez | CRABS CLAW and SPATULA Genes Regulate Growth and Pattern Formation during Gynoecium Development in Arabidopsis thaliana[END_REF], and previous work shows that CRC conserves this expression pattern in ANA-grade angiosperms [START_REF] Fourquin | Functional Conservation between CRABS CLAW Orthologues from Widely Diverged Angiosperms[END_REF][START_REF] Fourquin | Evidence that CRABS CLAW and TOUSLED have conserved their roles in carpel development since the ancestor of the extant angiosperms[END_REF], and was most probably part of the carpel gene regulatory network of the MRCA of flowering plants (Pfannebecker et al., 2017b).

It furthermore seems that the origin of the CRC lineage coincides with the origin of angiosperms, after the Epsilon whole genome duplication event [START_REF] Becker | A molecular update on the origin of the carpel[END_REF].

However, there are still unanswered questions surrounding CRC and its contribution to carpel origin and evolution. For instance: which other YABBY lineage is paralogous to CRC, what confers on CRC its specificities in carpel development, and how were these acquired? Further pertinent questions include: what are CRC's direct target genes, and how were these recruited? Similar questions can be asked of many further genes of importance to the carpel GRN. Clearly, as we continue in the evolutionary study of flowering plants, integrative approaches will be necessary to better comprehend the contribution of each candidate gene to carpel origin and evolution. These approaches will need to integrate many levels of study, from morphological analyses and structural biology to the epigenetic mechanisms that operate on gene expression to bring about developmental processes.

  Thanks to my Magical Scientific Advisors, Gwyneth Ingram and Francois Parcy from Comité de Suivi de Thèse, their wisdom has guided my work and has helped me grow as a researcher and as a person. And thank you to my jury members: Cristina Ferrandiz Maestre, Annette Becker, James Tregear and François Roudier, who have kindly agreed to review my work. Special mentions to: All the scientific collaborators of my work. Those from other laboratories (IPS2-Institute of Plant Sciences Paris-Saclay; UMR 1318 INRAe-AgroParisTech; Centro Nacional de Biotecnología-Consejo Superior de Investigaciones Científicas), they have been an indispensable part of my thesis advancement.

  According to this work, AG-and SEP-like genes were present in the ancestral flower and specified carpel identity, together with CRC. In early angiosperms, adaxial-abaxial polarity was already under the control of ETT and CRC, in combination with other transcription factors. ETT, SPT and HEC probably acted together to control apical/basal polarity in the carpel. The regulation of median and apical tissues was probably brought about through the action of ETT, LEUNIG (LUG), SEUSS (SEU), STYLISH (STY) and NGATHA (NGA), in combination with CRC and the bHLH factors SPT and HEC (Figure9).

  important external reference point to study the evolutionary processes that led to the origin of the flower. Even if many of the traits of the typical gymnosperm reproductive axis differ from those of angiosperms, numerous orthologs of floral development genes are known to be conserved in gymnosperms. Analyses of the molecular mechanisms of male and female cone development in gymnosperms are possible using a number of methods. The techniques available include in situ hybridization, in-vitro studies of the protein-protein and protein-DNA interactions involving transcription factors, and in-vivo functional analyses by the complementation of model-angiosperm mutants by genetic transformation using gymnosperm coding sequences. Also very useful is the increasing availability of gymnosperm transcriptomic data, in particular datasets from reproductive structures.

5.

  In-vitro and heterogeneous in-vivo methods: Questions in evolution and development of the flower could be answered by combining methods that measure protein-DNA or protein-protein interactions with genomic-scale analyses and modelling approaches. Such approaches could be used to describe the networks that controls reproductive development at the posttranscriptional, transcriptional and epigenetic levels. Such methods, which do not depend on functional-genetic capabilities, can help to elucidate the evolution of developmental regulatory mechanisms in non-model plants(Vialette-Guiraud et al., 2016a) that are important for the study of angiosperm origin. 6. "Protein Resurrection": ancestral sequence reconstruction from key stages in plant evolution, can be used to study the biophysical and biochemical properties of ancestral molecules that form part of the regulatory network of the reproductive axis.

  from answering the question of how the flowering plants first appeared. There remains some controversy on the characteristics of the ancestral flower, and much uncertainly concerning the gymnosperm group from which the flowering plants emerged. Also, little is known of the molecular changes that were required for the origin of angiosperms.In this work, I focus on the origin and evolution of female reproductive tissues of angiosperms. The central question I address is: "what is the molecular basis of the evolution of the carpel?" In attempting to answer this question, I have divided my work two parts, each of them responding to a more specific question derived from the above central question. The questions, aims and strategies of my work are specified in the next section.

2 .

 2 When was the transcription factor SPATULA recruited to its function in the carpel, and what were the molecular changes that occurred to integrate SPT into this molecular pathway? These questions involved two different approaches, and as a consequence, I will present my PhD work in two parts: PART I: in which I analyze transcriptomic data, combining our own unpublished datasets with data available from other work, to explore a new method for inter-species analysis using the R-package, WGCNA. The initial aim of this work was to identify genetic modules that control female reproductive development and are conserved between the ANA-grade angiosperm Amborella trichopoda and the core-eudicot model angiosperm Arabidopsis thaliana.PART II: in which I centered the analyses on one transcription factor that is heavily involved in A. thaliana gynoecium development. In this work, I analyzed the coding and upstream sequences of the bHLH transcription factor SPATULA and its orthologues in diverse plant species.In-vitro and in-vivo experiments were performed to study the interactions and functions of SPT across flowering plants and beyond. These studies aimed to determine the molecular changes which were responsible for the acquisition of SPT's role in the A. thaliana carpel, and when, during angiosperm evolution, these changes occurred.

  this study and on RNA-seq datasets[START_REF] Jiao | Cell-type specific analysis of translating RNAs in developing flowers reveals new levels of control[END_REF] from two stages of Arabidopsis flower development, as defined bySmyth et al. (1990). These Arabidopsis datasets represent the translatome (i.e. the population of mRNAs attached to ribosomes) from three overlapping regions of flower buds in which are expressed, respectively, the A, B-and C-class MADS-box floral homeotic regulators: APETALA1 (AP1, expressed in whorls 1-2), APETALA3 (AP3, expressed in whorls[2][3]

  analyzed their intersections (SI Tab 5) using the hypergeometric test to produce a list of 182 upper-tail p-values representing the probability of each positive (over-represented) intersection arising by chance (SI Tab 6). In Fig3, we illustrate relationships between modules with a highly significant p-value of <0.01, which includes a total of 24 pairwise intersections (dotted lines). At this level of statistical significance, a few modules appear to be not closely related to any module in the other species under comparison, including the closely similar red and tan modules of Arabidopsis. These two modules both show high eigengene expression in the AP3 (B-function)-expression domain, which includes the developing stamens. Stamen or staminode tissues were not sampled from Amborella in our study, which may explain the lack of a closely similar Amborella module to these two Arabidopsis modules.Several other cases exist in our study in which only one of two very similar modules in one species shows a highly significant (p<0.01) intersection with a module from the other. Examples of this phenomenon include the Amborella tan and green modules, both of which are characterized by high eigengene expression in the ovary wall. Of these two modules, only Atr_tan appears closely related (p=0.0028, SI Tab 6) in orthogroup-content to Ath_magenta, which shows high eigengene expression in the central zone of the Arabidopsis flower bud at Stage 6-7 and is thus coherent with the expression of the Atr_tan and Atr_green eigengenes. However, the full list of module-comparisons (SI Tab 6)

  intersecting modules listed in SI Tab 7, other than through their module eigengenes, which are shown in Fig 3.However, it is important to note that several different types of relationship can be found between orthologs from intersecting modules, and between those orthologs and their respective module eigengenes. In Fig.4, we illustrate the main types of relationship observed using three examples from each of two module intersections: Atr_blue vs. Ath_blue, both of whose eigengenes are highly expressed in female reproductive tissues (Fig.4A-C); and Atr_turquoise vs. Ath_black, both of whose eigengenes are highly expressed in perianth tissues (Fig 4D-F). Orthologous genes encoding a DEAD-box RNA helicase (Fig 4A) show high expression in young carpel and ovule tissues in Amborella, and in the AG-expressing central zone of the Arabidopsis flower at both of the developmental stages analyzed. These patterns are therefore in close agreement with the Atr_blue and Ath_blue module eigengenes (Fig. 3). Similarly, orthologous WUSCHEL-like homeobox genes (Fig. 4B) show highly congruent expression patterns in the two species under study, and with their respective module eigengenes. However, orthologous GATA-family transcription factors that group within the Atr_blue and Ath_blue modules (Fig. 4C) show an apparently inverse set of expression dynamics to those of the examples given in Figs 4A and B. This orthogroup clearly fits within the Atr_blue and Ath_blue modules as a negatively-regulated component, and was presumably included by WGCNA in the same intersecting modules as the previous two examples (4A and B) due to the unsigned nature of the clustering procedure.The Atr_turquoise and Ath_black modules both show high eigengene values in perianth tissues, including both of the developmental stages analyzed in Arabidopsis (Fig.3). Similarly to the examples shown in Figs4A and B, orthologs in these two modules, encoding a B7-domain transcription factor (Fig.4D) and a circadian-clock regulated transcript (Fig.4E), show expression patterns very close to each other, and to their respective module eigengenes. By contrast, a third example of an orthogroup shared between the Atr_turquoise and Ath_black modules, encoding a ENTH-domain protein (Fig.4F), reveals a novel set of expression relationships in which the Amborella ortholog shows a similar expression pattern to its Atr_turquoise module eigengene, but the Arabidopsis ortholog shows a broadly opposite expression pattern to those of both the Amborella ortholog and the Ath_black module eigengene. Thus, whereas in the first five examples described above (Figs4A-E), the orthologs of each orthogroup examined show similar expression patterns in their respective species, in this last example the two orthologous genes examined show broadly opposite patterns, the Amborella gene being highly expressed only in the perianth, while the Arabidopsis gene is highly expressed in all domains of the floral bud except the perianth. As for the example shown in Fig 4C,this Arabidopsis ortholog was presumably included in the same module as genes with an opposite expression profile due to the unsigned nature of the clustering procedure. In this case however, and in contrast to that shown in Fig 4C, it appears there has been a change in the expression pattern of one or other of the two orthologs under consideration since the MRCA of living angiosperms.

  ), very high PaxSPT expression persists in the placenta and developing ovules, and is also clearly observed in the L1 cell-layer of the developing stigma, petals and stamens. Weaker expression of PaxSPT is apparent around the loculi of the anthers and in the stylar transmitting tissue. PaxALC is less strongly and widely expressed than PaxSPT (Fig. 7G-L), showing clearest expression in the L1 cell-layer of the gynoecial primordium and at later stages in the L1 cell-layer of the upper style and stigma.

  Groszmann et al. (2010) had already compared three Brassicaceae SPT promoters and defined from these eight conserved regions. Most of the ten motifs identified in the present work fit within the Regions 1, 2, 5 and 6 identified by Groszmann et al. (2010), as shown in SI Fig 3. However, Motifs 4, 9 and 10 are newly identified motifs that occur in a well-conserved cluster between the Regions 5 and 6 of Groszmann et al. (2010).

  including P. axilaris), we identified lines carrying transposon insertions in both the Pax_SPT and Pax_ALC genes. Homozygous single Pax_spt-1 or Pax_alc-1 mutants showed no detectable phenotypic differences to wild-type plants (data not shown). However, a double homozygous mutant containing the same two alleles in the Mitchell genetic background showed a gynoecium phenotype in which the stigmatic surface lacked the normal papillae present in wild-type plants (Fig 12). This phenotype is distinct from the gynoecium phenotype of Arabidopsis spt or spt alc mutants, in which stigmatic tissues may be reduced, but some stigmatic papillae are still typically present. It is, however, in good agreement with the expression patterns of PaxSPT and PaxALC, which overlap in the L1 celllayer in the stigma (Fig 6L). The same two alleles Pax-spt-1 and Pax-alc-1, combined in the W138 (transposon line) genetic background, showed a more subtle stigmatic phenotype in which stigmatic papillae were present, though with some areas of the stigmatic surface devoid of papillae. However, a similar phenotype was observed also in wild-type W138 plants, so this phenotype cannot be ascribed to mutations in PaxSPT and PaxALC. A second mutant allele of each gene was then obtained, Pax_spt-2 and Pax_alc-2, and transferred into both the Michell and W115 genetic backgrounds by genetic crossing. Homozygous double mutants carrying these alleles, however, showed no mutant phenotype.At present, it is unclear whether a third gene may have been mutated, and contributed to the clear stigma phenotype originally observed in the Pax_spt-1 Pax_alc-1 double mutant in the Mitchell background. It is also not sure that all of the alleles investigated produced null mutant phenotypes. It is furthermore possible that environmental conditions (e.g. ambient light conditions) may have had an impact on allelic penetrance and have varied subtly between experiments. These questions will require further careful analyses in several mutant lines and strictly controlled environmental conditions to

  Groszmann et al. (2010) previously showed that a conserved, proximal region of the Arabidopsis SPT promoter, containing closely spaced G-box and AuxRE motifs, was essential to stylar and replum expression. Interestingly, such hybrid motifs are typically present also in the SPT promoters of Solanaceae species, and also occur in clusters, though with a different order and position relative to the proximal end of the promoter. Therefore, we tentatively suggest that structural rearrangements to the SPT promoter, combined with the introduction of a CAAT box, may have been involved in the acquisition by SPT of novel gynoecium expression patterns in a common ancestor of Brassicaceae.

(

  AT5G67110) were placed by WGCNA in the Ath_red and Ath_blue modules, respectively, while AtrSPT was placed in the Atr_turquoise module (Manuscript 1, SI Tab 3). Subsequent statistical analyses using the hypergeometric test gave upper-tail (for positive correlations) p-values of p=0.92 and p=0.63 for comparisons of Atr_turquoise with Ath_red and Ath_blue, respectively (Manuscript 1, SI Tab 6). This means there was no significant positive correlation between either of the modules in Arabidopsis that contained AthSPT and AthALC with the Amborella module that contained AtrSPT.

Table 1 .

 1 Summary of highly significant positive module intersections (p< 0.01) between WGCNA clustering analyses of Amborella and Arabidopsis, ranked by p-value.

							Eigengene
			No. of		Arabidopsis module	Amborella module	expression
	Arabidopsis	Amborella	common		eigengene expression	eigengene	congruent
	module	module	orthologs p-value	>0.25	expression >0.25	between species?
	Ath_turquoise	Atr_yellow	365	2.73E-12 AP1 St. 4	Carpel St. 5-6	N
	Ath_black	Atr_turquoise	245	6.32E-08 AP1 St. 4, AP1 St. 6	Tepal St. 5-6	Y
	Ath_pink	Atr_turquoise	163	6.22E-07 AGs4	Tepal St. 5-6	N
	Ath_magenta	Atr_turquoise	124	3.70E-06 AGs6	Tepal St. 5-6	N
	Ath_purple	Atr_red	23	1.45E-05 AGs4	Carpel St. 5-6	Y
	Ath_yellow	Atr_turquoise	223 0.000102605 AP1s6	Tepal St. 5-6	Y
	Ath_blue	Atr_blue	1310 0.00012267 AGs4, AGs6	Ovule St. L7	Y
	Ath_purple	Atr_magenta	11 0.000266973 AGs4	Ovary St. L7	Y
	Ath_magenta	Atr_black	11 0.000419318 AGs6	Ovary St. L7	Y
	Ath_purple	Atr_turquoise	87 0.000534048 AGs4	Tepal St. 5-6	N
	Ath_pink	Atr_greenyellow	10 0.000622646 AGs4	Carpel St. 5-6	Y
	Ath_salmon	Atr_black	7 0.000622887 AP1s4	Ovary St. L7	N
	Ath_purple	Atr_purple	12 0.000670362 AGs4	Ovary St. L7	Y
	Ath_magenta	Atr_purple	14 0.001492205 AGs6	Ovary St. L7	Y
	Ath_turquoise	Atr_red	202 0.001722988 AP1s4	Carpel St. 5-6	N
	Ath_pink	Atr_black	12 0.001746861 AGs4	Ovary St. L7	Y
	Ath_yellow	Atr_salmon	6 0.001839423 AP1s6	Ovule St. L7	N
	Ath_green	Atr_black	18 0.002593529 AP1s6	Ovary St. L7	N
	Ath_magenta	Atr_tan	6 0.002927334 AGs6	Ovary St. L7	Y
	Ath_salmon	Atr_magenta	7 0.003261332 AP1s4	Ovary St. L7	N
	Ath_yellow	Atr_purple	21	0.0086795 AP1s6	Ovary St. L7	N
	Ath_purple	Atr_cyan	2 0.00931224 AGs4	Ovule St. L7	Y
	Ath_pink	Atr_purple	15 0.009741206 AGs4	Ovary St. L7	Y
	Ath_green	Atr_brown	135 0.009886491 AP1s6	Carpel apex St. L7 N

  1 , Aurélie C. Vialette 1 , Amélie Andres-Robin 1 , Pierre Chambrier 1 , Loïc Bideau 1 , Patrice Morel 1 , Michiel Vandenbussche 1 , Roberto Solano 2 , Jose Manuel Franco-Zorrilla 2 and Charlie P. Scutt 1 * 1. Laboratoire Reproduction et Développement des Plantes, Univ. Lyon, ENS de Lyon, UCB Lyon-1, CNRS, INRAe, F-69342 Lyon, France 2. Centro Nacional de Biotecnología-Consejo Superior de Investigaciones Científicas, Campus Universidad Autónoma, 28049 Madrid, Spain

  within Brassicales.However, more recent studies[START_REF] Pabon-Mora | Evolution of fruit development genes in flowering plants[END_REF][START_REF] Ortiz-Ramirez | Expression and function of the bHLH genes ALCATRAZ and SPATULA in selected Solanaceae species[END_REF] have identified putative ALC orthologs from Solanaceae and other asterids and propose the ALC lineage to have separated from that of SPT in a common ancestor of core eudicots, probably at around the time of the At-γ genome triplication event. According to this latter hypothesis, both the SPT and ALC

	lineages would have been conserved in asterids (including Solanaceae) and rosids (including
	Brassicaceae).
	It has been suggested that long-branch attraction (LBA) in Bayesian phylogenies may have distorted
	the phylogenetic position of ALC genes, specifically in Brassicacae (Ines Ortiz-Ramirez et al., 2019).

  from databases given in SI Tab. 1. Amino-acid alignments were performed with MUSCLE in SeaView[START_REF] Gouy | SeaView Version 4: A Multiplatform Graphical User Interface for Sequence Alignment and Phylogenetic Tree Building[END_REF], using default parameters. Alignments were used to generate maximum likelihood phylogenies in PhyML, employing the LG evolutionary model. Branch support was provided by the aLRT method (Anisimova Arabidopsis thaliana wild-type Col-0 plants, or spt-11 mutants[START_REF] Ichihashi | The bHLH Transcription Factor SPATULA Controls Final Leaf Size in Arabidopsis thaliana[END_REF] in the Col-0 background, were used in genetic transformation experiments. Plants were grown under long-day

	Genetic transformations.
	conditions (18h light/6h dark cycles) at 22°C. Homozygous spt-11 mutants to be used in
	transformation procedures were additionally illuminated using far-red LEDs (24x 1W, 760 nm), from
	five weeks after germination until seed-set, to generate a low red/far-red light ratio that reduced the
	negative effect of the spt-11 mutation on fertility. Plants were transformed by standard "floral dip"
	procedures (Clough & Bent, 1998) and transformants selected on kanamycin (250 µg/L)-containing
	media.
	Transposon-mutant screening.
	Caledonia) and then maintained in Lyon in a greenhouse under conditions of ~70% relative humidity,
	18-30°C, and natural daylight attenuated by artificial shade-covering. Plants of Nymphaea caerulea,
	Selaginela moellendorffii and Picea abies were obtained from commercial sources. RNA was
	extracted from flower tissues for RT-PCR amplifications and other procedures using Trizol Reagent
	(Thermo-Fischer Scientific) according to the manufacturer's instructions. Genomic DNA was
	extracted from leaf/shoot tissues for PCR amplifications using a Nucleon PhytoPure kit (Thermo-
	Fischer Scientific).

[START_REF] Anisimova | Approximate likelihood-ratio test for branches: A fast, accurate, and powerful alternative[END_REF]

. Conserved motifs in promoters and coding sequences were identified using MEME Suite 5.1.1 (http://meme-suite.org/tools/meme). Plant material and nucleic acid extraction. Mutant and wild-type Arabidopsis thaliana seed accessions were obtained from Nottingham Arabidopsis Stock Centre (NASC). Nymphaea thermarum plants were generously donated by Paula Rudall and Carlos Magdalena, Royal Botanic Gardens, Kew, London. Pinus taeda needle material was a kind gift from Charles Dana Nelson, University of Kentucy, Lexington KY, USA. Amborella plants were obtained in 2010 in collaboration with Gildas Gâteblé and Bruno Fogliani (IAC, St Michel, New Petunia hybrida plants were grown under 18h/6h light/dark cycles at 22°C. A transposon mutant collection of P. hybrida W138 was screened for insertions in two SPT/ALC homologues as described by Vandenbussche et al. (2008). Primers used for genotyping, by PCR-amplification across transposon borders, are given in SI Tab. 4. Mutant alleles of interest were backcrossed into Mitchell and WII5 genetic backgrounds and combined by crossing and PCR-screening of F2 populations to detect double mutant homozygotes. A double-mutant homozygote in the W138 (transposon) line was also constructed.

Table 2 .

 2 Complementation of Arabidopsis spt-11 mutants using diverse coding sequences (p-values <0.05 underlined).

			Gynoecium fusion	%	p-value (comparison to pSPT::SPT)	Fruit size	%	p-value (comparison to pSPT::SPT)
	pSPT::SPT	21	21	100	N/A	18	85.7	N/A
	pSPT::AtrSPT	25	20	80	0.0536	11	44	0.0054
	pSPT::NcaSPT	23	21	91.3	0.4894	18	78.3	0.701
	pSPT::PabSPT	25	21	84	0.1142	0	0	<0.0001
	pSPT::PaxSPT	22	19	86.4	0.2326	4	18.2	<0.0001
	pSPT::PIF5	25	19	76	0.0247	13	52	0.026
	pSPT:: ΔPIF5	22	20	90.9	0.4884	13	59.1	0.0883
	pSPT::SmoPIF7/SPT	25	12	48	<0.0001	8	32	0.0003
	pSPT:: SmoΔPIF7/SPT 19	15	78.9	0.0424	6	31.6	0.0009
	pSPT::AthALC	20	19	95	0.4878	16	80	0.6965
	pSPT::PaxALC	16	16	87.5	1	13	81.3	1

Table 3 .

 3 Gus reporter assays in transgenic Arabidopsis

	Promoter fragment	No. of transformants	GUS +	Tissues showing GUS staining
	Ath pSPT_6253 bp (positive control)	17	14	sepals, petals, carpel margins, ovary wall, style, stigma
	Nca pSPT_6277 bp	25	7	young floral bud, anthers
	Pax pSPT_5006 bp	19	11	sepals, ovary wall
	Pta pSPT_1659 bp	25	18	sepals, petals, base of pedicel

Table 4 .

 4 Petunia transposon-mutant analyses.

	Gene	Allele	Position of insertion in coding sequence.
	PaxSPT	Pax_spt-1	437 bp
	PaxSPT	Pax_spt-2	18 bp
	PaxALC	Pax_alc-1	252 bp
	PaxALC	Pax_alc-2	267 bp
	Background	Allele combination	Mutant phenotype
	Mitchell	Pax_spt-1/alc-1	Stigmatic papillae absent
	W138	Pax_spt-1/alc-1	None
	Mitchell	Pax_spt-2/alc-2	None
	W115	Pax_spt-2/alc-2	None

Acknowledgements

This work was supported by ANR grant "ORANGe" (2013-18) and through core funding from CNRS, INRAe, ENS-Lyon and Université Claude-Bernard Lyon-1. ACRS was supported by a BECAL studentship from the Government of Paraguay. We thank Gildas Gâteblé and Bruno Fogliani (IAC, St Michel, New Caledonia) for generously supplying plant material.

Acknowledgements

This work was supported by ANR grant "ORANGe" (2013-18) and through core funding from CNRS, INRAe, ENS-Lyon and Université Claude-Bernard Lyon-1. ACRS was supported by a BECAL studentship from the Government of Paraguay. We thank Gildas Gâteblé, Bruno Fogliani, Paula Rudall, Carlos Magdalena and Charles Dana Nelson for generously supplying plant material.

AIMS AND STRATEGIES

RESULTS

PART I: TRANSCRIPTOMIC DATA FOR CARPEL EVOLUTIONARY STUDIES

Abstract

Gene-expression clustering from transcriptomic data can help elucidate the gene regulatory networks (GRNs) that control developmental processes in model organisms. In parallel, transcriptomic data are becoming available from an ever-increasing number of species that occupy key phylogenetic positions, of potentially great use to evo-devo studies aimed at reconstructing the evolution of developmental GRNs. However, such large-scale datasets are often sub-optimally exploited in evo-devo studies, in part because few methods are yet available to compare gene expression dynamics between distantlyrelated species on a genomic scale, rather than on a simple gene-by-gene basis. Here, we use lasercapture microdissection (LCM) to contribute the first detailed transcriptomic study of female flower development in the likely sister to all other living angiosperms, Amborella trichopoda. We use the data obtained to address two questions: Firstly, can transcriptomic data be used to identify entire genetic modules whose expression in floral tissues has been conserved over deep evolutionary time?

Secondly, to what extent can transcriptomic datasets, generated using diverse methods in independent studies, be usefully compared in plant evo-devo analyses? We use a gene-expression clustering method, followed by a custom procedure to compare the resulting genetic modules on the basis of orthology relationships at the genomic scale between Amborella and the molecular-genetic model angiosperm Arabidopsis thaliana. Using this approach, we have succeeded in identifying 13 pairs of modules that show both a highly significant intersection in orthogroup-content and congruent expression dynamics between the two species under comparison. We conclude that transcriptomic datasets resulting from independent studies can be used efficiently to identify genetic modules conserved at least since the most recent common ancestor of living angiosperms, which is believed to have lived at least 149 million years ago.

Author contributions

ACRS performed most of the bioinformatics work and co-wrote the manuscript, AAR performed the LCM work, ACV collaborated with ACRS in read-mapping and figure preparation, JJ provided advice and supervision in bioinformatics work, ALA performed the library construction and high-throughput sequencing, BD supervised the LCM work, and CPS coordinated the project and co-wrote the manuscript. SI Tab 1. Table of gene-orthology between Amborella and Arabidopsis SI Tab 2. Normalized counts from RNA-seq analyses of laser-microdissected female Amborella flower tissues SI Tab 3. Contents of gene-expression modules in Amborella and Arabidopsis following WGCNA analyses SI Tab 5. Lists of orthogroups in intersections between modules in pairwise comparisons (Arabidopsis vs. Amborella) SI Tab 6. Hypergeometric test of significance of between-species module intersections.

Supplementary information SI Tables

SI Tab 7. Module intersections Ath vs. Atr

SI Methods

SI Methods 1. PERL_CODES

PART II: STUDY OF A TRANSCRIPTION FACTOR INVOLVED IN CARPEL DEVELOPMENT

Abstract SPATULA (SPT) encodes a bHLH transcription factor in Arabidopsis thaliana that plays important roles in the development of the style, stigma and replum tissues of the gynoecium. Though the regulation, expression pattern, interactions and functions of SPT have been well-described in Arabidopsis, comparable data are largely lacking for SPT orthologs in other angiosperm groups. The main objective of this work is to provide evidence for the timing of SPT's recruitment to the development of apical and marginal gynoecium tissues, and to identify molecular changes which might have been responsible for this novel role. For this, we have chosen species that occupy key phylogenetic positions to reconstruct the evolution of the SPT lineage from early vascular plants to present-day Arabidopsis. We situate an early structural change in the SPT protein, which may have been a prerequisite for its current developmental roles, to a common ancestor of euphyllophytes (ferns+seed plants). We also demonstrate widespread conservation in the biochemical capacities of angiosperm SPT proteins, and in the identities of the target-genes they regulate. We report expression differences between SPT orthologs in diverse angiosperm taxa, and suggest elements of SPT-promoter structures which may be linked to these. We conclude the Arabidopsis SPT lineage to have acquired its current role in the style and septum in a common ancestor of Brassicaeae, mainly through changes to its expression pattern, rather than to its coding sequence or to the identities of its direct transcriptional targets. We furthermore attempt to clarify questions of gene orthology between SPTlike sequences from diverse core-eudicot families.

Arabidopsis, Amborella and Picea were overexpressed in E. coli as protein fusions to a Maltose Binding Protein domain, and analyzed using Protein Binding Microarrays [START_REF] Godoy | Improved protein-binding microarrays for the identification of DNA-binding specificities of transcription factors[END_REF] Twenty six target genes have been proposed as direct or immediate targets of Arabidopsis SPT [START_REF] Girin | INDEHISCENT and SPATULA Interact to Specify Carpel and Valve Margin Tissue and Thus Promote Seed Dispersal in Arabidopsis[END_REF][START_REF] Reymond | A Light-Regulated Genetic Module Was Recruited to Carpel Development in Arabidopsis following a Structural Change to SPATULA[END_REF][START_REF] Reyes-Olalde | The bHLH transcription factor SPATULA enables cytokinin signaling, and both activate auxin biosynthesis and transport genes at the medial domain of the gynoecium[END_REF]), all but one of which show Gboxes (CACGTG) in their putative promoter regions. In agreement with this observation, all three topscoring 8-mers for each of the three factors compared were also found to contain G-boxes (Fig. 4).

Previous studies of SPT targets in Arabidopsis have considered entire intergenic regions as potential promoter regions. However, this approach is not feasible in Amborella or Picea as average intergenic distances are much greater in these species than in Arabidopsis. Also, in Picea, the current genome assembly is far from complete and would not, in most cases, be expected to yield full intergenic regions.

To make a direct comparison between the three species under analysis, we therefore defined "promoters" as the 3 kb of sequence immediately upstream of the initiation codon in each gene of interest, regardless of the length (known or unknown) of the intergenic region concerned. Orthologs of putative Arabidopsis SPT-targets from Amborella and Picea were identified using BLAST searches, and their putative orthology was confirmed using Maximum Likelihood phylogenetic analyses in SeaView [START_REF] Gouy | SeaView Version 4: A Multiplatform Graphical User Interface for Sequence Alignment and Phylogenetic Tree Building[END_REF]; results available on request). Promoter sequences were obtained for these orthologs from Phytozome, or from Congenie in the case of Picea. The required 3-kb of upstream sequence could be obtained for all but three of the Picea genes of interest. All sequences were scanned in RSAT [START_REF] Nguyen | RSAT 2018: Regulatory sequence analysis tools 20th anniversary[END_REF] using the MotifScan program, using an arbitrary cut-off value of 6.0 to define hits. Overlapping sites, sites identified on both DNA strands, and sites identified using more than one of the three top-scoring matrices for each factor were reduced to a single site in the scan reports provided in SI Tab. 2 and summarized further to indicate only the number of high-scoring sites detected in each promoter in the summary given in Tab. 1. Interestingly, almost one-third (30/95) of the sites detected (SI Tab. 2) do not contain a full consensus G-box, despite giving a score of 6.0 or more in the analysis. Some Arabidopsis promoters of interest were not confirmed as targets in this analysis, in some cases because their only G-box-containing motifs were to be found at >3 kb upstream of the initiation codon, and in others because these motifs yielded scores of <6.0. Accordingly, 18 of 24 putative Arabidopsis target promoters analyzed were found to contain at least one high-affinity

Author contributions

ACRS performed the phylogenetic and bioinformatics analyses, most of the in-situ hybridizations, the molecular cloning and genetic transformations, all phenotypic characterizations and microscopy, helped prepare recombinant proteins for in vitro studies, and wrote the manuscript. ACV cloned and prepared recombinant proteins and tutored ACRS and LB. AAR performed Y2H analyses and in situ hybridizations on Amborella tissues. PC prepared recombinant proteins, tutored ACRS and cared for growing plant material. LB performed phylogenies of SPT-target genes and an earlier version of the RSAT analysis. PM and MV screened for Petunia spt mutants and supervised their phenotypic characterization by ACRS. RS and JMFZ performed PBM analyses. CPS coordinated the work and corrected the manuscript.