Frédéric Jiguet
email: frederic.jiguet@mnhn.fr

Kevin J Kardynal

Keith A Hobson

Stable isotopes reveal captive vs wild origin of illegally captured songbirds in France

Keywords: Bird, Emberiza hortulana, Illegal hunting, Ortolan Bunting, Poaching, seized birds Highlights

Naturally occurring stable isotopes have become important tracers in the study the wildlife ecology including the identification of origins for migration research, investigations of trophic relationships, and in the forensic determination of illegally harvested and captive fauna. Extensive illegal trapping for use as decoys and in cuisine has contributed to drastic declines of Ortolan Bunting Emberiza hortulana populations breeding and migrating through France. We contrasted feather hydrogen isotope (δ 2 Hf) values in illegally captured and subsequently confiscated Ortolan Buntings (n = 234), including feathers known to have grown in captivity (n = 34) or of unknown growing environment (wild or cage), with δ 2 Hf values in birds legally caught (ringed and released) in the wild (n = 40). We sought to determine if these bird groups could be differentiated based on this single isotope. Feathers grown in captivity had considerably lower δ 2 Hf relative to feathers of wild birds, which is potentially indicative of tap water consumption and the use of different diets in captive birds. Further, applying mixing models to δ 2 Hf values revealed similar proportions of captive vs. wild origins for birds illegally captured in 2012, 2014 and 2015, and a larger proportion of individuals with feathers grown in the wild in 2013. This potentially mirrors the confiscation of birds at poaching sites only in the former years, but also of recently caught wild buntings kept captive in 2013. Our results show that even a single stable isotope (δ 2 H) with good association with origins where feather keratins are produced may be useful in understanding origins of captive birds and may advance the monitoring of illegally captured birds.

Introduction

Naturally occurring patterns of stable isotopes across large geographic areas enable the tracking of bird migration. This is based on the principle that stable isotope ratios in food and water consumed by birds can be correlated with those values in growing tissues (e.g. feathers, blood) following welldescribed processes. Protium ( 1 H) and its heavier isotope, deuterium ( 2 H), occur naturally in environmental waters, and the ratio of the heavier to lighter isotope ( 2 H/ 1 H measured as δ 2 H) in precipitation has been shown to change predictably at continental scales (i.e. "isoscapes") [1]. Indeed, δ 2 H values in precipitation (δ 2 Hp) generally have a latitudinal structure across continents [START_REF] Bowen | Global application of stable hydrogen and oxygen isotopes to wildlife forensics[END_REF] and these patterns form the basis of tracing animal movements because such patterns are transfered up foodwebs [START_REF] Hobson | Tracking Animal Migration using Stable Isotopes[END_REF]. Foodweb isotopic signatures are reflected in the tissues of organisms, and such signatures can vary spatially based on a variety of biogeochemical processes. Organisms moving between isotopically distinct foodwebs carry with them information on the location of previous feeding (Hobson 1999). Migration connectivity can thus be studied by tracking chemicals contained in tissue, notably stable isotopes in keratins (Hobson & Wassenar 2008). As feathers grow during a short time period during a precise phase of the annual cycle, they are preferred to the continuously growing claws for inferring geographical origins. Determination of migratory origins using isotopes does not require sacrificing individuals, just sampling a feather of known growth timing. Migrants wearing feathers grown on breeding grounds can be attributed to probabilistic breeding areas by comparing the concentrations of some stable isotopes in their feathers and the geographical variations of the same isotopes in the natural environments where the feathers could have grown. The same holds true if analyzing feathers of breeding individuals that have molted on their wintering grounds.

Inferring the origin of migratory animals in terrestrial systems can be based on various stable isotopes, especially nitrogen, hydrogen and carbon. Nitrogen ( 15 N/ 14 N) stable isotopes are mainly used to determine the trophic level of a species. Hydrogen ( 1 H) and deuterium ( 2 H) has revolutionized stable isotope analysis in the study of animal migration. Deuterium ratios vary strongly with weather conditions, resulting in highly predictable spatial variation across continents (Hobson & Wassenaar 2008). Constructing continental maps that predict deuterium levels is relatively straightforward, given the large amount of existing data on continental weather patterns, and biologists further use such maps to determine probabilistic migratory origins of species. Deuterium ratios in feathers are effective indicators of breeding latitude in European birds (Hobson & Wassenaar 2008, Bearhop et al. 2003).

Carbon isotopic signatures ( 13 C/ 12 C) are used to distinguish plants that use C3, C4 and CAM modes of photosynthesis, as the C4 and CAM pathways lead to lower carbon fractionation than C3 photosynthesis (Karasov & Martínez del Rio 2007). Carbon isotopes can thus be used to reconstruct migratory routes, if the geographical distribution of C3, C4 and CAM plants and the diet preferences of the study species are known (Hobson 1999). As C3 and C4 plants have not the same occurrence rate north and south of the Sahara, carbon isotopic signatures in winter-grown feathers can discriminate individual birds that have overwintered south and north of the Sahara. Clearly, these isotopes provide rougher estimates of origins than deuterium, while C and N should be avoided when studying farmland species growing their feathers or hair in environments receiving agricultural fertilizers, disturbing natural concentrations of their isotopes.

The Ortolan Bunting Emberiza hortulana is a long-distance migrant Palaearctic songbird, that breeds across Eurasia and winters south of the Sahara. It breeds mainly in farmed habitats, while its migratory strategy has been confirmed by isotopic investigations of deuterium concentrations in winter-grown feathers [START_REF] Jiguet | Unravelling migration connectivity reveals unsustainable hunting of the declining ortolan bunting[END_REF]. The species is protected in the European Union and listed in Appendix I of the Birds Directive (1979/409/EEC), and formally protected in France since 1999. However, this species was illegally harvested in southwest France for culinary interest, as migrants have the outstanding capacity to double their mass, primarily as fat, within a short period prior to their fall migration. While bird conservation organizations have repeatedly denounced this practice, hunters, local politicians and leading French Michelin-starred chefs [START_REF] Darroze | Touch' pas mon ortolan[END_REF] have defended the persistence of this hunting and gastronomic practice as part of traditional cultural heritage. The European Commission referred France to the Court of Justice of the European Union (EU) in December 2016 on the charge of illegal hunting of Ortolan Buntings, as France could not prove it was satisfactorily taking action to stop the poaching activities. In this context, the French nature police intervened in the field by interrogating some alleged offenders and seizing captive Ortolan Buntings. These illegally captured birds were taken into care and later released in the wild before the end of the migration season.

In all birds, the renewal of feathers through moult is necessary to maintain the functions of these keratinous appendages. Songbirds typically renew flight feathers once each year and body feathers twice each year. Adult Ortolan Buntings moult the body feathers twice each year [START_REF] Jiguet | Stable isotopes reveal the systematic winter moult of central tail feathers in the Ortolan Bunting Emberiza hortulana[END_REF], on the breeding grounds in June to August, before the autumn migration to Africa, and on the wintering grounds before the spring migration. Their flight feathers are moulted only once, during the postbreeding moult in Europe. First-year individuals have feathers grown in the nest on the breeding grounds. Hence, during the fall migration, all individuals have a complete set of feathers recently grown on their breeding grounds regardless of their age.

Bird feathers grown at a given location generally have δ 2 H values (δ 2 Hf) proportional to local amount-weighted mean δ 2 Hp [START_REF] Hobson | Linking Hydrogen (δ 2 H) Isotopes in Feathers and Precipitation: Sources of Variance and Consequences for Assignment to Isoscapes[END_REF]. However, this is not necessarily true for feathers grown in captivity.

Captive birds are assumed to be provided tap water and commercial food of unknown and varied origins, which can have different δ 2 H values than local precipitation or wild grown seeds. This discrepancy in δ 2 H is especially true if tap water does not correspond well to amount-weighted growing season precipitation where feathers are grown and if food provided to the captive birds is not issued from local production [START_REF] Bowen | Global application of stable hydrogen and oxygen isotopes to wildlife forensics[END_REF], [START_REF] Bowen | A review of land-sea coupling by groundwater discharge of nitrogen to New England estuaries: Mechanisms and effects[END_REF]. Under a global research program dedicated to unravel the migration strategy of the Ortolan Bunting [START_REF] Jiguet | Unravelling migration connectivity reveals unsustainable hunting of the declining ortolan bunting[END_REF], we had access to numerous individuals captured illegally during four consecutive years. These birds included recently captured wild individuals and caged decoys placed on hunting sites. Caged decoys could be either individuals kept year-long in captivity (i.e. with captive-grown feathers), or recently captured wild individuals (i.e. wild-grown feathers) placed in cages to complement/renew the stock of long-captive decoy birds. All seized first-calendar individuals (i.e. hatch year) were of conclusively wild origin, thus having wild-grown feathers, since captive breeding of the species is not possible. Most other individuals could have feathers of wild or captive origin, but the plumage of a few adults displayed color aberration with either a lack of or an excess of dark pigmentation (leucism or melanism), signaling captive-grown feathers [START_REF] Sage | The incidence of albinism and melanism in British birds[END_REF].

Within the poached and seized birds, our main aim was to study δ 2 Hf values in order to estimate the proportion of individuals that were recently captured compared to those individuals captured in earlier years and further kept in captivity. All seized birds hatched in the wild, as there is no captive breeding of the species, so our aim was to address the question of the temporality of their capture, either recent or older. By first considering obvious caged buntings, we first assessed δ 2 Hf values for cage-grown feathers to determine if δ 2 Hf could be used to separate recently captured and caged birds. We expected birds with suspected cage-grown feathers to have potentially different δ 2 Hf, either lower or higher. As most Ortolan Buntings migrating by southwest France come from Poland and Scandinavia [START_REF] Jiguet | Unravelling migration connectivity reveals unsustainable hunting of the declining ortolan bunting[END_REF] where deuterium concentrations in the environment are lower than in France, we expected higher δ 2 Hf in caged birds. We then analyzed the distributions of δ 2 Hf in feathers collected from Ortolan Buntings illegally captured during four consecutive hunting seasons to estimate the proportions of individuals of wild vs captive origins. We finally propose possible interpretations for the observed inter-annual variations in these proportions, in terms of confiscation efforts by the police.

Materials and methods

Sample collection and hydrogen isotopic measurements

We collected feathers from Ortolan Buntings seized by the nature police in Landes department, southwestern France, when interrogating owners of illegal trapping sites. Seized birds were placed in wildlife care centers where licensed forensic ringers processed them before release. Seized birds could include caged and recently trapped birds found at capture sites, and potentially birds kept in captivity to be fattened for consumption. All surviving confiscated individuals were ringed and feather-sampled prior to release in the wild before the end of the autumn migration season. We organized the collection of one tail feather from each bird cared for in 2012 (n=50), 2013 (n=18), 2014 (n=53) and 2015 (n=113).

Individual birds were classified into three groups: 1) decoys (n=34) that were illegally captured birds with plumage abnormalities or highly developed feet scabies, 2) illegally captured birds from each year (2012, 2013, 2014 and 2015; these groups include the previously cited obvious decoys, but also illegally captured first-calendar year individuals), and 3) wild birds legally captured and ringed in autumn (n=40). For birds with tail feathers that were too heavily damaged from being in captivity, we instead sampled one tertial feather. We stored feathers in individual envelopes at ambient temperature until isotopic analysis.

Feathers were cleaned in 2:1 chloroform:methanol solvent rinse and prepared for δ 2 H analysis at the Stable Isotope Laboratory of Environment Canada, Saskatoon, Canada. The δ 2 H of the nonexchangeable hydrogen of feathers was determined using the method described by Wassenaar & Hobson [START_REF] Wassenaar | Comparative equilibration and online technique for determination of non-exchangeable hydrogen of keratins for use in animal migration studies[END_REF] based on two calibrated keratin hydrogen-isotope reference materials (CBS: -197 ‰, KHS:

-54.1 ‰). We performed hydrogen isotopic measurements on H2 gas derived from high-temperature (1350 °C) flash pyrolysis (Eurovector 3000; Milan, Italy) of 350 ± 10 ug feather subsamples and keratin standards loaded into silver capsules. Resultant separated H2 was analysed on an interfaced Isoprime (Crewe, UK) continuous-flow isotope-ratio mass spectrometer. Measurement of the two keratin laboratory reference materials corrected for linear instrumental drift were both accurate and precise with typical within-run measurement error < 2 ‰. All results are reported for non-exchangeable H expressed in the typical delta notation, in units of per mil (‰), and normalized on the Vienna Standard Mean Ocean Water-Standard Light Antarctic Precipitation (VSMOW-SLAP) standard scale.

Statistical analyses

We first tested if δ 2 Hf values measured from each bird group followed normal distributions by performing Shapiro-Wilk tests. We also performed a general linear model to compare δ 2 Hf values among five groups (wild birds and the four annual datasets). We then applied the normalmixEM function of the 'mixtools' package in R v3.5.2 [START_REF] Benaglia | Mixtools: An R Package for Analyzing Finite Mixture Models[END_REF] to the datasets for each year (2012, 2013, 2014 and 2015) to obtain the mixing proportions, means and standard deviations of both estimated mixed normal distributions. We could have pre-defined the means and standard deviations of the two mixed normal distributions using values estimated from the obvious wild and decoy birds. However, we did not do so as we expected that inter-annual variations in meteorological conditions during migration (e.g. predominance of eastern vs. western winds) and relative breeding productivity across the range (i.e. origins) should create variation in the annual distribution of δ 2 Hf in wild birds migrating through southwestern France. This expectation was supported by a year effect in an analysis of variance predicting δ 2 Hf values by year of capture for birds captured and ringed in the wild by forensic ringers (F2,37 = 28.3, P < 0.001).

Results

Normality

We report the mean ± s.d. values of δ 2 Hf for the wild individuals and obvious decoys in Table 1, while

Mixing proportions

Fitting normal mixture densities to the annual datasets, we identified for each year two mixed normal distributions closely matching those of obvious decoys and of wild individuals (Table 1 and Fig. 2). The estimated proportion of birds with captive-vs. wild-grown feathers were relatively balanced in 2013 (45% vs 55%) but were skewed in 2012 (61% vs 39%), 2014 (75% vs 25%) and 2015 (72% vs 28%), with a larger proportion of caged birds.

Discussion

Ortolan Buntings illegally captured in France in 2012-2015 included some individuals that were obvious decoys with plumage abnormalities, leucism or melanism, which are likely linked to nutrient deficiencies [START_REF] Sage | The incidence of albinism and melanism in British birds[END_REF], [START_REF] Hill | Proximate Basis of Variation in Carotenoid Pigmentation in Male House Finches[END_REF]. All of these individuals were captive for long periods prior to sampling and so they grew their feathers in captivity. The feathers sampled on these birds provided a good opportunity to obtain estimates of the δ 2 Hf concentrations in captive-grown feathers, and further compare them to similar measures obtained from Ortolan feathers grown in the wild. The latter came from active migrants captured in nature by licensed forensic ringers. The decoys had notably lower δ 2 Hf concentrations than wild birds which is consistent with the expectation that captive birds, presumably drinking tap water and eating commercial food, can have different δ 2 Hf than those of wild birds derived from diets and drinking water driven by local precipitation at the site of feather development. Here, by analyzing δ 2 Hf concentrations of seized Ortolans, we were able to predict their wild or captive origin, as long as the captive birds consumed only minimal rainwater and wild-grown seeds while growing their feathers. For birds that have not molted feathers while in captivity, analysis of δ 2 H in other chemically active tissues (e.g. toenails, blood) with differing turnover periods may provide further insights into their capture history and requires further study [START_REF] Mazerolle | Patterns of differential migration in white-throated sparrows evaluated with isotopic measurements of feathers[END_REF]. However, the expectation of a difference of isotopic concentrations in feathers of wild vs. captive individuals will be entirely case specific and depend very much on the molt origin of wild birds compared to captive birds and their artificial diets [START_REF] Bowen | Global application of stable hydrogen and oxygen isotopes to wildlife forensics[END_REF], [START_REF] Bowen | A review of land-sea coupling by groundwater discharge of nitrogen to New England estuaries: Mechanisms and effects[END_REF], [START_REF] Jameel | Tap water isotope ratios reflect urban water system structure and dynamics across a semiarid metropolitan area[END_REF].

Within the datasets obtained for the different years, 2013 appears distinct, with balanced proportions of wild and captive birds, while the other three years included more captive than wild birds. The difference could arise from variations in confiscation efforts and procedures, concerning mainly long-lasting or recently captured decoys present on field poaching stations, possibly complemented by additional wild and recently captured individuals moved indoors for fattening. If the police operated mainly at field capture sites, confiscated birds would concern only caged decoys, with a majority of long-caged individuals with feathers grown in captivity, and fewer recently captured individuals kept in the field to complement the available decoys surviving from the precedent year. If the police further confiscated the wild individuals captured recently but then moved indoor for fattening, we would expect a higher proportion of individuals wearing wild-grown feathers. The first scenario would produce a larger proportion of birds of captive origin (see 2012,2014,2015), while the second scenario would produce a comparatively larger proportion of wild individuals (as in 2013).

While isotope profiling or fingerprinting are often used in food quality controls or criminal cases [START_REF] Meier-Augenstein | Stable Isotope Forensics: An Introduction to the Forensic Application of Stable Isotope Analysis[END_REF], there is a growing literature showing that stable isotope analyses are a powerful tool in forensic tracing of the origins of legally or illegally traded animals and plants [START_REF] Retief | Can stable isotopes and radiocarbon dating provide a forensic solution for curbing illegal harvesting of threatened cycads?[END_REF], [START_REF] Oliveira | Wild animals seized in a state in Northeast Brazil: Where do they come from and where do they go?[END_REF]. Studying the African Grey Parrot Psittacus erithacus, Alexander et al. [START_REF] Alexander | Stable isotope analyses as a forensic tool to monitor illegally traded African grey parrots[END_REF] found a systematic difference in feather carbon (δ 13 Cf) and deuterium (δ 2 Hf) values for known wild and captive birds, and a match between isotopic ratios obtained for imported birds that deceased in captivity and for wild birds, attesting that these imported birds had been captured illegally in the wild. Castelli and Reed [START_REF] Castelli | Use of stable isotopes to distinguish wild from pen-raised northern bobwhite[END_REF] identified unique patterns of three isotopic ratios (δ 13 C, δ 15 N and δ 34 S) to distinguish wild and pen-raised Northern Bobwhite Colinus virginianus. Dittrich et al. [START_REF] Dittrich | Stable isotope analyses-A method to distinguish intensively farmed from wild frogs[END_REF] investigated the isotopic composition (δ 13 C, δ 15 N and δ 18 O) of legally traded frog legs from suppliers in Vietnam and Indonesia, and segregated naturally born from intensively farmed frogs. Indeed, the use of stable isotope analyses can definitely improve the monitoring of legally and illegally traded wildlife species across the globe.

Analysis of stable isotopes in recently grown feathers of illegally captured birds in Europe is a promising forensic tool to help authorities identify wild poached individuals of protected species that are otherwise bred legally in captivity. This could be highly efficient to segregate legally from illegally traded Goldfinches Carduelis carduelis, which suffers from a long-term poaching pressure that has resulted in a drastic range retraction in North Africa [START_REF] Khelifa | Unravelling the drastic range retraction of an emblematic songbird of North Africa: potential threats to Afro-Palearctic migratory birds[END_REF]. International illegal trade of poached Goldfinches has also recently increased across southern Europe, following the decline in North African breeding numbers [START_REF] Khelifa | Unravelling the drastic range retraction of an emblematic songbird of North Africa: potential threats to Afro-Palearctic migratory birds[END_REF]. Indeed, French authorities have recurrently seized Goldfinches in recent years, and analyzing the stable isotope concentrations (notably δ 2 H) in their feathers could help to determine the captive vs wild origin of seized birds, and the geographic origin of wild seized individuals [START_REF] Kelly | Stable hydrogen isotope analysis as a method to identify illegally trapped songbirds[END_REF]. As the principal strategy used to deal with animals recovered from illegal traffic is to release them back into nature [START_REF] Magroski | Where to release birds seized from illegal traffic? The value of vocal analyses and ecological niche modeling[END_REF], this would allow rapid release of individuals with recently wild-grown feathers, and give longer recovery time to individuals having been captive for longer durations.

As a conclusion, we acknowledge that differences in isotopic concentrations in bird feathers grown in captivity and in the wild are generally case specific, though if attested as was the case for ortolan buntings in southwest France, they can provide a useful tool to determine the duration of previous captivity for seized birds, and therefore adapt their rehabilitation period before an optimal release back in the wild. The information on the relative proportion of wild and recently captured individuals, vs. individuals captured since at least one year, can further inform on the efficiency of the confiscation efforts to target wild birds whose captures imped directly the fate of wild populations [START_REF] Jiguet | Unravelling migration connectivity reveals unsustainable hunting of the declining ortolan bunting[END_REF].

Figure 1

 1 Figure1shows the boxplot of the different groups studied here (including obvious decoys). The boxplot

Figure 1 .

 1 Figure 1. Boxplot of deuterium concentration values in Ortolan Bunting feathers (δ 2 Hf, in ‰) for

Figure 2 .

 2 Figure 2. Distributions of deuterium concentrations in Ortolan Bunting feathers (δ 2 Hf, in ‰) for (A)


Maxime Y Monin

Lyes Rahmouni

Francesco P Andriulli

A Hybrid Integral Equation Approach to Solve the Anisotropic Forward Problem in Electroencephalography

The boundary element method provides a computationally efficient solution to the Electroencephalography (EEG) forward problem on piece-wise homogeneous head models by using surface integral equations. However, realistic modeling of the head medium requires a proper account of the anisotropic electric nature of the human skull, which cannot be handled by standard surface integral equations. This work addresses this issue by presenting a new formulation which perturbs the standard Lippmann-Schwinger approach with volume elements within the skull. The resulting surface/volume integral equation can handle computations of fully realistic modeling for both skull and white matter. Numerical results will confirm the validity of the approach as well as its applicability to real case scenarios.

I. INTRODUCTION

Electroencephalography (EEG) is a standard method of recording the brain activity used for a wide range of medical and research applications. In particular, the accurate solution of the so-called forward EEG problem is a crucial step to obtain insight on the source activity which generates the electric potential measured on scalp electrodes. A standard numerical technique to solve the forward problem is the Boundary element method (BEM) [START_REF] Kybic | A common formalism for the integral formulations of the forward eeg problem[END_REF]. Boundary integral techniques feature several advantages over differential formulations, among which the convenient need to discretize only the boundary of the studied medium. A major drawback of standard BEM is the requirement for the conductivity to be isotropic and homogeneous. Thus, it cannot account for anisotropic human tissues in the head. The skull for instance, is known to be ten times more conductive in the tangential direction to its surface than in the normal direction [START_REF] Hallez | Review on solving the forward problem in eeg source analysis[END_REF]. To bypass this constraint, the authors of [START_REF] Rahmouni | Two volume integral equations for the inhomogeneous and anisotropic forward problem in electroencephalography[END_REF] have derived an adequate formulation which handles anisotropy and inhomogeneity of the conductivity in the EEG problem by adapting a volume integral technique previously used in high frequency electromagnetic solvers [START_REF] Kobidze | Integral equation based analysis of scattering from 3-d inhomogeneous anisotropic bodies[END_REF]. Consequently however, this formulation requires the discretization of the whole head model and the computational efficiency of the BEM is partially lost. This costly requirement can nonetheless be restricted. In fact, we show that the volume discretization can be reduced to only the anisotropic and inhomogeneous regions, whereas surface discretization is sufficient for regions with constant scalar conductivity. This is achieved by suitable definition of a conductivity contrast which reduces the inhomogeneous problem to an equivalent piecewise homogeneous problem. Accordingly, we introduce a new formulation to solve the forward problem which can efficiently handle an arbitrary number of isotropic and nonisotropic layers. The validity of the approach is confirmed by numerical results which show the practical relevance of the newly developed formulation.

II. BACKGROUND AND NOTATION

The head medium Ω is modelled as a superposition of nested regions Ω i , i = 1, ..., N with boundary Γ i . In each region, the 3 × 3 conductivity tensor σ(r) is either homogeneous and isotropic ( σ(r) = σ i I) or inhomogeneous and anisotropic. In the latter case we still define an arbitrary background scalar conductivity σ i = σ b . The forward EEG problem then consists in the determination of the unknown electric potential φ(r), r ∈ Γ N in the presence of a source dipole located in r 0 ∈ Ω 1 inducing a primary current J p (r).

In the quasi-static regime, Maxwell's system reduces to Poisson's equation

∇ • ( σ(r)∇φ(r)) = ∇ • J p (r) r ∈ Ω (1)
with the boundary conditions

φ(r)| - i = φ(r)| + i r ∈ Γ i (2a) n(r) σ(r)∇φ(r)| - i = n(r) σ(r)∇φ(r)| + i , r ∈ Γ i<N (2b) n(r) σ(r)∇φ(r) = 0, r ∈ Γ N (2c)
where n(r) is a unit vector normal to Γ i . Define the following surface and volume operators

S(f s )(r) = Γ G(r, r )f s (r )dΓ (3)
D * (f s )(r) = Γ n(r) • ∇G(r, r )f s (r )dΓ (4)
S * v (f v )(r) = Ω G(r, r )∇ • f v (r )dΩ (5)
D * v (f v )(r) = Ω n(r) • ∇G(r, r )∇ • f v (r )dΩ (6)
where

G(r, r ) = 1 4π|r -r | (7)
is the static Green's function.

III. A HYBRID FORMULATION

We introduce the conductivity contrast χ(r), piecewisedefined as:

χ(r) = (σ i I -σ(r)) σ-1 (r) r ∈ Ω i . (8)
In particular, we have χ(r) = 0 in homogeneous isotropic layers. Equation ( 1) is then rewritten as:

σ i ∆φ(r) = ∇ • (J p (r) + χ(r) σ(r)∇φ(r)) r ∈ Ω i . (9)
Thus the second term on the right-hand side χ(r) σ(r)∇φ(r)) = J eq (r) can be seen as an equivalent volume current source originated from the anisotropic behaviour of the conductivity. Because of our definition of the contrast, the volume unknown exists only in anisotropic regions. In these conditions, we can then follow a single-layer approach [START_REF] Kybic | A common formalism for the integral formulations of the forward eeg problem[END_REF] to obtain surface equations for each unknown single-layer potential ξ Γi (r)

σ i + σ i+1 2(σ i+1 -σ i ) ξ Γi (r) - k D * (ξ Γ k )(r) + 1 σ b D * v (J eq )(r) = - 1 σ 1 D * v (J p )(r), r ∈ Γ i . (10)
The potential can then be computed as

φ(r) = k S(ξ Γ k )(r)- 1 σ b S * v (J eq )(r)- 1 σ 1 S * v (J p )(r) (11)
but to retrieve it, another volume equation is required which we can get by taking the gradient of (11) in the anisotropic regions, obtaining

k ∇S(ξ Γ k )(r) -(σ b I -σ(r)) -1 J eq (r) - 1 σ b ∇S * v (J eq )(r) = - 1 σ 1 ∇S * v (J p )(r), r ∈ Ω, σ(r) = σ i . (12)
In order to obtain a discrete matrix system, the surface unknowns are discretized with pyramidal basis functions and the volume unknown with SWG functions [START_REF] Schaubert | A tetrahedral modeling method for electromagnetic scattering by arbitrarily shaped inhomogeneous dielectric bodies[END_REF]. Following a Galerkin approach, the equations are tested with the corresponding basis functions. It must be noted that with this scheme, only the anisotropic regions ( σ(r) = σ i ) need full volume discretization, while homogeneous ones still lead to efficient surface contributions.

IV. NUMERICAL RESULTS

A first set of test has been focusing on multi-layer spherical geometries since analytic solutions are available as a benchmark in this case. The model used is then a 3-layer sphere accounting for brain, skull and scalp media. The brain and scalp have a scalar constant conductivity, whereas the skull has inhomogeneous and anisotropic conductivity [START_REF] Hallez | Review on solving the forward problem in eeg source analysis[END_REF]. Fig. 1 shows the relative error with respect to the analytic solution as a function of the average mesh size h. From the figure it is clear that our scheme is converging to the analytic reference. To show the applicability of our new scheme to

Fig. 1 .Fig. 2 .

 12 Fig. 1. Relative error as a function of the mesh refinement


Julie Boisard

Isabelle Florent
email: isabelle.florent@mnhn.fr

Keywords: Apicomplexa, Evolutionary history, Genomics, Parasitology, Protozoa Scannning Electron Microscopy; TEM: Transmission Electron Microscopy

Gregarines, a polyphyletic group of apicomplexan parasites infecting mostly non-vertebrates hosts, remains poorly known at taxonomic, phylogenetic and genomic levels. However, it represents an essential group for understanding evolutionary history and adaptive capacities of apicomplexan parasites to the remarkable diversity of their hosts. Because they have a mostly extracellular lifestyle, gregarines have developed other cellular developmental forms and host-parasite interactions, compared to their much better studied apicomplexan cousins, intracellular parasites of vertebrates (Hemosporidia, Coccidia, Cryptosporidia). This review highlights the promises offered by the molecular exploration of gregarines, that have been until now left on the side of the road of the comparative -omic exploration of apicomplexan parasites. Elucidating molecular bases for both their ultrastructural, functional and behavioral similarities and differences, compared to those of the typical apicomplexan models, is expected to provide entirely novel clues on the adaptive capacities developed by Apicomplexa over evolution. A challenge remains to identify which gregarines should be explored in priority, as recent metadata from open and host-associated environments has confirmed how underestimated is our current view on true gregarine biodiversity. It is now time to turn to gregarines to widen the currently highly skewed view we have of adaptive mechanisms developed by Apicomplexa.

Introduction

Apicomplexa are unicellular eukaryotes (protists) collectively corresponding to ~350 genera and ~6000 named species, the wide majority of which have adopted a strict parasitic lifestyle in a very wide diversity of metazoan hosts [START_REF] Adl | Revisions to the Classification, Nomenclature, and Diversity of Eukaryotes[END_REF][START_REF] Portman | The flagellar contribution to the apical complex: a new tool for the eukaryotic Swiss Army knife?[END_REF]. Apicomplexa, together with the two sister groups Dinoflagellata and Ciliata, form the supergroup Alveolata, at the base of which are Rhizaria and Stramenopiles / Phaeophyta [START_REF] Adl | Revisions to the Classification, Nomenclature, and Diversity of Eukaryotes[END_REF]. Apicomplexa are mostly known for comprising infamous intracellular parasites of vertebrates responsible for important human diseases such as malaria due to Plasmodium spp., cryptosporidiosis due to Cryptosporidium spp. and toxoplasmosis due to Toxoplasma gondii. Apicomplexa also comprise diverse other intracellular parasites of vertebrates, with economical or veterinary importance such as Eimeria spp., Babesia spp. and Theileria spp. These apicomplexan parasites have simple to very complex life cycles. Some are restricted to single hosts (monoxenous parasites, e.g. Cryptosporidium, Eimeria), other alternate between two successive hosts (dixenous parasites such as Plasmodium, Babesia and Theileria, completing sexual reproduction in various insects or arthropods and asexual phases in various tissues of vertebrates) and few have the capacity to infect multiple hosts (polyxenous parasites such as Toxoplasma, completing its sexual reproduction in cats and several asexual phases in various tissues of diverse warm blooded vertebrates). Due to their medical, veterinary or economic importance, and because it has been possible to cultivate most of them in laboratory conditions, their genomes have been deciphered (~100, deposited into the EupathDB database [START_REF] Aurrecoechea | EuPathDB: the eukaryotic pathogen genomics database resource[END_REF], Figure 1) and are major references for medical investigations, comparative genomics studies and exploration of evolutionary history of apicomplexan parasites [START_REF] Janouskovec | Apicomplexan-like parasites are polyphyletic and widely but selectively dependent on cryptic plastid organelles[END_REF][START_REF] Janouskovec | Factors mediating plastid dependency and the origins of parasitism in apicomplexans and their close relatives[END_REF][START_REF] Kwong | A widespread coralinfecting apicomplexan with chlorophyll biosynthesis genes[END_REF]Woo et al., 2015).

But Apicomplexa also comprise another group of organisms collectively known as "gregarines", that are principally monoxenous parasites of a wide diversity of non-vertebrate metazoan hosts, ranging from Polychatea annelids to tunicates, arthropods and mollusks, in which they develop mostly extracellularly -contrary to the above mentioned parasites -in the intestinal and coelomic cavities of their hosts [START_REF] Desportes | Treatise on Zoology -Anatomy, Taxonomy, Biology. The Gregarines[END_REF]. These endoparasites are mostly considered as being nonpathogenic, with a few reported cases of recognized pathogenicity, while it is clear that experimental studies focusing on gregarine pathogenicity are mostly lacking (Rueckert et al., 2019a).

Gregarines: well described Apicomplexa though forgotten at molecular level.

There are several consequences to the mostly extracellular lifestyle displayed by gregarines, a feature that indeed distinguishes them from their intracellular parasites relatives [START_REF] Desportes | Treatise on Zoology -Anatomy, Taxonomy, Biology. The Gregarines[END_REF][START_REF] Schrevel | Gregarines[END_REF]. First, gregarines can reach very large sizes even for unicellular eukaryotes, from less than a µm to more than a mm for respectively the zoite and trophozoite forms of the marine eugregarine Porospora gigantea, intestinal parasite of the lobster Homarus gammarus [START_REF] Desportes | Treatise on Zoology -Anatomy, Taxonomy, Biology. The Gregarines[END_REF][START_REF] Schrevel | Gregarines[END_REF]. Insect eugregarines, such as Gregarina garnhami, intestinal parasite of the desert locust Schistocerca gregaria, display trophozoite forms reaching a dozen to several hundred µm [START_REF] Desportes | Treatise on Zoology -Anatomy, Taxonomy, Biology. The Gregarines[END_REF][START_REF] Schrevel | Gregarines[END_REF]. Archigregarine trophozoites, such as Selenidium pendula, intestinal parasite of the polychaete Scolelepis (Nerine) squamata also reach several dozen µm [START_REF] Schrevel | Ultrastructure of Selenidium pendula, the Type Species of Archigregarines, and Phylogenetic Relations to Other Marine Apicomplexa[END_REF]. The large sizes of these developmental stages and their very common occurrence in a large diversity of non-vertebrate metazoan hosts have facilitated the discovery and This article is protected by copyright. All rights reserved. biological studies on gregarines, resulting in an abundant literature on their morphologies, ultrastructures and life cycles, that have been examined by photonic and electron microscopy (SEM, TEM) imaging or by using cryoelectron microscopy or immunofluorescence (see [START_REF] Desportes | Treatise on Zoology -Anatomy, Taxonomy, Biology. The Gregarines[END_REF] for exhaustive bibliography). Dynamic recordings are also available notably concerning their movements or behaviors [START_REF] Desportes | Treatise on Zoology -Anatomy, Taxonomy, Biology. The Gregarines[END_REF]. This rich literature offers a wide panorama of the adaptive capacities of these organisms to their hosts and environments, awaiting now exploration by -omic approaches to decipher the molecular bases of their functioning and variations, as it has been developed so far for their intracellular apicomplexan cousins (Rueckert et al., 2019b). Indeed, the genomic knowledge on apicomplexan is currently highly biased in favor of intracellular parasites of vertebrate hosts, belonging to Haematozoa, Coccidia and to a lesser extent, Cryptosporidia (Figure 1). Gregarines have been so far mostly excluded from thisomic exploration, to the exception of (unpublished) genome of the terrestrial insect eugregarine Gregarina niphandrodes (accessible at EupathDB [START_REF] Aurrecoechea | EuPathDB: the eukaryotic pathogen genomics database resource[END_REF], section CryptoDB), intestinal parasite of Tenebrio molitor, very partial genomic data on insect eugregarine Ascogregarina taiwanensis [START_REF] Templeton | A genome-sequence survey for Ascogregarina taiwanensis supports evolutionary affiliation but metabolic diversity between a Gregarine and Cryptosporidium[END_REF] intestinal parasite of Aedes albopictus and partial and recently emerging transcriptomic data for a dozen of terrestrial and marine gregarine species [START_REF] Janouskovec | Apicomplexan-like parasites are polyphyletic and widely but selectively dependent on cryptic plastid organelles[END_REF][START_REF] Mathur | Multiple Independent Origins of Apicomplexan-Like Parasites[END_REF][START_REF] Omoto | Expressed sequence tag (EST) analysis of Gregarine gametocyst development[END_REF] (see Figure 1 for illustration on Apicomplexa genomic data knowledge).

There are several reasons why the acquisition of -omic knowledge on gregarines is lagging behind that of their intracellular vertebrate parasite cousins. (1) As there are infecting "only" nonvertebrates hosts and are mostly considered non-pathogenic (Rueckert et al., 2019a), they have been neglected. (2) The current lack of in vitro culture methods for these parasites complicates the isolation of biological material in adequate amounts and quality for accurate usage in molecular investigations. Indeed, gregarine biological studies must rely on field collections, mostly from infected hosts (alternatively their feces), which exposes the collected material to contaminations by host cells and environmental microorganisms. The ability to maintain the hosts of a diversity of gregarines in laboratory conditions, offers a good compromise as it allows regular access to different developmental stages amenable to a variety of cellular (microscopy, test of inhibitors) and molecular (-omics) studies (see [START_REF] Desportes | Treatise on Zoology -Anatomy, Taxonomy, Biology. The Gregarines[END_REF] for exhaustive descriptions). For example, Neanthes (Nereis) divesicolor, the host of the marine eugregarine Lecudina tuzetae, can be maintained for several months in natural or artificial sea water in the laboratory [START_REF] Desportes | Treatise on Zoology -Anatomy, Taxonomy, Biology. The Gregarines[END_REF][START_REF] Kuriyama | Dynamic organization of microtubules and microtubule-organizing centers during the sexual phase of a parasitic protozoan, Lecudina tuzetae (Gregarine, Apicomplexa)[END_REF]. Also, several insect raising facilities may be used to get access to their infecting gregarines [START_REF] Clopton | Phylogenetic Relationships, Evolution, and Systematic Revision of the septate gregarines (Apicomplexa:Eugregarinorida:Septatorina)[END_REF][START_REF] Desportes | Treatise on Zoology -Anatomy, Taxonomy, Biology. The Gregarines[END_REF]. Also, it can be expected that the concomitant progression of -omics and microscopic methodologies, allowing using increasingly reduced amounts of biological material, will also facilitate in a near future the bridging of this molecular knowledge gap, between the currently very poorly documented non cultivable gregarines and the increasingly well documented cultivable intracellular parasites of vertebrates [START_REF] Gawad | Single-cell genome sequencing: current state of the science[END_REF]. This article is protected by copyright. All rights reserved.

Why should we study gregarines?

By why should we focus on gregarines at molecular level? What could they tell us that we do not already know about apicomplexan parasites?

Because gregarines are Apicomplexa, although particular ones, with unique differences notably their mostly extracellular life mode and its biological consequences [START_REF] Adl | Revisions to the Classification, Nomenclature, and Diversity of Eukaryotes[END_REF][START_REF] Desportes | Treatise on Zoology -Anatomy, Taxonomy, Biology. The Gregarines[END_REF]Rueckert et al., 2019a;[START_REF] Schrevel | Gregarines[END_REF]. Like all Apicomplexa, gregarines present at least once during their life cycle a development form called zoite, a polarized cell comprising the so-called "apical complex" composed of scaffolding cytoskeletal elements enclosing specialized apical organelles (rhoptries, micronemes, dense granules) that gave the name to this phylum replacing the former Sporozoa [START_REF] Adl | Revisions to the Classification, Nomenclature, and Diversity of Eukaryotes[END_REF][START_REF] Morrissette | Cytoskeleton of apicomplexan parasites[END_REF][START_REF] Tardieux | Reassessing the mechanics of parasite motility and host-cell invasion[END_REF]. Gregarines (notably, Archigregarines) do have a conoid, composed of spirally arranged array of microtubules as found in Coccidia and Cryptospodia (forming Conoidasida), but secondarily lost in Haemosporidia (forming Aconoidasida) ( [START_REF] Adl | Revisions to the Classification, Nomenclature, and Diversity of Eukaryotes[END_REF][START_REF] Portman | The flagellar contribution to the apical complex: a new tool for the eukaryotic Swiss Army knife?[END_REF], Figure 1). In Apicomplexa having intracellular development phase(s) this "apical complex" has been clearly involved in the recognition and invasion of host cells, allowing parasites establishment and development in this novel ecological niche [START_REF] Hakimi | Toxoplasma Effectors Targeting Host Signaling and Transcription[END_REF][START_REF] Tardieux | Reassessing the mechanics of parasite motility and host-cell invasion[END_REF]. In Apicomplexa displaying extracellular lifestyle as most intestinal gregarines do, this "apical complex" appears to have a different role. Yet involved in parasite attachment to host cells at sporozoite stage, it subsequently appears mostly used for parasite feeding, sustaining spectacular growth phases, and not for achieving tissue penetration or parasite internalization within host cells [START_REF] Gentekaki | Largescale phylogenomic analysis reveals the phylogenetic position of the problematic taxon Protocruzia and unravels the deep phylogenetic affinities of the ciliate lineages[END_REF][START_REF] Schrevel | Ultrastructure of Selenidium pendula, the Type Species of Archigregarines, and Phylogenetic Relations to Other Marine Apicomplexa[END_REF][START_REF] Valigurova | Morphological analysis of the cellular interactions between the eugregarine Gregarina garnhami (Apicomplexa) and the epithelium of its host, the desert locust Schistocerca gregaria[END_REF]. This to the notable exception of coelomic (eu)gregarines capable of intestinal barrier crossing and neogregarines, capable of reaching intracellular niches in some of their hosts tissues [START_REF] Desportes | Treatise on Zoology -Anatomy, Taxonomy, Biology. The Gregarines[END_REF][START_REF] Schrevel | Gregarines[END_REF].

There are several consequences to this extracellular growth of these trophozoite forms in gregarines: 1) an extremely wide diversity of shapes and sizes, as mentioned above and largely used for taxonomical purposes [START_REF] Desportes | Treatise on Zoology -Anatomy, Taxonomy, Biology. The Gregarines[END_REF]; 2) a sexual phase (gamogony followed by sporogony) that also occurs extracellularly, producing developmental forms that are particular to gregarines, starting with the syzygy [START_REF] Desportes | Treatise on Zoology -Anatomy, Taxonomy, Biology. The Gregarines[END_REF][START_REF] Schrevel | Gregarines[END_REF].

Syzygy is the named given to the development stage that precludes the gregarine sexual reproduction. It corresponds to the bi-association of two trophozoites after they have detached from host cell and which are future gamonts (Figures 2,stages (a) and (b/b')). Although morphologically similar both in size and shape, the two partners of the syzygy are committed to evolve into respectively male and female gamonts (Figures 2,stages (c)). Depending on the species, the bi-association may be caudo-caudal (Figure 2A, stage (c)), lateral (Figure 2B, stage (c)), frontofrontal (not shown, see [START_REF] Desportes | Treatise on Zoology -Anatomy, Taxonomy, Biology. The Gregarines[END_REF]) or caudo-frontal (Figure 2C, stage (c)). In this latter case, found in Gregarina garnhami, the anterior partner of the syzygy is called primite the other satellite (Figure 2C, stage (c)). Occasionally, syzygy associations may involve more than two partners but the evolution of such "ménage à trois" has not been yet examined at molecular level [START_REF] Desportes | Treatise on Zoology -Anatomy, Taxonomy, Biology. The Gregarines[END_REF]. The evolution of the syzygy bi-association in a globular structure called gametocyst, initially composed of two hemispheres of similar shape and volume around which a thick wall is elaborated (Figures 2,stages (c

)). Series of nuclear divisions with final cytokinesis

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(gamogony) then occur within each hemisphere producing male gametes within one hemisphere and female gametes within the other one (Figures 2,stages (d)). A clear anisogamy is commonly observed between male gametes -more pyriform and usually flagellated -and female gametesmore globular and non-flagellated (Figures 2A 2B,stages (d')). It is therefore only after complete gametes production that the "sex" of gamonts may be deduced. Numerous imaging recordings have been performed to study the cellular events occurring during this first phase of the gregarine sexual reproduction called gamogony, with a remarkable confocal imaging analysis performed in the case of the marine eugregarine Lecudina tuzetae, intestinal parasite of the Polychaeta Hediste (Nereis) diversicolor, in which ~5000 male and as many female gametes are produced per gametocyst [START_REF] Kuriyama | Dynamic organization of microtubules and microtubule-organizing centers during the sexual phase of a parasitic protozoan, Lecudina tuzetae (Gregarine, Apicomplexa)[END_REF].

It is interesting to notice that, depending on the species, the length of the syzygy phase might be particularly long; in the atypical case of Diplauxis hatti, coelomic parasite of the Polychaeta Perinereis cultrifera, this bi-association remains stable for more than two years, awaiting host's sexual maturation to engage into gamogony [START_REF] Prensier | The unique adaptation of the life cycle of the coelomic gregarine Diplauxis hatti to its host Perinereis cultrifera (Annelida, Polychaeta): an experimental and ultrastructural study[END_REF].

Once the male and female gametes have been produced within their respective compartments, their mixing occurs within the gametocyst. Thousands of fertilizations then take place simultaneously during a process called "gamete dance" in L. tuzetae, which lasts ~4 hours and produces ~5000 zygotes per gametocyst [START_REF] Kuriyama | Dynamic organization of microtubules and microtubule-organizing centers during the sexual phase of a parasitic protozoan, Lecudina tuzetae (Gregarine, Apicomplexa)[END_REF] (Figure 2B stage (e)). Sporogony then begins and the gametocyst takes the name of sporokyst (Figures 2B and2C, stages (e/e')). Each zygote secretes a cyst wall (stage called immature sporocyst) and undergoes meiosis and additional mitosis (in eugregarines and neogregarines) leading to sporozoites formation (Figures 2, stages (f) to (i)). Each spore, also called sporocyst, therefore possesses a thick wall and is the form of dissemination of the gregarine in the environments [START_REF] Desportes | Treatise on Zoology -Anatomy, Taxonomy, Biology. The Gregarines[END_REF][START_REF] Schrevel | Gregarines[END_REF]. Its contains 4 sporozoites in the case of archigregarines and 8 sporozoites in eugregarines and neogregarines ( [START_REF] Desportes | Treatise on Zoology -Anatomy, Taxonomy, Biology. The Gregarines[END_REF][START_REF] Schrevel | Gregarines[END_REF], see also Figures 2 stages (i)). In Coccidia, sporozoites are also formed within a spore that however is called oocyst while in gregarines it is called sporocyst [START_REF] Desportes | Treatise on Zoology -Anatomy, Taxonomy, Biology. The Gregarines[END_REF][START_REF] Schrevel | Gregarines[END_REF]. In marine eugregarines, spores are eventually released into the environments with the breaking down of the sporokyst (Figure 2B stage (e) in the case of L. tuzetae). In terrestrial gregarines, spores are released in the environment via sporoducts that are formed at the surface of the sporokyst (see for example Gregarina garnhami Figure 2B stage (e')). The progeny in terms of number of spores, resulting from the evolution of a single syzygy, is considerable and can reach few thousand to several million depending on the gregarine species [START_REF] Desportes | Treatise on Zoology -Anatomy, Taxonomy, Biology. The Gregarines[END_REF][START_REF] Schrevel | Gregarines[END_REF]. Sporokysts are therefore obviously a material of choice to isolate genomic DNA. Sporocysts on the other hand, are the developmental forms that are likely collected within soils and sediments from environments (see below).

Once ingested by hosts, the spores undergo dehiscence after passage through the host's digestive system and sporozoites are released (4 in the case of archigregarines, 8 in the case of eugregarines and neogregarines, Figures 2 stages (j)). These will be able to attach to their host's intestinal cells (Figure 2C,(j')), using their apical end displaying typical "apical complex" features, and these attached sporozoites will start to grow dramatically, evolving into trophozoites. In the case of G. garnhami for example, trophozoites grow from less than 10µm to over 400µm in length within a single host. The size range of trophozoites may therefore cover 2 to 3 orders of magnitude This article is protected by copyright. All rights reserved. depending on the species [START_REF] Desportes | Treatise on Zoology -Anatomy, Taxonomy, Biology. The Gregarines[END_REF][START_REF] Schrevel | Gregarines[END_REF]. This gigantism, achieved for a large number of gregarine species, offers a remarkable material for cell biology explorations and immunofluorescence imaging for example [START_REF] Kuriyama | Dynamic organization of microtubules and microtubule-organizing centers during the sexual phase of a parasitic protozoan, Lecudina tuzetae (Gregarine, Apicomplexa)[END_REF][START_REF] Valigurova | The enigma of eugregarine epicytic folds: where gliding motility originates?[END_REF]. This radical difference in lifestyle including gamogonic and sporogonic phases, contrasting from those observed in Hematozoa and Coccidia, results also in very different interactions between gregarines and their hosts, which raises many questions about the adaptive pathways they have developed, as apicomplexan parasites, to survive over the course of evolution. Which molecular solution have they then developed to survive within the host's intestinal tracts or other cavities, maintain survival, acquire nutriments, complete (a)sexual reproduction, with a remarkable success if one considers their wide occurrence in such a high diversity of endoparasitic non-vertebrate hosts contexts? [START_REF] Desportes | Treatise on Zoology -Anatomy, Taxonomy, Biology. The Gregarines[END_REF][START_REF] Schrevel | Gregarines[END_REF] In order to widen our view on host-adaptive modes in Apicomplexa, we propose thereafter some possible exploration topics focusing on gregarines, which are obviously not exhaustive as the number of biological questions that may be asked is far more important.

3.1. Gregarines, as apicomplexan parasites, do possess a fully developed apical complex, at least in some developmental stages (sporozoites, trophozoites) [START_REF] Desportes | Treatise on Zoology -Anatomy, Taxonomy, Biology. The Gregarines[END_REF]. Biological and morphological studies have established that in gregarines, this apical complex is used for host cell attachment to allow the parasite to feed from its host cell by a process known as myzocytosis [START_REF] Schrevel | Ultrastructure of Selenidium pendula, the Type Species of Archigregarines, and Phylogenetic Relations to Other Marine Apicomplexa[END_REF][START_REF] Simdyanov | Fine structure and putative feeding mechanism of the archigregarine Selenidium orientale (Apicomplexa: Gregarinomorpha)[END_REF]. The host cell penetration by the parasite is not complete as the gregarine remains extracellular with only its apical end intimately engaged in a host-parasite interplay that has been studied at microscopic level but whose molecular actors are poorly defined [START_REF] Schrevel | Ultrastructure of Selenidium pendula, the Type Species of Archigregarines, and Phylogenetic Relations to Other Marine Apicomplexa[END_REF][START_REF] Simdyanov | A new view on the morphology and phylogeny of eugregarines suggested by the evidence from the gregarine Ancora sagittata (Leuckart, 1860) Labbe, 1899 (Apicomplexa: Eugregarinida)[END_REF][START_REF] Valigurova | An ultrastructural comparison of the attachment sites between Gregarina steini and Cryptosporidium muris[END_REF]. See also [START_REF] Desportes | Treatise on Zoology -Anatomy, Taxonomy, Biology. The Gregarines[END_REF] for exhaustive descriptions of several additional examples. Therefore, the biological function of "gregarine apical complex" only partly overlaps the biological function currently attributed to "apicomplexan parasite apical complex" i.e. recognition and invasion of their host cell [START_REF] Tardieux | Reassessing the mechanics of parasite motility and host-cell invasion[END_REF].

Several questions therefore emerge: to which extend does the molecular architecture of "gregarine apical complexes" compares to that of their best known cousins, i.e. Toxoplasma and Plasmodium [START_REF] Boucher | The apicomplexan glideosome and adhesins -Structures and function[END_REF][START_REF] Tardieux | Reassessing the mechanics of parasite motility and host-cell invasion[END_REF])? Are the "scaffolding", "recognition", "invasive" and "nutrition" functions fulfilled by the same molecular components of by other ones? What are the composition and functional roles of micronemes, rhoptries and dense granules in gregarines? In intracellular apicomplexan parasites of vertebrates (Plasmodium and Toxoplasma), these secretory organelles are documented to intervene sequentially in an orchestrated manner, with first micronemes secreting parasite proteins involved in host cell recognition proteins as well as AMA-1, which, when combined to a defined cortege of RON proteins (secreted by the neck of rhoptries) will assemble in the so-called mobile junction essential to the invasion process of host cells by Plasmodium merozoites or Toxoplasma tachyzoites (reviewed in [START_REF] Tardieux | Reassessing the mechanics of parasite motility and host-cell invasion[END_REF]). Subsequently, ROP proteins (secreted by the bulb of rhoptries) and GRA proteins (secreted by the dense granules) will allow establishing the intracellular niche for these parasites, either at the parasitophorous vacuole level (facilitating metabolite exchanges) or beyond this border to manipulate the host cell program to the benefit of the parasite [START_REF] Hakimi | Toxoplasma Effectors Targeting Host Signaling and Transcription[END_REF]. Although it may This article is protected by copyright. All rights reserved. be expected that gregarine micronemes, rhoptries and dense granules will have their own protein repertoires (there is currently very limited overlaps of these repertoires between currently described apicomplexan genus [START_REF] Boucher | The apicomplexan glideosome and adhesins -Structures and function[END_REF][START_REF] Counihan | Plasmodium rhoptry proteins: why order is important[END_REF][START_REF] Hakimi | Toxoplasma Effectors Targeting Host Signaling and Transcription[END_REF])) it will be interesting to decipher their specific roles and how they contribute (or not) to establishing also the host-parasite interface. It is important here to indicate that there are alternative invasive modes in apicomplexan parasites such as Theileria and Cryptopsoridium that differ from the better described Toxoplasma/Plasmodium mode [START_REF] Gubbels | Evolution of apicomplexan secretory organelles[END_REF]. What are the molecular similarities between the T. gondii and P. falciparum parasitophorous make up and the food vacuole of gregarines, that forms at the gregarine-host cell interface [START_REF] Schrevel | Ultrastructure of Selenidium pendula, the Type Species of Archigregarines, and Phylogenetic Relations to Other Marine Apicomplexa[END_REF][START_REF] Simdyanov | A new view on the morphology and phylogeny of eugregarines suggested by the evidence from the gregarine Ancora sagittata (Leuckart, 1860) Labbe, 1899 (Apicomplexa: Eugregarinida)[END_REF][START_REF] Valigurova | An ultrastructural comparison of the attachment sites between Gregarina steini and Cryptosporidium muris[END_REF]? Or is the similarity stronger to the feeder organelle of epicellular Cryptosporidium? [START_REF] Barta | What is Cryptosporidium? Reappraising its biology and phylogenetic affinities[END_REF][START_REF] Bartosova-Sojkova | Epicellular Apicomplexans: Parasites "On the Way In[END_REF].

Their mostly extracellular development.

A first morphological and biological consequence of this particular behavior selected over evolution is the fact mentioned above that gregarine trophozoites can reach very large sizes contrary to intracellular parasites of vertebrates (1-10µm at most). A second is that their sexual phase is also extracellular, starting with the syzygy that evolves into gametocysts then sporokysts producing sporocysts (Figure 2). These developmental forms are strikingly distinct from the developmental forms encountered in Toxoplasma, Plasmodium and even Cryptosporidium [START_REF] Aly | Malaria parasite development in the mosquito and infection of the mammalian host[END_REF][START_REF] Bouzid | Cryptosporidium pathogenicity and virulence[END_REF][START_REF] Robert-Gangneux | Epidemiology of and diagnostic strategies for toxoplasmosis[END_REF]. Interestingly, the oocysts forms of Toxoplasma and Cryptosporidium, which are also extracellular and disseminated with their hosts' feces as resistant forms in the environments, are however elaborated intracellularly, within their hosts' intestinal cells [START_REF] Bouzid | Cryptosporidium pathogenicity and virulence[END_REF][START_REF] Robert-Gangneux | Epidemiology of and diagnostic strategies for toxoplasmosis[END_REF]. Indeed, in Toxoplasma and Cryptosporidium the gamogony remains intracellular, whereas it is extracellular in gregarines. An important consequence is that gregarines thus display totally different types of host-parasite interactions, having other constraints to face such as surviving in host-gut environment. Several studies have explored the permeability of the trophozoite membrane in link with the question of their nutrition mode [START_REF] Desportes | Treatise on Zoology -Anatomy, Taxonomy, Biology. The Gregarines[END_REF][START_REF] Schrevel | Gregarines[END_REF]. This questions the molecular nature and the biological role of their inner membrane complex (imc), which, interestingly, may be acting as a continuous "shield" all around the trophozoite as it is only interrupted at the conoidal opening through which nutrition occurs (see [START_REF] Schrevel | Ultrastructure of Selenidium pendula, the Type Species of Archigregarines, and Phylogenetic Relations to Other Marine Apicomplexa[END_REF] for the case of S. pendula or [START_REF] Kuriyama | Dynamic organization of microtubules and microtubule-organizing centers during the sexual phase of a parasitic protozoan, Lecudina tuzetae (Gregarine, Apicomplexa)[END_REF] for the case of L. tuzetae). Interestingly, as the syzygy evolves towards the gametocyst form, this imc appears to be disassembled concomitantly with the secretion, by the gametocyst, of the protective cyst wall (see [START_REF] Kuriyama | Dynamic organization of microtubules and microtubule-organizing centers during the sexual phase of a parasitic protozoan, Lecudina tuzetae (Gregarine, Apicomplexa)[END_REF] for details on L. tuzetae). This suggests that in gregarines, one form of shielding (imc) in trophozoites and syzygies gives place to another form of shielding (cyst wall) during gametogony then sporogony, both of which being intended to isolate the gregarine from its hostile (gut) environment. Obviously, the molecular exploration of such a parasite-environment (host) interplay will reveal novel adaptive features developed by Apicomplexa over evolution. To which extend the host-gut environment is less hostile in an invertebrate host rather than in a vertebrate one, notably regarding immune system response and microbiota regulation, should certainly be taken into account. First, invertebrate hosts rely mainly on innate immunity to fight intruders while vertebrates also have adaptive defense mechanisms [START_REF] Buchmann | Evolution of Innate Immunity: Clues from Invertebrates via Fish to Mammals[END_REF]. In addition, the co-existing microbiome is notoriously less complex and diverse in invertebrates than vertebrates [START_REF] Bahrndorff | The Microbiome of Animals: Implications for Conservation Biology[END_REF]. These differences could explain the capacity of gregarines to self-maintain in such hostgut environment for an extended life cycle times while parasites of vertebrates have been This article is protected by copyright. All rights reserved. constrained to invade host cells to achieve their maintenance in hosts. Further studying not only gregarines but also their host's immune and microbiota responses will certainly clarify the contribution of these host-specific features to the diversity of gregarines behaviors and life traits over evolution.

Gregarines have developed a wider diversity of motility and mobility modes

than what is mostly described (and deeply studied at molecular level) for intracellular parasites of vertebrates: the gliding motility [START_REF] Frenal | Gliding motility powers invasion and egress in Apicomplexa[END_REF]. This movement, governed by an acto-myosin motor, involves dozens of proteins that appear (so far) well conserved between apicomplexan species [START_REF] Boucher | The apicomplexan glideosome and adhesins -Structures and function[END_REF][START_REF] Frenal | Gliding motility powers invasion and egress in Apicomplexa[END_REF][START_REF] Mueller | Functions of myosin motors tailored for parasitism[END_REF]. Whether the gliding components are conserved also in gregarines that move by gliding (i.e., most eugregarines) remains to be established. However, gregarines do display other modes of motility such as rolling, bending (notably archigregarines [START_REF] Desportes | Treatise on Zoology -Anatomy, Taxonomy, Biology. The Gregarines[END_REF]), the molecular bases of which are currently totally unknown. Do these alternative modes involve molecular components shared with those of the glideosome? Do they involve other components, inherited from the putative ancestor and possibly lost secondarily in intracellular parasites of vertebrate [START_REF] Füssy | Reductive Evolution of Apicomplexan Parasites from Phototrophic Ancestors[END_REF][START_REF] Janouskovec | Factors mediating plastid dependency and the origins of parasitism in apicomplexans and their close relatives[END_REF]Woo et al., 2015)? Or do they involve novel components, repositioned from the ancestor heritage or acquired by horizontal gene transfer? All this remains to be established for the diversity of known and to-be-discovered gregarines. Answers to these questions will be precious to understand how such a diversity of motility/mobility modes may have emerged for apicomplexan from a common ancestral genetic heritage, as apicomplexan derive from ancestral biflagellated organisms with repositioning of some of the former flagellar components into the apical complex structure and functioning [START_REF] Füssy | Reductive Evolution of Apicomplexan Parasites from Phototrophic Ancestors[END_REF][START_REF] Janouskovec | Factors mediating plastid dependency and the origins of parasitism in apicomplexans and their close relatives[END_REF]Woo et al., 2015). This knowledge will also be precious to widen our current knowledge of the adaptive capacities to hosts developed by these remarkable apicomplexan parasites. Indeed, evolutionary molecular studies on this point have established that gliding components partially existed in the common ancestor of Apicomplexa [START_REF] Füssy | Reductive Evolution of Apicomplexan Parasites from Phototrophic Ancestors[END_REF][START_REF] Janouskovec | Factors mediating plastid dependency and the origins of parasitism in apicomplexans and their close relatives[END_REF]Woo et al., 2015) but have been repositioned to be functionally operational in intracellular parasites of vertebrates. What paths of specialization did they then follow to generate such a diversity of movements in gregarines? And, corollary to these observations, can we formulate the hypothesis that the lack of host-cell fully invasive capacities of gregarines may be due either to absence of gliding capacities despite a developed / expected to be functional apical complex (in the case of archigregarines) or, conversely, to an under-developed apical complex despite operational gliding capacities (in the case of eugregarines)?

It is therefore now time to turn to gregarines to explore these adaptive traits at molecular level, but the next question is; which ones to select and from which extent of diversity? Indeed, a recent convergence of novel data clearly indicates that the current inventory of the true gregarine diversity is dramatically underestimated -and therefore, corollary, all the relevant biological models may have not yet been discovered.

The true gregarine biodiversity.

Our current understanding of gregarine biodiversity comes from three sources of information that only partly overlap : 1) the number of formally inventoried species [START_REF] Portman | The flagellar contribution to the apical complex: a new tool for the eukaryotic Swiss Army knife?[END_REF], 2) the number of species theoretically computable based on the inventory and diversity of their hosts This article is protected by copyright. All rights reserved. [START_REF] Desportes | Treatise on Zoology -Anatomy, Taxonomy, Biology. The Gregarines[END_REF]; 3) environmental or host-associated metagenomic or metabarcoding approaches, that have revealed novel molecular signatures, sufficiently related to gregarines to allow taxonomic affiliation to this group but sufficiently divergent to strongly suggest novel taxonomic species [START_REF] De Vargas | Ocean plankton. Eukaryotic plankton diversity in the sunlit ocean[END_REF][START_REF] Del Campo | Assessing the Diversity and Distribution of Apicomplexans in Host and Free-This article is protected by copyright[END_REF][START_REF] Mahe | Parasites dominate hyperdiverse soil protist communities in Neotropical rainforests[END_REF]. The regular cross-referencing of these three sources of data leads to a permanent readjustment of both the taxonomy and phylogeny of these species, so that it is safe to say that the current biodiversity of gregarines is a field of investigation under construction, whose physiognomy is likely to evolve considerably in the coming years.

Formally described species.

Regarding the first point, there are currently ~1770 formally described gregarine species, unequally distributed between archigregarines (~20), eugregarines (~1700) and neogregarines (~50) [START_REF] Portman | The flagellar contribution to the apical complex: a new tool for the eukaryotic Swiss Army knife?[END_REF]. In parallel, taxonomic and phylogenetic revisions concerning gregarines is a currently very active -but quite unstable -field with a diversity of successive proposals regarding their phylogenetic inter-relations as well as with other apicomplexan parasites [START_REF] Cavalier-Smith | Gregarine site-heterogeneous 18S rDNA trees, revision of gregarine higher classification, and the evolutionary diversification of Sporozoa[END_REF][START_REF] Rueckert | Archigregarines of the English Channel revisited: New molecular data on Selenidium species including early described and new species and the uncertainties of phylogenetic relationships[END_REF][START_REF] Schrevel | Ultrastructure of Selenidium pendula, the Type Species of Archigregarines, and Phylogenetic Relations to Other Marine Apicomplexa[END_REF][START_REF] Simdyanov | A new view on the morphology and phylogeny of eugregarines suggested by the evidence from the gregarine Ancora sagittata (Leuckart, 1860) Labbe, 1899 (Apicomplexa: Eugregarinida)[END_REF][START_REF] Simdyanov | First Ultrastructural and Molecular Phylogenetic Evidence from the Blastogregarines, an Early Branching Lineage of Plesiomorphic Apicomplexa[END_REF]. Molecular phylogenies are nowadays mostly based on usage of SSU rDNA marker, more rarely complete ribosomal loci [START_REF] Diakin | Morphology and Molecular Phylogeny of Coelomic Gregarines (Apicomplexa) with Different Types of Motility: Urospora ovalis and U. travisiae from the Polychaete Travisia forbesii[END_REF][START_REF] Diakin | Description of Ganymedes yurii sp. n. (Ganymedidae), a New Gregarine Species from the Antarctic Amphipod Gondogeneia sp. (Crustacea)[END_REF][START_REF] Simdyanov | First Ultrastructural and Molecular Phylogenetic Evidence from the Blastogregarines, an Early Branching Lineage of Plesiomorphic Apicomplexa[END_REF]. Studies based on the 18S SSU rDNA marker alone are fairly effective in defining monophyletic groups at the genus or family levels, but fail to robustly resolve the respective branches' relationships at higher taxonomic level. Attempts to improve phylogenies using the full ribosomal marker (18S SSU + 28S LSU rDNA) have provided some progress, but have the important disadvantage to be currently available for only very few gregarine species (~20) [START_REF] Diakin | Morphology and Molecular Phylogeny of Coelomic Gregarines (Apicomplexa) with Different Types of Motility: Urospora ovalis and U. travisiae from the Polychaete Travisia forbesii[END_REF][START_REF] Diakin | Description of Ganymedes yurii sp. n. (Ganymedidae), a New Gregarine Species from the Antarctic Amphipod Gondogeneia sp. (Crustacea)[END_REF][START_REF] Simdyanov | First Ultrastructural and Molecular Phylogenetic Evidence from the Blastogregarines, an Early Branching Lineage of Plesiomorphic Apicomplexa[END_REF]. Phylogenies relying on multiple genes (or more accurately proteins) sequences derived from genome / transcriptome investigations are now emerging but remain restrained to a dozen of gregarine species [START_REF] Janouskovec | Apicomplexan-like parasites are polyphyletic and widely but selectively dependent on cryptic plastid organelles[END_REF][START_REF] Mathur | Multiple Independent Origins of Apicomplexan-Like Parasites[END_REF]. Although they are indeed expected to be more resolving, the number of concerned species is even smaller and ambiguities remain in the interrelationships between groups, since the position of the genus Cryptosporidium is for example unstable between the two studies [START_REF] Janouskovec | Apicomplexan-like parasites are polyphyletic and widely but selectively dependent on cryptic plastid organelles[END_REF][START_REF] Mathur | Multiple Independent Origins of Apicomplexan-Like Parasites[END_REF]. These genome/transcriptome studies have however also shown another interest: some species historically described as gregarines (Platyproteum sp. and Digyalum oweni) do not actually appear anymore to be part of Apicomplexa [START_REF] Janouskovec | Apicomplexan-like parasites are polyphyletic and widely but selectively dependent on cryptic plastid organelles[END_REF][START_REF] Mathur | Multiple Independent Origins of Apicomplexan-Like Parasites[END_REF]. .

Extrapolation based on host diversity and host-parasite behaviors.

Concerning the second point, it is clearly documented that gregarines parasite virtually all nonvertebrate metazoan groups, from Polychatea annelids to tunicates, arthropods and mollusks [START_REF] Desportes | Treatise on Zoology -Anatomy, Taxonomy, Biology. The Gregarines[END_REF][START_REF] Schrevel | Gregarines[END_REF]. For a long time, experts in the gregarine field have argued that, given the currently documented diversity of gregarines, their lifestyle principally monoxenous and their apparently narrow host-range specificity, the real biodiversity of gregarines is probably several orders of magnitude underestimated in particular for those infecting insects (~half of metazoan diversity) [START_REF] Desportes | Treatise on Zoology -Anatomy, Taxonomy, Biology. The Gregarines[END_REF][START_REF] Schrevel | Gregarines[END_REF]. In addition, most known hosts are infected by several gregarine species, as for example the mealworm Tenebrio molitor that is infected by at least 3 gregarine species (Gregarina cuneata, Gregarina polymorpha and Gregarina steini) [START_REF] Clopton | Host stadium specificity in the gregarine assemblage parasitizing Tenebrio molitor[END_REF] and also G.

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niphandrodes. In consequences, some experts have even proposed that the real gregarine diversity should exceed that of their hosts, making it one of the most widespread groups of organisms in the environments [START_REF] Desportes | Treatise on Zoology -Anatomy, Taxonomy, Biology. The Gregarines[END_REF][START_REF] Schrevel | Gregarines[END_REF].

Environmental metadata uncovering a wide and mostly undescribed diversity.

Thirdly, several environmental and host-associated metagenomics/metabarcoding surveys have recently not only confirmed the gregarine experts' predictions: they have started to document this tremendous diversity at molecular level [START_REF] De Vargas | Ocean plankton. Eukaryotic plankton diversity in the sunlit ocean[END_REF][START_REF] Del Campo | Assessing the Diversity and Distribution of Apicomplexans in Host and Free-This article is protected by copyright[END_REF][START_REF] Mahe | Parasites dominate hyperdiverse soil protist communities in Neotropical rainforests[END_REF]. A very wide diversity of apicomplexan-related sequences (~80% of which appears more closely related to gregarines) is present in terrestrial soils and marine sediments [START_REF] Clopton | Host stadium specificity in the gregarine assemblage parasitizing Tenebrio molitor[END_REF][START_REF] Del Campo | Assessing the Diversity and Distribution of Apicomplexans in Host and Free-This article is protected by copyright[END_REF][START_REF] Mahe | Parasites dominate hyperdiverse soil protist communities in Neotropical rainforests[END_REF]. This diversity may be paralleled to the biological cycles of gregarines for which cyst forms, enclosing each thousand to millions of sporocyst progenies, are frequently released with the feces of their infected hosts, easily contaminating therefore such soils. In addition, the high resistance of these sporocyst forms to environmental conditions certainly explains the high abundance and maintenance (in environments) of their enclosed DNA/RNA [START_REF] Desportes | Treatise on Zoology -Anatomy, Taxonomy, Biology. The Gregarines[END_REF][START_REF] Schrevel | Gregarines[END_REF]. However, and even more remarkably, the diversity of apicomplexan-related sequences with gregarine-like affinities is also high in marine environments (both pelagic and benthic), suggesting the presence of either developing or resistant (sporocyst) forms of these gregarines in association with planktonic elements -biological or even mineral -that remain to be identified or "freely" floating in these marine environments [START_REF] De Vargas | Ocean plankton. Eukaryotic plankton diversity in the sunlit ocean[END_REF]. However, the flip side of these discoveries is that there is a remarkably high number of gregarine-like molecular data that cannot be related to formally described species [START_REF] Del Campo | Assessing the Diversity and Distribution of Apicomplexans in Host and Free-This article is protected by copyright[END_REF]. This is in part due to the very low molecular knowledge we currently have of taxonomically and morphologically described species. Only talking about the most commonly used molecular marker, SSU rDNA, it is available in databases for only about one hundred of the ~1770 formally described gregarine species, which corresponds to just ~5% of them, not taking into account the many gregarine species to be still discovered. So not only to we need to generate a much higher number of molecular markers for these known species, but we must also deploy appropriate strategies to morphologically and biologically describe the increasing number of "molecular-species" the existence of which is clearly emerging from metagenomics studies, pointing to entirely novel phylogenetic groups within gregarines [START_REF] De Vargas | Ocean plankton. Eukaryotic plankton diversity in the sunlit ocean[END_REF][START_REF] Del Campo | Assessing the Diversity and Distribution of Apicomplexans in Host and Free-This article is protected by copyright[END_REF][START_REF] Mahe | Parasites dominate hyperdiverse soil protist communities in Neotropical rainforests[END_REF].

Missing data blurs apicomplexan phylogeny

The missing information regarding gregarines is not restricted to this lack of connection between taxonomic data derived from morphological studies and molecular data emerging from metagenomics approaches. The confrontation of these data indicates that our understanding of the apicomplexan biodiversity remains very limited, biased because we have too long neglected the large spectrum of their pathogenicity focusing preferentially on the deadliest ones and ignoring the vast majority of poorly pathogenic ones (Rueckert et al., 2019a). This biased position has probably blurred not only our comprehension of the extent of their extraordinary host-adaptive capacities, but altogether a full section of their evolutionary history. So that we still do not know where the emergence of these species is taking root; is it within a group of non-pathogenic symbionts that has become even more diversified than we imagine, advocating for multiple emergence within a This article is protected by copyright. All rights reserved. radiation that is still incompletely understood [START_REF] Janouskovec | Apicomplexan-like parasites are polyphyletic and widely but selectively dependent on cryptic plastid organelles[END_REF][START_REF] Kwong | A widespread coralinfecting apicomplexan with chlorophyll biosynthesis genes[END_REF][START_REF] Mathur | Multiple Independent Origins of Apicomplexan-Like Parasites[END_REF]Rueckert et al., 2019b)?

A close examination of the recent taxonomy of Apicomplexa clearly mentions that most groups are currently polyphyletic or paraphyletic, notably: Aconoidasida, Coccidia, Gregarinasina, Archigregarinorida and Eugregarinorida [START_REF] Adl | Revisions to the Classification, Nomenclature, and Diversity of Eukaryotes[END_REF]. This is mostly due to lack of sufficiently informative and resolving data concerning these organisms, still mostly based on SSU rRNA phylogenies (see [START_REF] Cavalier-Smith | Gregarine site-heterogeneous 18S rDNA trees, revision of gregarine higher classification, and the evolutionary diversification of Sporozoa[END_REF][START_REF] Simdyanov | A new view on the morphology and phylogeny of eugregarines suggested by the evidence from the gregarine Ancora sagittata (Leuckart, 1860) Labbe, 1899 (Apicomplexa: Eugregarinida)[END_REF][START_REF] Simdyanov | First Ultrastructural and Molecular Phylogenetic Evidence from the Blastogregarines, an Early Branching Lineage of Plesiomorphic Apicomplexa[END_REF] to give few examples) and recent, emerging, phylogenomic analyses [START_REF] Janouskovec | Apicomplexan-like parasites are polyphyletic and widely but selectively dependent on cryptic plastid organelles[END_REF][START_REF] Kwong | A widespread coralinfecting apicomplexan with chlorophyll biosynthesis genes[END_REF][START_REF] Mathur | Multiple Independent Origins of Apicomplexan-Like Parasites[END_REF]. Certainly, insufficient sampling also prevents a solid and integrated vision of phylogenetic inter-relationships for all of these species [START_REF] Del Campo | Assessing the Diversity and Distribution of Apicomplexans in Host and Free-This article is protected by copyright[END_REF][START_REF] Janouskovec | Apicomplexan-like parasites are polyphyletic and widely but selectively dependent on cryptic plastid organelles[END_REF][START_REF] Kwong | A widespread coralinfecting apicomplexan with chlorophyll biosynthesis genes[END_REF][START_REF] Mathur | Multiple Independent Origins of Apicomplexan-Like Parasites[END_REF].

Conclusion.

With just over 100 genomes deciphered for ~350 genera and ~6000 described species, Apicomplexa is a group for which there is still a lot to discover even if, it is not the poorest documented branch of the tree of life [START_REF] Sibbald | More protist genomes needed[END_REF]. It is far from having revealed all the secrets of the diversity of its molecular innovations, developed during its evolutionary history as it had to adapt to such a wide diversity of hosts and environments [START_REF] Desportes | Treatise on Zoology -Anatomy, Taxonomy, Biology. The Gregarines[END_REF][START_REF] Schrevel | Gregarines[END_REF]. As to date, molecular exploration of apicomplexan parasites have mainly concerned a very small number of species that have in common (1) to infect humans causing threatening and global diseases such as malaria or less threatening but worldwide spread diseases such as toxoplasmosis and cryptosporidiosis; (2) to be cultivable in the laboratory, at least for some developmental stages; (3) to have been the subject of extremely sophisticated methodological developments such as genetic manipulation, -omics in all their variations and static and dynamic microscopy.

In this panorama, gregarines, full members of the Apicomplexa phylum, have been so far left on the side of the road for exactly corollary reasons: (1) they do not infect humans, (2) they are not easy to cultivate and (3) while there is a very abundant literature of their life cycles, morphologies and ultrastructure, they are almost unknown at the genomic/transcriptomic levels and have been the subject of very few biochemical studies. But their future is now open for exploration.

The terrain is marked out for the emergence of genomic data. A first move should be in favor of known species, selected either for their biological characteristics (intestinal, coelomic, motile, nonmotile) or their particular phylogenetic position (archigregarines vs eugregarine as a broad distinction). They have the potential to teach us novel adaptive aspect of the group (relative to intracellular parasites of vertebrates), particularly in relation to their extracellular mode of living and the associated specific constraints to survival capacities. Such exploration should also reveal a part of the ancestral heritage, now possibly lost in the other branches of Apicomplexa. Studying gregarines can also allow understanding finer adaptive mechanisms, for example regarding the functionalization of the apical complex, which obviously, compared to intracellular parasites of vertebrates displays common properties (recognition) with additional functions (nutrition) and missing ones (full invasion), the molecular bases of which are to be discovered. But it cannot be overlooked that the currently documented diversity of gregarines is lagging, by probably several orders of magnitude, far beyond the true diversity of these organisms in open and host-associated This article is protected by copyright. All rights reserved.

environments [START_REF] Del Campo | Assessing the Diversity and Distribution of Apicomplexans in Host and Free-This article is protected by copyright[END_REF][START_REF] Desportes | Treatise on Zoology -Anatomy, Taxonomy, Biology. The Gregarines[END_REF][START_REF] Mahe | Parasites dominate hyperdiverse soil protist communities in Neotropical rainforests[END_REF][START_REF] Mathur | Multiple Independent Origins of Apicomplexan-Like Parasites[END_REF][START_REF] Schrevel | Gregarines[END_REF]. This diversity is also likely, once it is explored and named (in terms of species and lifestyle) to reveal even more original and unknown adaptive mechanisms for this fascinating group, the Apicomplexa. This article is protected by copyright. All rights reserved.

Figure legends

Figure 1. The genomic panorama of Apicomplexa. On this schematic representation inspired by [START_REF] Portman | The flagellar contribution to the apical complex: a new tool for the eukaryotic Swiss Army knife?[END_REF][START_REF] Portman | The flagellar contribution to the apical complex: a new tool for the eukaryotic Swiss Army knife?[END_REF][START_REF] Portman | The flagellar contribution to the apical complex: a new tool for the eukaryotic Swiss Army knife?[END_REF] and using the most recent taxonomy by Adl et al., 2019[START_REF] Adl | Revisions to the Classification, Nomenclature, and Diversity of Eukaryotes[END_REF], we have compiled the genomic information currently available on Apicomplexa (104 genomes) and proto-Apicomplexa (Chromerida, 2 genomes), mostly available from EupathDB [START_REF] Aurrecoechea | EuPathDB: the eukaryotic pathogen genomics database resource[END_REF]. For each group of data (6 in total) we have indicated: the number of available genomes, specifying the concerned species, the number of protein-coding genes, the nuclear genome size in Mb and the presence (or absence in Cryptosporidium spp; the question being still open in the case of the (unpublished) G. niphandrodes genomic data), of mitochondrial or plastid genomes. The question mark symbolizes the currently unresolved branching order of the various apicomplexan groups.

This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

With just over 100 genomes deciphered for ~6000 described species, the -omic exploration of Apicomplexa has so far mainly concerned a limited number of intracellular pathogens of vertebrates. Gregarines, full members of this phylum infecting non-vertebrates, mostly extracellular and nonpathogenic, have been so far left on the side of the road while their study offers strong promises to reveal novel, original and unknown adaptive mechanisms and evolutionary history of Apicomplexa.

  We deeply thank Professor J. Schrével, Emeritus Professor, UMR7245 CNRS MNHN for careful reading and editing of text and figures as well as valuable suggestions. The authors declare they have no conflict of interest 7. Funding. This was supported by a grant from Agence Nationale de la Recherche [LabEx ANR-10-LABX-0003-BCDiv], in the program "Investissements d'avenir" [ANR-11-IDEX-0004-02], as well as several interdisciplinary Programs of the MNHN (ATM-Microorganismes, ATM-Génomique et Collections, ATM-Emergence, AVIV department), and by CNRS (PhD fellowship to Julie Boisard, 2018-2021). 8. Authorship. IF conducted the study, JB elaborated all illustrations, IF and JB wrote the manuscript. Mourier, R. Naeem, M. Nair, A.K. Panigrahi, N.D. Rawlings, E. Padron-Regalado, A. Ramaprasad, N. Samad, A. Tomcala, J. Wilkes, D.E. Neafsey, C. Doerig, C. Bowler, P.J. Keeling, D.S. Roos, J.B. Dacks, T.J. Templeton, R.F. Waller, J. Lukes, M. Obornik, and A. Pain. 2015. Chromerid genomes reveal the evolutionary path from photosynthetic algae to obligate intracellular parasites. eLife. 4:e06974.

Figure 2 .

 2 Figure 2. Representative development cycles for 3 gregarines. The developmental cycles of: A. the (marine) archigregarine Selenidium pendula, intestinal parasite of the polychaeta Scolelepis (Nerine) squamata, adapted from (Schrevel and Desportes, 2015); B. the marine eugregarine Lecudina tuzetae, intestinal parasite of the Polychaeta Hediste (Nereis) diversicolor, adapted from (Schrevel and Desportes, 2015); C. the terrestrial eugregarine Gregarina garnhami (C), intestinal parasite of the desert locust Schistocerca gregaria (C), based on (Canning, 1956) and personal observations. The drawings use identical "legend letters" to designate similar developmental stages across the three cycles. A. Selenidium pendula: (a) detached trophozoite; (b) caudal syzygy; (b') particularity in syzygy for this species (nuclear modifications before encystment); (c) gametocyst undergoing gamogony; (d) gametocyst with fully differentiated gametes; (d') details of male (flagellated) and female (ovoid) gamete; (e) zygote ready to undergo sporogony yielding stages with 2 nuclei (g), then 4 nuclei (h); (i) spore containing 4 sporozoites ; (j) release sporozoite (in host) starting vegetative phase. B. Lecudina tuzetae. (a) detached trophozoite; (b) lateral syzygy; (c) gametocyst undergoing gamogony; (d) gametocyst with fully differentiated gametes; (d') details of male (flagellated) and female (ovoid) gamete; (e) sporokyst enclosing ~5000 zygotes ready to evolve into spore (f) eventually undergoing sporogony yielding stages with 2 nuclei (g), 4 nuclei (h); (i) spore containing 8 sporozoites ; (j)