Dr Hector Escriva
email: hescriva@obs-banyuls.fr

My Favorite Animal, Amphioxus: Unparalleled for Studying Early Vertebrate Evolution

Keywords: amphioxus, cephalochordates, Evo-Devo, invertebrate chordate, lancelet, vertebrates

Amphioxus represents the most basally divergent group in chordates and probably the best extant proxy to the ancestor of all chordates including vertebrates. The amphioxus, or lancelets, are benthic filter feeding marine animals and their interest as a model in research is due to their phylogenetic position and their anatomical and genetic stasis throughout their evolutionary history. From the first works in the 19th century to the present day, enormous progress is made mainly favored by technical development at different levels, from spawning induction and husbandry techniques, through techniques for studies of gene function or of the role of different signalling pathways through embryonic development, to functional genomics techniques. Together, these advances foretell a plethora of interesting developments in the world of research with the amphioxus model. Here, the author review the discovery and development of amphioxus as a superb model organism in evolutionary and evolutionary-developmental biology.

My First Contact with Amphioxus

It was 1989 and I was studying my third year of biology at the University of Valencia in Spain. In the zoology course, we started the study of the chordate phylum, and in particular the cephalochordate group (i.e., the amphioxus), which was described by our teacher as our closest relative in evolutionary terms. This close evolutionary relationship with vertebrates was established since Gabriel Costa in 1834 [START_REF] Costa | Annuario Zoologico, Tipografia di Azzolino e Comp[END_REF] described the presence of a notochord in amphioxus. In addition, numerous studies later showed the substantial conservation at the anatomical level between amphioxus and vertebrates. Likewise, the urochordates (i.e., including the ascidians), which are the third group of chordates, are extremely divergent in anatomical terms (and today we know that that is also the case at the genomic level). [START_REF] Schubert | [END_REF] As a result suggested by recent phylogenomic studies, we now know that these phylogenetic relationships are not correct, because the sister group of vertebrates are the urochordates and not the cephalocordates, which represent the most basally divergent clade in the phylum (Figure 1). [3][4][5] At that point in our zoology course, a strike began, and it lasted the rest of the year. This prevented me from studying amphioxus because the chordate phylum was excluded from the final exam. However, I personally studied what the Brusca and Brusca [START_REF] Brusca | Invertebrates[END_REF] text book said about amphioxus, which started to become "my favourite animal."

Years later, during my PhD at the Pasteur Institute in Lille, France, our paths crossed again. This second contact with amphioxus was only "molecular," because I was studying the evolution of the nuclear receptor (NR) gene family and I received an Eppendorf tube with some amphioxus genomic DNA to amplify NR fragments by PCR. Later, the results of this work [START_REF] Escriva | [END_REF] motivated my postdoctoral research at the "Ecole Normale Superieure de Lyon" to study the function of some of these NRs during the embryonic development of amphioxus. That's how, in 1999, I got to go for the first time to Tampa, Florida, where I met colleagues from all the Western laboratories interested in amphioxus studies. Why in Tampa? Because it was the place where Nicholas and Linda Holland, the couple of researchers who can be considered the "parents" of modern amphioxus research, discovered how to obtain amphioxus embryos with a simple electric shock to mature adults. [8] So, it was there, at the Courtney Campbell Causeway in Tampa, that we used to meet every summer to collect amphioxus adults, to induce their spawning and to obtain embryos. It was also there that I saw for the first time this peculiar animal with a dorsal notochord that, unlike vertebrates, persists in the adult, with an extremely simple dorsal neural tube but slightly thickened in the anterior part called the cerebral vesicle. I could also observe its V-shaped myotomes, its ventral segmented gonads and gill slits, its hepatic caecum-the homologue of our liver-and its unique frontal eye-also homologue of our eyes (Figure 2). I was also fascinated by the beauty of the amphioxus embryos, which develop quite fast (from the egg to the larvae in about 72 h) (Figure 3) and which are small and transparent, allowing classical experiments of developmental biology such as whole-mount in situ hybridization (ISH), or immunolocalization, to be easily performed.

From Florida to Banyuls sur Mer

There are about 30-35 amphioxus species described around the world, all of which are benthic filter feeding animals. [9] They live in shallow waters (from just a few centimetres to about 50 m deep, although there are some much deeper examples, [10,11] ).

They always live in sandy bottoms but the granulometry depends on the species and the site. Amphioxi are gonochoric animals and reproduce only during their spawning season, which varies slightly between species but which usually corresponds to spring and summer months. Although all amphioxus species spawn shortly after sunset, their spawning behavior also varies between species. Thus, while in the Florida species (Branchiostoma floridae) the animals spawn synchronously about once every 2 weeks, [12] in the Mediterranean species (Branchiostoma lanceolatum) spawning occurs gradually during the spawning season, without any conspicuous synchronization. [13] As I said before, Nicholas and Linda Holland had developed a method to induce spawning of adult amphioxus in the laboratory in Tampa. [8] This method consists of a simple electric shock to individuals separated one by one in small plastic cups filled with sea water. But this method only works if it is applied on the day of natural spawning (I remember here that for the Florida species all animals from a given site spawn the same day in a synchronized way). Thus, a classic working day consists in collecting amphioxus on the beach every afternoon (simply with a shovel and a sieve) and after sunset, in the laboratory, one can try the electric shock. If it is not the natural spawning day, no embryos are obtained and everything starts again the next day. This method, although tedious and unpredictable (the natural spawning day cannot be predicted), allowed numerous laboratories to perform the first Evo Devo studies with amphioxus. However, for the European research teams, the work in Tampa represented an enormous investment of time and money since in a month's work and hopefully we could get embryos only 3 or 4 nights, which slowed down the development of the different technical approaches and research projects. For this reason, and given that the presence of amphioxus near the Banyuls sur Mer marine station in the south of France had been described, [14] I organized in December 2001 a meeting in this marine station to which I invited all the European teams interested in amphioxus to try to develop the necessary techniques to work with the Mediterranean species, Branchiostoma lanceolatum. Most of the groups accepted the challenge and we started working with this species in Banyuls sur Mer.

From the Electric Shock to the Thermal Shock

In Banyuls sur Mer, unlike Tampa, collection of adult amphioxus with a shovel and a sieve was impossible because the animals live at 4-50 m depth. In the year 2002, we began our study trying to reproduce the way it was working in Tampa. For this, we used a boat from the marine station, which could collect amphioxus with a Charcot Picard dredge once a week, and we tried to induce the spawning with an electric shock every collection day at sunset. However, collecting animals once a week posed a problem. In 2002, we did not know the spawning behavior of B. lanceolatum, so if the Mediterranean amphioxus spawned synchronically like B. floridae and the natural spawning day was not the day of animal collection, we would never get spawning in the laboratory, and that is what happened. Therefore, the Figure 1. Schematic representation of the phylogenetic relationships between deuterostome phyla. The cephalochordates (i.e., amphioxus) are the earliest divergent lineage within chordates (blue box), which also comprise the urochordates (including ascidians) and their sister group, the vertebrates. The asterisk indicates the moment when at least two complete genome duplications occurred during evolution. The sister group of chordates, the Ambulacraria (brown box), include the echinoderms (i.e., sea urchins, starfish, etc.) and hemichordates (i.e., acorn worms).

Figure 2. The amphioxus shares with vertebrates a large number of characteristics (chordate synapomorphies). A) Photograph of an adult amphioxus (Branchiostoma lanceolatum) from the Mediterranean (collected at the Racou beach, close to Banyuls sur Mer, France). The front part is on the left and the dorsal part is at the top. B) Schematic representation of an adult amphioxus. cv, cerebral vesicle; nt, hollow neural tube; ms, V shape myotomes; not, notochord; pat, post-anal tail; m, mouth; gs, gill slits; go, gonads; at, atriopore; an, anus; hc, hepatic caecum; g, gut. The blastula (F) develops at 5 hpf, and quickly the vegetal pole invaginates during gastrulation to form the mesendoderm (G, early gastrula at 6 hpf; H, mid-gastrula at 8 hpf; I, late gastrula at 11 hpf). Neurulation begins with the closure of the blastopore at the posterior part of the embryo generating the tailbud (J, early neurula, 15 hpf; K, early-mid-neurula, 17-18 hpf; L, mid-neurula, 24 hpf; and M, late-neurula, also called pre-mouth stage, 36-40 hpf). In the late neurula, the pharynx swells where the mouth and first gill slit will develop (M). The larval stage begins when the mouth is opened, from 60 to 70 hpf (N). The amphioxus larvae are left-right asymmetric. Some examples of this asymmetry are the mouth, which opens on the left side, and the gill slits that develop ventrolaterally on the right side. The somites also develop asymmetrically, with the left somites displaced half segment to the front compared to the right somites. The larvae continue to grow accumulating gill slits (depending on the amphioxus species this period varies quite a bit from a couple of weeks in B. floridae to 2-3 months in B. lanceolatum). Thus, the larva that is presented in N, has developed three gill slits. At 2-3 months the larvae undergo a process of metamorphosis, the most important aspects of which are a considerable reduction in asymmetry (the mouth migrates and eventually positions medially, the gill slits duplicate and are placed in pairs on each side of the pharynx, but the muscle blocks derived from the somites are still asymmetrical). In O a larva that has begun the metamorphosis is shown. The development of the metapleural folds is observed in O. In P the larva is in the middle of metamorphosis, the mouth is already located medially, the cirri begin to develop as well as the hepatic caecum. In Q the metamorphosis has culminated and the juvenile already has all the typical structures of the adult. Another interesting aspect of the metamorphosis process is the change in lifestyle from a pelagic life for the larva to a benthic life for the juvenile and adult. All the pictures from G to Q show the embryos with the anterior part to the left. tb, tailbud; ph, pharynx; gs, gill slits; mt, metapleural folds; m, mouth; c, cirri; hc, hepatic caecum. Scale bar in A represents 50 μm and applies from A to L. Scale bar representing 50 μm in M applies also to N. Scale bar in O represents 1 mm and applies from O to Q.

following year, in May 2003, we decided to rent a boat with which we were able to collect animals every day with a Van Veen dredge. Our first results showed two things, i) the electric shock, despite inducing spawning as in Tampa, activated the eggs, which prevented in vitro fertilization and ii) the spawning behavior of B. lanceolatum differed from that of the Florida species, since the animals did not spawn eggs synchronically.

Since the electric shock technique could not be used, we tried the way in which embryos were obtained at that time in the Asian species of amphioxus (B. belcheri). [15] This technique, of simple observation of the adults in culture, involved waiting for the natural spawning in the tanks to recover the embryos from the water. Luckily, or by serendipity, while the month of June is warm in Banyuls, and therefore the temperature in the aquariums was quite constant around 22-23 C, one day there was a strong storm (the so-called "tramuntana") that induced a drop in temperature of about 5 C that persisted for more than 24 h. Two days later, all the animals in the aquariums had spawned. After this observation, using new animals, I was able to repeat the experiment, this time in a controlled way (I kept the animals at 23 C for a week and I changed the temperature of the tanks to 18 C in the afternoon of day À1), and again all the animals spawned during the night of day 0 showing that a long thermal stress (24 h) was able to induce spawning during the night of the second day. [16] Actually, spawning occurs naturally, without any other kind of induction than the temperature change, unlike what happens with the electric shock in the Florida species. In fact, it has been described that B. floridae spawning only takes place when the second meiotic division has occurred. So unfertilized eggs are arrested in metaphase of the second meiotic division. [17] In B. lanceolatum it seems that what the temperature change induces is the second meiotic division of the oocytes, a division that is necessary and sufficient for the animals to spawn naturally at sunset. This method has been improved since then and today we do not use a drop in temperature but an increase (from 17 to 23 C) because this allows us to keep adult animals at a lower temperature, which avoids contamination with pathogenic bacteria. On a more personal level, the discovery of this spawning induction method had a major impact, since it made me decide to move and to establish my own research team in the Banyuls sur Mer marine station in 2005.

From Comparative Anatomy to Functional Genomics

Ernst Haeckel proclaimed: "We begin with the lancelet-after man the most important and interesting of all animals. Man is at the highest summit, the lancelet at the lowest root, of the vertebrate stem." [START_REF] Haeckel | The Evolution of Man; a Popular Scientific Study[END_REF] Haeckel probably exaggerated with his assumption, which, moreover, is wrong in evolutionary terms, but despite the fact that amphioxus is not a model species used in many laboratories (probably due to the difficulty in getting access to embryos), since the beginning of the 20th century about 2000 articles have been published on amphioxus (according to the Web of Science in May 2018, searched for: TOPIC: (amphioxus) OR TOPIC: (lancelet) OR TOPIC: (cephalochordate); Timespan: All years (starting in 1900).

Indexes: SCI-EXPANDED, SSCI, A&HCI, CPCI-S, CPCI-SSH, BKCI-S, BKCI-SSH, ESCI, CCR-EXPANDED, IC. (see Figure 4). As it can be observed in Figure 4, I have divided the history of research using the amphioxus model into three main, partly overlapping, periods.

First Period: Comparative Anatomy

The first period, quite long and with a low but constant number of publications per year, corresponds to the period during which numerous researchers described the anatomy of a number of metazoans (including amphioxus), and used these descriptions to establish evolutionary relationships between the different groups using comparative anatomy. Figure 4 shows only the number of publications from 1900 onwards, but this first period began earlier, in the middle of the 19th century. Some of the most important researchers and works of this period, before the 20th century, described the adult anatomy, but soon the embryology of amphioxus was also characterized. I will cite here some of the most important publications of this period that although not exhaustive, illustrate the type of research that was carried out at that time. As examples of description of adult anatomy and histology we can cite Alexander Kowalevsky, who noted that amphioxus shares key anatomical features with vertebrates, such as a hollow dorsal nerve tube, an endostyle, a segmented body derived from somites and a postanal tail. [START_REF] Kowalevsky | [END_REF] De Quatrefages, who described the nervous system of amphioxus, [START_REF] De Quatrefages | Libraires-editeurs[END_REF] E. Ray Lankester who also described the general morphology of adult amphioxus, [START_REF] Lankester | [END_REF] and some other early researchers on amphioxus include Heinrich Rathke [START_REF] Rathke | Bemerkungen uber den Bau des Amphioxus lanceolatus eines Fisches aus der Ordnung der Cyclostomen, Gebrüder Bornträger[END_REF] or John Goodsir. [START_REF] Goodsir | [END_REF] Concerning amphioxus embryology, even though some descriptions of larvae collected from plankton were made by Schultze and Leuckart, [24,25] the first complete description of amphioxus embryos was done by Kowalevsky. [START_REF] Kowalevsky | [END_REF] Other important contributions to amphioxus embryology include the work of Hatschek or Conklin [START_REF] Hatschek | The Amphioxus and Its Development[END_REF][START_REF] Conklin | [END_REF] or many years later the experimental embryology work of Tung (reviewed by S. Yan in 1999 [28] ).

Second Period: The Amphioxus Become an EvoDevo Model

The second period starts when Nicholas and Linda Holland described a method of obtaining amphioxus embryos by induction of spawning in captivity and in vitro fertilization in 1989. [8] This second period is characterized by a first subperiod during which using whole-mount in situ hybridization (ISH) on embryos, the study of gene expression was used to establish body plan homologies between amphioxus and vertebrates, [START_REF] Holland | Molecular Zoology: Advances, Strategies, and Protocols[END_REF] and a second subperiod, during which the role of different cellular signals in the control of embryonic development were studied using pharmacological treatments capable of activating or inhibiting these signals (reviewed in Bertrand et al. [START_REF] Bertrand | [END_REF] ). This second period extends until today, as many studies continue to use comparative gene expression and pharmacological treatments to establish homologous relationships of organs or even cell types between amphioxus and vertebrates and to study the role of different signals during amphioxus development, respectively.

Obviously there is a considerable amount of publications corresponding to the first subperiod, but here I would like to highlight just a few of them. The first work of this subperiod in which the technique of whole-mount ISH was used to get insights into the relationship between the amphioxus and vertebrate body plans focused on the expression of a gene from the Hox family (Hox3), which is a classic in the world of EvoDevo. [31] This work allowed to establish the existence of a conservation in the anteroposterior patterning of the central nervous system in chordates and suggested the presence of a hindbrain in amphioxus. The study of gene expression of many other genes followed this first work. Different publications suggested conservation of CNS patterning. [32,33] Other genes that have been used to establish homology relationships between amphioxus and vertebrates' structures include Hand, Csx (Nkx-2.5), and Tbx4/5, which expression suggests the presence in amphioxus of an homologue of the heart. [34] A putative homologue of the vertebrate adenohypophysis was also established through the conserved expression of the nuclear receptor NR4A [35] together with the expression of the amphioxus orthologs of Pit-1, Eya, Islet, Lhx, Pitx, Six1/2, and Six3/6. [36][37][38][39] The homology between the vertebrate eyes and the frontal eye in amphioxus was proposed given the conserved expression of Pax4/6 [40] as well as the expression of other genes such as Rx, Otx, Pax4/6, Mitf. [41] Also, homology between the endostyle and the thyroid gland was established through the conserved expression of different transcription factor and enzyme genes. [42][43][44][45] The conservation of gene expression has also been used as an evidence of homology at the cellular level, such as the expression of Pax3/7 in some amphioxus cells putatively homologous of the vertebrate satellite cells. [46] In this case, instead of ISH, immunolocalization was used to study expression.

As far as the second subperiod is concerned, I will not go into details because most if not all published papers in which a cellcell communication signal has been functionally studied using pharmacological treatments have recently been reviewed by Bertrand et al. [START_REF] Bertrand | [END_REF] However, I would like to say that the use of different drugs has been, and still is, an extremely powerful tool to perform functional studies in amphioxus, due to the experimental simplicity and the large number of embryos that can be obtained in one spawning night (a medium size female may spawn 2-3 thousand eggs), which allows one to perform numerous treatments at different drug concentrations in a fast, simple and reproducible way.

Third Period: Functional Genomics

Finally, the third period starts in 2008, when the first complete sequence of an amphioxus genome (Branchiostoma floridae) is published. [47] This period also extends until today and it includes on the one hand numerous studies in genomics and comparative genomics, and, on the other hand, as a result of the development of new techniques, different functional studies establishing the role of several cellular signals throughout embryonic development. The green line indicates the period of time that began after developing a method of spawning induction. Research during this period was based first on comparative studies of gene expression, (using the technique of whole-mount in situ hybridization), and later on functional studies using pharmacological treatments. The blue line indicates the period starting after the publication of the first complete genome sequence of an amphioxus (B. floridae), and includes both comparative genomic studies and functional studies using new techniques allowing the specific analysis of the function of a given gene (morpholinos, knock out animal lines using TALEN), or high throughput techniques for the study of chromatin structure.

The first complete sequence of an amphioxus genome was published for the American species (B. floridae) in 2008 [47] and was followed a few years later by the publication of the genome of the Asian species (B. belcheri) [48] and, recently, of the Mediterranean species (B. lanceolatum). [49] Susumu Ohno hypothesized in 1970 the existence of a double duplication of the vertebrate genome. [START_REF] Ohno | Evolution by Gene Duplication[END_REF] Since then, this question has been one of the most discussed issues in the field of evolutionary genomics. In 1994, Jordi Garcia-Fernandez and Peter Holland carried out a work that was the starting point for numerous subsequent studies. [START_REF] Garcia-Fernandez | [END_REF] In this work, they demonstrated the presence of a single cluster of Hox genes in the amphioxus genome, suggesting that the double duplication of the genome must have occurred after the evolutionary divergence of cephalochordates. Although the demonstration of the existence of this double duplication of the genome (the socalled 2R) in the ancestor of vertebrates (and therefore after the divergence of cephalochordates and urochordates) (Figure 1) was carried out in 2005, [52] the complete sequence of the amphioxus genome corroborated this study, closed the discussion about the timing of the 2R and allowed the reconstruction of the ancestral karyotype of chordates. [47] Access to the complete genome sequence of amphioxus allowed the realization of a great number of studies on the evolutionary history of different gene families such as the homeobox, [53,54] the tyrosine kinase receptor, [55] the nuclear receptor [53,56] or the opsin gene families. [53,57] Other studies have focused more on the presence/absence of genes involved in a given function, such as, for example, genes known to be involved in immunity [58] or in the neural crest gene regulatory network. [59] Apart from the specific interest of these studies in the context of the evolution of the different gene families, we can extract an important trait of the amphioxus genome, which is its high conservation in terms of gene content. In fact, it seems that amphioxus has retained a gene content very close to the gene content that was probably present in the ancestor of all chordates. [60] This characteristic is unique within chordates since both urochordates and vertebrates lost numerous genes during their evolutionary histories. However, this trait does not mean that the amphioxus genome is equivalent to the genome of the chordate ancestor (in terms of gene content) since numerous lineage-specific gene duplications occurred in cephalochordates.

In this last period of publications, I would like to highlight a separate group of studies, which represents only a few articles. However, these publications use new techniques with a huge potential for functional studies in amphioxus and I consider they are the first of a "new era" in amphioxus research. Some of these works concern the development of aquaculture techniques, which allowed the development of new approaches. For example, to study regeneration in adult amphioxus, a process that takes between 3 to 4 months in B. lanceolatum, it was necessary to develop an aquaculture system allowing keeping adult individuals separately for such a long period in small water volumes. [61] Other techniques that have represented great advances are the induction of spawning by temperature change that I already mentioned above, [16] which allows one to obtain embryos on a daily basis during the spawning season, or the development of husbandry techniques with artificial sea water, [62] which allows laboratories inland to work with amphioxus. Obtaining the complete life cycle of amphioxus in captivity [63][64][65][66] has also been a very important step, particularly to create knockout lines of amphioxus using transcription activator-like effector nucleases, (TALEN)-based knockout. [67] This knockout technique allowed Li and collaborators to demonstrate the master role of the Nodal antagonist Cerberus in the control of left-right asymmetries in amphioxus. [67] Other techniques allowing functional studies developed by various laboratories include the use of morpholinos to knockdown gene expression or the overexpression of a given gene through microinjection of capped-mRNA. These studies include the overexpression of chimeric transcription factors acting as constitutive repressors as a result of the fusion of the Drosophila Engrailed repressor domain to the sequence of a given transcription factor. [67] The first morpholino microinjected in amphioxus embryos was a morpholino blocking the expression of Hox1. This study showed a posteriorized phenotype in knockdown embryos and demonstrated that Hox1 mediates the role of RA signaling in setting the posterior limit of the pharynx. [68] Other studies using morpholino approaches include knockdown of Chordin and Blimp, [69] Dkk3, [70] and Tbx1/10. [71] The first of these studies showed that Nodal/Vg1 and Bmp signaling act in opposition for the specification and maintenance of the dorso-ventral and antero-posterior axes. [69] The second one focused on the role of Dkk3, a secreted protein of the Dickkopf family, in the control of development of anterior structures in amphioxus, showing that the knockdown of Dkk3 produces a headless phenotype similar to the phenotype produced by upregulation of Wnt/βcatenin signalling. [70] Then, the third study showed how a regulatory network involving the mutual inhibition of retinoic acid (RA) and Tbx1/10 signaling is involved in the control of pharyngeal segmentation. [71] Lastly, microinjection of reporter constructs to test the enhancer/promoter activity of cisregulatory DNA and particularly cross-species regulatory comparisons on key developmental genes is an extremely powerful approach to gain insight into the cis-regulatory mechanisms underlying developmental evolution. The first study using this approach, by Yu et al., [72] showed the reliability of the technique and was followed by many other studies that have been reviewed in Kozmikova et al. [73] Since 2015 other studies have used this technique to describe the regulatory activities of SoxE [74] and Gsx [75] upstream regulatory elements and their evolutionary implications, or more recently to show the putative enhancer activity of non-coding sequences in the vicinity of the amphioxus hox cluster. [49] Finally, the development of high throughput techniques such as chromosome conformation capture followed by high throughput sequencing (4C-seq) or Assay for Transposase-Accessible Chromatin with high throughput sequencing (ATACseq), have recently allowed to study the amphioxus chromatin conformation and the presence of presumptive active enhancers in the genome. [49] This study has shown important differences in the 3D structure of the chromatin around the Hox cluster between amphioxus and vertebrates. It is well known that the 3D structure of this locus in vertebrates is very important for its function in the patterning of the limbs. [76] The loci of the HoxA and HoxD complexes of vertebrates are organized in a threedimensional bipartite structure (in two so-called topologically associated domains, TADs). These TADs contain distant regulatory elements on both sides of the Hox complex that are essential for the regulation of Hox genes expression in the limbs. However, in amphioxus its unique Hox cluster is structured into a single TAD that contains only a few regulatory elements. Thus, this study suggests that the evolution of the three-dimensional structure of chromatin must have played a central role in the appearance and evolution of vertebrate limbs.

The Future of Research Using the Amphioxus Model

The techniques that I have just presented here, and in particular those that allow us to understand the specific function of different genes in developmental processes (such as knockdown and knockout techniques), or how gene expression is regulated (such as the 4C-seq and ATAC-seq techniques), will allow remarkable progress to be made in the coming years. Without any doubt, the use of these techniques will allow notable advances in the understanding of the mechanisms that regulate the embryonic development of amphioxus. But despite the enormous technical advances that have taken place in the research world of the amphioxus model, we always have to keep in mind one thing: that there is always a model in biology to answer a specific question. The amphioxus model has been, is, and probably will be, the best existing model to understand the evolutionary changes that occurred during chordate evolution and that allowed the appearance of the vertebrates, including humans.

Figure 3 .

 3 Figure 3. The amphioxus life cycle. The pictures show different developmental stages of the Mediterranean amphioxus. After spawning, the first polar body can be clearly observed next to the egg (see asterisk in A). The first division (B) occurs approximately 75 min post fertilization (at 19 C). The following divisions at 19 C occur approximately every 45 min (C, four cells stage; D, eight cells stage in which 4 micromeres and four macromers are observed). The morula stage or cleavage stage (E) is observed at 4 h post fertilization (hpf).The blastula (F) develops at 5 hpf, and quickly the vegetal pole invaginates during gastrulation to form the mesendoderm (G, early gastrula at 6 hpf; H, mid-gastrula at 8 hpf; I, late gastrula at 11 hpf). Neurulation begins with the closure of the blastopore at the posterior part of the embryo generating the tailbud (J, early neurula, 15 hpf; K, early-mid-neurula, 17-18 hpf; L, mid-neurula, 24 hpf; and M, late-neurula, also called pre-mouth stage, 36-40 hpf). In the late neurula, the pharynx swells where the mouth and first gill slit will develop (M). The larval stage begins when the mouth is opened, from 60 to 70 hpf (N). The amphioxus larvae are left-right asymmetric. Some examples of this asymmetry are the mouth, which opens on the left side, and the gill slits that develop ventrolaterally on the right side. The somites also develop asymmetrically, with the left somites displaced half segment to the front compared to the right somites. The larvae continue to grow accumulating gill slits (depending on the amphioxus species this period varies quite a bit from a couple of weeks in B. floridae to 2-3 months in B. lanceolatum). Thus, the larva that is presented in N, has developed three gill slits. At 2-3 months the larvae undergo a process of metamorphosis, the most important aspects of which are a considerable reduction in asymmetry (the mouth migrates and eventually positions medially, the gill slits duplicate and are placed in pairs on each side of the pharynx, but the muscle blocks derived from the somites are still asymmetrical). In O a larva that has begun the metamorphosis is shown. The development of the metapleural folds is observed in O. In P the larva is in the middle of metamorphosis, the mouth is already located medially, the cirri begin to develop as well as the hepatic caecum. In Q the metamorphosis has culminated and the juvenile already has all the typical structures of the adult. Another interesting aspect of the metamorphosis process is the change in lifestyle from a pelagic life for the larva to a benthic life for the juvenile and adult. All the pictures from G to Q show the embryos with the anterior part to the left. tb, tailbud; ph, pharynx; gs, gill slits; mt, metapleural folds; m, mouth; c, cirri; hc, hepatic caecum. Scale bar in A represents 50 μm and applies from A to L. Scale bar representing 50 μm in M applies also to N. Scale bar in O represents 1 mm and applies from O to Q.

Figure 4 .

 4 Figure 4. Graphic representation of the number of publications focusing on amphioxus since 1900. The two key dates (1989 and 2008) in the evolution of the studies using this animal model are indicated. The red line indicates the period of studies based on anatomical descriptions and comparative anatomy.The green line indicates the period of time that began after developing a method of spawning induction. Research during this period was based first on comparative studies of gene expression, (using the technique of whole-mount in situ hybridization), and later on functional studies using pharmacological treatments. The blue line indicates the period starting after the publication of the first complete genome sequence of an amphioxus (B. floridae), and includes both comparative genomic studies and functional studies using new techniques allowing the specific analysis of the function of a given gene (morpholinos, knock out animal lines using TALEN), or high throughput techniques for the study of chromatin structure.

Acknowledgements

The author thanks Stephanie Bertrand for proofreading and her continuous collaboration in our common laboratory. The author also thanks the CNRS and the ANR (ANR16-CE12-0008-01 to HE) for funding our research projects.

Conflict of Interest

The author declares no conflict of interest.


Luc Behaghel

Karen Macours

Julie Subervie

Can RCTs help improve the design of CAP?

Keywords: Common agricultural policy, Impact evaluation, Policy design, Field experiments JEL codes: C93, Q18

We illustrate how randomized controlled trials (RCTs) could be used as a learning tool to shed light on various aspects of the Common Agricultural Policy (CAP). RCTs are quasi-absent from the CAP evaluation toolbox, despite their frequent use to evaluate other European Union policies, or agricultural policies in developing countries. We draw upon existing debates on the role of RCTs in policy-making to derive a list of points of attention. We then consider four specific examples of evaluation questions for the CAP, and based on examples drawn from agricultural and social policies in developing and developed countries, argue that the RCT toolbox has the potential to significantly add to existing approaches to evaluating and designing components of the CAP.

Introduction

Despite the renewed demand for evaluating agricultural policies in the European Union and the revival of randomized controlled trials (RCTs) in the evaluation of social policies in Europe since the mid-2000s, RCTs have not been applied to the evaluation of the Common Agricultural Policy (CAP) [START_REF] Colen | Economic experiments as a tool for agricultural policy evaluation: Insights from the European CAP[END_REF]. This may seem surprising given the role played by RCTs during the "glory days" of agricultural economics until the 1960s (Herberich, Levitt and List, 2009). There are undoubtedly many reasons for this absence, both on the demand side (e.g. the type of evaluations requested or accepted by various stakeholders such as governments, international agencies, farmers,…) and on the supply side (e.g. the incentives available to the consultants and academics operating in this area). This paper abstracts from these constraints -each of which undoubtedly deserve careful analyses of their own --and asks a preliminary question: are RCTs an appropriate tool to address issues relevant to the design of today's CAP?

While evaluation can serve as both an accountability measure and as a learning tool, this paper focuses on its role as the latter, as RCTs can be particularly powerful instruments in improving understanding about the effectiveness of policies.1 For many aspects of the CAP, the important policy question to address is not whether a certain type of policy ought to exist, but rather whether specific changes to the design of an existing policy could lead to better efficiency and/or equity outcomes. To the extent that such questions are of interest, RCTs can help provide rigorous answers by randomly introducing potentially relevant variations in the design, and testing their implications for a policy's effectiveness.

We consider the two pillars of the CAP2 , and highlight a (non-exhaustive) list of policy-relevant design issues that could benefit from rigorous empirical evidence: (i) How could the uptake of agrienvironmental measures by farmers be increased? (ii) What schemes can induce coordination among farmers on environmental issues in which one farmer's defection can jeopardize the efforts of all the others ? (iii) What implications could different mechanisms for direct payments have on efficiency and equity? (iv) How to design agri-environmental contracts to ensure that farmers receive sufficient compensation for their conservation efforts without inducing large efficiency losses? We then discuss how RCTs can shed light on these questions.

These questions illustrate that RCTs should not be primarily considered as tools to evaluate the impact of existing policies that are already being implemented universally to the entire target population. As such, they do not necessarily provide an alternative to the typical ex-post evaluations based on observational data. Instead, they can be particularly powerful to empirically test whether and to what extent potential policy changes lead to anticipated (and potentially unanticipated) impacts when implemented in real-life contexts, but prior to the universal application of the policy change. As such, they can help inform decisions regarding potential policy (design) options and provide credible and rigorous empirical evidence of impacts that complement the typical theoretical and modeling based ex-ante approaches.

The four policy design questions are meant to demonstrate the adaptability of RCTs, and show that they can be applied both to test the adequacy of the policy design ('doing the right thing') and for the adequacy of the policy implementation ('doing things right'). It may seem hardly surprising that, transposing and extending lessons drawn from the lab by behavioral economists, experiments on information-based interventions or different types of nudges can shed light on a farmer's propensity to adopt agri-environmental contracts. One may be skeptical, however, about what can be learned from RCTs with respect to three other issues, namely coordination failures, equity -efficiency tradeoffs, and market design. Despite this, we believe that one of the key lessons that emerges from the recent literature is the strong adaptability of the RCT toolkit and its usefulness in addressing questions that had been deemed out of reach only a few years ago. To illustrate this claim, we complement the discussion of hypothetical experimental designs that could be applied to the CAP with actual examples from two related policy domains: agricultural policies in developing countries, and social policies in Europe and other contexts.

Our exercise contributes to a small prospective literature on the role that experiments can play in the evaluation of agricultural policies. Heberich et al. (2009) build on observations over the past century to argue that agricultural economists could take "a new train at the depot," in the form of field experiments inspired by laboratory experiments and economic theory. [START_REF] Colen | Economic experiments as a tool for agricultural policy evaluation: Insights from the European CAP[END_REF] provide a detailed review of the state of the art in the evaluation of the CAP, and discuss the comparative advantages of different experimental methods, and their complementarities with other methods. Both papers take an optimistic view of the potential of the RCT toolbox while acknowledging that this potential has not yet been realized. To complement these discussions, we highlight potentially fruitful examples of evaluations, inspired by recent successes in neighboring areas. The next section provides more background on the debates regarding RCTs and policy-making. The subsequent sections consider the four policy design questions in turn, and the last section offers several concluding remarks.

Background: The debated role of RCTs and policy-making

Existing CAP evaluation relies on a wide range of methods: simulation models, statistical and econometric analysis of survey data, qualitative evaluations, and, more recently, experimental methods. [START_REF] Colen | Economic experiments as a tool for agricultural policy evaluation: Insights from the European CAP[END_REF] argue that the new policy instruments introduced over the past three decades call for an increased reliance on experimental methods, which are particularly adapted to analyze farmers' heterogeneous responses to decoupled payments, or enrolment decisions in voluntary schemes such as agri-environmental contracts, for instance. Moreover, the greater discretion granted to Member States in the 2014-20 CAP programming period gives room to regional or State-level policy experiments that can be accompanied with experimental evaluation. [START_REF] Colen | Economic experiments as a tool for agricultural policy evaluation: Insights from the European CAP[END_REF] provide several examples of how discrete choice experiments and laboratory experiments have been used in that context to help design and evaluate the PAC. There also exist a large number of positive mathematical programming models covering individual European States or regions; and a number of them have full EU coverage. 3 Randomized controlled trials, however, have remained absent from the PAC evaluation toolbox. As noted above, this comes in sharp contrast with the rise of RCTs in the evaluation of agricultural policies in developing countries, and in the evaluation of social policies in the European Union. If the law of diminishing marginal returns applies, this fact suggests that introducing a little bit of RCTs to evaluate the PAC may have high returns. Yet another fact calls for caution: the rising popularity of RCTs in policy-making has come with continuing debates on the pros and cons of the approach:4 does this mean that RCTs have been overvalued in other policy areas, providing a good reason not to use them for agricultural policy-making? While a complete summary of the debates on RCTs is beyond the scope of this section, we use them to derive "points of attention" when designing RCTs, which we then apply to the specific examples considered in this paper: what threats against the validity of RCTs should we consider? What are the conditions for their feasibility and acceptability?

Internal and external validity of RCTs for policy-making

Following [START_REF] Roe | Internal and external validity in economics research: Tradeoffs between experiments, field experiments, natural experiments and field data[END_REF], RCTs can be viewed as an intermediate approach to the fundamental tradeoff between internal and external validity in empirical research. 5 While laboratory experiments achieve high internal validity by fully controlling the context of the experiment, the specificities of the laboratory context limit its external validity. At the other end of the spectrum, naturally occurring data can be representative of the context of interest, so that results may have high external validity, but their internal validity may be questionable in the absence of exogenous sources of variation to identify causal effects. By contrast, RCTs -when well-designed and carefully implemented -may provide both relatively high internal and external validity: in RCTs, the researcher manipulation of a naturally occurring context induces the exogenous variation needed for causal identification while the context remains policy-relevant, and is more likely than a laboratory to resemble other policy-relevant contexts. However, naturally occurring contexts cannot be fully controlled, which can pose threats to internal validity; and unless the RCT is implemented at the same scale as the policy of interest, without further assumptions external validity is limited to its context, as in any empirical research (see Deaton and Carwright, 2018). Viewing RCTs as an intermediate approach in the tradeoff between internal and external validity has two important consequences. First, it implies that ranking RCTs against other empirical methods will involve putting weights on these two criteria, which is not straightforward. As noted by [START_REF] Athey | The Econometrics of Randomized Experiments[END_REF], claims in favor of RCTs based on the primacy of internal validity ("without internal validity causal studies have little value") have been challenged by other researchers arguing that, from a statistical decision perspective, there is no particular priority for internal validity over external validity [START_REF] Manski | Public Policy in an Uncertain World: Analysis and Decisions[END_REF]. Second, we should not expect all RCTs to reach the same point in this tradeoff. Trivially, large-scale RCTS are more likely to have high external validity, but this may come at the expense of internal validity if compliance with the design is less controlled. More generally, the degree of internal and external validity of an RCT depends on the context of the study, the experimental design, and the execution of the experiment.

Among the different threats to validity identified in the literature, let us discuss two that have direct consequences for the specific examples of CAP impact evaluations considered below: violation of the "Stable unit treatment value assumption" (SUTVA) and differential attrition in follow-up data. 6 SUTVA requires that the (potential) outcomes of control units be unaffected by the fact that other units get treated, so that they reflect what would have occurred to treatment units in the absence of the program evaluated. This rules out general equilibrium effects, peer effects and any sort of externalities. However, SUTVA is by no way required for RCTs: when it holds, simple experimental designs (e.g. simple individual randomization) can be used, but alternative designs can be used in cases where it is which a causal relationship holds over variation in persons, settings, treatments, and outcomes" (p. 83). See also [START_REF] Athey | The Econometrics of Randomized Experiments[END_REF]. 6 A more complete list would notably include imperfect compliance with treatment assignment (and the distinction between intent-to-treat effects and local average treatment effects), and "experimental effects" such as the Hawthorne and Henry effects, by which treatment and control units change their behavior when they are aware of participating in an experiment.

likely to be violated. For instance, peer effects and other externalities can be taken into account in a "partial population design" [START_REF] Moffitt | Policy Interventions, Low-Level Equilibria And Social Interactions[END_REF] that introduces random variation in the exposure of nontreated units to interactions with treated units; applications include [START_REF] Miguel | Worms: Identifying Impacts on Education and Health in the Presence of Treatment Externalities[END_REF] in the context of health in Kenya, or Avvisatti et al. (2014) in French public schools. General equilibrium effects can also be estimated in designs that consider local markets as the units of randomization (see [START_REF] Crépon | Do Labor Market Policies have Displacement Effects? Evidence from a Clustered Randomized Experiment[END_REF], for an active labor market program in France). Differential attrition 7 threatens the validity of RCTs as survey response behavior potentially reintroduces selectivity: the subsamples of the treatment and control groups for which a follow-up can be completed may not be comparable anymore, even though the initial two samples were comparable due to randomization. Selective attrition is a pervasive issue. It is often neglected or only corrected on the basis of observable covariates in panel surveys. By contrast, evaluators conducting RCTs have paid increasing attention to it both as a threat to representativeness and to causal identification. More and more RCTs use exhaustive administrative data that do not suffer from the problem; in addition, robust statistical methods have been developed to estimate or bound the effects under minimum assumptions in the presence of differential attrition (Lee, 2009;[START_REF] Behaghel | Please Call Again: Correcting Non-Response Bias in Treatment Effect Models[END_REF].

Feasibility and acceptability

Another possible reason why RCTs have not been used to evaluate the CAP may be that they are simply not feasible, or not acceptable from a political or ethical perspective. Technically, RCTs are feasible under two minimum conditions: assignment to treatment 8 can be randomized, and both the "control" and "treatment" groups that are thus created can be followed. As illustrated in the next section, randomization can be conducted in ways that minimize disruption and may not even ever be perceived by individuals participating in the experiment. Also, the follow-up can rely upon administrative data that are collected for other purposes. In such cases, there is little doubt on the technical feasibility of RCTs. But it clearly depends on the question at stake, and some policies will probably never be evaluated with an RCT (think of major macroeconomic or fiscal reforms). Our take of the literature is, however, that many evaluation questions for which RCTs were deemed not feasible have been actually successfully addressed by RCTs. Take for instance the evaluation of the structure of peer effects in education. An active researcher in that field, Caroline Hoxby, had been putting forward quasiexperimental results, arguing that experiments that would artificially generate significant random 7 Differential attrition is characterized by the fact that outcome measures are missing in different proportions in the treatment and control groups. 8 Note that random assignment to treatment does not mean that all members of the treatment group enroll in the program, and that no member of the control group enrolled (which would define perfect compliance to the assignment). It is enough to randomly send an encouragement to enter the program ("encouragement design"); this random encouragement is then used as an instrumental variable to identify the impact of program enrollment.

differences in class composition would not be politically feasible. A few years later, at least two experiments of that sort had been run: one in Kenya testing the effectiveness of tracking [START_REF] Duflo | Peer Effects, Teacher Incentives, and the Impact of Tracking: Evidence from a Randomized Evaluation in Kenya[END_REF]; one in the US, testing the optimal composition of squadrons at the naval academy [START_REF] Carrell | From Natural Variation to Optimal Policy? The Importance of Endogenous Peer Group Formation[END_REF]. Concerning measurement and follow-up, innovative ways of measuring outcomes have been found --see for instance the measurement of corruption in [START_REF] Olken | Monitoring Corruption: Evidence from a Field Experiment in Indonesia[END_REF]. In short, what RCTs can technically do must be judged empirically, not a priori. This does not mean that RCTs come at no cost: the sheer fact that access to a program needs to be manipulated for evaluation purposes may complicate the program operations. As discussed by List (2011), this may require long-run investments by evaluators to gain trust within implementing partners' organizations. A related concern is that only a subset of programs run by selected implementing partners may be evaluated with RCTs [START_REF] Ravallion | Should the Randomistas (Continue to) Rule?[END_REF]: RCTs would solve the issue of selection into treatment, but would do so only for selected programs, which would limit their external validity. While this concern is real, it is unclear if it is a concern against RCTs, or a call to run more of them. Feasibility is endogenous: methodological innovations increase the range of policies that can be evaluated with RCTs, and their diffusion increases the readiness of implementing organizations and authorities to rely on them, pushing to their use in more and more varied contexts.

Ethical concerns are harder to assess, as they cannot be resolved by simply looking at what RCTs are run in practice: perhaps some RCTs have been run that should not have been run. A full discussion of the ethics of RCTs is again beyond the scope of this paper. [START_REF] Ravallion | Should the Randomistas (Continue to) Rule?[END_REF] insists that ethical concerns should be taken more seriously, building upon guidelines elaborated for medical trials, such as the principle of equipoise, by which experimenting (and depriving the control group from treatment) is only ethical as long as we are sufficiently ignorant about treatment impact. What however "sufficient ignorance" entails is debatable. From a positive perspective, [START_REF] Banerjee | Improving Spatial Coordination Rates under the Agglomeration Bonus Scheme: A Laboratory Experiment with a Pecuniary and a Non-Pecuniary Mechanism (NUDGE)[END_REF] precisely argue that RCTs have developed as a way to respond to "adversarial audiences" that would not accept "reasonable" priors. Applying this discussion to the PAC, a tension may appear between two agendas: a "learning" agenda that would use RCTs to answer questions for which uncertainty is high, and a "costcutting" agenda that would use RCTs as a disciplinary device through which any intervention would have to prove its (cost-)effectiveness, somehow imposing the burden of the proof to farmers involved in the experiments. Healthy scepticism fosters good science, but should not be used to deny existing evidence.

Overall, our take of this quick review is that RCTs have proven a useful learning tools in many policy areas, and experience has accumulated on how to use them best, so that RCTs could usefully enter the CAP evaluation toolkit, with due methodological and ethical care. We try to outline how this may occur by considering specific examples in the next sections.

Inducing behavioral changes: nudges and information-based experiments

Farmers are involved in decisions with complex trade-off dynamics and sometimes lack relevant information when considering engaging in new agricultural practices. The complexity and changing nature of some of the environmental regulations and policies, as well as the heterogeneity in conditions and constraints among many farmers in the EU, may well imply that some farmers are not necessarily aware of all the information and practices relevant for their particular situation. This potentially is a bigger constraint among smaller (potentially part-time) farmers or certain farmers in newer member states. Whether such information constraints actually exists, and if so for whom, is clearly an empirical question. Targeting the provision of information regarding eligibility for certain benefits, or advantages of shifting practices, to certain types of farmers could help evaluate such questions.

Even when farmers have access to all the relevant information, the complex and dynamic trade-offs they need to consider for many of their decisions may foster the use of heuristics and a susceptibility to behavioral inertia and the status quo in agricultural practices. Insights from behavioral science suggests that "nudges" [START_REF] Thaler | Nudge: Improving Decisions about Health, Wealth, and Happiness[END_REF] can then be powerful tools to shift decision making.

A nudge is not a cash payment. It may consist, for instance of the provision of information about the social norm (about what others think or do). It can also simply consist of the way in which a choice is presented. Empirical evidence shows that nudges can be effective in changing consumer behavior, in particular pro-environmental behaviors (see [START_REF] Schubert | Green nudges: Do they work? Are they ethical?[END_REF] for a review). The question then arises: is it possible to nudge farmers to change their practices, and possibly adopt more pro-environmental (or "greener") practices?

There are indeed some reasons to believe that agri-environment scheme (AES) uptake could be improved with the use of nudges. Some studies suggest that nudges can affect farmers' intentions to (re-)enroll in AESs [START_REF] Kuhfuss | Nudges, Social Norms, and Permanence in Agri-environmental Schemes[END_REF][START_REF] Chen | Linking social norms to efficient conservation investment in payments for ecosystem services[END_REF]. There is also evidence of a "social identity" mechanism behind individual decisions [START_REF] Goldstein | A Room with a Viewpoint: Using Social Norms to Motivate Environmental Conservation in Hotels[END_REF], by which an individual adheres to the descriptive norms of the group of which he considers himself a member. This effect may well apply to farmers, especially among members of a cooperative. Thus, there is reason to believe that this type of psychological lever could be useful in improving the design of agri-environmental policies.

In recent years, behavioral economics research using lab experiments has grown rapidly and has provided evidence of a large number of tools that can be used to encourage green practices. Because these results cannot always be directly extrapolated to the field, lab-in-the field experiments 9 are sometimes used to validate results derived from the lab. RCTs could be considered as the next logical step in the chain, and the insights from the lab-in-the field regarding context can indeed help to subsequently design a RCT. The RCT in turn allows study whether and to what extent the nudge affects actual real-life decisions. Despite a recent craze for nudges in both the academic and the public sphere (in UK and US in particular 10 ), there are almost no experimental studies providing evidence of the impact of nudges on farmers' decisions in the real world. One exception is a study by [START_REF] Messer | Behavioral nudges in competitive environments: a field experiment examining defaults and social comparisons in a conservation contract auction[END_REF], who ran an RCT in which farmers from Texas, Delaware, and Maryland competed in an auction of conservation contracts that required them to adopt practices that reduced nutrient run-off. The authors show that changing the default option can result in larger bids, and that providing information about what others think of the required practice increases the likelihood that a producer participates in the auction. [START_REF] Wallander | Addressing participant inattention in federal programs: A field experiment with the conservation reserve program[END_REF] and [START_REF] Chabé-Ferret | Can Nudges Induce Changes in Farmers' Choices of Agricultural Practices? Evidence from a Randomized Controlled Trial with French Winegrowers[END_REF] also use social norms in field experiments with the aim of inducing greater farmer participation in AESs, and they make use of RCTs to test the effectiveness of these nudges. Both studies failed to detect a statistically significant impact of these interventions on farmer participation in the agri-environmental schemes, demonstrating the importance of testing the theoretical attractive instruments in real life conditions. Many alternative nudges remain to be tested in the field and could help improve the design of agrienvironmental policies. [START_REF] Ferraro | Applying Behavioral Insights to Improve Water Security[END_REF], for example, suggest using a default option in order to increase farmer participation in the US Conservation Reserve Program. The authors also suggest presenting payments in agri-environmental programs using a loss frame, specifically by stating the maximum payment the participant could earn and how much he would lose for every practice not adopted. Both of these suggestions could also work in the European context. Previous evidence thus suggests that insights from behavioral sciences, combined with rigorous empirical tests of the concepts, could indeed be used to improve the design of the second pillar of the CAP.

For a variety of information interventions or nudges, the evaluation of the scheme's effectiveness using a RCT seems quite feasible, since large samples can be easily reached and as long as the design assures that the Stable Unit Treatment Value Assumption (SUTVA) is likely to hold. One difficulty of such experiments, however, is that small (albeit valuable) effects may be hard to detect, as the record of 9 Lab-in-the field experiments differ from other laboratory experiments by recruiting participants from the field for which a measure is experimented (e.g., farmers rather than students when considering AESs) and by framing the experiment to represent the field context ("contextualized" experiment). [START_REF] Suter | Towards an Understanding of the Performance of Ambient Tax Mechanisms in the Field: Evidence from Upstate New York Dairy Farmers[END_REF] have run ambient tax experiments with student subjects and dairy farmers and found that the two groups may produce different results. 10 Several governments in developed countries have constituted 'behavioral insights teams' within their civil services. In 2014, the U.S. Department of Agriculture (USDA) created the Center for Behavioral and Experimental Agri-Environmental Research (CBEAR). Galizzi (2017) also identifies initiatives by the governments of Australia, Canada, Denmark, Finland, France, Israel, the Netherlands, New Zealand, Norway and Singapore.

the "What Works Centres" in UK shows: experimenters must therefore make sure that they have sufficient statistical power. One limitation is that the interpretation of the effects is not always straightforward in the presence of a variety of plausible "behavioral" models and heterogeneous types of behavior (e.g. Duflo et al., 2011). But even if the impact of a nudge is small, it may remain quite costeffective, as these types of interventions tend to require very little in terms of implementation costs. This is all the more true in countries such as France, where the online application for agricultural subsidies has been mandatory since 2016, and the (marginal) cost of providing information or nudges through pop-up windows in the Telepac website would thus be small. Take, by way of example, potential measures to reduce contamination by pesticides from agriculture, a source of water quality degradation in several countries in the European Union. In France, during the 2007-2014 CAP programming period, AESs have been used to fulfill the objectives of the European Union water framework directive and have been implemented in catchment areas where water quality improvement has been identified as a priority from 2007. As has been the case for many other measures, the participation of farmers has been extremely low [START_REF] Kuhfuss | Do European Agri-environment Measures Help Reduce Herbicide Use? Evidence From Viticulture in France[END_REF]. One reason for this may be that many farmers actually lack all of the relevant information when considering reducing the use of pesticides. Since 2008, the Dephy farm network of the French Ecophyto program has been experimenting with techniques that reduce the use of pesticides and herbicides without affecting crop productivity and profitability and recent analyses suggest that they have been successful in many cases (Lechenet et al., 2017). The dissemination of these innovative practices is a major challenge faced by the program. But as long as this information can be embedded in customized messages,11 dissemination to a very large number of randomly selected farmers via the Telepac application should be quite feasible.

Evaluating the impact of such intervention would also require collecting data on farmers' pesticide use after they have received information about the innovative techniques. This could be done with no additional cost through the "Pratiques Culturales" survey run every three years by the French Department of Statistics of the Ministry of Agriculture (SSP), which collects data on phytosanitary treatments in French farms from the four main categories of crop production. Many other similar information-based experiments could be envisaged, in France and presumably in other European countries.

Incentives to coordinate: experimenting with collective bonuses

As illustrated in the previous section, a number of different AESs have been put in place at the level of individual farms. However, the recent literature has emphasized the potential gains that could be realized by adopting an approach that is characterized by a larger, landscape-wide scale. 12 Accordingly, AES compensation payments are now allowed by EU regulation to be paid to groups of farmers (Regulation N° 1305/2013, article 28, cited in [START_REF] Westerink | Collaborative governance arrangements to deliver spatially coordinated agri-environmental management[END_REF]. Such payments, often referred to as "collective bonuses", raise important design questions. Contract theory highlights the various potentially counterproductive mechanisms at play. An obvious rationale for setting incentives at the landscape level is many of the outcomes for which the schemes are designed (e.g., wildlife conservation, water quality and storage) are also applicable at the landscape level. While actions at the individual farm level could certainly contribute to these objectives, their impact on landscape-level results would arguably be weaker, thus yielding imperfectly aligned incentives. Employing "multitasking" models [START_REF] Holmstrom | Multitask Principal-Agent Analyses: Incentive Contracts, Asset Ownership, and Job Design[END_REF] may also induce farmers to focus on betterincentivized tasks at the expense of other tasks that could be more important with respect to the environmental objectives identified. Collective bonuses can also create collective action problems, such as free-riding. The free-rider problem can, however, be mitigated to some extent by institutional arrangements or peer pressure. In short, the appropriate design of collective bonuses is a difficult theoretical question involving numerous plausible mechanisms whose effects may be context dependent. Empirical evidence is needed in order to weigh the relative strengths of these mechanisms and to assess their net effect. Relatedly, given the evidence in several instances that incentives may have perverse effects, transparent and convincing evidence that collective bonuses have the expected positive effects in the contexts of interest will be key to their political viability.

Although to our knowledge RCTs have not yet been used to evaluate agri-environmental schemes at the landscape level, 13 two examples suggest that they may prove a useful and feasible tool in this regard. In Uganda, [START_REF] Jayachandran | Cash for carbon: A randomized trial of payments for ecosystem services to reduce deforestation[END_REF] analyze the impact of financial incentives for forest owners to maintain the integrity of their forestland, thus providing experimental evidence of the effectiveness and cost-effectiveness of Payments for Ecosystem Services. Even though the incentives 12 A number of lab experiments have shed some light on the likely interactions between heterogeneous agents of the same group in a context of non-point source pollution [START_REF] Cason | Empowering neighbors versus imposing regulations: An experimental analysis of pollution reduction schemes[END_REF][START_REF] Miao | The impact of information on behavior under an ambient-based policy for regulating nonpoint source pollution[END_REF][START_REF] Poe | Exploring the Performance of Ambient-Based Policy Instruments When Nonpoint Source Polluters Can Cooperate[END_REF][START_REF] Spraggon | Testing ambient pollution instruments with heterogeneous agents[END_REF] or in the management of shared groundwater resources [START_REF] Suter | Behavior in a Spatially Explicit Groundwater Resource: Evidence from the Lab[END_REF]. 13 Banerjee (2018) uses a laboratory experiment to examine the role of various mechanisms in incentivizing spatially coordinated land uses under the Agglomeration Bonus scheme. Using lab and artefactual field experiments, [START_REF] Fooks | Conserving Spatially Explicit Benefits in Ecosystem Service Markets: Experimental Tests of Network Bonuses and Spatial Targeting[END_REF] evaluate the relative effectiveness of collective bonuses and spatial targeting (which provides the government with a means to select contiguous parcels over non-contiguous parcels when enrollment is budget-constrained) for achieving optimal contiguity of parcels in the landscape. in this study are set at the farm level, where each individual is given the opportunity to enroll to receive payments if she refrains from clearing trees, the randomization of treatment assignments takes place at the village level such that the impacts that are measured encompass any "leakages" (externalities) and collective dynamics within villages. The authors also use random variation in the proximity between treatment and control villages to account for potential spillovers across villages. As a result, outcomes in this study are measured at the landscape level. Though there is no evidence of spillovers and limited evidence of leakages, program enrollment is low (32 percent), seemingly due to limited program awareness, leading the authors to question whether the program could be better marketed.

Collective incentives could indeed be a way to do so, as they could induce farmers to the program to other farmers in their village. In other words, everything in this experimental design is in place to analyze alternative collective schemes, and the results suggest that such schemes are indeed worth trying.

The second example comes from the incentive literature in the economics of education. Quite naturally, given the collective nature of the education production function at the classroom and school level, researchers have been experimenting with various forms of individual and collective bonuses for teachers and students as incentives to increase student performance. Empirical evaluations in this literature underscore the complexity of the mechanisms at play, and specifically their dynamic nature.

For instance, Muralidharan and Sundararaman (2011) compare the effectiveness of teacher-and school-level incentives in India: while the two compare well in the first year, teacher-level incentives outperform school-level incentives in the second year, suggesting collective action problems might have come into play. Other systematic investigations such as those conducted by Roland Fryer or John List suggest that the literature in the economics of education remains on the steep segment of the learning curve regarding how to best harness the power of incentives in this area (e.g., [START_REF] Fryer | Enhancing the Efficacy of Teacher Incentives through Loss Aversion: A Field Experiment[END_REF]. Much could certainly be learned from similar efforts with respect to AES.

Technically, an RCT of collective bonuses does not pose specific design difficulties. The number of farmers involved needs to be large, so that randomization takes place by clusters (e.g., randomization units at the landscape levels). Importantly, administrative data should be used as much as possible, and the monitoring of compliance with AESs should be strictly identical in the treatment and control groups: as many outcomes would be measured at the landscape level, there should be no attrition issues. Political feasibility may be more of a problem. Under which conditions would experiments with collective bonuses be acceptable in the field? A first condition is the acceptability of the bonuses themselves. If the scheme that one wants to test is faced with strong opposition, control and treatment groups will be affected by the conflicting views, and may distort their behavior to prove the policy wrong or right. A proposed evaluation of collective "project grants" at the class level thus had to be abandoned at the pilot stage: it had been the subject of heated debates in the medias, so that the conditions for a valid RCT were not met [START_REF] Behaghel | Programme expérimental "Bourse aux projets de classe" : bilan de la phase pilote du point de vue de l'évaluateur[END_REF]. Clearly, however, this had less to do with RCTs as such than with the intervention itself, and the way it was framed in the media. The pilot helped clarify what schemes would be acceptable and feasible, but it came too late. The role of piloting interventions before reaching the stage of a full RCT should not be underestimated. This pilot stage can and should build on a variety of evaluation tools: qualitative methods (focus groups, interviews, observation) and lab-in-the-field experiments with stakeholders, for instance.

The second condition for acceptability is to answer fairness concerns that arise if, for example, the expected gains of treated individuals are higher than the gains of control individuals. It is however possible to design experiments in which the expected gains of the different experimental groups are similar --see for instance [START_REF] Fryer | Enhancing the Efficacy of Teacher Incentives through Loss Aversion: A Field Experiment[END_REF], where incentives are framed in two treatment arms, but are financially equivalent. As suggested by Moravetz (2014), control farmers may also receive as a lump sum the same payment as the expected or average payments made to farmers in the treatment group.

Fighting farmer poverty: experimenting with transfers?

The first pillar of the CAP has long-consisted of direct payments to farmers, helping farmers cope with volatile prices. Such subsidies are a core component of the CAP and are likely to be sustained in the foreseeable future as a key principle of the program. Prima facie, it may therefore appear that there is no useful role for RCTs in the evaluation of these policies. Yet beyond the normative questions regarding the principle of the payments per se, many questions exist regarding the specific design of the different interventions within the first pillar, over which member countries do exercise some degree of freedom. Ensuring that transfers reach the targeted groups without inducing large efficiency losses matters for the sustainability of the CAP over the long term. The equity considerations underlying the payments of the first pillar are also receiving more attention given differences in allocations between older and newer member states.

Incidentally, RCTs have proved to be a very useful tool in shedding light on the advantages and disadvantages of direct payments to farmers and other rural households in developing countries. The evaluation of a conditional cash transfer program in Mexico (PROGRESA, now named Prospera) was one of the first large-scale RCTs conducted in the developing world, and the experimental evidence of its impacts was crucial in assuring continued support through changing political climates. In fact, the program rapidly expanded nationwide in Mexico and triggered similar programs across Latin America, where they now reach 25 percent of the total population. Such programs have also spread to Africa, Asia, and even Europe and the United States over the last 20 years (Fizsbein and Schady, 2009;Robles, Rubio and Stampini, 2015).

The initial evaluation of the program in Mexico, which tested the effectiveness of one particular design, was followed by a large literature experimentally comparing alternative design variations. These studies helped policymakers understand, for instance, whether making transfers conditional, labeling them, or making them entirely unconditional impacted their effectiveness (Baird, Ozler and McIntosh, 2011;[START_REF] Benhassine | Turning a Shove into a Nudge? A "Labeled Cash Transfer" for Education[END_REF]. They also tested whether varying the recipient in the household (men vs. women) affected outcomes [START_REF] Akresh | Evidence from a randomized evaluation of the household welfare impacts of conditional and unconditional cash transfers given to mothers or fathers[END_REF]Almas et al 2018), and whether making payments conditional on actions or achievements was more effective [START_REF] Barrera-Osorio | Improving the Design of Conditional Transfer Programs: Evidence from a Randomized Education Experiment in Colombia[END_REF]. Such studies have now been carried out in many different settings (e.g. Malawi, Morocco, Burkina Faso, Bosnia, Colombia), and frequently in close collaboration with government ministries. The agencies implementing these studies often have valid questions regarding how to optimally design a transfer program for a specific objective or context.

Once it is acknowledged that the answers to such questions are not obvious beforehand, experimentation becomes an ethical policy procedure. While such RCTs do not call into question the benefits themselves, nor the justification for the policy goals they aim to achieve, they are well suited to provide empirical evidence on potential trade-offs between different objectives that alternative design options may imply. Learning from these experiences suggests that RCTs have potential as useful instruments in comparing alternative direct payment schemes under the first pillar of the CAP, and in investigating how different designs may imply different trade-offs between the possible objectives therein. A potential application could be to use a RCT to compare different methods of monitoring farmer's compliance with greening. Green direct payments account for 30% of EU countries' direct payment budgets. Farmers receiving an area-based payment have to make use of various straightforward, non-contractual practices that benefit the environment and the climate. Whether they use such practices then of course need to be monitored and paying agencies are required to inspect at least 5% of declarations, via field visits and photointerpretation of High Resolution satellite imagery (European Union, 2013). Recently, using much cheaper remote sensing techniques has been suggested as a possible alternative [START_REF] Sitokonstantinou | Scalable Parcel-Based Crop Identification Scheme Using Sentinel-2 Data Time-Series for the Monitoring of the Common Agricultural Policy[END_REF]. Clearly the method of monitoring affects both the costs and the potential effectiveness and trade-offs may include the accuracy and frequency of the measure. This in turn can affect farmers' compliance behavior. Indeed, the problem is quite similar to that posed for payments of CCT in developing countries, where some evidence suggest that hard monitoring may not be needed to change behavior. Hence experimenting with the compliance monitoring method could help answer an important question. Such experimentation appears quite feasible, as files of different farmers could be assigned to different monitoring methods.

Targeting under adverse selection: experimenting with innovative agri-environmental contract designs

Voluntary agri-environmental contracts that offer fixed payment schemes suffer from two major problems. First, farmers who face the lowest costs for complying with environmental requirements are more likely to enter the program; in cases where the program would pay some farmers for doing nothing differently from what they would have done in the absence of payment, adverse selection may induce large windfall effects [START_REF] Chabé-Ferret | How much green for the buck? Estimating additional and windfall effects of French agro-environmental schemes by DID-matching[END_REF]. Second, those with the highest costs of participating are less likely to enter the program, though may precisely be those whose engagement would have the greatest contribution to the program's effectiveness [START_REF] Kuhfuss | Préférences individuelles et incitations collectives : quels contrats agroenvironnementaux pour la réduction des herbicides par les viticulteurs ?[END_REF].

One option for reducing the efficiency losses due to adverse selection is to shift from fixed payment schemes to auction mechanisms. Procurement auctions have been in place in other countries for many years (e.g. the US Conservation Reserve Program, established in the 1980s, as well as several pilot programs in Australia). However, the context in which conservation auctions are implemented may impact their effectiveness [START_REF] Lundberg | Context Matters: Exploring the Costeffectiveness of Fixed Payments and Procurement Auctions for PES[END_REF][START_REF] Ferraro | Asymmetric information and contract design for payments for environmental services[END_REF]. It is therefore impossible to anticipate the additional gains from auctions (compared to fixed payment contracts) in the European context; here again, RCTs could provide a useful way to address this question.

For several decades, researchers have used experimental auctions to estimate consumer willingnessto-pay for new products [START_REF] Corrigan | Comparing Open-Ended Choice Experiments and Experimental Auctions: An Application to Golden Rice[END_REF]. In a demand-revealing auction mechanism similar to the [START_REF] Vickrey | Counterspeculation, Auctions, and Competitive Sealed Tenders[END_REF] and Becker-DeGroot-Marschak (BDM) (1964) auctions, bids indeed provide a direct measure of auction participants' willingness-to-pay for the good being sold. There is a substantial literature dealing with the implementation of such designs in university experimental economics labs (Berry et al. 2015 and references therein). More recently, a number of studies in developing countries have also demonstrated that these insights can have broader relevance for addressing design tradeoffs in actual policy applications. [START_REF] Hoffmann | Do Free Goods Stick to Poor Households? Experimental Evidence on Insecticide Treated Bednets[END_REF] used a BDM design to measure the gap between willingness-to-pay and willingness-to-accept for bed nets in Uganda. [START_REF] Berry | Eliciting and Utilizing Willingness to Pay: Evidence from Field Trials in Northern Ghana[END_REF] also use a BDM mechanism to estimate the willingness to pay for water filters in Ghana, while Guiteras and Jack (2017) use the same mechanism to investigate how workers respond to different contractual arrangements in the context of informal day labor markets in rural Malawi.

Finally, and more closely related to the topic we address here, Jack (2013) uses a Vickrey auction to explicitly take into account landholders' willingness-to-accept an afforestation contract in Malawi. She moreover runs a RCT to demonstrate that landholders who received a tree planting contract as a result of bidding in the auction kept significantly more trees alive over a three-year period than did landholders who received the contract through a lottery. Much can certainly be learned from a systematic analysis of the demand for AESs in European countries using a similar approach, especially taking into account the heterogeneity that is likely to be present with respect to this demand.

In fact, recently in France, environmental auctions à la [START_REF] Vickrey | Counterspeculation, Auctions, and Competitive Sealed Tenders[END_REF] have been implemented in a pilot program run by the Water Agency Artois-Picardie [START_REF] Kuhfuss | Une alternative originale pour l'allocation de contrats agro-environnementaux : l'appel à projets de l'Agence de l'eau Artois-Picardie[END_REF]. This pilot program was implemented in a way akin to the afforestation program evaluated by [START_REF] Jack | Private Information and the Allocation of Land Use Subsidies in Malawi[END_REF] in Malawi: auction participants were given a thorough explanation of the auction rules, then sealed bids were collected and ranked to determine both the auction clearing price based on the available budget and the rejected bids. Unfortunately, contrary to the Malawian program, the French pilot has not been implemented through random assignment of participants; therefore, it was not possible to assess how far it had outperformed (or not) the usual system of14 This initiative suggests, however, that there is a demand for experimentation by the practitioners and public authorities, and also that it is de facto feasible to pilot such innovative approaches with some farmers and not others, so that there is only one step left to reach the stage of evidence-based evaluation of innovative schemes.

Concluding comments

The lessons from this prospective exercise can be summarized as follows. First, many insights from laboratory and field experiments in the behavioral sciences could be used to improve the design of the second pillar of the CAP, and RCTs would be useful in evaluating how well these approaches can contribute to achieving higher participation rates in real world conditions. Second, the value of collective incentives is a challenging theoretical question due to the many mechanisms that are potentially at play in determining the ultimate impacts of these incentives. RCTs may therefore be of particular use in weighing the relative strengths of plausible mechanisms in specific field settings.

Importantly, the acceptability of experiments involving (collective) bonuses needs to be ensured: this requires preliminary work and evaluation using other methods on the scheme and its framing; it also requires paying attention to fairness concerns and providing control farmers with appropriate alternatives. Third, ensuring that transfers reach the targeted groups without inducing large efficiency losses is an important consideration in fostering the sustainability of the CAP; this question, too, can be usefully addressed by RCTs. Finally, the potential for adverse selection is high in current schemes where farmers receive the same payment regardless of the opportunity costs of conservation they face. Here again, RCTs can be used for both eliciting individual willingness-to-accept an agrienvironmental contract and for separately identifying the effects of farmer selection and payment on the provision of environmental services.

In this paper, we strongly advocate for experimentation, aiming to demonstrate that RCTs have significant potential for improving the design of the CAP. We do not view RCTs as a way of evaluating the CAP per se, but rather as a tool for evaluating different design alternatives for which there are no obvious ex ante expectations. We moreover argue that RCTs could be used as a tool to test new schemes with complementary interventions in an effort to enable the CAP to reach its target audience and objectives. For several years in France, the legislative framework has authorized social experiments that aim at testing the effectiveness of public policies through pilot programs [START_REF] Gomel | L'expérimentation sociale aléatoire en France en trois questions[END_REF] and in fact, a number of RCTs have already been implemented since then, in particular in the fields of education [START_REF] Behaghel | Ready for Boarding? The Effects of a Boarding School for Disadvantaged Students[END_REF]) and employment [START_REF] Behaghel | Private and Public Provision of Counseling to Job Seekers: Evidence from a Large Controlled Experiment[END_REF]).

It cannot be denied that researchers and policymakers would face many real-world challenges when designing and implementing randomized evaluations within the CAP. Fortunately, lots has been learned about how to prevent and/or specifically account for such effects in trial design and implementation, and the accumulated experience and development of best-practice will be useful to address similar challenges to tackle key questions in the European agricultural and environmental context.

Sceptics of RCTs often point to the ethical problems that randomized experiments could create. Such issues can arise when randomized assignment implies creating a group of individuals who will be denied a program that is clearly beneficial, and who otherwise would have benefitted. For many components of the CAP, however, this question appears to be of less concern. Agri-environmental schemes, for instance, do not enrich the farmers -but instead to improve or preserve the quality of the environment by changing the behaviour of the participants without impoverishing them.

Moreover, it has been more than twenty years since voluntary schemes to support the change of agricultural practices were proposed to French farmers and only a very small proportion of them finally took part in those programs (less than 6 per cent of them subscribed to an agri-environmental measure during the 2007-2014 CAP period). Experimentation to increase uptake of programs that area already available to all, therefore do not pose obvious ethical problems.

The lack of randomized experiments in the field of agricultural policies may thus indicate the presence of political economic issues -such as the fear that the tool may do a disservice to its promoters and damage the credibility of CAP itself, rather than potential legal, technical or ethical obstacles. As such, it could be useful to follow the guidance offered by Campbell and Stanley (1969)

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As such, even though RCTs belong to the ex post evaluation approach (as they are based on observational data, measuring outcomes after they are realized, as opposed, e.g., to ex ante simulation methods), they can be used "ex ante", i.e. before scaling up actual policies.

The first pillar consists of direct payments to farmers and market measures, and the second pillar is a rural development policy aimed at balancing territorial development with sustaining a farming sector that is environmentally sound, while simultaneously fostering competitiveness and innovation(European Commission, 2013).

See for example to the CAPRI-FT model[START_REF] Gocht | EU-wide farm type supply models in CAPRI --How to consistently disaggregate sector models into farm type models[END_REF], the AROPAj model[START_REF] De Cara | Marginal abatement costs of greenhouse gas emissions from European agriculture, cost effectiveness, and the EU non-ETS burden sharing agreement[END_REF] or the IFM-CAP model[START_REF] Louhichi | Does the crop diversification measure impact EU farmers' decisions? An assessment using an Individual Farm Model for CAP Analysis (IFM-CAP)[END_REF][START_REF] Louhichi | Economic impacts of CAP greening: application of an EU-wide individual farm model for CAP analysis (IFM-CAP)[END_REF].

 4 For recent conflicting views, see[START_REF] Deaton | Understanding and misunderstanding randomized controlled trials[END_REF] and the comments in[START_REF] Imbens | Comments On: Understanding and Misunderstanding Randomized Controlled Trials By Cartwright and Deaton[END_REF] as well as in the special issue of Social Science & Medicine 210 (August 2018); Ravallion (2018) also revisits the debates

.5 FollowingShadish, Cook and Campbell (2002), a study has internal validity if the observed relationship between a treatment and an outcome reflects a causal relationship; and external validity "concerns inferences about the extent to

Interaction between the control and treatment groups can lead to a number of challenges that can threaten the internal validity a randomized evaluation. In our example, the control group may benefit from the treatment if treated farmers share the information they receive with untreated farmers. Sending customized messages (of no apparent interest to other farmers) may limit such spillovers.

In an auction setting à la[START_REF] Vickrey | Counterspeculation, Auctions, and Competitive Sealed Tenders[END_REF], it is possible to estimate the effectiveness of the contract using a regression discontinuity design (RDD), which is a quasi-experimental method that allows estimating the causal effect of an intervention by assigning a threshold above or below which the intervention is assigned. By comparing observations lying closely on either side of the threshold, it is possible to estimate the average treatment effect in environments in which randomization is unfeasible. This method however requires that the number of participants around the threshold is very large, which is rarely the case.

conflicts and misperceptions: real decisions often imply choosing between plan A and plan B rather than putting the global architecture of a policy in question; when such choices can be made following a documented and transparent process, the global architecture may be continuously improved, and the policy reinforced.