Philippe Steemans

Nisan Sariaslan

Borja Cascales-Miñana

Martin R Langer

Wilfried Meyenbrock

Thomas Servais
email: thomas.servais@univ-lille.fr

A diverse Early Devonian palynoflora from the Waxweiler Lagerstätte (Klerf Formation, Rhenish Massif, western Germany): palaeobotanical implications

Keywords: Devonian, miospores, early land plants, Psilophyton, Rhenish Massif, Klerf Formation, Germany

The Lower Devonian Klerf Formation is an exceptional Konservat-Lagerstätte, exposed at multiple sites in the Waxweiler region in the Eifel area, western Germany. It has been studied for its various fossils, mainly arthropods, fishes, plants, molluscs, brachiopods, and crinoids. At Waxweiler, the sediments are palaeoecologically interpreted as a prograding deltaic depositional system elongated from NW to SE in the Ardenno-Rhenish area. The Klerf Formation has, however, not been studied in full detail in terms of its microflora and microfauna. Our study of the sediments of the formation from two different quarries in the Waxweiler area yielded fairly diverse miospore assemblages dominated by abundant organic matter of varying degrees of coalification. The miospore assemblages are mainly composed of classical Lower Devonian taxa of the Old Red Continent (Laurussia). These belong, among others, to the genera Ambitisporites, Apiculiretusispora, and Retusotriletes. Biostratigraphically more important species recovered include Acinosporites lindlarensis, Apiculiretusispora brandtii, Cymbosporites asymetricus, Diatomozonotriletes franklinii, Emphanisporites annulatus, Verruciretusispora dubia and Verrucosisporites polygonalis. In addition, Emphanisporites foveolatus, which is known only from a limited area in the Ardenno-Rhenish region, is also identified, indicating an earliest Pragian to middle Emsian age for the composite section. These assemblages are found to be accompanied by reworked phytoplankton to a much lesser extent. Our results reveal a much larger palaeobotanical diversity from the Rhineland outcrops than previously known, indicating a well-developed Psilophyton-type vegetation with related plants. The results further suggest a likely presence of plants such as Leclercqia and Pertica.

Introduction

The west German Eifel Mountains are part of the Rhenish Massif and constitute the peneplain remains of a Variscan truncated upland formed by uplifting and erosion of the Variscan mountains. Most of its rocks comprise Devonian slates, sandstones and limestones, that were deposited along the southern margin of the Old Red Sandstone Continent (western part of Laurussia), a mountain chain that was formed by the Caledonian Orogeny during the Silurian. Situated at the interface between marine and terrestrial ecosystems, the Lower Devonian Eifel sediments comprise transitional facies of erosional debris washed in from the northern Laurussian continent, and shallow marine Rhenish facies deposited in estuaries and lagoons. Based on the analyses of sediments and fossil biotas, a low relief deltaic complex with a variety of sub-environments was inferred (e.g., [START_REF] Fuchs | Upper Siegenian and lower Emsian in the Eifel hills[END_REF][START_REF] Schweitzer | Die Unterdevonflora des Rheinlandes, 1. Teil[END_REF][START_REF] Poschmann | Arthropods and trace fossils from the Lower Devonian (Emsian)[END_REF][START_REF] Kanzler | Die Geologie des Raumes Waxweiler -Lünebach (NE-Teil der Daleider Muldengruppe/SW-Eifel) unter besonderer Berücksichtigung der Sedimentologie und Palökologie der Klerfer Schichten[END_REF][START_REF] Rebske | Over enkele fossielen uit de Klerf-Schichten (Onder-Emsien) bij Waxweiler (Eifel, BRD) en hun leefmilieu[END_REF][START_REF] Stets | Depositional environments in the Lower Devonian siliciclastics of the Rhenohercynian Basin (Rheinisches Schiefergebirge, W-Germany) -case studies and a model[END_REF]. The subenvironments reported include deltaic floodplains, interdistributary bays, ephemeral lakes, lagoons, fluvial channels, crevasse splays and point bars, indicating a limited marine, brackish-to fresh-water, tidally influenced shallow-water setting. Despite being folded and overthrust in the Variscan Orogeny, the Lower Devonian sediments preserve an exceptionally rich record of diverse biotas including e.g. fungi, algae, psilo-and filicophyta, bivalves, annelids, brachiopods, ostracods, eurypterids, arthropods, crinoids, cephalopods, trilobites, fishes, and bryozoans (for an extensive list, see [START_REF] Franke | Die Klerf-Schichten (Unter-Devon) im Großherzogtum Luxemburg, in der Westeifel (Deutschland) und im Gebiet von Burg Reuland (Belgien): fazielle und biostratigraphische Deutungen[END_REF]. Compared to reports on the biostratigraphy and palaeoecology of the Lower Devonian Eifel Mountains macrofossil assemblages, palynological studies on the microflora of the region are rare (e.g. [START_REF] Riegel | Sporenformen aus den Heisdoft-, Lauch-, und Nohn-Schichten (Emsium und Eifelium) der Eifel, Rheinland[END_REF][START_REF] Riegel | Palynological sequence from Lower Emsian to Givetian of the Eifel Region[END_REF][START_REF] Riegel | Palynological aspects of the Lower/Middle Devonian transition in the Eifel Region[END_REF][START_REF] Schultz | Eine unterdevonische Mikroflora aus den Klerfer Schichten der Eifel (Rheinisches Schiefergebirge)[END_REF][START_REF] Huwe | Unentdeckte Schätze im Sammlungs Archiv: Palynomorphen im Unterdevonder Eifeler Kalkmulden[END_REF]. Furthermore, such studies mainly focus on the Ardenne, the western extension of the Eifel in Belgium and Luxembourg [START_REF] Leclercq | Sporomorphes du Dévonien de Belgique[END_REF][START_REF] Streel | Miospore contribution to the upper Famennian-Strunian event stratigraphy[END_REF][START_REF] Streel | Spore stratigraphy and correlation with faunas and floras in the type marine Devonian of the Ardenne-Rhenish regions[END_REF][START_REF] Steemans | Rapport d'analyse de 60 echantillons de l'Emsien du nord du Grand-Duché de Luxembourg[END_REF][START_REF] Steemans | Preliminary survey of the palynological content of the Lower Devonian of the Oesling, Luxembourg[END_REF] or the Hunsrück Mountains south of the Eifel region [START_REF] Brocke | Palynological data from sediments of the Hunsrückschiefer type, Lower Devonian of the SW Hunsrück, Germany[END_REF].

Over the past years, special attention has been paid to the in situ spore fossil record, especially among Early Devonian plants (e.g. [START_REF] Wellman | Palaeoecology and palaeophytogeography of the Rhynie chert plants: further evidence from integrated analysis of in situ and dispersed spores[END_REF][START_REF] Bek | Paleozoic in situ spores and pollen[END_REF][START_REF] Bek | Paleozoic in situ spores and pollen[END_REF][START_REF] Cascales-Miñana | Further observations on the spores of the Rhynie chert plant Horneophyton lignieri (Kidston & Lang) Barghoorn & Darrah, 1938: Implications for palaeodiversity studies[END_REF].

This interest comes from the fact that in situ spores provide the sole evidence to link plant macrofossils to dispersed spores, and consequently an unequivocal way for discerning parent plants.

Isolated spore diversity further provides important information for vegetation reconstruction (e.g. [START_REF] Gibson | The use of spore-pollen assemblages to reconstruct vegetation changes in the Permian (Lopingian) Zechstein deposits of northeast England[END_REF]. Hence, the detailed investigation of the palynoflora from the Eifel region provides new insights into the Rhineland vegetation during the late Early Devonian.

Among the key localities for Lower Devonian biotas of the Eifel mountains are outcrops at Waxweiler (Köppen Quarries, Rhineland-Palatinate), where the Emsian-aged Klerf Formation is found in a prograding deltaic depositional system [START_REF] Fuchs | Upper Siegenian and lower Emsian in the Eifel hills[END_REF][START_REF] Stets | Depositional environments in the Lower Devonian siliciclastics of the Rhenohercynian Basin (Rheinisches Schiefergebirge, W-Germany) -case studies and a model[END_REF]. The Klerf Formation takes its name from the Luxembourg locality "Clervaux" and was first described by the German palaeontologist Rudolf Richter [START_REF] Richter | Zur Stratigraphie und Tektonik der Oesling-Eifel-Mulde. I. Über den Muldenabschnitt südlich der Schneifel[END_REF]; see also [START_REF] Gosselet | Note sur le Taunusien dans le bassin de Luxembourg et particulièrement dans le golfe de Charleville[END_REF]. Is is widely exposed in the Waxweiler area, where its outcrops are among the most diversified in the Rhenish Massif in terms of fossil content (e.g. [START_REF] Schweitzer | Zwei neue Pflanzen aus dem Unterdevon der Eifel[END_REF][START_REF] Schweitzer | Neue Pflanzenfunde im Unterdevon der Eifel (Deutschland)[END_REF][START_REF] Rebske | Over enkele fossielen uit de Klerf-Schichten (Onder-Emsien) bij Waxweiler (Eifel, BRD) en hun leefmilieu[END_REF][START_REF] Anderson | On the Emsian (Lower Devonian) arthropods of the Rhenish Slate Mountains: 2. The synziphosurine Willwerathia[END_REF][START_REF] Basse | Marine Faunen aus dem frühen Unteremsium (Unterdevon) des Givonne-Oesling-Antiklinoriums (Luxemburg)[END_REF][START_REF] Dunlop | On the Emsian (Lower Devonian) arthropods of the Rhenish Schiefergebirge: 1. Xenarachne, an enigmatic arachnid from Willwerath, Germany[END_REF][START_REF] Poschmann | Chelicerate arthropods, including the oldest Phalangiotarbid arachnid, from the early Devonian (Siegenian) of the Rhenish Massif, Germany[END_REF][START_REF] Poschmann | The eurypterid Adelophthalmus sievertsi (Chelicerata: Eurypterida) from the Lower Devonian (Emsian) Klerf Formation of Willwerath, Germany[END_REF][START_REF] Poschmann | Arthropods and trace fossils from the Lower Devonian (Emsian)[END_REF][START_REF] Poschmann | On the Emsian (Lower Devonian) arthropods of the Rhenish Slate Mountains: 5. Rare and poorly known eurypterids from Willwerath, Germany[END_REF][START_REF] Braddy | Giant claw reveals the largest ever arthropod[END_REF][START_REF] Poschmann | Wurzelstrukturen früher Gefäßpflanzen aus der Klerf-Formation (Unterdevon, höchstes Unter-Emsium) von Waxweiler (SW-Eifel, Rheinland-Pfalz, SW-Deutschland)[END_REF][START_REF] Poschmann | Telltale eyes: the lateral visual systems of Rhenish Lower Devonian eurypterids (Arthropoda, Chelicerata) and their palaeobiological implications[END_REF][START_REF] Poschmann | A new sea scorpion (Arthropoda, Eurypterida) from the Early Devonian of Willwerath (Rhineland-Palatinate, SW Germany)[END_REF]. They are composed of dark grey to black thinly laminated, organic-and metal-rich shales and siltstones that have been slightly metamorphosed during the late and post-Caledonian tectonic phases. Waxweiler can be considered a "Konservat Fossil-Lagerstätte", with a high disparity and abundance of various fossils including early fish (jawless heterostracans, jawed placoderms, see [START_REF] Blieck | The Waxweiler project, Eodevonian heterostracan pteraspidomorphs and mythology of the big five[END_REF]).

Here we document the palynological microfloral assemblages from Waxweiler in light of recent studies concerning the stratigraphic, palaeogeographic and ecological distribution of early land plants with the objective to provide a better understanding of the palaeoenvironmental context of the region in the frame of early land plant evolution in a deltaic depositional system.

Materials and methods

Samples for this study were collected from two quarries in the Waxweiler region (Figure 1). These sediments consist of the above-described deltaic complex deposits manifested in the alternation of sub-to supratidal facies. The Klerf Formation has been assigned to the lower Emsian (Lower Devonian; e.g. [START_REF] Solle | Gliederung und Aufbau der Klerfer Schichten am Nordrand der Olkenbacher Mulde (Unterdevon, Sudost-Eifel)[END_REF][START_REF] Braddy | Giant claw reveals the largest ever arthropod[END_REF][START_REF] Poschmann | Wurzelstrukturen früher Gefäßpflanzen aus der Klerf-Formation (Unterdevon, höchstes Unter-Emsium) von Waxweiler (SW-Eifel, Rheinland-Pfalz, SW-Deutschland)[END_REF][START_REF] Dejonghe | The lithostratigraphy of the lower Devonian formations of the Eisleck region (northern Luxembourg). Comparison with their Belgian lateral equivalents[END_REF]. In ascending order, eleven samples were collected from the Köppen quarry in Waxweiler (locality 1 in Fig. 1). All of them provided palynomorphs WAX1-11. Two additional samples were collected from the Köppen quarry in Merlscheid (locality 2 in Fig. 1), of which one provided palynomorphs (Eifel II-1). The stratigraphical position between the two quarries is unclear, but all samples provide palynomorphs of a same assemblage that is described below. The samples were processed in the palynological laboratory of the Palaeontology research team of the UMR 8198 CNRS unit Evo-Eco-Paleo at the University of Lille (France). Following standard procedures, palynomorphs were extracted from fine-grained samples such as shale and siltstone, using standard palynological techniques. This involved cold hydrochloric (HCl), cold hydrofluoric (HF) and hot hydrochloric (HCl) acid maceration to remove carbonates, silicates, and fluorosilicates, respectively. The organic residues were sieved through 15 μm nylon meshes. Nine of the processed samples contain palynomorphs, with most abundant land-plant derived spores, but also few acritarchs and scolecodonts, in varying abundances and states of preservation, but generally moderately to poorly preserved. Each palynological slide was observed using a ZEISS Axioskop optical microscope equipped with x40, x63 and x100 objectives. All rock samples, palynological slides and residues are stored in the palaeontological collections of the of the UMR 8198 CNRS of the University of Lille (France).

Additional palynological slides are also stored in the collections of the University of Liège (Eddy Laboratory). Palynological constituents have been recorded and identified to species level wherever possible. The relative abundances have been recorded as "rare, common or abundant" to shed more light on the environmental setting (Table 1). For the identification and biostratigraphic correlation, we followed e.g. [START_REF] Breuer | Devonian spore assemblages from northwestern Gondwana: taxonomy and biostratigraphy[END_REF], [START_REF] Richardson | Middle Old Red Sandsone spore assemblages from the Orcadian Basin northeast Scotland[END_REF][START_REF] Richardson | Silurian and Devonian spore zones of the Old Red Sandstone Continent and adjacent regions[END_REF][START_REF] Streel | Spore stratigraphy and correlation with faunas and floras in the type marine Devonian of the Ardenne-Rhenish regions[END_REF].

Results and discussion

Assemblage composition, biostratigraphy and sedimentology

A total of 28 species and 14 genera of miospores were identified from the samples analyzed (Table 1; Plate 1). In addition to the miospores, numerous phyto-and zooclasts have been observed. The description of these elements is not the withing the aim of the present study. Among the identifiable palynomorphs are also very few acritarchs (Plate 1).

The preservation of the palynomorphs, including the miospores, varies from sample to sample and ranges from moderate to poor. In terms of palynofacies, the samples show numerous types of organic matter of differing degrees of coalification, dominating the other main components in the assemblages. However, there are still sufficient numbers of miospores in most of the samples (Table 1). These miospores are usually brown to dark brown in color, and many of them are fragmented.

Most of the miospore taxa identified belong to the genera Ambitisporites, Apiculiretusispora, and Retusotriletes. The majority of these are typical forms dominating the Early Devonian assemblages of the Old Red Sandstone Continent. However, biostratigraphically more important species have also been identified (Figure 2). They include: Acinosporites lindlarensis, Apiculiretusispora brandtii, Cymbosporites asymetricus, Diatomozonotriletes franklinii, Emphanisporites annulatus, Emphanisporites foveolatus, Verruciretusispora dubia and Verrucosisporites polygonalis. The last is a well-known worldwide-distributed species appearing in the lowest part of the Pragian [START_REF] Richardson | Silurian and Devonian spore zones of the Old Red Sandstone Continent and adjacent regions[END_REF][START_REF] Steemans | Etude palynostratigraphique du Dévonien inférieur dans l'Ouest de l'Europe[END_REF][START_REF] Streel | Spore stratigraphy and correlation with faunas and floras in the type marine Devonian of the Ardenne-Rhenish regions[END_REF].

The remaining taxa are typical of lower/middle Emsian strata, except for A. brandtii, which appears close to the Pragian/Emsian boundary, either just above or just below. According to the authors, E. annulatus appears at the Pragian/Emsian boundary [START_REF] Richardson | Silurian and Devonian spore zones of the Old Red Sandstone Continent and adjacent regions[END_REF] or just above [START_REF] Breuer | Devonian spore assemblages from northwestern Gondwana: taxonomy and biostratigraphy[END_REF]. Emphanisporites foveolatus and Verruciretusispora dubia both characterise the FD Oppel Biozone [START_REF] Streel | Spore stratigraphy and correlation with faunas and floras in the type marine Devonian of the Ardenne-Rhenish regions[END_REF] above the AB Oppel Biozone in the lower and middle Emsian. Emphanisporites foveolatus is the eponymous species of the Interval Zone Fov, the lowest biozone of the Oppel Biozone FD. Verruciretusispora dubia has been very rarely recorded from Gondwana, but it is known from Saudi Arabia [START_REF] Breuer | Devonian spore assemblages from northwestern Gondwana: taxonomy and biostratigraphy[END_REF]) from the lindlarensis-sextantii up to the svalbardiae-eximius zones, which are respectively correlated with the upper part of the FD Biozone up to the upper part of the AP Biozone of [START_REF] Streel | Spore stratigraphy and correlation with faunas and floras in the type marine Devonian of the Ardenne-Rhenish regions[END_REF]. Therefore, the Waxweiler samples correspond to the early/middle Emsian. However, both species, in particular B. foveolatus, are not well known outside the Ardenno-Rhenish area.

Besides locally known taxa, a species outside of NW Gondwana observed for the first time,

Cymbosporites asymetricus, appears higher in the Emsian of Saudi Arabia [START_REF] Breuer | Devonian spore assemblages from northwestern Gondwana: taxonomy and biostratigraphy[END_REF], but also in the late Pragian of Brazil (Mendlowicz [START_REF] Mauller | Resultados palinoestratigráficos do Devoniano da Sub-Bacia de Alto Garças (Bacia do Paraná -Brasil)[END_REF]. The poor quality of the available material and incomplete data on the stratigraphical distribution of the taxon do not allow to provide more precision. Cymbosporites asymetricus, Apiculiretusispora brandtii and Rhabdosporites minutus (the eponymous species of the ORSC Min Interval Zone) seem to be related and have been included in the same morphon [START_REF] Breuer | Devonian spore assemblages from northwestern Gondwana: taxonomy and biostratigraphy[END_REF] making long distance correlations difficult.

The miospore assemblages are representatives of typical shallow-water habitats (e.g. [START_REF] Cascales-Miñana | A new highly diverse palynoflora from the Lower Devonian Nogueras Formation of the Iberian Peninsula[END_REF][START_REF] Daners | Pragian-Emsian palynomorphs from the Cordobés Formation, Norte Basin, Uruguay: stratigraphically restricted and regionally correlative palynological events in the cool-water Malvinokaffric Realm[END_REF] with autochthonous open marine phytoplankton (acritarchs) being absent in the slides. The assignment to this environment is supported by abundant sedimentological and palaeontological evidence showing e.g. fine-grained, parallel bedded slabs and well preserved imprints of Undichna trails. Evidence for endogenic bioturbators is lacking but the association with Diplichnites trackways influenced by currents also points to a very shallow depositional environment, possibly influenced by tides and situated on a deltaic floodplain [START_REF] Poschmann | Arthropods and trace fossils from the Lower Devonian (Emsian)[END_REF].

Palaeobotanical associations

The miospore assemblages also give insights into the palaeovegetation. Acinosporites lindlarensis is a miospore that can be related, at least, to two Leclercqia macroplant species: Leclercqia complexa and Leclercqia uncinata (Xu et al., 2011;[START_REF] Bek | Paleozoic in situ spores and pollen[END_REF]. Likewise, the miospore Apiculiretusispora brandtii has been related to the plant species Psilophyton charientos and P. forbesii [START_REF] Gensel | Devonian in situ spores: a survey and discussion[END_REF]), but also to Pertica dalhousii [START_REF] Edwards | Review of in situ spores in early land plants[END_REF]. Evidence of the presence of Pertica is indirectly provided by Apiculiretusispora plicata [START_REF] Granoff | A new species of Pertica from the Devonian of eastern Canada[END_REF][START_REF] Edwards | Review of in situ spores in early land plants[END_REF].

However, arguably, the most interesting fact is that A. plicata is also documented from Rhynia gwynne-vaughanii (Wellman 2017), a plant that has never been found outside of the Rhynie chert assemblage, despite the fact that A. plicata is widely distributed around the world. The same context is observed for the miospore Emphanisporites rotatus, also globally known, that has recently been related to Horneophyton lignieri [START_REF] Cascales-Miñana | Further observations on the spores of the Rhynie chert plant Horneophyton lignieri (Kidston & Lang) Barghoorn & Darrah, 1938: Implications for palaeodiversity studies[END_REF]. Other spore taxa with implications for vegetation reconstruction are Retusotriletes rotundus, documented from the plant genera Crenaticualis and Sawdonia [START_REF] Gensel | Devonian in situ spores: a survey and discussion[END_REF][START_REF] Hotton | Early terrestrial plant environments: An example from the Emsian of Gaspé, Canada[END_REF], and Retusotriletes cf. triangulatus, documented from Asteroxylon, Psilophyton and Tricherophyton [START_REF] Gensel | Devonian in situ spores: a survey and discussion[END_REF][START_REF] Wellman | Palaeoecology and palaeophytogeography of the Rhynie chert plants: further evidence from integrated analysis of in situ and dispersed spores[END_REF][START_REF] Neregato | Reinvestigation of the enigmatic Carboniferous sphenophyte strobilus Cheirostrobus Scott and implications of in situ Retusotriletes spores[END_REF].

Reconstruction of vegetation and palaeoenvironmental interpretation

The current knowledge of the Lower Devonian (early Emsian) deposits of the Eifel region shows a fairly diverse flora formed by at least 17 plant species belonging to 11 genera (see Schweitzer 1990, table 3, for details). This flora includes six cosmopolitan genera and one endemic genus. Concerning According to current plant classification, the (early) Emsian flora of the Eifel contains many different basal forms of tracheophytes [START_REF] Capel | A factor analysis approach to modelling the early diversification of terrestrial vegetation[END_REF][START_REF] Capel | The Silurian-Devonian terrestrial revolution: Diversity patterns and sampling bias of the vascular plant macrofossil record[END_REF]. Such forms include rhyniopsids (e.g. Renalia), paratracheophytes (former Rhyniaceae, Gerrienne et al. 2006, i.e. Stockmansella) and zosterophyllopsids (e.g., Sawdonia), barinophytopsid-like plants, as well as basal euphyllophytes (former trimerophytes sensu Banks 1968, i.e. Psilophyton) and lycopsids (i.e. Drepanophycus).

Hence, the floristic composition of this Rhineland flora is characterized by an important richness of zosterophyllopsids and the absence of well-defined rhyniopsids such as typical rhyniopsids, i.e.

Cooksonia-like plants.

Rhyniopsids experienced a great diversification through the earliest Early Devonian although many taxa do not extend into the Pragian (middle Lower Devonian; [START_REF] Capel | A factor analysis approach to modelling the early diversification of terrestrial vegetation[END_REF]. Interestingly, the closely coeval Emsian flora of Wépion (Belgium) is more dominated by rhyniopsids and Psilophyton-like plants than by zosterophylls (see Gerrienne 1993, for summary), which likely reflects the phytogeographic importance of the Rhineland flora. Moreover, in a larger framework, the Emsian floras of the Rhineland represent the early steps of the Eophytic turnover from which extant floras turn from a zosterophyllopsid-dominated to a lycopsid-dominated vegetation. This scenario suggests an early Eophytic character to the described Rhineland assemblage. However, the findings of our study suggest the presence of a well-developed Psilophyton-type vegetation and related plants.

It further reveals a hidden diversity from the Rhineland outcrops, with a likely presence of Leclercqia and Pertica. Moreover, the presence of in situ spores from the Rhynie chert, a plant diversity interpreted as belonging to hot springs environment [START_REF] Channing | Yellowstone hot spring environments and the palaeo-ecophysiology of Rhynie chert plants: towards a synthesis[END_REF], would suggest a more complex palaeoenvironment than the coastal and shallow waters context previously proposed to the Rhineland flora by Schweitzer (1990, fig. 11).

Of particular interest for biostratigraphy, palaeogeography and palaeobotany is

Emphanisporites foveolatus, a taxon that is only known in a small area of the Ardenno-Rhenish region and in the Emsian of Poland (i.e. Lanninger 1968;[START_REF] Rebske | Over enkele fossielen uit de Klerf-Schichten (Onder-Emsien) bij Waxweiler (Eifel, BRD) en hun leefmilieu[END_REF][START_REF] Riegel | Sporenformen aus den Heisdoft-, Lauch-, und Nohn-Schichten (Emsium und Eifelium) der Eifel, Rheinland[END_REF][START_REF] Riegel | Palynological sequence from Lower Emsian to Givetian of the Eifel Region[END_REF][START_REF] Riegel | Palynological aspects of the Lower/Middle Devonian transition in the Eifel Region[END_REF][START_REF] Steemans | Etude palynostratigraphique du Dévonien inférieur dans l'Ouest de l'Europe[END_REF][START_REF] Streel | Spore stratigraphy and correlation with faunas and floras in the type marine Devonian of the Ardenne-Rhenish regions[END_REF][START_REF] Turnau | Size distribution in some Middle Devonian dispersed spores and its bearing on the problem of evolution of heterospory[END_REF][START_REF] Turnau | Spore stratigraphy of Lower Devonian and Eifelian (?), alluvial and marginal marine deposits of the Radom-Lublin area (Central Poland)[END_REF]. This species could be the witness of a phytogeographic province, more or less equivalent to the S-Z province [START_REF] Steemans | The Moesian Terrane during the Lochkovian -A new palaeogeographic and phytogeographic hypothesis based on miospore assemblages[END_REF].

Reworking observed in the samples

All acritarchs identified in this study are of late Cambrian to Early Ordovician age and therefore, reworked. Reworked acritarchs are common in the Ardenne (Belgium) and the Siegerland (Germany) [START_REF] Vanguestaine | Remaniements d'acritarches dans le Siegenien et l'Emsien (Dévonien Inférieur) du Synclinorium de Dinant (Belgique)[END_REF][START_REF] Steemans | Etude palynostratigraphique du Dévonien inférieur dans l'Ouest de l'Europe[END_REF]. Schematically, in the Ardenne, it has been observed on the northern side of the Dinant Synclinorium that reworked Silurian acritarchs are abundant and Cambro-Ordovician specimens are rare. On the contrary, on the southern side of the Dinant Synclinorium, the Cambro-Ordovician acritarchs dominate the assemblages [START_REF] Steemans | Etude palynostratigraphique du Dévonien inférieur dans l'Ouest de l'Europe[END_REF]. Furthermore, [START_REF] Steemans | Etude palynostratigraphique du Dévonien inférieur dans l'Ouest de l'Europe[END_REF] noted that reworked acritarchs in Belgium have been eroded from at least seven different horizons, ranging from the Cambrian to the Early Devonian. The studies in Belgium suggest at least two different sources of sediments to the basin. A northern source, the Brabant Massif, would have delivered Silurian sediments, and a southern source, the "Mittel Deutsche Schwelle", would have provided reworked palynomorphs from Cambro-Ordovician sediments into the basin (see also [START_REF] Vanguestaine | Remaniements d'acritarches dans le Siegenien et l'Emsien (Dévonien Inférieur) du Synclinorium de Dinant (Belgique)[END_REF]. The results obtained in the Waxweiler locality only include patchy observations, with very few acritarchs recorded, that are clearly reworked from Cambro-Ordovician sediments.

Reworking of late Cambrian-Early Ordovician acritarchs and their subsequent deposition in the Waxweiler sediments are indicative for active erosional surfaces during the Early Devonian. These findings are in line with studies obtained from sedimentary petrography, provenance analysis of minerals and zircon ICP-MS laser ablation ages [START_REF] Wierich | Orogene Prozesse im Spiegel synorogener Sedimente, korngefügekundliche Liefergebietsanalyse siliziklastischer Sedimente im Devon des Rheinischen Schiefergebirges[END_REF][START_REF] Schäfer | accretion and exhumation at a Variscan active margin, recorded in the Saxothuringian flysch[END_REF][START_REF] Stets | Depositional environments in the Lower Devonian siliciclastics of the Rhenohercynian Basin (Rheinisches Schiefergebirge, W-Germany) -case studies and a model[END_REF]Schäfer 2002, Franke and[START_REF] Franke | Back to sender: tectonic accretion and recycling of Baltica-derived Devonian clastic sediments in the Rheno-Hercynian Variscides[END_REF], showing (1) that the exhumation of the basement of the Rhenish Massif lead to substantial erosion and reworking, and ( 2) that synorogenic clastic sediments in the prograding Ardenno-Rhenish delta are derived from the northern continent Baltica to be ultimately deposited in the Rheno-Hercynian Belt of Germany in the Rhenish Massif [START_REF] Franke | Back to sender: tectonic accretion and recycling of Baltica-derived Devonian clastic sediments in the Rheno-Hercynian Variscides[END_REF].

Conclusions

The new palynological analysis from the Devonian Klerf Formation deposits at Waxweiler revealed assemblages from deltaic and nearshore coastal settings comprising abundant phyto-and zooclasts, together with palynomorphs, mostly miospores and rare acritarchs. The poorly to moderately wellpreserved taxa provide the following results:

(1) The assemblages from the Waxweiler outcrops reveal a much more diverse palynoflora than those formerly known from the Lower Devonian of the German part of the Rhenish Massif, comprising 28 miospore species from 14 genera, indicating a well-developed Psilophytontype vegetation and related plants, with a likely presence of Leclercqia and Pertica.

(2) Some of the spores found in this study (e.g. Apiculiretusispora plicata and Emphanisporites rotatus) have been found in situ in the sporangia of plants previously regarded to be exclusive of the Rhynie Chert (e.g. Rhynia gwyne-vaughanii and Horneophytopn lignieri). These plants have been described to be associated with hot spring systems, and would be indicative of a more complex palaeoenvironment than what was previously considered to represent the Rhineland flora, or, on the other hand, indicate that some of the typical plants of the Rhynie chert had a much wider geographical distribution.

(3) Emphanisporites foveolatus could correspond to a phytogeographic province; more or less equivalent to the S-Z phytogeographic province of [START_REF] Steemans | The Moesian Terrane during the Lochkovian -A new palaeogeographic and phytogeographic hypothesis based on miospore assemblages[END_REF].

  widely distributed genera, the flora is composed of Taeniocrada (Göppert) Kräusel & Weyland, Zosterophyllum Penhallow, Distichophytum Mägdefrau, Sawdonia Hueber, Drepanophycus Göppert, and Psilophyton Dawson. Endemicity is represented by Tursuidea Schweitzer. Interestingly, numerous taxa of the Emsian flora constituents are monospecific, including Stockmansella langii (Stockmans) Fairon-Demaret, Sartilmania jabachensis (Kräusel & Weyland) Fairon-Demaret, Sawdonia spinosissima Schweitzer, Distichophytum mucronatum Mägdefrau, Drapnophycus spinaeformis Göppert, and Tursuidea paniculata Schweitzer. The species diversity is completed by Taeniocrada, Renalia and Psilophyton with two species each: Taeniocrada decheniana (Göppert) Kräusel & Weyland and T. dubia Kräusel & Weyland, Renalia graberti Schweitzer and R. major Schweitzer, as well as Psilophyton arcuatum (Halle) Schweitzer and P. burnotense (Gilkinet) Kräusel & Weyland (=Psilophyton goldschmidtii Halle). Zosterophyllum is present with three species: Zosterophyllum rhenanum Kräusel & Weyland, Z. spectabile Schweitzer and Z. fertile Leclercq). To this list some tentative taxonomic assignations can be added, such as ?Protobarinophyton sp. and "Drepanophycus" gaspianus.

( 4 )

 4 Very few acritarchs recovered from the samples are all of Cambro-Ordovician age, and thus clearly not in situ, and represent reworked material derived from older sediments. This finding is in line with scenarios of active erosional surfaces, petrographic provenance studies and laser ablation ages of zircon minerals. (5) Future examination of the abundant phyto-and zooclasts, in terms of detailed palynofacies analysis, could provided interesting results. Xu HH, Berry CM, Wang Y, Marshall JE. 2011. A new species of Leclercqia Banks, Bonamo et Grierson (Lycopsida) from the Middle Devonian of North Xinjiang, China, with a possible climbing habit. International Journal of Plant Sciences. 172:836-846.

FigureFigure 1 .

 1 Figure, Plate, and TableCaptions :

Figure 2 .Plate 1 .

 21 Figure 2. Biostratigraphic comparison chart (a-b: annulatus-bellatulus).

Acknowledgements

We acknowledge the useful comments and suggestions by Paweł Filipiak and an anonymous referee, as well as from the chief-editor James Riding. We are grateful to Agemar Siehl (Bonn) for literature information on the source area, to Sylvie Régnier (Lille) for her support in the palynological laboratory, and to Christoph Steinhoff and Georg Heumann (Bonn) for assistance with the microscopy. P.S is a Senior Research Associate of the Belgium NFSR. B.C.M. and T.S. were supported by EARTHGREEN project (ANR-20-CE01-0002-01) and N.S. and M.R.L. by the University of Bonn.

Declaration of Interest

No potential conflict of interest was reported by the authors.


Yehuda Izraeli

David Lepetit

Shir Atias

Netta Mozes-Daube

Gal Wodowski

Oded Lachman

Neta Luria

Shimon Steinberg

Julien Varaldi

Einat Zchori-Fein

Elad Chiel
email: elad-c@sci.haifa.ac.il

Genomic characterization of viruses associated with the parasitoid Anagyrus vladimiri (Hymenoptera: Encyrtidae)

Keywords: biocontrol agent, dicistrovirus, endosymbiont, iflavirus, mealybug, reovirus, virome

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

Introduction

Numerous arthropod species host symbiotic microorganisms that are integral to their life history, ecology and evolution [START_REF] Zchori-Fein | Manipulative Tenants; Bacteria Associated with Arthropods[END_REF]. Such microorganisms engage in a wide range of symbiotic relationships, from parasitism to obligate mutualism, and may affect various biological features of their hosts [START_REF] Zchori-Fein | Manipulative Tenants; Bacteria Associated with Arthropods[END_REF][START_REF] Drew | Microbial evolution and transitions along the parasitemutualist continuum[END_REF]. While the diverse interactions of bacteria, and to a lesser extent of fungi, with insects have been extensively studied, viruses associated with insects are much less explored. This is mainly due to paucity of knowledge of a great fraction of virus diversity as attested by recent large-scale or more focused surveys that unravelled numerous new virus lineages [START_REF] Shi | Redefining the invertebrate RNA virosphere[END_REF][START_REF] Webster | Twenty-Five new viruses associated with the Drosophilidae (Diptera)[END_REF][START_REF] Wu | Abundant and diverse RNA viruses in insects revealed by RNA-Seq analysis: Ecological and evolutionary implications[END_REF].

Among insects, it appears that endoparasitoids have special relationships with viruses. Those insects develop within an arthropod host, ultimately causing its death [START_REF] Godfray | Parasitoids : Behavioral and Evolutionary Ecology[END_REF]. Most parasitoids belong to the order Hymenoptera. Apart from pathogenic viruses, they often harbour inherited viruses that may affect their phenotype. For instance, some viruses may affect their behaviour, as observed in the solitary parasitoid Leptopilina boulardi. In that case, the virus forces infected females to superparasitize (i.e., laying eggs in already parasitized hosts), thus favouring its horizontal transmission within the superparasitized host [START_REF] Varaldi | Infectious behavior in a parasitoid[END_REF][START_REF] Varaldi | Cost induced by viral particles manipulating superparasitism behaviour in the parasitoid Leptopilina boulardi[END_REF]. The ssRNA iflavirus DcPV is also injected by the parasitoid Dinocampus coccinellae into its ladybeetle host during oviposition. The virus then replicates in the beetle's brain which may participate in turning the ladybeetle into a 'zombie' that guards the parasitoid cocoon from predation [START_REF] Dheilly | Who is the puppet master? Replication of a parasitic wasp-associated virus correlates with host behaviour manipulation[END_REF]. Other true free-living viruses, such as the entomopoxvirus DlEPV found in some parasitoid wasps, may protect the wasp offspring from host immune reaction [START_REF] Coffman | A mutualistic Poxvirus exhibits convergent evolution with other heritable viruses in parasitoid wasps[END_REF]. On the farther end of the pathogenic-mutualist axis, are some domesticated viruses that became integrated into the insect genome. The best-known example of this phenomenon is the case of polydnaviruses (PDVs) which are associated with wasps that parasitize caterpillars of butterflies and moths. The endogenized viral genes allow the production of viral-like particles which are injected together with the eggs inside the caterpillar host and suppress its immune system, hence allowing the successful development of the wasp larvae [START_REF] Herniou | When parasitic wasps hijacked viruses: genomic and functional evolution of polydnaviruses[END_REF]. Evidently then, viruses have diverse and important effects on the phenotype and ecology of endoparasitoids, and there are likely many more virus-insect associations that are yet to be unveiled. In the current study the viruses associated with Anagyrus vladimiri Triapitsyn (Hymenoptera: Encyrtidae), a solitary endoparasitoid wasp attacking mealybugs (Hemiptera: Pseudococcidae), were identified and characterized. Anagyrus vladimiri is mass-reared for commercial use in biological control programs to manage two major global pest species, which attack plants in dozens of families: the citrus mealybug, Planococcus citri (Risso) and the vine mealybug P. ficus (Signoret) [START_REF] Bugila | Suitability of five mealybug species (Hemiptera, Pseudococcidae) as hosts for the solitary parasitoid Anagyrus sp. nr. pseudococci (Girault) (Hymenoptera: Encyrtidae)[END_REF]. The female parasitoid lays an egg into the body of the mealybug host, which continues to develop while the wasp larva feeds and develops inside it. After a few days the mealybug host dies, its cuticle hardens and turns into a 'mummy' and the parasitoid larva pupates inside.

Eventually, the adult parasitoid chews a hole in the mummified host and emerges [START_REF] Avidov | Studies on an Israel strain of Anagyrus pseudococci (Girault) [Hym., Encyrtidae]. II. Some biological aspects[END_REF].

Recently, we reported an analysis of the fungal and bacterial portion of microbiome inhabiting this population of A. vladimiri. We found that the symbiont Wolbachia is dominating the bacterial community and under some conditions it has minor negative effects on the fecundity of the parasitoid [START_REF] Izraeli | Wolbachia influence on the fitness of Anagyrus vladimiri (Hymenoptera: Encyrtidae), a bio-control agent of mealybugs[END_REF]. The current research was launched to characterize the virome of A. vladimiri, annotate the viral genomes, assess their phylogenetic position, and test the prevalence of the various viruses in lab and field populations of the wasp.

Results

Electrophoresis characterization of Anagyrus vladimiri virome

Viral nucleic acids (VNAs) were purified from a pool of adults (from the mass-reared line, see Table 1 for details) and treated by RNaseA and DNaseI. At least nine bands ranging in size from ~1.5Kbp to ~4Kbp were clearly observed when VNAs were separated on non-denaturating gel by electrophoresis (Fig. 1, lane 3). VNAs were successfully digested by RNaseA (Fig. 1, lanes 8-11), but not by DNaseI (Fig. 1, lanes 13), suggesting that the virome of A. vladimiri is composed of RNA viruses only. For both our positive control (Phi6 dsRNA) and the VNAs from A. vladimiri, the RNase digestion was mitigated in high concentration of sodium citrate buffer (=high ionic strength) with low concentration of RNase, which is the expected outcome when the RNA is double-, and not single-stranded [START_REF] Sorrentino | Ionic control of enzymic degradation of double-stranded RNA[END_REF][START_REF] Khramtsov | Virus-like, double-stranded RNAs in the parasitic protozoan Cryptosporidium parvum[END_REF]. These results suggest that the tested strain of A. vladimiri harbours at least one, dsRNA virus with nine segments (or more, since segments of similar size may co-migrate at the same position), or several nonsegmented or segmented dsRNA viruses.

Sequencing the RNA virome of A. vladimiri

After reverse transcription, the VNAs of A. vladimiri were sequenced on an Illumina platform. We obtained 16 million of high quality paired-end reads.High sequence duplication levels were observed (>96%), suggesting that the genome sequence is complete (except for the extremities of untranslated regions), and as expected from the apparent low complexity of the sample (Fig. 1) which totalled approximately 20-30Kbp. De novo assembly of this dataset, resulted in only 30 contigs, 18 of which were shorter than 1,500bp.

Homology (by amino acid sequence identity) of 11 out of the 12 contigs longer than 1,500bp, as identified by BLASTx and PSI-BLASTp, were found to three RNA virus families; Dicistroviridae, Iflaviridae (both positive strand ssRNA, order: Picornavirales) and Reoviridae (segmented linear dsRNA, order: Reovirales) (Table 2, Fig. 2).

One contig was most similar to a Dicistroviridae member named Black queen cell virus infecting honeybees, with an average of 41% amino acid sequence identity. The contig is 8,726bp long, which is in the expected range for Dicistriviridae in general (8.5-10.2kb) and very close to the genome length of Black queen cell virus (8,550bp). We propose to name this newly discovered virus: Anagyrus vladimiri dicistrovirus (AnvDV).

One contig, 10,031bp long, had the highest similarity to different members of the family Iflaviridae, with 46% amino acid identity with the iflavirus member Dinocampus coccinellae paralysis virus used as a reference. The contig length is well within the expected range for Iflaviridae (8.8-9.7kb). We propose to name this newly discovered virus: Anagyrus vladimiri iflavirus (AnvIFV).

Nine contigs were identified as homologous to nine different segments of a reovirus infecting the winter moth Operophtera brumata (ObIRV). Protein alignment to the known ObIRV segments as a reference by BLASTp, revealed that the coverage between the nine contigs to the nine segments ranges between 20-98%, while the amino acid sequence identity ranges between 19% and 37% in all segments (Table 2). The one remaining contig out of the 12 'long' ones, had no similarity according to BLASTx and Psi-BLASTp searches (contig k199_18). However, its length was similar to other contigs assigned to the reovirus, therefore it was added to the screening assay for all reovirus segments in individuals. Results of that assay (see below) strongly support the assumption that this contig is part of the reovirus genome, and together with the nine contigs identified by BLAST, this genome consists of 10 segments. We propose to name this newly discovered segmented virus: Anagyrus vladimiri reovirus (AnvRV).

Six out of the 18 contigs shorter than 1,500bp (contigs k119_1, k119_3, k119_5, k119_8, k119_9 and k119_13; Fig. 2), were also assigned to the Iflaviridae family by the BLASTx search (Table 2, Fig. 2). Aligning those to the long AnvIFV contig showed that the small contigs are actually parts of this long contig with >96% nucleotide identity. They were discarded from the analysis.

The nearly-complete gnome sequence of the three novel RNA viruses is available in GeneBank under BioProject PRJNA641546, with the following BioSample accession numbers __.

Annotation

Analysis of the recovered contigs predicted that all 12 'long' (>1,500bp) ones have a large open reading frame (ORF), covering >90% of their length, suggesting that they encode true functional genes. In contrast, most of the 18 'short' (<1,500bp) contigs had large portions of un-translated regions (UTRs), with the ORFs covering on average less than 60% (Table 2, Fig. 2).

Interestingly, the contig identified as AnvDV harbours only one ORF, unlike most known viruses of this family, which harbour two ORFs. Furthermore, no 'internal ribosome entry site' (IRES)an RNA element typically found between the two ORFs of dicistroviruses (and enables the translation initiation of the second ORF) -was found along this contig. The genomes of the three RNA viruses were further annotated by searching the predicted ORFs for conserved domains using the NCBI CDD tool. Both the AnvIFV and AnvDV were found to each harbour five domains encoding for: RNA-dependent RNA polymerase (RdRP) (accession: cd01699), RNA helicase (accession: pfam00910), capsid proteins (CRPV_capsid superfamily, found in many dicistroviruses) (accession: cl07393) and two 'rhv-like' domains (part of a capsid protein, named as 'drug-binding pocket' found in poliovirus, also a member of Picornavirales order) (accession: cd00205) (Fig. 3a).

No conserved domains were found for the 10 AnvRV segments. Nevertheless, two genes were inferred from the BLASTx searches by homology with the ObIRV genome: RdRP on segment no.1 and a polyhedrin protein on segment no.4 (Fig. 3b).

One conserved sequence motif (GAAGAKC) was found in the 3' end termini of the five out of 10 contigs assigned to AnvRV (supp. fig 2). No conserved sequences were found in the 5' end, suggesting that a few bases in the extremities of the contigs may be missing in our current assembly.

Phylogeny

AnvRV. Phylogenetic analysis of the RdRP encoding segment of the AnvRV revealed that its closest known relative is the 'Zoersel tick virus', identified from the tick Ixodes ricinus. This virus, together with three other close relatives, have no genus taxonomic classification. The closest classified virus is the ObIRV, which belongs to the genus Idnoreovirus (Fig. 4). Pairwise alignments of the AnvRV to those five viruses reveals a 34-43% RdRP amino acid sequence identity, while it's amino acid identity with all other viruses compared in the phylogenetic analysis was 27% and lower. As the common cut-off between ICTV verified genera is 30% amino acid identity [START_REF] Matthijnssens | ICTV Virus Taxonomy Profile: Reoviridae[END_REF], this suggests that the AnvRV, together with the four other unclassified relatives, belongs to the genus Idnoreovirus (Fig. 4).

AnvDV. The phylogenetic analysis for the AnvDV clearly show that this virus belongs to the genus Triatovirus, together with the Black queen cell virus (51% amino acid RdRP identity) and other relatives (Fig. 5).

AnvIFV. The phylogenetic analysis for the AnvIFV place this virus near the Nasonia vitripennis virus [accession ACN94442.1], Aedes vexans iflavirus [QGW51140.1](61-62% amino acid RdRP identity), and others. The unique Dinocampus coccinellae paralysis virus [YP_009111311.1], also clusters on the same branch with AnvIFV, and has 58% amino acid RdRP identity with it (Fig. 5).

Prevalence of viruses in lab and field samples

To assess the prevalence of the three RNA virus candidates in the different A. vladimiri populations, diagnostic PCRs with specific primers were used (Supp. table 1) on 20 individual females from each of the Wolbachia-carrying (W + ) and Wolbachia-free (W -) lab-reared lines [START_REF] Izraeli | Wolbachia influence on the fitness of Anagyrus vladimiri (Hymenoptera: Encyrtidae), a bio-control agent of mealybugs[END_REF]. In addition, 24 field-collected individuals were screened (see Table 1 for details).

All 40 lab-reared individuals harboured the AnvRV. The AnvIFV and the AnvDV were found in 33 and 25 specimens out of 40 individuals respectively (Fig. 6). All three viruses were also detected in field individuals, although the prevalence was lower, with three females carrying AnvRV (12.5%), 10 carrying AnvDV (42%), and two carrying AnvIFV (8%) out of 24 individuals (Fig. 6).

Interestingly, out of 10 field females carrying the AnvDV, only three lacked Wolbachia, and seven out of the eight females carrying Wolbachia, also carried the AnvDV. The co-occurrence of those two microbes was statistically significantly correlated (Fisher's exact test: p=0.002), suggesting a positive interaction between Wolbachia and AnvDV within A. vladimiri populations. However, AnvDV was present both in the Wolbachia positive and the Wolbachia negative strains in the lab.

Screening for the 10 Reovirus segments in individual A. vladimiri wasps

To test whether all the 10 Reovirus segments (nine identified by sequence homology, and onecontig K119_18 -suspected by its length and UTR/contig length ratio) belong to the same AnvRV genome, rather than to different RNA viruses, 24 individuals from two wasp lines were screened by a set of diagnostic PCRs. Those included 16 individuals from the W + RV + line -a line harbouring the AnvRV as confirmed by PCR with primers targeting the RdRP segment, and 8 individuals from the 'Field-RV -' line, as negative controls. Generally, the results were as expected (table 3); in 9 W + RV + individuals all the segments were amplified, while in 6 'Field-RV -' individuals, none were amplified, suggesting that all 10 segments belong to the same virus. However, in 7 individuals, one or two segments failed to amplify, and in two individuals from the negative population one single segment was detected (Table 3).

Presence of viruses in the mealybugs

To test whether any of the three RNA viruses originated from the mealybug hosts, four pools were tested for their presence: i) Mass-reared (MR) W + A. vladimiri, ii) Field-collected A. vladimiri line without the AnvRV (RV -), iii) un-parasitized P. citri, and iv) P. citri, four days after parasitization by MR A. vladimiri, (see Table 1 for details). AnvRV was not detected in the un-parasitized mealybugs, nor in the Field-RV -A. vladimiri, which were reared under the same conditions as the MR A. vladimiri, indicating that this virus was not acquired from the mealybugs, and is associated with A. vladimiri. In contrast, AnvIFV and AnvDV were detected in both A. vladimiri and unparasitized mealybugs (albeit the bands for the mealybug samples were weak, they were confirmed by Sanger sequencing, with >99% identity), suggesting that those viruses may be acquired from the mealybugs or from the rearing environment, possibly the potato sprouts that the mealybugs feed on [START_REF] Cheng | Novel Dicistroviruses in an unexpected wide range of invertebrates[END_REF], or even the various fungi that develop on this substrate (supplementary Fig. 1).

Virus particles in A. vladimiri ovaries

To validate the presence of viruses in the females' reproductive tissues, transmission electron microscopy (TEM) was carried on sections of A. vladimiri ovaries (from the W + RV + line, see Table 1 for details). Images obtained from the three specimens tested showed clusters of round shaped viral particles, 60-65nm in diameter, organized either in viroplasm structures, or in dense clusters in the perinucleus area in the cytoplasm of follicle cells (Fig. 7). This pattern is typical to Reoviridae viruses [START_REF] Shah | Genome packaging of reovirus is mediated by the scaffolding property of the microtubule network[END_REF].

Discussion

The viral community of the parasitoid A. vladimiri, an agriculturally important biological control agent of mealybugs, is dominated by two positive ssRNA viruses from the order Picornavirales, which have sequence similarity to viruses of economically important insects, and a dsRNA segmented genome, assigned to the Idnoreovirus genus of the Reoviridae family. The latter was localized in the ovaries of the A. vladimiri, and was not found in the mealybug hosts, suggesting it specifically infects the wasp.

As most insect viromes are yet to be explored, it was not surprizing to discover new viruses in this host (e.g. [START_REF] Shi | Redefining the invertebrate RNA virosphere[END_REF]. Nevertheless, to the best of our knowledge, other than this study there is only one available detailed report on viruses in wasps of the family Encyrtidae, which reported the genome sequence of one Picorna-like virus in the parasitoid Diversinervus elegans [START_REF] Wu | Genome sequence of a novel member of the order Picornavirales from the endoparasitoid wasp Diversinervus elegans[END_REF].

Most reoviruses reported to infect insects were found in Dipterans and in Lepidopterans (most of them in species that are important agricultural pests), and are assigned to the genera Cypovirus (16 accepted species, which include >75 isolates), and Idnoreovirus (7 accepted species, >10 isolates) [START_REF] Matthijnssens | ICTV Virus Taxonomy Profile: Reoviridae[END_REF]. Some are reported to cause chronic diarrheal disease to larvae, with mild-severe symptoms, while many others have no apparent symptoms on their host [START_REF] Matthijnssens | ICTV Virus Taxonomy Profile: Reoviridae[END_REF].

Out of the few reports on reoviruses infecting parasitoids, even fewer have tested for phenotypic effects of the virus on its host. [START_REF] Renault | Commensal and mutualistic relationships of reoviruses with their parasitoid wasp hosts[END_REF] and [START_REF] Graham | Sequence analysis of a reovirus isolated from the winter moth Operophtera brumata (Lepidoptera: Geometridae) and its parasitoid wasp Phobocampe tempestiva (Hymenoptera: Ichneumonidae)[END_REF] both found no evidence for phenotypic effects of reoviruses inhabiting Diadromus pulchelus and Phobocampe tempestiva respectively. However, in some cases, reoviruses evidently cause significant effects on their parasitoid host. [START_REF] Renault | The cypovirus Diadromus pulchellus RV-2 is sporadically associated with the endoparasitoid wasp D. pulchellus and modulates the defence mechanisms of pupae of the parasitized leek-month, Acrolepiopsis assectella[END_REF] reported that during parasitization Diadromus pulchellus females inject a reovirus into the lepidopteran host together with the egg, where like the PDV effect, it inhibits the immune response and allows the full development of the parasitoid larva. In contrast, the cypovirus that infects both Campoletis sonorensis parasitoid and its lepidopteran host (in which it cannot replicate), seems to be beneficial to the lepidopteran as it reduces percent of mortality from parasitism [START_REF] Deacutis | The characterization and biological effects of a novel cypovirus on the Heliothis virescens and Campoletis sonorensis host-parasitoid system[END_REF].

Viruses of the families Dicistroviridae (15 accepted species, which include many more isolates) and Iflaviridae (15 accepted species, >80 isolates) are more commonly associated with insects, but similarly to reoviruses, in most cases their phenotype in the insect host is unknown (Valles et al., 2017a(Valles et al., , 2017b)). Reported effects of viruses from those families are mostly pathogenic (e.g., Acute bee paralysis virus, Drosophila C viruse, Cricket paralysis virus, for Dicistroviridae, and Varroa destructor virus1, Deformed wing virus for Iflaviridae). Conversely, at least one symbiotic relationship of those viruses with parasitoids was reported. The iflavirus DcPV aids the Dinocampus coccinellae to paralyze its ladybeetles host, as it replicates in the ladybeetle brain possibly leading to behaviour changes that protect the developing parasitoid [START_REF] Dheilly | Who is the puppet master? Replication of a parasitic wasp-associated virus correlates with host behaviour manipulation[END_REF].

Another symbiotic relationship was found between a cripavirus (Dicistroviridae) and Drosophila flies, where the virus seems to increase the fecundity of individuals that carry it [START_REF] Zhang | A novel cripavirus of an ectoparasitoid wasp increases pupal duration and fecundity of the wasp's Drosophila melanogaster host[END_REF].

As mentioned above, in most cases the relationship of the virus with its insect host is unknown.

As A. vladimiri wasps are reared in our lab for many generations, with no apparent disease symptoms, it can be assumed that under the tested rearing conditions (especially the AnvRV, which is fixed in the mass-reared population), the virus is at least not pathogenic, or even may be beneficial to the wasp. Nonetheless, all scenarios are to be taken into consideration when speculating on the relationship of those three novel viruses with A.vladimiri, including the possibility that those viruses don't have any phenotypic effect, and are spread in the population through efficient vertical and or horizontal transmission (e.g. [START_REF] Lüthi | A Novel RNA Virus in the parasitoid wasp Lysiphlebus fabarum: Genomic structure, prevalence, and transmission[END_REF][START_REF] Martinez | Additional heritable virus in the parasitic wasp Leptopilina boulardi: Prevalence, transmission and phenotypic effects[END_REF][START_REF] Perreau | Genetic innovations in animal-microbe symbioses[END_REF] . Future study should address these possibilities.

Screening the wasp populations showed that while the two ssRNA viruses are not present in all individuals, the AnvRV is fixed in the lab-reared lines, suggesting either efficient transfer of this virus, whether vertical, horizontal or both (e.g. [START_REF] Varaldi | A newly discovered virus manipulates superparasitism behavior in a parasitoid wasp[END_REF]. It may also suggest that the specific rearing conditions favour the carrying of the virus (e.g. [START_REF] Himler | Rapid spread of a bacterial symbiont in an invasive whitefly is driven by fitness benefits and female bias[END_REF][START_REF] Coffman | A mutualistic Poxvirus exhibits convergent evolution with other heritable viruses in parasitoid wasps[END_REF]. However, due to the low prevalence of the AnvRV in field populations of A. vladimiri, we conclude that the interaction between the two is not obligatory. Additionally, while the two ssRNA viruses were detected in virion extractions of unparasitized mealybugs, the AnvRV was not, meaning it is a wasp symbiont. On the other hand, the two ssRNA viruses may originate from the mealybug, or they can infect both the wasp and its host (e.g. [START_REF] Zhang | A novel cripavirus of an ectoparasitoid wasp increases pupal duration and fecundity of the wasp's Drosophila melanogaster host[END_REF]).

The TEM images of virus particles in follicle cells of A. vladimiri, resemble the typical pattern found in viruses from the family Reoviridae, which are known to be found in the cytoplasm, to be round shaped, 60-70nm long, and may be occluded in viroplasms [START_REF] Shah | Genome packaging of reovirus is mediated by the scaffolding property of the microtubule network[END_REF]. All species of this genus were detected only in insects [START_REF] Matthijnssens | ICTV Virus Taxonomy Profile: Reoviridae[END_REF] .Such a localization of a microbe in the ovary, strongly suggests vertical maternal transfer, which in turn may hint on long evolutionary relationships, and perhaps mutualism, between the two [START_REF] Zchori-Fein | Manipulative Tenants; Bacteria Associated with Arthropods[END_REF][START_REF] Perlmutter | Microorganisms in the reproductive tissues of arthropods[END_REF].

The evolutionary arms race between parasitoids and their hosts have led for several defence mechanisms that hosts use to escape parasitism. In response to invasion by an endo-parasitoid egg (or other foreign organisms), immune haemocytes in mealybugs may aggregate around the inserted egg, and form a melanized capsule, which eliminates its development [START_REF] Blumberg | Parasitoid encapsulation as a defense mechanism in the Coccoidea (Homoptera) and its importance in biological control[END_REF]. The rates of this encapsulation response to A. vladimiri parasitism is known to differ between P. citri and P. ficus hosts species [START_REF] Blumberg | Response by encapsulation of four mealybug species (Homoptera: Pseudococcidae) to parasitization by Anagyrus pseudococci[END_REF][START_REF] Suma | Encapsulation rates of the parasitoid Anagyrus sp. nr. pseudococci, by three mealybug species (Hemiptera: Pseudococcidae)[END_REF]. it can be speculated that AnvRV aids the wasp to overcome encapsulation, like DpRV2 does in the Diadromus pulchellus -Acrolepiopsis assectella system [START_REF] Renault | The cypovirus Diadromus pulchellus RV-2 is sporadically associated with the endoparasitoid wasp D. pulchellus and modulates the defence mechanisms of pupae of the parasitized leek-month, Acrolepiopsis assectella[END_REF], but with different efficiency in different Planococcus hosts. If this speculation is indeed true then the different prevalence between lab and field lines can be explained by the fact that the field population originated in vineyards where P. ficus is the main host for A. vladimiri, whereas the lab population develops in P. citri hosts.

Overall results of the screening of all ten segments in A. vladimiri specimens, were conclusive and indicate that all segments belong to the same virus. However, four segments failed to amplify in between one-four out of 16 specimens tested. This might suggest that those four segments have lower copy numbers than the other five. The detection of segment number four (k119_19) in two specimens of the negative population can be explained by either technical error, or by contamination.

From the pattern of Wolbachia and AnvDV prevalence in the 24 field collected females, where the presence of those two microorganisms is somewhat correlated, it can be speculated that some interaction takes place between those two. Reports on Wolbachia interaction with viruses focus on associations with human viral pathogens vectored by insects and insect diseases caused by viruses.

For example, Wolbachia protects Drosophila flies from the Drosophila C virus (Family: Dicistroviridae) (e.g. [START_REF] Hedges | Wolbachia and virus protection in insects[END_REF], and inhibits the Dengue Fever and Chikungunya pathogens in humans (DENV and CHIKV, both also positive ssRNA viruses), (e.g. [START_REF] Hoffmann | Successful establishment of Wolbachia in Aedes populations to suppress dengue transmission[END_REF][START_REF] Walker | The wMel Wolbachia strain blocks dengue and invades caged Aedes aegypti populations[END_REF]. This effect is also detected in natural populations of Drosophila and is however virus-specific [START_REF] Cogni | Wolbachia reduces virus infection in a natural population of Drosophila[END_REF]. Those inhibitions were generally correlated with lower viral copy number of the viruses. In other cases, endosymbionts do correlate positively with viral presence and transmission (e.g [START_REF] Gottlieb | The transmission efficiency of tomato yellow leaf curl virus by the whitefly Bemisia tabaci is correlated with the presence of a specific symbiotic bacterium species[END_REF][START_REF] Kliot | Implication of the bacterial endosymbiont Rickettsia spp. in interactions of the whitefly Bemisia tabaci with tomato yellow leaf curl virus[END_REF], such as reported here for A. vladimiri, where a significant positive correlation was found between the presence of the bacterium and the AnvDV in the field population, and also a higher prevalence in the lab strain infected by Wolbachia. This is intriguing since it could suggest a positive effect of Wolbachia on virus transmission, may further indicate that this virus and Wolbachia interact and clearly needs to be further investigated.

Interestingly, in oppose to most species of Dicistroviridae members, which have a 'Di-cistronic' genome (the trait which is the source of the family's name), only one polypeptide ORF could be identified in AnvDV. This is more similar to other families in the Picornavirales order, such as Iflaviridae and Picornaviridae, however in those families the genes are in opposite order to the order that was found in the AnvDV, where non-structural proteins are closer to the 3' end [START_REF] Bonning | Dicistroviruses[END_REF]. As far as we know, such genomic arrangement was found only in a few viruses, most of which are from aquatic invertebrates (Shi et al., 2016, see supplementary data #33;[START_REF] Cheng | Novel Dicistroviruses in an unexpected wide range of invertebrates[END_REF][START_REF] Wu | Genome sequence of a novel member of the order Picornavirales from the endoparasitoid wasp Diversinervus elegans[END_REF]. This rare finding suggests that this family has a broader diversity than previously known.

Conclusion

Most studies on viruses in insects focus on pathogenic interactions or transient in vectors to plants or animals. This is one of the first attempts to characterize the virome of a biocontrol agent, that overall seems to be healthy. The discovery of three new RNA viruses in a single population with no apparent diseases, highlights the richness of this hidden community of viruses. This community certainly deserves further investigation in order to clarify its transmission along generations and the phenotypic effects associated. If one (or more) of the three viruses influences the efficiency of A. vladimiri as a natural enemy, whether positively or negatively, this knowledge can be applied to improve the biological control of mealybug pests.

Methods

Insect rearing and field collections of parasitoids

Anagyrus vladimiri wasps were obtained from 'BioBee Sde Eliyahu Ltd' mass-rearing (MR) facility (Sde Eliyahu, Israel), and used to establish a Wolbachia-positive line (W + ), from which a Wolbachia-free line (W -) was established by antibiotic treatments [START_REF] Izraeli | Wolbachia influence on the fitness of Anagyrus vladimiri (Hymenoptera: Encyrtidae), a bio-control agent of mealybugs[END_REF]. The two lines were reared under controlled conditions of 26±1°C, 60±20% RH, and 16L:8D photoperiod regime on the citrus mealybugs Planococcus citri, feeding on sprouted potatoes Solanum toberosum.

Additional A. vladimiri individuals were collected in the field during the summer of 2020, using funnel insect traps placed in an unsprayed vineyard in northern Israel (32. 7200N, 35.1903E). The traps (n=10), baited with mealybugs-infested potatoes, were retrieved from the field after one week and parasitized mealybugs were incubated in the lab. The A. vladimiri adults which emerged (emergence was achieved from three out of the 10 traps, the other seven were empty), were used either for screening viruses' prevalence (n=24, results in Fig. 3), and others were let to parasitize in the lab, establishing the 'Field' line (n=~20 female foundresses). One generation later, eight females of this Field line were individually placed in cups and allowed to oviposit in mealybugs.

Mothers and three of their emerged offspring were then tested for the presence of the AnvRV and Wolbachia. All the cups in which both symbionts were not detected (n=7, since Wolbachia was detected in one mother) were pooled to establish an uninfected A. vladimiri line (termed hereafter 'Field-RV -' line).

The origins of the five wasp lines, and the various experiments they were used for are summarized in Table 1.

Enzymatic characterization of A. vladimiris' virome

To characterize the viral community inhabiting A. vladimiri, viral nucleic acids (VNAs) were purified following [START_REF] Martinez | Additional heritable virus in the parasitic wasp Leptopilina boulardi: Prevalence, transmission and phenotypic effects[END_REF]. Briefly, viral capsids were purified from 0.5 g of MR A. vladimiri wasps (pool of >1,500 individuals), by filtration, nuclease treatment (RNaseA and DNaseI) and centrifugation. To classify the genome type of potential viruses (RNA/DNA, double/single strand), VNAs were then extracted by SDS, subjected to different nuclease treatments, and then examined on an 0.8% agarose gel [START_REF] Martinez | Additional heritable virus in the parasitic wasp Leptopilina boulardi: Prevalence, transmission and phenotypic effects[END_REF]. A non-treated VNA sample, a dsRNA ladder (Phi6, Invitrogen, MA, USA), and a DNA ladder (1Kb+, Invitrogen, MA, USA) were used as controls.

Sequencing strategy

To further reveal all viral candidates inhabiting the wasp, VNAs were extracted from purified viral capsids using the All-In-One DNA/RNA Miniprep Kit (Biobasic, ON, Canada). Since the gel images produced in this process showed no indication of viral DNA molecules, only samples with RNA VNAs were further examined and sequenced. Libraries were prepared by the TruSeq RNA Ribo-Zero kit (Illumina, CA, USA) and sequenced by Illumina novaseq 6000 with 100 bp pairedend reads.

Since the sequence yield was very high (~16 million paired-end reads, where suspected viruses' genome size is up to 30 thousand bp), the data was randomly subsampled to three smaller datasets, including 10^5 paired reads, 10^6 paired reads and 2*10^6 paired reads. The subsampled datasets were de-novo assembled using 'Megahit' version 3.4 with default parameters. Comparing assembly results of the three subsampled datasets to the original full dataset, revealed that most of the contigs are identical, with a few additional small contigs and the 10^6 paired reads dataset was chosen as the most suitable for further analysis (average fold (sometimes termed 'depth') per bp = 4.2*10^4). The resulting contigs were searched for homologous similarity against both the nr and the swissprot databases using BLASTx, targeting viruses, bacteria and Hymenoptera (taxids 10239, 2, 7399 respectively). The following parameters were changed from default to increase sensitivity: matrix -BLOSUM45, Gap costs -12;2. An additional PSI-Blastp analysis, using the protein sequences predicted by the ORFfinder online tool (RRID:SCR_016643), was used to identify similarity of contigs that Blastx failed to identify.

Annotation

All contigs were subjected to an open reading frame (ORF) search using ORFfinder online tool (RRID:SCR_016643), with start codon including alternative initiation codons. The length of the un-translated regions (UTR), included the 5' and 3'UTRs, was calculated as contig length minus the ORF length. As the ORFfinder tool may predict also false proteins, a calculation of UTR divided by the contig length was done to estimate the likeliness of the contig to harbour a 'true' protein, the higher the value the lower chance to have a 'true' protein.

The UTRs of the ten identified segments of AnvRV were searched for conserved sequence motifs in the 5' and 3' end termini. This was done by subjecting those UTR sequences to a BLAST search against themselves, with the automatic adjusted parameters for short sequences of NCBI BLASTn suite.

Further annotation analysis of the viral genomes was done by subjecting all the ORFs to the NCBI batch-CDD tool [START_REF] Marchler-Bauer | CDD: NCBI's conserved domain database[END_REF] , against the CDD v.3.19 database to search for conserved domains. For relevant viruses a search for internal ribosome entry sites (IRES) was done by the IRESite online tool (IRESite: The database of experimentally verified IRES structures, 2022) using the NUC4.4 matrix.

Phylogeny

To construct phylogenetic trees of the three novel viruses, the RdRP amino acid sequence of each virus was aligned together with ~20 sequences of other viruses from the corresponding family.

Representative sequences were chosen from a BLASTp search and from the supplementary material of [START_REF] Shi | Redefining the invertebrate RNA virosphere[END_REF], to represent the whole family (or subfamily for the AnvRV). As the AnvDV and AnvIFV belong to the same Picornavirales order, one tree was constructed for both of them together. Multiple sequence alignment was conducted by Clustal Omega and a maximum likelihood tree was constructed with the LG substitution model using PhyML V.3 [START_REF] Sievers | Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega[END_REF] . Branch support was measured by approximate likelihood ratio tests (SH-aLRT; [START_REF] Guindon | New algorithms and methods to estimate maximum-likelihood phylogenies: Assessing the performance of PhyML 3.0[END_REF]. The outgroups chosen for the AnvRV tree were two mammal infecting viruses from the subfamily Sedoreovirinae, and a poliovirus sequence was used for the Picornavirales tree.

Virion purification from Anagyrus vladimiri and Planococcus citri

To test whether any of the three RNA viruses originate from the mealybug hosts, virions were purified from pools of: i) Mass-reared (MR) W + A. vladimiri (~0.7 g), ii) Field-collected A. vladimiri line without the AnvRV (RV -) (~0.2 g), iii) un-parasitiezed P. citri,(~0.7 g) and iv) P. citri, four days after parasitization by MR A. vladimiri, (~0.3 g) (see Table 1 for details).

Purification was done according to [START_REF] Luria | Isolation and characterization of a novel cripavirus, the first Dicistroviridae family member infecting the cotton mealybug Phenacoccus solenopsis[END_REF], with minor modifications. Briefly, the insects were homogenized in a TBE buffer, supernatant was filtered through a 0.45 µm membrane, placed on a 30% sucrose cushion, and subjected to ultracentrifugation at 242,922 g for 2 h. Then, RNA was extracted from the pellet using the Viral RNA extraction kit (Bioneer, Daejeon, South Korea). Detection of the three viruses (AnvRV, AnvIFV and AnvDV) in those samples was carried by cDNA synthesis and PCRs as described below.

PCR detections of the three viruses

Detection of RNA viruses in A. vladimiri individuals was obtained by RNA extraction (RNeazy plus kit, Qiagen, Düsseldorf, Germany) according to manufacturer protocol, with addition of Dithiothreitol to homogenizing buffer, to inhibit degradation by RNase. RNA samples were reverse-transcribed to cDNA using the RT-PCRbio kit (PCR Biosystems, London, UK), with notemplate and no-enzyme controls. cDNA was used as templates for diagnostic PCR to identify presence/absence of the three viruses. Specific primers were designed for each virus and each viral genomic segment according to the contig sequences identified from the RNA-seq data (Supp. table 1).

Transmission electron microscope (TEM)

Anagyrus vladimiri females from the W + RV + line (see Table 1 for details) were used to test for the presence of viruses in the reproductive tissues of the parasitoids. Dissected A. vladimri ovaries were fixed overnight with 1% Paraformaldehyde, 2.5% Glutaraldehyde in 0.1 M Cacodylate buffer pH 7.4 containing 5 mM CaCl2, washed in 0.1 M cacodylate, post fixed for 1 hour in with 1% OsO4 and 5 mM CaCl2 in Cacodylate buffer. Then the samples went through dehydration in ethanol and embedded in epon 812 (EMS). The blocks were cut with a diamond knife using a UC7 ultramicrotome (Leica,Wetzlar, Germany). Sections of ~75nm were collected on grids, contrast stained for 10 min with 2% uranyl acetate and visualized in a TEM (L120C, Talos, Thermo Fisher Scientific, MA, USA). group. The icons of organisms on the tips represent the taxonomic group of the host in which the virus was found (however, in some cases the viruses can be found in hosts from additional arthropod orders, such as Cricket paralysis virus). The 'star of David' symbol signs the virus that is reported to have a mutualistic relationship with its insect host. The phenotype of all other viruses is either unknown or pathogenic. The tree was inferred by maximum-likelihood using the LG substitution model, the numbers on branches indicate the results of approximate likelihood ratio tests for branch support (aLRT-SH test; [START_REF] Guindon | New algorithms and methods to estimate maximum-likelihood phylogenies: Assessing the performance of PhyML 3.0[END_REF]. Scale bar: 0.5 substitutions per site. 1 for all primer pairs used). W+: wasps from the Wolbachia-infected line, W-: wasps from Wolbachia-uninfected line. See table 2 for more details about each contig. The human poliovirus (order: Picornavirales, family: Picornaviridae) was used as an outgroup. The icons of organisms on the tips represent the taxonomic group of the host in which the virus was found (however, in some cases the viruses can be found in hosts from additional arthropod orders, such as Cricket paralysis virus). The 'star of David' symbol signs the virus that is reported to have a mutualistic relationship with its insect host. The phenotype of all other viruses is either unknown or pathogenic. The tree was inferred by maximum-likelihood using the LG substitution model, the numbers on branches indicate the results of approximate likelihood ratio tests for branch support (aLRT-SH test; [START_REF] Guindon | New algorithms and methods to estimate maximum-likelihood phylogenies: Assessing the performance of PhyML 3.0[END_REF]. Scale bar: 0.5 substitutions per site.

Fig 1 .

 1 Fig 1.Electrophoresis pattern (using a 0.8% agarose gel) of VNAs extracted from A. vladimiri, which were treated by RNase and DNase separately. 'AV' denotes VNAs of A. vladimiri. 'SSC' denotes sodium citrate buffer, which was used in 0.01x and 2x ionic strengths. '10'/'0.1' denotes high/low concentration (μg/ml) of RNase respectively. Details by lane numbers: 1-3; no digestion treatment. 1; 1Kb ladder (1KB+, Invitrogen, MA, USA), 2; Phi6 -a ladder made of viral dsRNA (Invitrogen, MA, USA), 3; A. vladimiri VNA. 4-11; Digestion by RNase A in four buffer/enzyme concentrations. 4-7; Phi6 ladder as a control for the enzymatic activity of RNase. 8-11; AV digestion. 12-13; Digestion by DNase I, 12; 1Kb -a DNA ladder as a control for the enzymatic activity of DNase. 13; AV digestion.

Fig 2 .

 2 Fig 2.Virome analysis of A. vladimiri. The assembled 30 contigs were assigned to seven taxonomic groups according to the best BLASTx hit. A) Average depth per taxonomic group, calculated as: ((sum number of reads*100bp)/sum length of contigs). B) Columns represent the size of each of the 30 contigs (bp length). Purple crosses represent the proportion of both 5' and 3' untranslated regions (UTRs) to the contigs' full length, as predicted by the ORF finder. High proportion suggests insufficient protein products, while low proportion suggests a true protein is translated from this contig. See table 2 for more details about each contig.

Fig 3 .

 3 Fig 3. Genome annotation of the three novel RNA viruses. A) AnvDV and AnvIFV. White box indicates location of ORF, colored boxes indicate identified protein domains. Numbers on scale are in bp according to the full contig. RdRP, RNA-dependent RNA polymerase. B) Annotation of the segmented AnvRV. The ten segments are drawn to scale, with boxes indicating the ORF location on the full segment, colors represent the proteins encoded from those segments as assigned from homology to known sequences of the ObIRV genome.

Fig 4 .

 4 Fig 4. Phylogenetic analysis of AnvRV (colored), based on amino acid sequence similarities of the RdRP genome segment (~1,200aa long). Representative viruses of all nine genera in the subfamily Spinareovirinae are shown. The icons of organisms on the tips represent the taxonomic group of the host in which the virus was found. The • symbol indicate viruses that have not been classified to the genus level.The human Rotavirus B and the bovine Bluetongue virus (Family: Reoviridae, subfamily: Sedoreovirinae) were used as an outgroup. The tree was inferred by maximum-likelihood using the LG substitution model, the numbers on branches indicate the results of approximate likelihood ratio tests for branch support (aLRT-SH test;[START_REF] Guindon | New algorithms and methods to estimate maximum-likelihood phylogenies: Assessing the performance of PhyML 3.0[END_REF]. Scale bar: 0.7 substitutions per site.

Fig 5 .

 5 Fig 5. Phylogenetic analysis of A. vladimiri iflavirus (AnvIFV) and A. vladimiri dicistrovirus (AnvDV) (both colored), based on amino acid sequence similarities of the RdRP gene (~300aa long). The human poliovirus (order: Picornavirales, family: Picornaviridae) was used as an out-

Fig 6 .

 6 Fig 6. The multi-infection status of three lines of A. vladimiri individuals by the three RNA viruses and Wolbachia. Boxes on the same horizontal axis denote the infection status of the four microbes in the same individual; blue fill=detected, no fill=undetected. Detection of the AnvRV, which has a segmented genome, was done with the primers targeted to the largest segment, encoding for the RdRp gene (see supp. table1for all primer pairs used). W+: wasps from the Wolbachia-infected

Fig 7 .

 7 Fig 7. Transmission electron microscope images of AnvRV particles in A. vladimiri follicle cells. A) a cluster of virus particles (black arrow), B) a viroplasm (Vp), C) high magnification of a small cluster of virions. N, nucleus; M, mitochondria. Scale bars; A) 500 nm, B) 500nm, C) 100nm.

Figures Fig 1 Fig 1 .

 11 FiguresFig 1

Fig 2 Fig 2 .

 22 Fig 2

Fig 3 Fig 3 .

 33 Fig 3

Fig 4 Fig 4 .

 44 Fig 4

Fig 5 Fig 5 .

 55 Fig 5

Fig 6 Fig 6 .

 66 Fig 6

Fig 7 Fig 7 .

 77 Fig 7

Tables 599 Table 1 .

 5991 Summary of Anagyrus vladimri lines used in this study.

	line name source

Table 2 .

 2 Virome analysis of A. vladimiri. Details for all 30 contigs resulted from RNAseq.

	600
	contig	contig	ORF	UTRs	UTR/contig	BLASTx hit	taxonomic	amino acid
	name	length	length	length	length		group	identity (%)
	k119_28	10031	9173	858	0.085535	YP_009111311.1	Iflaviridae	47
	k119_29	8726	7829	897	0.102796	NP_620564.1	Dicistroviridae 38
	k119_27	4250	4148	102	0.024	YP_392501.1	Reoviridae	33
	k119_21	3936	3788	148	0.037602	QBA09478.1	Reoviridae	29
	k119_25	3620	3521	99	0.027348	APG79179.1	Reoviridae	25
	k119_19	3388	3257	131	0.038666	YP_392504.1	Reoviridae	45
	k119_14	2163	1985	178	0.082293	YP_392505.1	Reoviridae	22
	k119_18	1963	1388	575	0.292919		Reoviridae*
	k119_22	1880	1814	66	0.035106	QYV43128.1	Reoviridae
	k119_20	1865	1739	126	0.06756	AWA82241.1	Reoviridae	24
	k119_12	1620	1526	94	0.058025	YP_392507**	Reoviridae
	k119_17	1535	1418	117	0.076221	QBA09478.1	Reoviridae	34
	k119_24	1067	107	960	0.899719	XP_017796731.1	Hymenoptera
	k119_30	887	223	664	0.748591		NA
	k119_9	809	743	66	0.081582	YP_009342053.1	Iflaviridae	39
	k119_10	782	311	471	0.602302		NA
	k119_16	711	0	711	1		NA
	k119_8	701	611	90	0.128388	YP_009342053.1	Iflaviridae	40
	k119_15	571	437	134	0.234676	CAB0037418.1	Hymenoptera
	k119_2	564	389	175	0.310284	XP_015596585.1	NA
	k119_4	560	230	330	0.589286	EZA52908.1	Hymenoptera
	k119_7	451	320	131	0.290466	CAA84692.1	Phage
	k119_13	432	203	229	0.530093	YP_009342053.1	Iflaviridae
	k119_6	412	83	329	0.798544		NA
	k119_5	394	356	38	0.096447	QFP98431.1	Iflaviridae	36
	k119_3	375	371	4	0.010667	YP_009342053.1	Iflaviridae
	k119_0	358	143	215	0.600559	ABD33943.1	Picornaviriales
	k119_23	351	86	265	0.754986	AGF91671.1	Phage
	k119_26	306	296	10	0.03268	QGJ03578.1	Phage
	k119_1	218	164	54	0.247706	YP_009342053.1	Iflaviridae
	601	*concluded as part of the genome of AnvRV by the segment screening assay (see table 3)
	602	**this hit was found only by using the PSI-BLASTp algorithm

Acknowledgements

The authors would like to thank Prof. A. Dombrovsky for useful advice, E. Erel for insect rearing consultancy and Dr. L. Shaulov for preparation and visualization of ovary slides in TEM.

Funding

This research was supported by the ISRAEL SCIENCE FOUNDATION (grant No. 397/21) to E. Z-F and E.C.

Data availability

Accession numbers to be received.

 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 1 2 3 4 6 7 8k119_27 segment1 + + + + + + + + + + + + + + + + 0 0 0 0 0 0 0 k119_21 segment2 + + + 0 + + + + + + 0 + + + 0 0 0 0 0 0 0 0 0 k119_25 segment3 + + + + + + + + + + + + + + + + 0 0 0 0 0 0 0 k119_19 segment4 + + + + + + + + + + + + + + + + + 0 0 0 0 0 + k119_14 segment5 + + + 0 + + + 0 + + + + 0 + + + 0 0 0 0 0 0 0 k119_20 segment6 + + + + + + + + + + + + + + + + 0 0 0 0 0 0 0 k119_12 segment7 + + + + + + + + + + + + + + + + 0 0 0 0 0 0 0 k119_17 segment8 + + + + + 0 + + + + + + 0 + + + 0 0 0 0 0 0 0 k119_22 segment10 + + + + + + + 0 + + + + + + + + 0 0 0 0 0 0 0 k119_18 Unknown + + + + + + + + + 0 + + + 0 0 0 0 0 0 0

Conflict of interest declaration

Author SS was employed by BioBee Sde Eliyahu Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.