Protist 172 (2021) 125806 http://www.elsevier.de/protis Published online date

ORIGINAL PAPER

A morpho-molecular perspective on the diversity and evolution of ()

⇑ Miguel M. Sandin a, , Tristan Biard b, Sarah Romac a, Luis O’Dogherty c, Noritoshi Suzuki d ⇑ and Fabrice Not a, aSorbonne Universite´, CNRS, AD2M-UMR7144 Station Biologique de Roscoff, 29680 Roscoff, France bUniv. Littoral Coˆte d’Opale, Univ. Lille, CNRS, UMR 8187, LOG, Laboratoire d’Oce´anologie et de Ge´osciences, F 62930 Wimereux, France cFacultad de Ciencias del Mar y Ambientales, Cadiz University, E-11510 Puerto Real, Spain dDepartment of Earth Science, Graduate School of Science, Tohoku University, Sendai 980–8578, Japan Submitted September 2, 2020; Accepted April 5, 2021 Monitoring Editor: B.S.C. Leadbeater

Spumellaria (Radiolaria, ) are holoplanktonic amoeboid , ubiquitous and abundant in the global ocean. Their silicified skeleton preserves very well in sediments, displaying an excellent fossil record extremely valuable for paleo-environmental reconstruction studies, from where most of their extant diversity and ecology have been inferred. This study represents a comprehensive clas- sification of Spumellaria based on the combination of ribosomal taxonomic marker genes (rDNA) and morphological characteristics. In contrast to established taxonomic knowledge, we demonstrate that symmetry of the skeleton takes more importance than internal structures at high classification ranks. Such reconsideration allows gathering different morphologies with concentric structure and spherical or radial symmetry believed to belong to other Radiolaria orders from the fossil record, as for some Entactinaria families. Our calibrated molecular clock dates the origin of Spumellaria in the middle Cambrian (ca. 515 Ma), among the first radiolarian representatives in the fossil record. This study allows a direct connection between living specimens and extinct morphologies from the Cambrian, bringing both a standpoint for future molecular environmental surveys and a better understanding for paleo-environmental reconstruction analysis. Ó 2021 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC- ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Key words: Spumellaria; rDNA; Molecular diversity; Barcoding; Molecular clock; Molecular evolution.

⇑ Corresponding authors. e-mail [email protected] (M.M. Sandin). https://doi.org/10.1016/j.protis.2021.125806 1434-4610/Ó 2021 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 2 M.M. Sandin et al.

Introduction extensive morphological criteria (De Wever et al. 2001; Noble et al. 2017; O’Dogherty et al. 2009)with Radiolaria are holoplanktonic amoeboid protists recent rDNA molecular studies (Matsuzaki et al. belonging to the Rhizaria lineage, one of the main 2015). An outcome from this later work, the current branches of the eukaryotic tree of life (Burki and classification scheme describes nine extant super- Keeling 2014). Along with they com- families: Actinommoidea (Haeckel 1862; pose the phylum , characterized by cytoplas- O’Dogherty 1994), Hexastyloidea (Haeckel 1882), mic prolongations and a skeleton composed of Liosphaeroidea (Haeckel 1882), Lithelioidea different minerals (Adl et al. 2019). Spumellaria rep- (Haeckel 1862; Petrushevskaya 1975), Pylonioidea resents an important order within the Radiolaria (Dumitrica 1989; Haeckel 1882), Saturnalioidea (Suzuki and Not 2015), extensively studied across (Deflandre 1953), Spongodiscoidea (De Wever the fossil record thanks to its opaline silica skeleton et al. 2001; Haeckel 1862), Sponguroidea (De (De Wever et al. 2001). Along with that of other radi- Wever et al. 2001; Haeckel 1862), Stylodictyoidea olarians, the Spumellaria fossil record dates back to (Haeckel 1882; Matsuzaki et al. 2015); and two the early middle Cambrian (ca. 509–521 Ma; undetermined families (Heliodiscidae, Haeckel, Aitchison et al. 2017; Suzuki and Oba 2015; 1887; De Wever et al. 2001; and Spongosphaeridae Zhang and Feng 2019) constituting an important tool Haeckel, 1862). In Matsuzaki et al. (2015) the for paleo-environmental reconstructions analysis authors stated the “overwhelmingly artificial” (e.g., Abelmann and Nimmergut 2005). In contem- spumellarian classification outweighed by morpho- porary oceans, molecular-based metabarcoding logical criteria in regard to the little amount of molec- surveys performed at global scale have shown that ular data available. Radiolaria contribute significantly to plankton com- Few studies have explored the genetic diversity munities (de Vargas et al. 2015; Pernice et al. of Spumellaria, essentially unveiling relationships 2016) and the silica biogeochemical cycle (Llopis- among higher rank taxonomical groups (Ishitani Monferrer et al. 2020), although very little is known et al. 2012; Krabberød et al. 2011; Kunitomo et al. about their ecology and diversity. Living Spumellar- 2006; Yuasa et al. 2009). To date, with a total of ia, as other Radiolaria, are characterized by a com- 35 sequences from morphologically described spec- plex protoplasmic meshwork of imens, covering 5 out of the 9 superfamilies extending radially from their skeleton (Suzuki and described, mismatches between morphological Aita 2011). With their pseudopodia, they actively and molecular characters are reported for two main capture prey, such as copepod nauplii or tintinnids, groups of Spumellaria among which different inner- by adhesion (Sugiyama and Anderson 1997). In most shell structures are found (Ishitani et al. 2012; addition to predation, many spumellarian species Yuasa et al. 2009). Despite such important insights, dwelling in the sunlit ocean harbour photosynthetic the understanding of the inner structure relation- algal symbionts, mainly identified as dinoflagellates ships with the rest of the families remains elusive. (Probert et al. 2014; Yuasa et al. 2016; Zhang et al. Time-calibration of radiolarian phylogenies thanks 2018). to the fossil record allows a better understanding Radiolaria classifications are historically based of relationships among extinct groups along with a on morphological criteria relying essentially on the contextualized evolutionary history (Decelle et al. initial spicular system, an early developed skeletal 2012a; Lewitus et al. 2018; Sandin et al. 2019). In structure considered to be the foundation of the sys- addition, the morphological description of single cell tematics at family and higher levels (De Wever et al. reference DNA barcodes establishes the basis for 2001). Traditionally, radiolarians with concentric further molecular ecology surveys, allowing us to structures and spherical or radial symmetry have infer the actual diversity and ecology in the extant been classified as Spumellaria or Entactinaria oceans through metabarcoding approaches (Biard depending on the absence or presence, respec- et al. 2017; Decelle et al. 2013; Nitsche et al. 2016). tively, of such a skeletal structure. Yet these taxo- Here we present an integrative morpho- nomic differences, based on the initial spicular molecular classification of Spumellaria obtained by system, have been recently questioned, finding combining ribosomal DNA genes (18S and 28S par- molecular evidence for the polyphyly of Entactinaria tial rDNA), widely used as taxonomical markers, and among Rhizaria (Nakamura et al. 2020). The latest imaging techniques (light and scanning electron spumellarian classification attempted to merge the microscopy). Phylogenetic placement of environ- Diversity and Evolution of Spumellaria (Radiolaria) 3 mental sequences provided insights in the extant molecular clades. All these morpho-molecular genetic diversity of Spumellaria in the contemporary clades are highly supported in both the 18S rDNA oceans, allowing access to undescribed diversity. In and 28S rDNA gene phylogenies, except clades B addition, the extensive fossil record of Spumellaria and E in the 28S rDNA gene phylogeny (Supple- allowed calibrating our phylogenetic analysis in time mentary Material Figure S1). In contrast to the clade and inferring their evolutionary history contextual- definition, the general topology and relationships ized with global scale geological and environmental between clades slightly disagree between the 18S changes from the Cambrian to present ecosystems. rDNA and the 28S rDNA gene phylogenies. Such discrepancies are basically due to the variable posi- Results tion of clade F that in the 18S rDNA gene phylogeny appears basal to all clades and in the 28S rDNA Comparative Molecular Phylogeny and gene appears basal to clades J, K, L and M, weakly Morphological supported as a sister group of clades H and G. Another example is, clades B, C, D and E, which Our final molecular phylogeny is composed of 133 are in the same highly supported group in the 18S distinct spumellarian sequences of the full 18S rDNA gene phylogeny, whereas in the 28S rDNA rDNA gene, completed with 55 sequences of the gene phylogeny these clades appear scattered at partial 28S (D1 and D2 regions) rDNA gene (Sup- basal positions regarding the rest of the clades. In plementary Material Table S1). From the 133 final the concatenation of the two genes clade F appears sequences of the 18S rDNA, 67 were obtained in highly supported as a group with the clades G to M this study, 58 were previously available sequences (Figure 1). associated to a morphologically described speci- Lineage I includes clades A, B, C, D and E and is men and 8 were environmental sequences not affil- phylogenetically distant to the other 3 lineages. iated to any morphology, added to increase Lineage I is characterized by the presence of one phylogenetic support in poorly represented clades. or two concentric shells or a full spongy test, some- Regarding the partial 28S rDNA, a total of 37 times non-distinguishable, where the main spines sequences were obtained in this study, where 18 grow from the innermost shell and go through the were previously available. The final alignment matrix outermost shell, when present (Figure 2A-E). Only has 25.45% of invariant sites. clade A and B shows two concentric shells with six Phylogenetic analyses show 13 different clades primary spines from the center (Figure 2A-B). Clade (cladesA,B,C,D,E,F,G,H,I,J,K,LandM)clearly A is composed of 1 novel and 2 previously available differentiated by BS and PP values distributed in 4 sequences and a common characteristic is the frag- main lineages (I, II, III, and IV) showing high BS ile and hexagonal mesh constituting the outermost (>97) and PP (1) and consistent position across shell (Figure 2A). Clade B is composed of two novel the different phylogenetic analyses (Figure 1). In and five previously available sequences clustered general, morphological classification agrees with with high BS and PP values despite their phyloge- molecular phylogeny at the clade level. Considered netic distance (Figure 1). All specimens of clade B together, morphological similarities and molecular show a more irregular and thicker mesh of the shell phylogenetic support established super-families compare to that of clade A, and, when present, very Hexastyloidea (spread in clades A, B and C), Spon- thin spines (generally called by-spines) coming out gosphaeroidea (clade D), Lithocyclioidea (clade E1) from all the surface of the shell (Figure 2Ba and and Spongodiscoidea (clade E2 and E3) in lineage I; Bb). Clade C is composed of five novel sequences Liosphaeroidea (clade F) in lineage II; Rhi- showing a large single double layered shell with no zosphaeroidea (clade G) and Centrocuboidea main spines but a large abundance of long and thin (clades H and I) in lineage III and Actinommoidea spines (Figure 2C). These three clades agree with (clade K), Stylodictyoidea (clade J), Spongopy- the definition of the Superfamily Hexastyloidea loidea (clade L2) and Pylonioidea (clade M) in lin- (Haeckel 1882; Matsuzaki et al. 2015) displaying eage IV with two unresolved families (clade L1, an innermost shell that, if present, always shows Litheliidae and Sponguridae). Superfamily and fam- pyriform with six radial beams. Three different fam- ily definitions are based in similarity of morphological ilies within Hexastyloidea correspond to: Hexastyli- characters, shared synapomorphies and phyloge- dae (Haeckel 1887) in clade A, Hexalonchidae netic clustering support into the so-called morpho- (Haeckel 1882) in clade B and Hollandosphaeridae 4 M.M. Sandin et al. Diversity and Evolution of Spumellaria (Radiolaria) 5

(Deflandre 1973) in clade C. The innermost shell if (Figure 2F2) where short spicules come out of the present always shows pyriform with six radial spines. Those specimens agree with the definition beams. Clades D and E are the more distal clades of the family Astrosphaeridae (Haeckel 1887; in this lineage and highly supported together (100 Matsuzaki et al. 2015) in the large hollow and fragile BS and 1 PP). The presence of spongy structures shell with the presence of long or short spicules differentiates these two clades from the others of lin- coming out at the far end of the spines. eage I (Figure 2D-E). Clade D is composed of four Within lineage III, including clades G, H and I, novel sequences obtained in this study. All speci- clade G is represented by five novel and three mens of this clade show a single, very small spher- previously available sequences. Members of this ical shell where several long and three bladed clade have a unique innermost shell, named spines grow interconnected by a spongy mesh “rhizosphaerid-type microsphere” or “microbursa” (Figure 2D). This definition agrees with the genus (Dumitrica et al. 2010) with a large and fragile spher- Spongosphaera belonging to the Superfamily Spon- ical skeleton (Figure 2Ga) or several cortical shells gosphaeroidea (Haeckel 1862). The last clade of lin- with a robust skeleton (Figure 2Gb). Either morphol- eage I (clade E) is composed of 30 sequences of ogy agree with the definition of the family Rhi- which 15 are novel. Members of this clade tend to zosphaeridae (Dumitrica 2017; Hollande and lose the concentric symmetry towards a cylindrical Enjumet 1960). Clade H is composed by one novel to ellipsoidal (sub-clade E1; Figure 2E1a and E1b) sequence with a morphological reference (Ses55, or flat symmetry (sub-clade E2, Figure 2E2a and Supplementary Material Figure S2)andtwoenvi- Eb; and sub-clade E3, Figure 2E3), and so does ronmental sequences not morphologically the spines and the shell. Spongy structures take described. The picture available for this specimen more importance complicating the inner structure. lacks taxonomic resolution, although the skeletal These morphologies agree with the definition of structure resembles that shown in Aita et al. the Family Artiscinae (Haeckel 1882) for E1 and (2009, pl. 23, Figure 3) specified as Centrocubus the Superfamily Spongodiscoidea (De Wever et al. cladostylus (Haeckel 1887). This species has 2001; Haeckel 1862) for E2 and E3. Within the latest characteristic interconnected spines and the highly clades there are representatives of two families supported position between clade G and I agrees Spongobrachiidae (Haeckel 1882) with moderate with the definition of the family Centrocubidae BS and PP values in sub-clade E2 and Eucthitoni- (Hollande and Enjumet 1960;emend.Dumitrica idae (Haeckel 1882) highly supported in sub-clade 1994). The last clade of this lineage (clade I) is rep- E3. resented by two novel sequences obtained in this Lineage II, including clade F alone, is composed study and 6 environmental sequences. Specimens of 13 sequences of which five were previously avail- of this clade show only spongy structures with radi- able and eight are novel. All specimens within this ated fibers and there is no evidence of either shell or clade are characterized by one large and hollow main spines (Figure 2I). shell where long spines grow from its surface (Fig- In lineage IV, clade J, K and L cluster together ure 2F1 and F2), that agree with the definition of with relatively high BS (88) and PP (0.98) values the Superfamily Liosphaeroidea (Haeckel 1882). as a sister group of clade M. Although relationships Members of the sub-clade F1 (Figure 2F1) share a between clades J, K and L remain still elusive due to robust shell compared to that of sub-clade F2 the short phylogenetic distance and the different

3 Figure 1. Molecular phylogeny of Spumellaria inferred from the concatenated complete 18S and partial 28S (D1-D2 regions) rDNA genes (135 taxa and 2459 aligned positions). The tree was obtained by using a phylogenetic Maximum likelihood method implemented using the GTR + c + I model of sequence evolution. PhyML bootstrap values (100 000 replicates, BS) and posterior probabilities (PP) are shown at the nodes (BS/ PP). Black circles indicate BS 99% and PP 0.99. Hollow circles indicate BS 90% and PP 0.90. Sequences obtained in this study are shown in bold. Thirteen main clades are defined based on statistical support and morphological criteria (A, B, C, D, E, F, G, H, I, J, K, L, M), divided in 4 main lineages (I, II, III, IV). For each morpho-molecular clade, capital letters represent the Superfamily name and small letters the Family name. Main skeletal traits are identified on the right for each spumellarian specimen. Fourteen Nassellaria sequences were assembled as outgroup. Branches with a double barred symbol are fourfold reduced for clarity. 6 M.M. Sandin et al.

A Ba Bb C D

E1a E1b E2a E2b E3

F1 F2Ga Gb I

J1J1’ J2 K

L1L1’ L2a L2b

M1 M2 M4a M4b Diversity and Evolution of Spumellaria (Radiolaria) 7 topology when using a Maximum Likelihood (Fig- Litheliidae. Sub-clade L2 is represented by either: ure 1) or a Bayesian (Figure 3) approach (BS: 49 a flatten circular test with a tunnel-like pylome and between clade K and L and PP: 0.38 between clade a test comprised by a very densely concentric J and K). All members of this lineage share a very structure (Figure 2L2a) attributed to Spongodis- small spherical inner most shell with four or more coidea; or by a spherical translucent protoplasm radial beams connecting to the second shell, where with an encrypted flat consolidated skeletal shell in 12 or more radial beams come out (Figure 2J-M). distal position and star-like solid soluble materials lineage IV differs from lineage I in the absence of (Figure 2L2b and Supplementary Material Fig- discrete radial beams from the second or later ure S2) assigned to . In clade M there shells. Clade J is characterized by five sequences are 13 novel sequences and seven previously avail- obtained in this study and three previously available able sequences. A morphological characteristic of sequences. Members of this clade share a flattened this clade is the broken, or fenestrated, outermost skeleton with two or more concentric shells and shell with the presence of pyloniid central structure radial spines from the innermost or second inner- (Figure 2M), corresponding to the Superfamily most shell (Figure 2J1, J10 and J2), agreeing with Pylonioidea (Haeckel 1882), thereafter, the symme- the definition of the Superfamily Stylodictyoidea try and the different opening of the outermost shell (Haeckel 1882; Matsuzaki et al. 2015). Morphologi- distinguishes the sub-clades. Yet, phylogenetic cal differences between sub-clade J1 (Figure 2J1) relationships within this clade remains elusive. The and J2 (Figure 2J2) were not possible to determine, first sub-clade (M1) consists of three basal yet they have been separated into two different sub- sequences poorly supported as a clade yet sharing clades due to high BS (96 in J1 and 100 in J2) and a cubic morphology and the two opposite and closed PP (0.98 in J1 and 1 in J2) values. Clade K contains gates (Figure 2M1), which agrees with the family one novel and four previously available sequences. Tholoniidae (Haeckel 1887). The three other sub- All specimens of this clade have three spherical clades have different morphologies (Figure 2M2; shells with more than eight main spines that extend Ses13, Ses53 and Vil437, Supplementary Material from the inner shell (Figure 2K), representing the Figure S2) agreeing with the definition of different Superfamily Actinommoidea (Haeckel 1862). Clade families within Pylonioidea. Sub-clade M2 (Fig- L is represented by five novel and 14 previously ure 2M2) has a spherical to ellipsoidal skeleton available sequences. Members of this clade without distinctive openings. On the other side, are morphologically distant. Sub-clade L1 has a sub-clades M3 and M4 are highly supported as a tightly concentric or coiled center with one thick pro- group with a flattened box-shaped skeleton for the toplasmic pseudopodium (axopodium) (AB61787) former sub-clade (Ses13, Ses53 and Vil437, or a rhomboidally inflated center (Figure 2L1 and Supplementary Material Figure S2) and a cubic L10), which is not observed in any other Spumellaria. skeleton characterized by big openings of the In the first case, this morphology is attributed to second shell for the later sub-clade (Figure 2M4a the family Sponguridae, and in the second to and 4b).

3 Figure 2. Scanning Electron Microscope (SEM) images of Spumellaria specimens used in this study for phylogenetic analysis or morphologically related to one of the morpho-molecular clades of Figure 1. Letters correspond to its phylogenetic clade in Figure 1. Scale bars = 50 mm. (A) Osh194: sp. (specimen not included in phylogeny). (Ba) Vil484: Stigmostylus ferrusi.(Bb) Mge17-15: Hexacontella? sp. (C) Vil174: Hollandosphaera hexagonia.(D) Mge17-82: Spongosphaera streptacantha.(E1a) Vil186: Didymocyrtis sp. (E1b) Vil458: Cypassis irregularis.(E2a) Osh48: gen et sp. Indet. A (E2b) Mge17-17: Spongobracium ellipticum.(E3) Vil246: Dictyocoryne koellikeri.(F1) Osh34: Heliosphaera bifurcus.(F2) Vil499: Cladococcus sp. A. (Ga) Vil240: Haliommilla capillacea (broken specimen). (Gb) Mge17-81: Rhizosphaera trigonacantha.(I) Vil210: Plegmosphaeromma?sp.(J1) Vil441: arachnea.(J10) detail of Vil441. (J2) Osh90: Stylodictya stellata.(K) Vil438: Actinomma trinacrium.(L1) Mge17-118: Trilobatum aff. acuferum.(L10) detail of Mge17-118. (L2a) Osh191: Schizodiscus spp. (specimen not included in phylogeny). (L2b) Vil296: Calcaromma morum.(M1) Vil200: Tholomura transversara.(M2) Osh16: Larcopyle aff. buetschlii.(M4a) Mge17-20: Tetrapyle octacantha.(M4b) Vil452: Tetrapyle octacantha (specimen not included in phylogeny). 8 M.M. Sandin et al.

Outgroup (14 sequences of Nassellaria) Vil282 - Hexacontella sp. A HQ651798 - Hexastylus nobilis HQ651795 - Hexastylus nobilis Vil484 - Stigmostylus ferrusae B Mge17-15 - Hexacontella sp HQ651796 - Hexacontium sp A HQ651797 - Hexacontium sp A HQ651794 - Hexacontium sp A AB284519 - Hexacontium cf hexagonum HQ651784 - Hexacontium sp A Vil454 - Hollandosphaera hexagonia C Vil174 - Hollandosphaera hexagonia Vil217 - Hollandosphaera hexagonia Vil448 - Hollandosphaera hexagonia Vil209 - Hollandosphaera hexagonia Oki25 - Spongosphaera streptacantha D Osh188 - Spongosphaera streptacantha Osh69 - Spongosphaera streptacantha Mge17-82 - Spongosphaera streptacantha Ses64 - Didymocyrtis tetrathalamus E1 AB193605 - Didymocyrtis tetrathalamus Ses19 - Didymocyrtis tetrathalamus Ses63 - Didymocyrtis tetrathalamus coronatus Vil458 - Didymocyrtis irregularis LC093106 - Didymocyrtis tetrathalamus coronatus Vil186 - Didymocyrtis sp AB439010 - Didymocyrtis irregularis Vil450 - Didymocyrtis oblongus AB246695 - Spongodiscus biconcavus E2 Osh79 - Spongodiscus sp. Osh174 - Spongodiscus sp. Osh48 - Spongodiscus sp. AB430760 - Spongodiscidae gen et sp indet D Mge17-17 - Spongocyclia elliptica Vil449 - Spongaster? toxon Mge17-74 - Dicytocoryne gegenbauri E3 Vil246 - Dicytocoryne koellikeri Vil293 - Dicytocoryne koellikeri AB430758 - Dictyocoryne truncata AB101541 - Dictyocoryne truncata AB430757 - Dictyocoryne truncata AB101542 - Spongaster tetras tetras AB246696 - Spongodiscus aculeatus Mge17-99 - Dictyocoryne sp. AB101540 - Dictyocoryne profunda AB179732 - Myelastrum elegans AB179733 - Myelastrum furcatum AB179734 - Myelastrum lobum LC093107 - Myelastrum aurivillii HQ651792 - Heliosphaera aff actinota F1 HQ651782 - Heliosphaera aff actinota AB284518 - Heliosphaera aff actinota Osh34 - Heliosphaera bifurcus AB490706 - Astrosphaera hexagonalis F2 AB490705 - Astrosphaera hexagonalis Vil499 - Cladococcus sp. A Vil173 - Cladococcus sp. A Vil497 - Arachnospongus varians Vil244 - Cladococcus viminalis Vil243 - Cladococcus viminalis Vil228 - Cladococcus scoparius Vil238 - Clacococcus irregularis Mge17-81 - Rhizosphaera trigonacantha G Vil476 - Rhizosphaera trigonacantha JQ706069 (Oki50) - Rhizosphaera trigonacantha AB246684 - Rhizosphaera sp. AB246686 - Rhizosphaera sp. Ses15 - Haliommilla capillacea Mge17-44 - Haliommilla capillacea Vil240 - Haliommilla capillacea Ses55 - Octodendron aff. cubocentron H GU822672 - Uncultured Rhizaria GU821394 - Uncultured Rhizaria KX532703 - Uncultured marine I KJ757677 - Uncultured marine eukaryote Vil210 - Spongoplegma? sp. Vil451 - Spongoplegma? sp. JX842018 - Uncultured marine eukaryote GU821097 - Uncultured Rhizaria GU822292 - Uncultured Rhizaria GU822590 - Uncultured Rhizaria HQ651788 - Actinomma boreale K HQ651780 - Actinomma boreale HQ651781 - Actinomma boreale Vil438 - Actinomma trinacrium HQ651789 - Actinomma boreale Vil441 - Stylodictya arachnea J1 Vil503 - Stylodictya sp AB860148 - Perichlamydium venustum AB246698 - Stylodictya subtilis J2 Osh90 - Stylodictya stellata Vil453 - Stylodictya arachnea Vil501 - Stylodictya arachnea AB860147 - Stylodictya stellata AB617584 - Lithelius cf. alveolina L1 AB617587 - Schizodiscus sp. Mge17-118 - Trilobatum aff. acuferum AB860151 - Spongopyle osculosa L2 AB246689 - Un-identifiable specimen AB860150 - Spongopyle osculosa AB860156 - Schizodiscus? spp. L3 AB860154 - Schizodiscus? spp. AB860155 - Schizodiscus? spp. AB860149 - Spongopyle osculosa AB860152 - Schizodiscus? spp. AB860153 - Schizodiscus? spp. AB617590 - Schizodiscus asymmetricus AB617588 - Schizodiscus stylotrochoides AB617589 - Spongotrochus beringianus Vil249 - Calcaromma morum Osh153 - Calcaromma morum Vil297 - Calcaromma morum Vil296 - Calcaromma morum Toy110 - Tholomura pilula M1 Vil200 - Tholomura transversara HQ651803 - Sphaeropylolena circumtexta AB246688 - Sphaerolarnacillium tanzhiyuani M2 AB617585 - Un-identifiable specimen Osh16 - Larcopyle aff. buetschli LC049344 - Larcopyle buetschli LC049343 - Larcopyle buetschli HQ651783 - Un-identifiable specimen M3 Vil437 - Cryptolarnaciinae gen. et sp. indet Ses13 - Pylodiscus spinulosus Ses53 - Pyloniidae gen et sp indet Mge17-20 - Tetrapyle octacantha M4 Vil298 - Tetrapyle octacantha Ses18 - Tetrapyle octacantha Vil432 - Tetrapyle octacantha AB246680 - Tetrapyle sp. Vil506 - Tetrapyle octacantha Vil231 - Tetrapyle octacantha Vil480 - Tetrapyle octacantha

700 600 500 400 300 200 100 Time (Ma) Figure 3. Time-calibrated tree (Molecular clock) of Spumellaria, based on alignment matrix used for phylogenetic analyses. Node divergences were estimated with a Bayesian relaxed clock model and the GTR + c + I evolutionary model, implemented in the software package BEAST. Nine different nodes were selected for the calibration (blue dots). Blue bars indicate the 95% highest posterior density (HPD) intervals of the posterior probability distribution of node ages.

Molecular Dating genetic relationships are doubtful, yet two radiation events at 277 (363–203) Ma and 252 (334–182) The molecular clock dated the diversification Ma separate clades J, K and L. clade L is the first between Nassellaria and Spumellaria (the root of of these clades diversifying at 184 (255–121) Ma, the tree) at a median value of 651 Ma (95% Highest followed by clade J 153 (228–88) Ma, clade K at Posterior Density -HPD-: between 789 and 540 Ma) 145 (185–105) Ma and clade M at 105 (123–88) (Figure 3). From here on, all dates are expressed as being the youngest clade of lineage IV. Lineage III median values followed by the 95% HPD interval. diverged soon after lineage IV, yet the next ramifica- The first diversification of Spumellaria happened tion was between clade G and clade I 229 (326– around 515 (659–382) Ma, followed by a rapid 145) Ma. It was not until 143 (162–123) and 135 branching of the lineages. Despite their dubious (202–76) Ma when clade G and I respectively radi- phylogenetic relationships, lineage II splits apart ate, and the latest diversification within this lineage 445 (571–332) Ma and lineage III and IV diversified corresponds to clade H 49 (107–12) Ma. Lineage I from each other 407 (526–306) Ma. The next diver- appeared 245 (264–226) Ma, and thereafter is char- sification events correspond to the first radiation of acterized by a series of relatively continuous diversi- lineage IV 319 (417–234) Ma and that of lineage fication events until the radiation of clades B, A, E, D III 307 (414–213) Ma. Within lineage IV, the phylo- and C at 142 (205–79), 131 (216–54), 153 (163–90), Diversity and Evolution of Spumellaria (Radiolaria) 9

34 (78–8) and 28 (68–7) Ma respectively. Regarding years1). The first diversification of extant Spumel- lineage II, it is the youngest of all the lineages laria corresponds to an early and fast divergence appearing at 215 (255–177) Ma despite its early of the different lineages from 515 to 407 Ma. branching from any other lineage. After that, the diversification of living groups remained at standstill until 313 Ma when lineage Post-hoc Analyses IV and III radiated. From 276 to 215 Ma all differ- ent groups diversified: clades K, J and L (within lin- The lineages through time analysis (Figure 4) eage IV) split apart; lineage I appeared and started a showed a classic exponential diversification slope, continued diversification; within lineage III the three with a 0.0102 rate of speciation (Ln(lineages)million clades split apart; and lineage II emerged. Another

Figure 4. Lineages Through Time (LTT) analysis based on the molecular clock results for Spumellaria, removing the outgroup (in black) and of each lineage independently: lineage I (purple), lineage II (light green), lineage III (dark green) and lineage IV (orange) and of Nassellaria (data taken from Sandin et al. 2019). The y- axis represents the number of lineages (N) expressed in logarithmic (base e) scale (Ln(N)) and in the x-axis it is represented the time in millions of years ago (Ma). Horizontal gray bars in black slope represent the 95% Highest Posterior Density (HPD) of molecular clock estimates. 10 M.M. Sandin et al. important diversification event happened from 157 Environmental Genetic Diversity of Spumellaria to 125 Ma when lineage IV greatly diversified, fol- lowed by lineage I and II. During the following years A total of 1165 environmental sequences affiliated there was a tiered diversification, where clade M to Spumellaria, and retrieved from NCBI public data- appeared in isolated events as well as the ramifica- base, were placed in our reference phylogenetic tion of clade L or the branching between sub-clades tree (Figure 5; Supplementary Material Table S3). E2 and E3. Finally, from 54 Ma onwards there was From these, 617 were closely related to clade F. a continuous diversification where the rest of the Two other clades with a high number of environmen- clades appeared or keep diversifying. During this tal sequences related to are clade I and clade M, time lineage III diversified notably and so did lineage with 145 and 47 sequences respectively. While up II. to 40 sequences were scattered between clades The ancestral state reconstruction analysis (Sup- E, L, B, G, D and J (17, 14, 3, 3, 1 and 1 respec- plementary Material Figure S3) established a spher- tively), no environmental sequences were clustered ical skeleton shape with a line of symmetry for the within clades A, C, H and K. The rest of the common ancestor to all Spumellaria, with a central sequences (317) where distributed across the tree, structure that can be either empty (state 0) or with mainly at basal nodes, with no possible assignation: an entactinarian-type microsphere or fused fibers 206 basal to clade F in lineage II, 70 in lineage I (of or soft spongy system (state 1), but with a wide which 41 basal to the lineage, 11 basal to clades C, space in between the central structure and the cor- D and E, 12 basal to clades D and E, 6 basal to tical shell, without spines arising from it and one dis- clade B and 1 basal to clade C), 26 in lineage IV tinctive cortical shell. The common ancestor of (of which 16 basal to clade J, 5 basal to clade M lineage I and that of lineages II, III and IV share a and 5 basal to the lineage), 4 in lineage III basal to spherical skeleton shape with a line of symmetry, clade G, 2 basal to lineages II, III and IV and 8 final a wide internal cavity and one cortical shell. The sequences basal to all Spumellaria. difference between these two ancestors are that To go beyond rapid phylogenetic placement the ancestor of lineage I is characterized by an using the pplacer tool, these 317 environmental entactinarian-type central structure or fused fibers sequences dubiously assigned, were later included or soft spongy system with more or less six spines, in a phylogenetic tree of the 18S rDNA gene whereas that of lineages II, III, IV could also have an (RAxML v8.2.10, GTR + G + I, with 1000 rapid boot- empty central structure and have no spines arising straps, Stamatakis 2014) after removing chimeras from the central structure. The common ancestor (52 sequences were detected as chimeric) and sus- for lineage II has the most ancestral state in every picious sequences (11 sequences with multiple phy- reconstructed trait (state 0). The common ancestor logenetic placements and/or long branches). Up to of lineages III and IV also have a spherical skeleton six different new clades were found (Env1 – 6; Fig- shape with a line of symmetry, like the common ure 6), six sequences scattered over the phyloge- ancestor of these two lineages independently. netic tree (one related to Env2, three sequences Although the common ancestor of lineages III and related to clade F and Env5 and two sequences clo- IV differs from that of lineage II in a higher complex- sely related to clade G) and two last sequences ity of the central structure, the appearance of struc- were clustered with high support basal to clade M tures in the internal cavity, the presence of (Figure 6; further details of sequence assignations significantly more than six spines and that it could can be found in Supplementary Material have three cortical shells. Lastly, the common Table S3). From these clades, Env5 gathers 149 ancestor of the lineage III and that of lineage IV sequences related to clade F in lineage II, yet low could also have one or three cortical shells with sig- bootstrap values (51) avoid a formal assignation. nificantly more than six radial spines. Yet in lineage Two clades (Env1 with 63 sequences and Env2 with III the internal cavity gets denser than that of lineage seven) are found at basal positions of lineage I. IV, whereas the central structure tends to be Another environmental clade appears in lineage IV entactinarian-type, fused fibers or soft spongy in lin- (Env4 with 16 sequences), and two final clades eage III and that of the common ancestor of lineage are found basal to lineages I, III and IV (Env6 with IV is undefined since it has equal probabilities every eight sequences) and basal to lineage IV (Env3 with character state. three sequences). Diversity and Evolution of Spumellaria (Radiolaria) 11 12 M.M. Sandin et al.

In order to improve the understanding of the content of Env1 also reveals comparable results to diversity of the environmental clades (Figure 6) the other clades, where Env1 has an average GC and try to explain the high chimeric ratio (13– content of 50.1% (±1.36% sd) and the average of 25%) observed, we performed a second phyloge- all the sequences analyzed is of 48.9% (±1.61% netic analysis (RAxML v8.2.10, GTR + G + I, with sd) (Supplementary Material Figure S5). Yet Env1 1000 rapid bootstraps, Stamatakis 2014). This anal- has the highest average GC content among its rela- ysis includes the dataset used to build Figure 6 and tives in lineage I and decreases towards more distal nine other sequences obtained from single-cell iso- groups, finding a similar pattern in lineage IV. lates but previously removed due to the contrasting morphological assignation in the clustered phyloge- Discussion netic clade. It turns out that these nine sequences clustered with moderate to high support among Morpho-molecular Classification of Spumellaria one of the environmental clades, clade Env1 (Fig- ure 7; Supplementary Material Table S1). These Our study shows that the symmetry of the skeleton nine specimens show a morphology that matches and the overall morphology are key features clades E (Osh87 and Osh174), F (Osh156), G describing spumellarian phylogeny at higher rank (Osh82, Osh97 and Osh114), I (Osh187), L classifications, as for other radiolarian groups such (Osh116) and a last specimen with a morphology as Acantharia (Decelle et al. 2012b) or Nassellaria not covered in our morpho-molecular framework (Sandin et al. 2019). Central structure patterns are (Osh108). Our morpho-molecular classification also well defined between clades helping to discern show that each molecular clade has a clear and similar symmetric trends in distantly related clades endorsed (two or more specimens) morphological (e.g. flat and radial symmetry in the super-families patterns distinct from one another. The combination Spongodiscoidea, Spongopyloidea and Stylodicty- of different morphologies within the same clade oidea). These results contrast the recognized impor- Env1 contrasts with this global pattern. In addition, tance of internal structures in previous morphology- one of the specimens from clade Env1 (Osh174) is based radiolarian classifications (Afanasieva et al. also found within clade E. Although in clade E it is 2005; De Wever et al. 2001) in which the initial spic- composed by the concatenation of the first part of ular system was the key element for taxonomic the 18S rDNA and the 28S rDNA gene (1489 bp in delimitation and characterization. Yet, overall shape total) and that of clade Env1 by the second part of has always played a significant role in the further the 18S rDNA gene (871 bp). description of different taxonomic groups. We there- To assess possible pseudogenes for clade Env1, fore establish the symmetry and the overall mor- we performed two analyses for the 18S rDNA: (i) an phology of the skeleton of Spumellaria as main entropy analysis of the alignment and (ii) the GC morphological markers, followed by central struc- content estimation of every sequence. No differ- ture patterns at finer taxonomic ranks. ences were found between the entropy of Env1 The morpho-molecular framework established and that of the different lineages (Supplementary herein understands the Spumellaria as Material Figure S4), showing an average Shannon radiolarians with concentric structure and a spheri- entropy of 0.07 (±0.16 standard deviation; sd) in cal or radial symmetry. This concept includes some Env1,0.08(±0.19sd)inlineageI,0.03(±0.08sd) living groups classified under the order Entactinaria in lineage II, 0.11 (±0.24 sd) in lineage III and 0.14 in De Wever et al. (2001) such as Hexastyloidea that (±0.26 sd) in lineage IV. The greatest entropy values were already suggested to be included in Spumel- correspond to the V4 hyper-variable region of the laria (Ishitani et al. 2012; Matsuzaki et al. 2015; 18S rDNA gene (positions 450-850). Other Yuasa et al. 2009). Our results also confirm the important regions correspond to the V2 (around inclusion of the Entactinaria super-families Cen- the position 200), V7 (1400) and V9 (1720) trocuboidea and Rhizosphaeroidea within Spumel- hyper-variable regions of the 18S rDNA gene. GC laria. The Radiolaria orders Spumellaria and

3 Figure 5. Pplacer phylogenetic placement of 1165 environmental sequences into a concatenated phyloge- netic tree of Spumellaria (complete 18S + partial 28S rDNA genes). Numbers at nodes represent the amount of environmental sequences assigned to a branch or a node. Diversity and Evolution of Spumellaria (Radiolaria) 13 14 M.M. Sandin et al.

Entactinaria were traditionally separated based on previously gathering morphologies from clades E the absence or presence of internal structures and L are polyphyletic (Matsuzaki et al. 2015). Dif- respectively (De Wever et al. 2001). Here we show ferent families included within Hexastyloidea have that the Entactinaria order in the sense of De Wever been classified as independent super-families in et al. (2001) is scattered into clades A, B, C (from lin- previous morphology-based taxonomic studies eage I) G, H and I (from lineage III). Recent (Afanasieva et al. 2005; De Wever et al. 2001), as advances in imaging techniques have suggested seen in the paraphyletic relationships of our phylo- the Spumellaria as a sub-order within the Entacti- genetic results. For instance, flat spumellarians naria based on findings of homologous structures have been classified all together on the basis of their with the initial spicular system in Spumellaria among flat symmetry, yet the previously stated polyphyletic primitive radiolarian forms (Kachovich et al. 2019). nature of this group is confirmed by our results, due Despite our results agreeing with Kachovich et al. to differences in the test and the overall morphology (2019) in the taxonomic inclusion of both orders, of the skeleton (De Wever et al. 2001). Despite our we consider Entactinaria as a polyphyletic group efforts, some uncertainties regarding specific fami- within the Rhizaria as shown in Nakamura et al. lies such as Heliodiscidae, Ethmosphaeridae or (2020). The prevalence of the overall shape at Saturnalidae remain yet to be genetically described. higher taxonomic levels would therefore place the This could be addressed by performing further sin- Spumellaria as the name of polycystines radiolari- gle celled morpho-molecular characterization analy- ans with concentric structure and a spherical or ses on targeted specimens from a broad variety of radial symmetry, in contrast to Kachovich et al. environments (e.g. deep water). Although these (2019). The formal inclusion of most entactinarian families are represented by very few species and families with extant Spumellaria provides a direct have similar symmetric and central structure pat- and documented link from the Cambrian to current terns to other families presented herein; they could ecosystems, improving the connection for paleo- be therefore emended within one of the described environmental reconstruction analyses. In addition clades. to our conclusions, there are no genetic evidences of a purely Entactinaria clade so far and at this point Diversity of Spumellaria we cannot exclude that other groups sharing these symmetric patterns might also be emended within The great environmental diversity found at early Spumellaria, as for the Centrocubidae and Rhi- diverging positions (i.e.; Env1, Env3, Env6 from Fig- zosphaeridae families. Despite the demonstrated ure 6) prevents a comprehensive exploration of the polyphyletic nature of Entactinaria, different appear- extant spumellarian morpho-molecular diversity and ance and extinction ranges along the fossil record challenges the reliability of such molecular clades. makes it a valuable taxon for biostratigraphic However, our analyses allowed us to hypothesizing studies. different morphologies for such environmental Extant Spumellaria included in our study can be clades, precisely Env1. Various lines of evidences divided in 13 morpho-molecular clades, grouped in led us to conclude the existence of two different four main evolutionary lineages based on the combi- and phylogenetically distant 18S rDNA gene copies nation of phylogenetic clustering support, morpho- within the same specimen: logical features and molecular dating. Our results (i) The notably high ratio of chimeric sequences confirm the monophyly of some families (e.g., Lio- obtained from each single cell analyzed (13– sphaeroidea, Rhizosphaeridae, Actinommoidea or 25%) that would reflect the presence of different Pylonioidea) while others such as Hexastyloidea rDNA genes amplified with the same Spumellaria- found in clades A, B and C or Spongodiscoidea specific primer sets from a single cell/specimen.

3 Figure 6. Molecular phylogeny of environmental sequences associated to Spumellaria. The tree was obtained by using 1000 rapid bootstrap RAxML GTR + c model of sequence evolution. Black circles at nodes indicate BS 99% and hollow circles indicates BS 90%. BS lower than 60 were removed for clarity. Branches with a double barred symbol are fourfold reduced for clarity. Black triangles represent clades which morphology is known (Figure 1, Figure 2). Gray triangles represent environmental clades. Sequence composition of each environmental clade is described in Supplementary Material Table S3. Diversity and Evolution of Spumellaria (Radiolaria) 15 16 M.M. Sandin et al.

(ii) The common relatively long branches within Heliozoa have appeared to be polyphyletic (Burki clade Env1 (Figure 7) traducing the decreased et al. 2016; Cavalier-Smith et al. 2015), as for fresh- accuracy and quality of sequences obtained by the water heliozoans (Nikolaev et al. 2004). Indeed, sequencing of a mix of rDNA gene copies. some groups of Heliozoa, such as Gymnosphaeri- (iii) Distantly related morpho-species unexpect- dae, have been moved to the Retaria lineage (pre- edly clustered in the same phylogenetic clade cisely within Radiolaria) based on ultra-structural Env1 (Figure 7), contrasting with the globally well features of the axopodial complex (Yabuki et al. supported morpho-molecular clades in Spumellaria. 2012). Therefore, one cannot rule out that some of (iv) The presence of the same single cell speci- these basal environmental clades are represented men in 2 different and highly supported clades by heliozoan-like organisms and some of them (Osh174 in Env1 and E2), when different amplifica- (clade Env1) developed within other tion and sequencing runs have been carried out. Spumellaria. Further analyses based on the entropy and GC The morpho-molecular framework of Spumellaria content of sequences clustering in Env1 showed established in this study allows an accurate phylo- similar trends compare to the rest of spumellarian genetic placement of environmental sequences for sequences, disregarding the possibility of a pseudo- which it would not be possible to infer their taxo- gene (e.g. Balakirev et al. 2014). Intra-genomic or nomic position otherwise. Most of the environmental intra-specific polymorphism may also be excluded (i.e. not morphologically described) sequences as possible causes since phylogenetic distances placed in our reference morpho-molecular frame- between families and superfamilies are of grater work are highly related to Liosphaeroidea, coming magnitude. Given the regular occurrence of symbi- primarily from deep environments (1500–2500 m) otic relationships within the SAR super-group ( (Edgcomb et al. 2011; Lie et al. 2014). Another Bjorbækmo et al. 2020; Stoecker et al. 2009, group displaying a large environmental diversity is 2016), the most likely explanation left for clade the family Excentroconchidae, mainly represented Env1 is the existence of a naked symbiotic group by sequences coming from anoxic environments of Spumellaria developing within another, (Edgcomb et al. 2011; Kim et al. 2012). These skeleton-bearing Spumellaria. The exact symbiotic results agree with the data obtained by morphologi- nature (parasitic, mutualistic or commensalistic) of cal observations from the fossil record (De Wever such group remains beyond the scope of this study et al. 2001) or from plankton and sediments traps and cannot be inferred from our data. It is worth and surface sediment materials (Boltovskoy et al. mentioning that the possibility of environmental 2010; Boltovskoy and Correa 2016), in which both DNA contamination cannot be excluded. All 9 spec- the Liosphaeroidea and the Excentroconchidae imens come from different samples within the water are among the most abundant groups. Representa- column (from surface down to 2000 m deep; Sup- tives of these families are characterized by a thin plementary Material Table S1) yet belonging to the and fragile skeleton with a dense protoplasm. As same water mass. Further imaging analysis on live previously suggested (Not et al. 2007), cell break- or fixed specimens (e.g. fluorescence in situ age may also contribute to the outstanding amount hybridization) could help settle this hypothesis by of sequences found among these taxonomic groups visualizing two cells/nucleus within the same skele- compared to other clades. ton or detecting skeleton-less Spumellaria. Similar associations have been already identified between Evolutionary History of Spumellaria benthic bathyal Foraminifera and dead tests of Xenophyophorea (Foraminifera) (Hughes and Our molecular clock results dated the first diversifi- Gooday 2004; Pawlowski et al. 2003). To our knowl- cation of Spumellaria at 515 (HPD: 659–382) edge the few molecular data available for marine Ma, in agreement with recent findings of the oldest

3 Figure 7. Detailed molecular phylogeny of the environmental clade Env1, showing the different morphotypes within the same phylogenetic clade. In brackets after the species name is indicated the expected clade based in the morpho-molecular framework established herein. RAxML bootstrap values (1000 replicates, BS) are shown at the nodes. Black circles indicate BS 99% and hollow circles indicates BS 90%. BS lower than 60 were removed for clarity. Branches with a double barred symbol are fourfold reduced for clarity. Diversity and Evolution of Spumellaria (Radiolaria) 17 radiolarian fossils with spherical forms, taxonomi- been reported in living Nassellaria inferred from the cally classified in the extinct spumellarian genus combination of the molecular clock and the direct Paraantygopora (early Cambrian, Series 2, ca. observation of the fossil record (De Wever et al. 521–509 Ma; Zhang and Feng 2019). This early 2006; Sandin et al. 2019); in which ancient forms, diversification separated the two contrasting lin- presumably extinct during the end-Permian extinc- eages I and IV, followed soon afterwards by the tion (Aitchison et al. 2017; De Wever et al. 2006; branching of linages II and III, during the so-called O’Dogherty et al. 2011; Sepkoski 1981; Takahashi Great Ordovician Biodiversification Event (Noble et al. 2009) led to the diversification of extant and Danelian 2004; Servais et al. 2016). At this groups. moment many different symmetrically spherical The second part of the is characterized radiolarians appeared in the fossil record by a stepped diversification of Spumellaria, as (Aitchison et al. 2017; Noble et al. 2017). Yet, poor already described for Nassellaria (Sandin et al. radiolarian-bearing rocks (Aitchison et al. 2017), dif- 2019) and Foraminifera (Hart et al. 2003; Leckie ferent topologies of the different marker genes and et al. 2002). This phenomenon is classically the short phylogenetic distance at these nodes in explained by the onset of a global oceanic anoxia the concatenated phylogenetic analysis make rela- during the (Jenkyns 1998) and a series of tionships between lineages obscure. From this per- Oceanic Anoxic Events during the Cretaceous iod until the end of the the diversification (Erbacher et al. 1996; Jenkyns 2010; Kemp and of extant lineages is low and basically restricted to Izumi 2014; Schalanger and Jenkyns 1976; Yilmaz the end of the (358.9 – 298.9 Ma) et al. 2012). In contrast to Nassellaria diversification, when lineages III and IV start diversifying. towards the end of the Jurassic (201.4–145 Ma) and All over the Paleozoic, extinct specimens previ- beginning of Cretaceous (145–66 Ma), there is a big ously attributed to Entactinaria show a great diver- increase in Spumellaria diversification where most sity that mostly became extinct towards the of the clades suddenly diversified. This trend is also beginning of the Mesozoic (De Wever et al. 2003, reported in the fossil record (De Wever et al. 2003; 2006). Some representatives of this group have Kiessling 2002). Such differences between diversifi- been tentatively considered as Nassellaria in latest cation patterns of Spumellaria regarding other radi- classifications and later suggested as ancestors of olarian groups is a question already raised in this order due to overall morphology and molecular recent studies (Kachovich et al. 2019). Both extant clock results (Noble et al. 2017; Sandin et al. Nassellaria and Spumellaria share similar environ- 2019). The morpho-molecular framework here pro- mental preferences (Suzuki and Not 2015). Yet posed allows the attribution of some radiolarian Spumellaria tend to possess a larger protoplasmic extinct groups with spherical or radial symmetry as volume (Des Combes and Abelmann 2009; possible ancestors of the extant Spumellaria, that Takahashi 1982). There is also a drastic diversifica- can be reflected in the long branches of clades tion of flat spumellarians, a feature that increases belonging to lineages II, III and IV (Figure 1). These the surface/volume ratio of a given organisms. Prob- phylogenetic patterns may be interpreted as a bot- ably such morphological adaptations provided an tleneck effect, in which many groups went extinct advantage to thrive during low nutrient availability before they had a chance to radiate. periods, as known to be case during the Jurassic At the beginning of the Mesozoic the first extant (Ca´rdenas and Harries 2010). Once the conditions representatives appear with the diversification of lin- became more favorable heterotrophic Spumellaria eage II (Liosphaeroidea) and Hexastyloidea. started a sustained diversification that followed Although, the polyphyletic nature of Hexastyloidea throughout the Cenozoic (66–0 Ma). Similar diversi- shows a later diversification within the clades (A, B fication patterns linked to climatic oscillations over and C), explaining why the Triassic (251.9– the Cenozoic were found in Nassellaria (Sandin 201.3 Ma) genera does not resemble that of the et al. 2019), Foraminifera (Hallock et al. 1991)and Cenozoic (66–0 Ma) (O’Dogherty et al. 2011). Dur- coccolithophores (Bown et al. 2004). ing the Triassic, lineage III also diversifies and all clades from lineage IV are already separated from Methods each other, yet their diversifications are not happen- Sampling and single cell isolation: Plankton samples were ing until the end of the Mesozoic. Similar diversifica- collected: 1- off Sendai (11 samples: 38° 0’ 28.8’’ N, 142° 0’ 28.8’’ E), tion patterns at the beginning of the Mesozoic have 2- Sesoko (8 samples: 26° 48’ 43.2’’ N, 73° 58’ 58.8’’ E), 3- the 18 M.M. Sandin et al.

Southwest Islands, South of Japan (2 samples: 28° 14’ 49.2’’ N, 129° 5’ 27.6’’ E) 4- in the bay of Villefranche-sur-Mer (38 samples: 43° 40’ 51.6’’ N, 7° 19’ 40.8’’ E) and 5- in the western Mediterranean Sea (9 samples: MOOSE-GE cruise) by net tows (20, 64 or 200 mm), Ver- tical Multiple-opening Plankton Sampler (VMPS) or Bongo net (24- 200 mm). Samples related metadata can be found in the RENKAN database (http://abims.sb-roscoff.fr/renkan). Spumellarian speci- mens were individually handpicked using Pasteur pipettes from the plankton tow sample and transferred 3 to 4 times into 0.2 mmfiltered seawater to allow self-cleaning from debris, particles attached to the cell or prey digestion. Images of live specimens were taken under an Medlin et al., 1988 Decelle et al., 2012b Ishitani et al. 2012 Ando et al., 2009 inverted microscope and thereafter transfer into 1.5 ml Eppendorf tubes containing 50 ml of molecular grade absolute ethanol and C Reference stored at -20 °C until DNA extraction. ° DNA extraction, amplification and sequencing: DNA was extracted using the MasterPure Complete DNA and RNA Purification Kit (Epicentre) following manufacturer’s instructions. Both 18S rDNA and partial 28S rDNA genes (D1 and D2 regions) were amplified by Polymerase Chain Reaction (PCR) using Radiolaria and Spumellaria specific and general primers (Table 1). For further details about rDNA amplification see: dx.doi.org/10.17504/protocols.io.xwvfpe6. PCR amplicons were visualized on 1% agarose gel stained with ethidium Direction Tm bromide. Positive reactions were purified using the Nucleospin Gel and PCR Clean up kit (Macherey Nagel), following manufacturer’s instructions and sent to Macrogen Europe for sequencing. Scanning Electron Microscopy (SEM): After DNA extraction, spumellarian skeletons were recovered from the eluted pellet and handpicked under binoculars or inverted microscope. After cleaning and preparing the skeletons (detailed protocol in dx.doi.org/10. 17504/protocols.io.ug9etz6) images were taken with a FEI Phenom tabletop Scanning Electron Microscope (FEI technologies). Single cell morphological identification: Spumellaria speci- mens were identified at the species level, referring to pictures of holotypes, through observation of live images and analysis of the skeleton by scanning electron microscopy when available (seeSup-

plementary Material Table S1 for taxonomic authority of specimens 0

included in our study). Further details in taxonomic assignment of the -3 0 specimens can be found in the Material and Methods of Sandin et. al. 2019. Phylogenetic analyses: After sequencing, forward and reverse sequences were checked and assembled using ChromasPro soft- ware version 2.1.4 (2017). Sequences were compared to the Gen- Bank reference database (GenBank) using the BLAST search tool integrated in ChromasPro to discriminate radiolarian sequences from possible contamination. Presence of chimeras, at a frequency as high as 13-25%, was detected by mothur v.1.39.3 (Schloss et al. 2009) against previously available reference sequences of Spumel- laria. Sequences not detected as chimeras, were in turn included in our reference database for future phylogenetic reconstruction and chimeric analysis. Two different datasets for each genetic marker (18S rDNA gene and partial 28S rDNA gene) were aligned separately using MAFFT v7.395 (Katoh and Standley 2013) with a L-INS-i algorithm (‘-- localpair’) and 1000 iterative refinement cycles for high accuracy. Each alignment was manually checked in SeaView version 4.6.1 (Gouy et al. 2010) and trimmed automatically using trimal (Capella- Gutie´rrez et al. 2009) with a 30% gap threshold. For both genes, the 18S rDNA (133 taxa, 1789 positions) and the 28S rDNA (55 taxa, S879V9RITSa4R Radiolaria CCA ACT GTC Radiolaria CCT ATC AAT CAT CCT TCY GCA GGT TCA TCA CCT CCA AC TCT TTC GGG TCC CGG CAT Reverse Reverse Reverse This study Romac (unpub.) 746 positions), phylogenetic analyses were performed indepen- dently. The best nucleotide substitution model was chosen following the corrected Akaike Information Criterion (AICc) using the modelT- est function implemented in the R version 3.6.0 (R Core Team 2014) Primer sequences and temperatures used for DNA amplification and sequencing. package phangorn version 2.5.5 (Schliep 2011). The obtained model (General Time Reversible with Gama distribution and proportion of Invariable sites, GTR + G + I) was applied to each data set in R upon Table 1. 28S (D1 + D2) 28S Rad2 Radiolaria TAA GCG GAG GAA AAG AAA Forward 52 18S (1st part)18S (2nd SA part) S32_TASN Radiolaria Eukaryotes CCA GCT CCA ATA AAC GCG CTG TAT GTT RC GAT CCT GCC AGT Forward Forward 57 56 Targeted gene Primer Specificity Sequence 5 Diversity and Evolution of Spumellaria (Radiolaria) 19 the packages APE version 5.3 (Paradis et al. 2004)andphangorn Blome 1980). Although it is not sure whether there is a continuity version 2.5.5 (Schliep 2011). A Maximum Likelihood (ML) method between members from the Mesozoic and Cenozoic (Matsuzaki (Felsenstein 1981) with 10 000 replicates of bootstrap (Felsenstein et al. 2015). 1985) was performed to infer phylogenies. 6. N(170, 20): The family Actinommidae appears Despite specific discrepancies in the topology of the two analyses for the first time in the fossil record in the Middle Jurassic (Aalenian: (Supplementary Material Figure S1), the two genes were concate- ca. 174.2–170.3 Ma; O’Dogherty et al. 2011) yet some morphologies nated in order to increase taxonomic coverage and improve phylo- from the Triassic resemblance this Superfamily (De Wever et al. genetic resolution. A final data set containing 133 taxa and 2535 2001). positions was used to infer phylogenies following the previous 7. Rhizosphaeroidea N(148, 10): The Rhizosphaeridae appeared methodology. Fourteen sequences of Nassellaria were assembled to during the late Jurassic (Tithonian: ca. 145–152.1 Ma) in the fossil form the outgroup as seen in previous classifications to be the sister record (Afanasieva and Amon 2006; De Wever et al. 2001; Dumitrica clade of Spumellaria (e.g., Cavalier-Smith et al. 2018; Krabberød 2017; Petrushevskaya 1975). et al. 2011). The best model obtained was GTR + G + I, with four 8. Pylonioidea N(97, 10): The family Larnacillidae are the first intervals of the discrete gamma distribution, and a ML method with representatives of the superfamily Pylonioidea appearing at the 100 000 bootstraps. In parallel, a Bayesian analysis was performed beginning of the Late Cretaceous (Cenomanian: ca. 100.5–93.9 Ma; using BEAST version 1.8.4 (Drummond et al. 2012)withthesame Afanasieva and Amon 2006; De Wever et al. 2001, 2003). model parameters over 100 million generations sampled every 9. Coccodiscoidea N(45, 10): The family Coccodiscoidea 1000 states, to check the consistency of the topology and to calculate appears for the first time in the fossil record in the Early Eocene posterior probabilities. The final tree was visualized and edited with (Afanasieva and Amon 2006; De Wever et al. 2001). This family cor- FigTree version 1.4.3 (Rambaut 2016). All sequences obtained in responds to clade E1 in Figure 1. this study and used for the phylogeny were submitted in GenBank Post hoc analyses: Two different analyses were performed a under the accession numbers: MT670437 - MT670549. posteriori: a diversification of taxa over time (Lineages Through Time: Morphological observations performed with light and scanning LTT) and an ancestral state reconstruction. The former analysis was electron microscopy (Supplementary Material Figure S2)allowed carried out with the ltt.plot function implemented in the package APE assigning these sequences to nine super-families (Actinommoidea, (Paradis et al. 2004) upon the tree obtained by the molecular dating Hexastyloidea, Liosphaeroidea, Lithocyclioidea, Pylonioidea, Spon- analyses after removing the outgroup. The second analysis used the godiscoidea, Spongopyloidea, Spongosphaeroidea and Stylodicty- resulting phylogenetic tree to infer the evolution of morphological oidea), two super-families considered to belong to Entactinaria characters. A numerical value was assigned to each state of a char- (Centrocuboidea and Rhizosphaeridae) and two unresolved families acter trait, being 0 for the considered ancestral state, and one to three (Litheliidae and Sponguridae). for the presumed divergence state. In total six traits were considered Molecular clock analyses: Molecular clock estimates were (Supplementary Material Table S2): the skeleton shape, the symme- performed according to Sandin et al. (2019). Nine nodes were cho- try, the central structure, the internal cavity (outside of the central sen to carry out the calibration. The selection of these nodes is structure), the number of spines arisen from the central structure explained below from the oldest to the newest calibration age, and and the number of distinctive cortical shells. Once the character given the name of the node for the taxa they cover: matrix was established a parsimony ancestral state reconstruction 1. Root: The calibration for the root of the tree corresponds to the was performed to every character independently in Mesquite version hypothesized last common ancestor between Nassellaria and 3.2 (Maddison and Maddison 2017). Spumellaria (De Wever et al. 2001). A uniform distribution (U) with a Environmental sequences: Each of the reference 18S rDNA minimum bound of 300 million years ago (Ma) and a maximum of and partial 28S rDNA spumellarian sequences available in our study 800 Ma (U[300, 800]) was set to allow uncertainty in the diversifica- was compared with publicly available environmental sequences in tion of both groups and to establish a threshold restricting the range GenBank (NCBI) using BLAST (as of May 2019). It allowed esti- of solutions for the entire tree. mating the environmental genetic diversity of Spumellaria and to 2. Nassellaria N(410, 20): The outgroup of the phylogeny is cal- assess the genetic coverage of our phylogenetic tree. A total of 1171 ibrated based in a consensus between the first appearance of and six environmental sequences affiliated to Spumellaria were nassellarian-like fossils in the Upper Devonian (ca. 372.2 Ma) in the retrieved for the genes 18S rDNA and 28S rDNA respectively. Out of fossil record (Cheng 1986) and the first diversification of Nassellaria the six 28S rDNA sequences, four were also found in the 18S rDNA dated with previous analysis of the molecular clock (ca. 423 Ma; 95% survey covering the full rDNA operon. These four sequences along HPD: 500–342 Ma; Sandin et al. 2019). Therefore, the node was nor- with the eight sequences already included in the phylogeny (two in mally distributed (N) with a mean of 410 and a standard deviation of clade H and eight in clade I) were therefore removed to avoid 20: N(410, 20). duplicates. Final dataset containing 1165 environmental sequences 3. Spumellaria U[700, 200]: Recent studies have found the oldest were aligned against the reference alignment used for the phyloge- spumellarian representatives in the early middle Cambrian (Zhang netic analysis using MAFFT v7.395 (Katoh and Standley 2013)with and Feng 2019). Yet, De Wever et al. (2001) have argued that the the “--add” option and placed in our reference phylogenetic tree using initial spicular system may be subjected to preservation bias. Since the pplacer software (Matsen et al. 2010). A RAxML (GTR + G + I) many spumellarians and entactinarians (presence of the initial spic- tree was built for the placement of the sequences with a rapid boot- ular system or not, respectively) are superficially similar and it is strap analysis and search for best-scoring ML tree and 1000 not possible to distinguish from one another (Suzuki and Oba bootstraps. 2015), a uniform distribution was set to allow uncertainty in the diver- In order to discriminate pseudo-genes among environmental sification of Spumellaria. clades due to a high frequency of chimeras encountered and long 4. Hexastyloidea N(242, 10): The family Hexastylidae is the first phylogenetic branches, two preliminary analyses were performed: a representative of the superfamily Hexastyloidea and has its first Shannon entropy analysis and an estimation of the GC content. appearance in the fossil record in the Middle Triassic (Late Anisian: Shannon entropy was calculated for every position of the resulting ca. 246.8–241.5 Ma; O’Dogherty et al. 2011). alignment matrix, without considering gaps due to differences in 5. Liosphaeroidea N(233, 20): The Liosphaeroidea seems to length of environmental sequences (script available on https:// have appeared in the Triassic (De Wever et al. 2001; Pessagno and github.com/MiguelMSandin/DNA-alignment-entropy). The GC con- 20 M.M. Sandin et al. tent was estimated for every sequence independently as follows: (G Cˇ epicˇka B, Chistyakova L, del Campo J, Dunthorn M, +C)/(G+C+A+T),whereG,C,AandTarethenumberofgua- Edvardsen B, Eglit Y, Guillou L, Hampl V, Heiss AA, nines, cytosines, adenines and thymines respectively. Analysis were Hoppenrath M, James TY, Karnkowska A, Karpov S, Kim performed in R version 3.6.0 (R Core Team 2014) and plotted with E, Kolisko M, Kudryavtsev A, Lahr DJ, Lara E, Le Gall L, the package ggplot2 version 3.2.1 (Wickham 2016). Lynn DH, Mann DG, Massana R, Mitchell EA, Morrow C, Park JS, Pawlowski JW, Powell MJ, Richter DJ, Rueckert Declaration of Competing Interest S, Shadwick L, Shimano S, Spiegel FW, Torruella G, Youssef N, Zlatogursky V, Zhang Q (2019) Revisions to the The authors declare that they have no known com- classification, nomenclature, and diversity of eukaryotes. J Eukaryot Microbiol 66:4–119 peting financial interests or personal relationships that could have appeared to influence the work Afanasieva MS, Amon EO (2006) Biotic crises and stages of reported in this paper. radiolarian evolution in the Phanerozoic. Paleontol J 40: S453–S467 Acknowledgements Afanasieva MS, Amon EO, Agarkov YU, Boltovskoy BS (2005) Radiolarians in the geological record. Paleontol J 39 This work was supported by the IMPEKAB ANR 15- (Suppl. 3):135–392 CE02-0011 grant and the Brittany Region ARED C16 Aita Y, Suzuki N, Ogane K, Sakai T, Lazarus D, Young J, 1520A01, the Japan Society for Promotion of Tanimura Y (2009) Haeckel Radiolaria Collection and the H. Science KAKENHI Grant No. K16K0-74750 for N. M.S. Challenger plankton collections. Nat Mus Natl Sci Suzuki and “the Cooperative Research Project with Monogr 40:35–45 the Japan Science and Technology Agency (JST) Aitchison J, Suzuki N, Caridroit M, Danelian T, Noble PJ and Centre National de la Recherche Scientifique (2017) Paleozoic Radiolarian Biostratigraphy. In Danelian T, (CNRS, France) “Morpho-molecular Diversity Caridroit M, Noble P, Aitchison JC (eds) Catalogue of Assessment of Ecologically, Evolutionary, and Geo- Paleozoic Radiolarian Genera. Geodiversitas 31:503–531 logically Relevant Marine Plankton (Radiolaria)”. Ando H, Kunitomo Y, Sarashina I, Iijima M, Endo K, We are grateful to the CNRS-Sorbonne University Sashida K (2009) Intraspecific variations in the ITS region of ABiMS bioinformatics platform (http://abims.sb-rosc- recent radiolarians. Earth Evol Sci 3:37–44 off.fr) for providing computational resources. 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Available online at: www.sciencedirect.com ScienceDirect Outgroup (14 sequences of Nassellaria) HQ651792 - Heliosphaera aff. actinota F1 AB284518 - Heliosphaera aff. actinota 62 HQ651782 - Heliosphaera aff. actinota 76 Osh34 - Heliosphaera bifurcus AB490706 - Astrosphaera hexagonalis F2 AB490705 - Astrosphaera hexagonalis 88 Vil173 - Cladococcus sp. A 89 Vil499 - Cladococcus sp. A Vil497 - Arachnospongus varians 97 Vil244 - Cladococcus viminalis 63 Vil243 - Cladococcus viminalis Vil228 - Cladococcus scoparius 65 Vil238 - Cladococcus irregularis Vil174 - Hollandosphaera hexagonia B Vil454 - Hollandosphaera hexagonia 62 Vil448 - Hollandosphaera hexagonia Vil217 - Hollandosphaera hexagonia Vil209 - Hollandosphaeragonia Vil282 - Hexacontella sp. A HQ651798 - Hexastylus nobilis HQ651795 - Hexastylus nobilis Vil484 - Stigmostylus ferrusi C Mge17-15 - Hexacontella? sp. 76 HQ651794 - Hexacontium sp. A HQ651797 - Hexacontium sp. A Outgroup (7 sequences of Nassellaria) HQ651796 - Hexacontium sp. A D Mge17-82 - Spongosphaera streptacantha AB284519 - Hexacontium cf. hexagonum E1 71 HQ651784 - Hexacontium sp. A Vil186 - Didymocyrtis sp. Mge17-82 - Spongosphaera streptacantha D E3 Mge17-74 - Dictyocoryne gegenbauri Osh69 - Spongosphaera streptacantha 64 Oki25 - Spongosphaera streptacantha Vil293 - Dictyocoryne koellikeri 86 88 Osh188 - Spongosphaera streptacantha AB430758 - Dictyocoryne truncata Osh174 - gen et sp. indet. E2 Osh48 - gen et sp. indet. AB430757 - Dictyocoryne truncata 87 AB430760 - gen et sp. indet. E2 Osh79 - Spongodiscoidea fam, gen et sp. indet. A 84 Mge17-17 - Spongobracium ellipticum Osh79 - gen et sp. indet. Osh174 - Spongodiscoidea fam, gen et sp. indet. A Vil449 - Spongasteriscus toxon AB430760 - Spongodiscoidea fam, gen et sp. indet. A AB246695 - gen et sp. indet. 63 Vil186 - Didymocyrtis sp. E1 Mge17-17 - Spongobracium ellipticum AB439010 - Cypassis irregularis C Vil217 - Hollandosphaera hexagonia Vil450 - Cypassis oblongus LC093106 - Didymocyrtis tetrathalamus coronatus Vil174 - Hollandosphaera hexagonia 86 97 60 Vil458 - Cypassis irregularis B Vil484 - Stigmostylus ferrusae Ses64 - Didymocyrtis tetrathalamus 80 AB193605 - Didymocyrtis tetrathalamus HQ651784 - Hexacontium sp. A Ses63 - Didymocyrtis tetrathalamus coronatus H 76 Ses55 - Centrocubus cladostylus 85 Ses19 - Didymocyrtis tetrathalamus Mge17-74 - Dictyocoryne gegenbauri E3 G Mge17-81 - Rhizosphaera trigonacantha 73 Vil293 - Dictyocoryne koellikeri Vil246 - Dictyocoryne koellikeri Vil476 - Rhizosphaera trigonacantha AB430758 - Dictyocoryne truncata Vil240 - Haliommilla capillacea 78 AB430757 - Dictyocoryne truncata 70 AB101541 - Dictyocoryne truncata Mge17-44 - Haliommilla capillacea AB246696 - Spongodiscus aculeatus Ses15 - Haliommilla capillacea 81 AB101542 - Spongaster tetras tetras F1 AB101540 - Dictyocoryne profunda HQ651782 - Heliosphaera aff. actinota Mge17-99 - Dictyocoryne sp. F2 Vil499 - Cladococcus sp. A 85 81 AB179732 - Myelastrum elegans Vil497 - Arachnospongus varians 67 AB179733 - Myelastrum furcatum LC093107 - Myelastrum aurivillii Vil228 - Cladococcus scoparius 81 AB179734 - Myelastrum lobum JQ706069 (Oki50) - Rhizosphaera trigonacantha G Vil243 - Cladococcus viminalis AB246684 - Rhizosphaera sp. Vil244 - Cladococcus viminalis AB246686 - Rhizosphaera sp. J1 Vil503 - Stylodictya sp. 63 Mge17-81 - Rhizosphaera trigonacantha 86 Vil476 - Rhizosphaera trigonacantha AB860148 - Perichlamydium venustum Ses15 - Haliommilla capillacea 65 Osh90 - Stylodictya stellata 66 Mge17-44 - Haliommilla capillacea 77 J2 96 Vil240 - Haliommilla capillacea AB860147 - Stylodictya stellata Ses55 - Centrocubus cladostylus H GU821394 - Uncultured Rhizarian Vil453 - Stylodictya arachnea 66 GU822672 - Uncultured Rhizarian Vil501 - Stylodictya arachnea 66 KX532703 - Uncultured marine eukaryote I K KJ757677 - Uncultured eukaryote HQ651780 - Actinomma boreale JX842018 - Uncultured marine eukaryote HQ651781 - Actinomma boreale Vil451 - Plegmosphaeromma? sp. L2 Vil210 - Plegmosphaeromma? sp. AB860156 - Schizodiscus? spp. 61 GU821097 - Uncultured Rhizaria AB860155 - Schizodiscus? spp. 79 GU822590 - Uncultured Rhizaria GU822292 - Uncultured Rhizaria AB860154 - Schizodiscus? spp. Vil441 - Stylodictya arachnea J1 AB860149 - Schizodiscus? spp. Vil503 - Stylodictya sp. AB860148 - Perichlamydium venustum L1 Mge17-118 - Trilobatum aff. acuferum AB246698 - Stylodictya subtilis J2 AB860151 - Spongopyle osculosa 86 Osh90 - Stylodictya stellata 82 AB860147 - Stylodictya stellata AB860150 - Spongopyle osculosa Vil453 - Stylodictya arachnea AB860152 - Schizodiscus? spp. Vil501 - Stylodictya arachnea 73 78 HQ651789 - Actinomma boreale K AB860153 - Schizodiscus? spp. Vil438 - Actinomma trinacrium Vil249 - Calcaromma morum 88 HQ651788 - Actinomma boreale HQ651780 - Actinomma boreale Vil297 - Calcaromma morum HQ651781 - Actinomma boreale Vil296 - Calcaromma morum 81 AB617584 - Lithelius cf. alveolina L1 AB617587 - Spongurus? pylomaticus M1 Vil200 - Tholomura transversara Mge17-118 - Trilobatum aff. acuferum M3 HQ651783 - Un-identifiable specimen AB860151 - Spongopyle osculosa L2 AB246689 - Un-identifiable specimen Ses53 - Pyloniidae gen et sp. indet AB860150 - Spongopyle osculosa Ses13 - Pylodiscus spinulosus AB860149 - Spongopyle osculosa 79 AB860154 - Schizodiscus? spp. M4 Vil298 - Tetrapyle sp. AB860155 - Schizodiscus? spp. Ses18 - Tetrapyle octacantha AB860156 - Schizodiscus? spp. AB860152 - Schizodiscus? spp. Vil432 - Tetrapyle octacantha 79 82 AB860153 - Schizodiscus? spp. Vil480 - Tetrapyle octacantha 79 AB617590 - Schizodiscus asymmetricus AB617589 - Un-identifiable specimen Vil506 - Tetrapyle octacantha AB617588 - Schizodiscus stylotrochoides 67 Vil249 - Calcaromma morum Vil297 - Calcaromma morum 0.09 64 Osh153 - Calcaromma morum 71 Vil296 - Calcaromma morum Vil200 - Tholomura transversara M1 HQ651803 - Sphaeropylolena circumtexta Toy110 - Tholomura pilula 82 AB246688 - Sphaerolarnacillium tanzhiyuani M2 AB617585 - Un-identifiable specimen Osh16 - Larcopyle aff. buetschli 72 LC049343 - Larcopyle buetschlii LC049344 - Larcopyle buetschlii 82 Ses13 - Pylodiscus spinulosus M3 Ses53 - Pyloniidae gen et sp indet Vil437 - Cryptolarnaciinae gen et sp HQ651783 - Un-identifiable specimen >= 90% BS Mge17-20 - Tetrapyle octacantha M4 Vil298 - Tetrapyle octacantha >= 99% BS Vil432 - Tetrapyle octacantha Ses18 - Tetrapyle octacantha : 25% of original length 88 67 AB246680 - Tetrapyle sp. 87 Vil506 - Tetrapyle octacantha Vil480 - Tetrapyle octacantha 0.06 Vil231 - Tetrapyle octacantha Vil282 Vil209 Vil448 Vil217

Vil174 Vil174 Vil454 Vil454

Vil484 Vil484 Mge17-15 Mge17-15

Mge17-82 Mge17-82 Osh69 Osh69

Osh188 Osh188Mge17-82 Oki25 Ses64

Vil186 Vil186 Ses19 Ses63 Vil450 Vil450 Vil458 Vil458

Vil449 Vil449 Osh79 Osh174

Mge17-17 Mge17-17 Osh48 Osh48

Mge17-74 Vil293 Vil246 Vil246

Mge17-99 Mge17-99 Osh34 Osh34

Vil499 Vil499 Vil173 Vil173 Vil497 Vil238 Vil243 Vil244

Vil228 Ses15 Mge17-44 Mge17-44

Vil476 Vil476 Vil240 Vil240

Mge17-81 Mge17-81 Vil210 Vil210

Vil451 Vil451 Ses55 Vil501

Vil441 Vil503 Vil503 Osh90 Osh90Mge17-20 Vil453 Vil453

Vil438 Vil438 Vil249 Osh153

Vil297 Vil297 Vil296 Vil296

Mge17-118 Mge17-118 Mge17-118 Toy110

Vil200 Vil200 Ses13 Ses53

Vil437 Osh16 Osh16 Vil298 Mge17-20 Mge17-20 Vil506 Ses18

Vil432 Vil231 Vil480 Vil480 Cortical shells Number of spines Internal cavity Central structure Symmetry Skeleton shape 1 Zero Wide space Empty Line symmetry Spherical 2 6 Space dividers Entactinaria-type, fused fibers or soft spongy Spherical or plane Spherical modified Dense 3 More or less than 6 Double medullary shell or highly dense centre Reflection and/or plane Cubic-, box-, dice-shaped Significantly more than 6 Dense with pylome More than 3 Complex Circular or rotational Flat Outgroup: 14 Nassellaria sequences

Vil282 - Hexacontella sp. HQ651795 - Hexastylus nobilis A HQ651798 - Hexastylus nobilis Vil484 - Stigmostylus ferrusi Mge17-15 - Hexacontella? sp. B HQ651794 - Hexacontium sp. A HQ651796 - Hexacontium sp. A HQ651797 - Hexacontium sp. A HQ651784 - Hexacontium sp. A AB284519 - Hexacontium cf. hexagonum Vil174 - Hollandosphaera hexagonia Vil217 - Hollandosphaera hexagonia C Vil209 - Hollandosphaera hexagonia Vil448 - Hollandosphaera hexagonia Vil454 - Hollandosphaera hexagonia Mge17-82 - Spongosphaera streptacantha Osh69 - Spongosphaera streptacantha D Osh188 - Spongosphaera streptacantha Oki25 - Spongosphaera streptacantha Vil186 - Didymocyrtis sp. AB439010 - Cypassis irregularis E1 Vil450 - Cypassis cf. oblogum LC093106 - Didymocyrtis tetrathalamus coronatus Vil458 - Cypassis irregularis Ses64 - Didymocyrtis tetrathalamus tetrathalamus AB193605 - Didymocyrtis tetrathalamus Ses19 - Didymocyrtis tetrathalamus Ses63 - Didymocyrtis tetrathalamus coronatus AB246695 - gen et sp. indet. Osh79 - gen et sp. indet. E2 Osh48 - gen et sp. indet. Osh174 - gen et sp. indet. AB430760 - gen et sp. indet. Vil449 - Spongasteriscus toxon Mge17-17 - Spongobrachium elliptica Mge17-74 - Dictyocoryne gegenbauri Vil293 - Dictyocoryne koellikeri E3 Vil246 - Dictyocoryne koellikeri AB430758 - Dictyocoryne truncata AB430757 - Dictyocoryne truncata AB101541 - Dictyocoryne truncata AB246696 - Spongodiscus resurgens AB101542 - Spongaster tetras tetras Mge17-99 - Dictyocoryne sp. AB101540 - Dictyocoryne profunda AB179732 - Myelastrum elegans AB179733 - Myelastrum furcatum LC093107 - Myelastrum aurivillii AB179734 - Myelastrum lobum HQ651792 - Heliosphaera aff actinota AB284518 - Heliosphaera aff actinota F1 HQ651782 - Heliosphaera aff actinota Osh34 - Heliosphaera bifurcus AB490706 - Astrosphaera hexagonalis AB490705 - Astrosphaera hexagonalis F2 Vil173 - Cladococcus sp. A Vil499 - Cladococcus sp. A Vil497 - Arachnospongus varians Vil244 - Cladococcus viminalis Vil243 - Cladococcus viminalis Vil228 - Cladococcus scoparius Vil238 - Cladococcus irregularis JQ706069 (Oki50) - Rhizosphaera trigonacantha Mge17-81 - Rhizosphaera trigonacantha G Vil476 - Rhizosphaera trigonacantha AB246684 - Rhizosphaera sp. AB246686 - Rhizosphaera sp. Ses15 - Haliommilla capillacea Vil240 - Haliommilla capillacea Mge17-44 - Haliommilla capillacea Ses55 - Centrocubus cladostylus GU821394 - Uncultured eukaryote H GU822672 - Uncultured eukaryote KX532703 - Uncultured marine eukaryote KJ757677 - Uncultured eukaryote I JX842018 - Uncultured marine eukaryote Vil451 - Plegmosphaeromma? sp. Vil210 - Plegmosphaeromma? sp. GU821097 - Uncultured Rhizaria GU822292 - Uncultured Rhizaria GU822590 - Uncultured Rhizaria Vil441 - Stylodictya arachnea AB860148 - Perichlamydium venustum J1 Vil503 - Stylodictya sp. Osh90 - Stylodictya stellata AB246698 - Stylodictya subtilis J2 AB860147 - Stylodictya stellata Vil453 - Stylodictya arachnea Vil501 - Stylodictya arachnea HQ651789 - Actinomma boreale HQ651788 - Actinomma boreale K Vil438 - Actinomma trinacrium HQ651780 - Actinomma boreale HQ651781 - Actinomma boreale AB617584 - Lithelius cf. alveolina AB617587 - Spongurus? pylomaticus L1 Mge17-118 - Trilobatum aff. acuferum AB860151 - Spongopyle osculosa AB246689 - Un-identifiable specimen L2 AB860150 - Spongopyle osculosa AB860156 - Schizodiscus? sp. AB860154 - Schizodiscus? sp. AB860155 - Schizodiscus? sp. AB860149 - Spongopyle osculosa AB860152 - Schizodiscus? sp. AB860153 - Schizodiscus? sp. AB617590 - Schizodiscus asymmetricus AB617588 - Schizodiscus stylotrochoides AB617589 - Un-identifiable specimen Vil249 - Calcaromma morum Vil296 - Calcaromma morum Vil297 - Calcaromma morum Osh153 - Calcaromma morum Vil200 - Tholomura transversa HQ651803 - Sphaeropylolena circumtexta M1 Toy110 - Tholomura pilula AB246688 - Sphaerolarnacillium tanzhiyuani AB617585 - Un-identifiable specimen M2 Osh16 - Larcopyle aff. buetschli LC049344 - Larcopyle buetschlii LC049343 - Larcopyle buetschlii HQ651783 - Un-identifiable specimen Vil437 - Cryptolarnaciinae gen et sp M3 Ses53 - Pyloniidae gen et sp indet Ses13 - Pylodiscus spinulosus Mge17-20 - Tetrapyle octacantha Vil298 - Tetrapyle octacantha M4 Ses18 - Tetrapyle octacantha AB246680 - Tetrapyle sp Vil432 - Tetrapyle octacantha Vil480 - Tetrapyle octacantha Vil231 - Tetrapyle octacantha Vil506 - Tetrapyle octacantha 1.5

● ● Lineage I ● Lineage II Lineage III ● ● ● ● Lineage IV ● Env 1 ● ● ● ● ● ● ●●● ● ● ● ● ● ● ● ●● ●● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ●● ●● ● ● ● ● ● ● ●● ● ● ● 1.0 ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ●● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ●● ● ●● ● ● ● ● ● ●●● ● ●● ● ● ●●● ● ● ● ●●● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ●● ● ●●●●● ●● ●● ● ● ● ● ● ●●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●●●● ● ● ● ● ● ● ● ●● ● ● ●● ● ● ● ● ● ● ● ●● ●● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ●●● ● ● ● ● ● ● ● ● ●●● ● ● ● ● ●●●●●●●●● ●●●●●● ●●● ● ● ● ●● ● ● ● ●● ●● ● ● ● ● ●●●● ● ● ● ● ● ● ● ● ●● ●●● ● ● ● ● ● ● ● ●●●●●●●● ● ●● ● ● ● ● ●●● ● ● ● ● ● ● ● ●● ● ●● ● ●●● ●●●●●● ● ● ● ●●●● ● ● ● ● ● ● ●● ● ●●● ● ● ● ● ● ●● ● ● ● ● ● ● ● ●●● ● ● ●●●● ●● ●● ● ● ● ● ● ● ● ●● ●● ● ● ● ● ● ● ● ●● ● ●●●● ●●●●● ● ● ● ● ● ● ●● ● ●●●● ● ● ● ● ● ●● ● ● ● ● ●●● ●● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●●● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ●● ● ● ● ●● ●● ● ●● ●● ● ● ● ●●● ● ● ● ● ●● ●● ●● ● ● ● ● ● ● ● Shannon entropy ● ● ● ●● ● ● ●● ● ● ●● ● ● 0.5 ● ● ● ●● ● ● ●● ● ● ●● ● ● ●● ● ● ● ●● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ●●● ● ● ● ● ●● ● ● ●● ●●● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ●● ●●● ● ● ● ● ● ● ● ● ● ● ●● ●●● ● ●● ● ●● ● ●● ● ● ● ● ●● ●●●● ● ● ● ● ● ● ● ●●● ● ● ● ● ●●● ● ● ● ●● ● ●● ● ● ● ● ● ● ● ● ● ● ●●● ●● ● ● ● ● ● ●●● ● ● ● ● ●● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ●● ●● ● ●● ● ● ●● ● ● ●● ● ●● ● ● ● ● ● ● ● ● ● ●● ●● ● ● ●● ●●● ● ● ● ●●● ●● ● ● ● ● ●●● ●● ●● ● ●● ● ● ● ●● ● ● ●● ● ●● ● ● ● ●● ● ●● ● ● ● ● ●● ● ●● ● ● ●●● ● ● ● ●● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ●● ●●● ● ●● ●● ● ● ● ●● ● ●● ● ● ● ● ●● ●●● ● ● ● ● ●●● ●● ●●● ● ● ● ●●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●●● ● ● ●● ●● ● ● ● ● ● ● ● ●●● ● ● ●● ● ●● ● ●● ● ● ●● ● ●● ● ●● ● ● ● ● ●● ● ● ●●●●● ● ● ● ● ● ● ●●●● ●● ●● ●● ● ●●●● ● ●● ● ●● ● ● ●●● ● ●●●●● ● ●●●● ● ● ● ● ●●●●●● ●●●● ●●●●●●● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●● ● ● ●●● ●● ● ● ●● ●●●●●● ●●● ● ● ● ●● ●● ● ● ● ● ●●●● ● ● ●● ● ● ●● ● ● ● ● ●●● ●● ● ●●● ●●●●● ●●● ● ● ● ● ● ● ● ●● ●● ●● ● ● ● ●●● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ●● ●● ● ●● ● ●●●●●● ● ● ● ● ● ● ●● ● ● ● ● ●●●●●● ●● ● ● ● ●● ●●● ● ●●● ● ● ● ●● ● ● ●● ● ●● ● ● ● ● ● ● ● ●● ● ● ●●●● ●● ● ● ● ●● ● ●● ●●●● ●●●● ●● ● ●● ● ● ● ●● ●●●● ●● ● ● ●● ●● ●● ●●●●●●●●● ● ● ● ● ● ●●●●●●●●●●●●●●●●●●● ●●●●● ● ● ● ●●●●●● ● ●● ●●●●●●●●● ●● ● ● ●●● ●●●●●●●● ●● ●●●● ●●●●●●●● ●●●●●●●●●● ● ● ●●● ● ● ● ●●●●●● ●●● ●●●● ● ● ●●● ●●●●●●● ● ●● ●●● ●●● ● ●● ● ●● ● ●●● ●●●●●●●●●●●●●● ●●● ● ●●●●●● ●●●●● ● ● ●●● ●●●●●● ● ● ● ● ●●●●●● ●● ●● ●●●●● ● ●● ● ● ● ●●●●● ● ● ●●●●●●● ●●●● ●●●●●● ● ● ● ●●●●● ● ●●● ●●● ●●●●●●●●●●● ● ● ●●●●● ●●● ● ●● ●●●● ●●●●●●●● ●●●●●●●●●●●●●● ●● ● ●●●●●● ● ●● ●● ●● ●●●● ●●● ● ● ●●●●●●●●●●●●●●●●● ●●●●●● ●●●●●●●●●●●●●●●●● ● ●●● ●●●●●●●●●●●●●● ● ●●●●●●●●●●●●●●●● ● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ● ●● ● ●● ●● ●●●●●●●●●●●● ● ●●●●● ●●●● ●●●●●●●●●●●●●●●●●●●●●●●● ●●●●●●●●● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●●●●● ●●●●● ●●●●●●●●●●● ●●●●●●●●● ●●●●●●●● ● ●● ●●●●● ●●● ●●●●●●● ● ●●● ●●●●●●●●●●●●●●●●●●●●●●● ●●●●●●●● ● ● ● ● ● ●● ●●● ● ●●● ● ●● ● ● ● ● ● ●● ●● ● ● ●● ● ● ● ● ● ● ● ● ● ● ●● ●●●●●●●●●● ●●●●●●●●●●●●●●●●●●●●● ●●●●●● ●● ● ●●●●●●●●●●●●●● ● ● ●●●●● ●●●●●●●●●●●●●●●●●●●●●●● ●●●●● ●● ●●●●●●●●● ●●●● ●●●●●●● ● ●●●●●●●●●●●● ●●●● ●●●●●●●●●●●●●●●●●●●●●●●●●●●● ●●●●●●●●●●●●● ●●●●●●● ●● ●● ●● ●●●●●●●●●● ● ● ●● ●●●●● ●●●●● ●●●●●● ●●● ●●●●●●●●● ●●● ●● ●●●●●●●●●●●●●●●●●●●●●●●●●●●●● ● 0.0

0 200 400 600800 1000 1200 1400 1600 1800 Alignment position 52.5 n=72 n=7 n=3 n=7 n=5 n=4 n=30 n=165 n=8 n=8 n=3 n=8 n=3 n=5 n=19 n=8 n=16 n=22

●

● ● ● ● 50.0 ● ● ● ● ● ● ● ● ●

●

● ●

●

47.5 ●

● GC content (%) ● ●

● ●

● 45.0

● ●

●

Lineage I Lineage II Lineage III Lineage IV

Env 1 Env 2 Env 6 Env 3 Env 4 Clade E LineageII Hexastylidae Pylonioidea Hexalonchidae Centrocubidae ActinommoideaSpongopyloideaStylodictyoidea Rhizosphaeroidea HollandosphaeridaeSpongosphaeroidea Excentroconchidae ID Clade Subclade Superfamily Family Genus Vil282 A A Hexastyloidea Hexastylidae Hexacontella HQ651795 A A Hexastyloidea Hexastylidae Hexastylus HQ651798 A A Hexastyloidea Hexastylidae Hexastylus Mge17-15 B B Hexastyloidea Hexalonchidae Hexacontella? AB284519 B B Hexastyloidea Hexalonchidae Hexacontium HQ651784 B B Hexastyloidea Hexalonchidae Hexacontium HQ651794 B B Hexastyloidea Hexalonchidae Hexacontium HQ651796 B B Hexastyloidea Hexalonchidae Hexacontium HQ651797 B B Hexastyloidea Hexalonchidae Hexacontium Vil484 B B Hexastyloidea Hexalonchidae Stigmostylus Vil174 C C Hexastyloidea Hollandosphaeridae Hollandosphaera Vil209 C C Hexastyloidea Hollandosphaeridae Hollandosphaera Vil217 C C Hexastyloidea Hollandosphaeridae Hollandosphaera Vil448 C C Hexastyloidea Hollandosphaeridae Hollandosphaera Vil454 C C Hexastyloidea Hollandosphaeridae Hollandosphaera Mge17-82 D D Spongosphaeroidea Spongosphaeridae Spongosphaera Oki25 D D Spongosphaeroidea Spongosphaeridae Spongosphaera Osh188 D D Spongosphaeroidea Spongosphaeridae Spongosphaera Osh69 D D Spongosphaeroidea Spongosphaeridae Spongosphaera AB439010 E E1 Lithocyclioidea Artiscinae Cypassis Vil458 E E1 Lithocyclioidea Artiscinae Cypassis Vil450 E E1 Lithocyclioidea Artiscinae Cypassis Vil186 E E1 Lithocyclioidea Artiscinae Didymocyrtis AB193605 E E1 Lithocyclioidea Artiscinae Didymocyrtis Ses19 E E1 Lithocyclioidea Artiscinae Didymocyrtis Ses64 E E1 Lithocyclioidea Artiscinae Didymocyrtis LC093106 E E1 Lithocyclioidea Artiscinae Didymocyrtis Ses63 E E1 Lithocyclioidea Artiscinae Didymocyrtis AB246695 E E2 Spongodiscoidea Spongobrachiidae gen_indet AB430760 E E2 Spongodiscoidea Spongobrachiidae gen_indet Osh174 E E2 Spongodiscoidea Spongobrachiidae gen_indet Osh48 E E2 Spongodiscoidea Spongobrachiidae gen_indet Osh79 E E2 Spongodiscoidea Spongobrachiidae gen_indet Vil449 E E2 Spongodiscoidea Spongobrachiidae Spongasteriscus Mge17-17 E E2 Spongodiscoidea Spongobrachiidae Spongobracium Mge17-74 E E3 Spongodiscoidea Euchitoniidae Dictyocoryne Vil246 E E3 Spongodiscoidea Euchitoniidae Dictyocoryne Vil293 E E3 Spongodiscoidea Euchitoniidae Dictyocoryne AB101540 E E3 Spongodiscoidea Euchitoniidae Dictyocoryne Mge17-99 E E3 Spongodiscoidea Euchitoniidae Dictyocoryne AB101541 E E3 Spongodiscoidea Euchitoniidae Dictyocoryne AB430757 E E3 Spongodiscoidea Euchitoniidae Dictyocoryne AB430758 E E3 Spongodiscoidea Euchitoniidae Dictyocoryne LC093107 E E3 Spongodiscoidea Euchitoniidae Myelastrum AB179732 E E3 Spongodiscoidea Euchitoniidae Myelastrum AB179733 E E3 Spongodiscoidea Euchitoniidae Myelastrum AB179734 E E3 Spongodiscoidea Euchitoniidae Myelastrum AB101542 E E3 Spongodiscoidea Euchitoniidae Spongaster AB246696 E E3 Spongodiscoidea Euchitoniidae Spongodiscus AB284518 F F1 Liosphaeroidea Astrosphaeridae Heliosphaera HQ651782 F F1 Liosphaeroidea Astrosphaeridae Heliosphaera HQ651792 F F1 Liosphaeroidea Astrosphaeridae Heliosphaera Osh34 F F1 Liosphaeroidea Astrosphaeridae Heliosphaera Vil497 F F2 Liosphaeroidea Astrosphaeridae Arachnospongus AB490705 F F2 Liosphaeroidea Astrosphaeridae Astrosphaera AB490706 F F2 Liosphaeroidea Astrosphaeridae Astrosphaera Vil238 F F2 Liosphaeroidea Astrosphaeridae Cladococcus Vil228 F F2 Liosphaeroidea Astrosphaeridae Cladococcus Vil173 F F2 Liosphaeroidea Astrosphaeridae Cladococcus Vil499 F F2 Liosphaeroidea Astrosphaeridae Cladococcus Vil243 F F2 Liosphaeroidea Astrosphaeridae Cladococcus Vil244 F F2 Liosphaeroidea Astrosphaeridae Cladococcus Mge17-44 G G Rhizosphaeroidea Rhizosphaeridae Haliommilla Ses15 G G Rhizosphaeroidea Rhizosphaeridae Haliommilla Vil240 G G Rhizosphaeroidea Rhizosphaeridae Haliommilla AB246684 G G Rhizosphaeroidea Rhizosphaeridae Rhizosphaera AB246686 G G Rhizosphaeroidea Rhizosphaeridae Rhizosphaera JQ706069 G G Rhizosphaeroidea Rhizosphaeridae Rhizosphaera Mge17-81 G G Rhizosphaeroidea Rhizosphaeridae Rhizosphaera Vil476 G G Rhizosphaeroidea Rhizosphaeridae Rhizosphaera Ses55 H H Centrocuboidea Centrocubidae Centrocubus GU821394 H H Centrocuboidea Centrocubidae Uncultured GU822672 H H Centrocuboidea Centrocubidae Uncultured Vil210 I I Centrocuboidea Excentroconchidae Plegmosphaeromma? Vil451 I I Centrocuboidea Excentroconchidae Plegmosphaeromma? KJ757677 I I Centrocuboidea Excentroconchidae Uncultured JX842018 I I Centrocuboidea Excentroconchidae Uncultured KX532703 I I Centrocuboidea Excentroconchidae Uncultured GU821097 I I Centrocuboidea Excentroconchidae Uncultured GU822292 I I Centrocuboidea Excentroconchidae Uncultured GU822590 I I Centrocuboidea Excentroconchidae Uncultured AB860148 J J1 Stylodictytoidea Stylodictyidae Perichlamydium Vil441 J J1 Stylodictytoidea Stylodictyidae Stylodictya Vil503 J J1 Stylodictytoidea Stylodictyidae Stylodictya Vil453 J J2 Stylodictytoidea Stylodictyidae Stylodictya Vil501 J J2 Stylodictytoidea Stylodictyidae Stylodictya AB860147 J J2 Stylodictytoidea Stylodictyidae Stylodictya Osh90 J J2 Stylodictytoidea Stylodictyidae Stylodictya AB246698 J J2 Stylodictytoidea Stylodictyidae Stylodictya HQ651780 K K Actinommoidea Actinommidae Actinomma HQ651781 K K Actinommoidea Actinommidae Actinomma HQ651788 K K Actinommoidea Actinommidae Actinomma HQ651789 K K Actinommoidea Actinommidae Actinomma Vil438 K K Actinommoidea Actinommidae Actinomma AB617584 L L1 unknown Litheliidae Lithelius AB617587 L L1 unknown Sponguriidae Spongurus? Mge17-118 L L1 unknown unknown Trilobatum Osh153 L L2 Spongopyloidea Cristallosphaeridae Calcaromma Vil249 L L2 Spongopyloidea Cristallosphaeridae Calcaromma Vil296 L L2 Spongopyloidea Cristallosphaeridae Calcaromma Vil297 L L2 Spongopyloidea Cristallosphaeridae Calcaromma AB617590 L L2 Spongopyloidea Spongopylidae Schizodiscus AB617588 L L2 Spongopyloidea Spongopylidae Schizodiscus AB860152 L L2 Spongopyloidea Spongopylidae Schizodiscus? AB860153 L L2 Spongopyloidea Spongopylidae Schizodiscus? AB860154 L L2 Spongopyloidea Spongopylidae Schizodiscus? AB860155 L L2 Spongopyloidea Spongopylidae Schizodiscus? AB860156 L L2 Spongopyloidea Spongopylidae Schizodiscus? AB860149 L L2 Spongopyloidea Spongopylidae Spongopyle AB860150 L L2 Spongopyloidea Spongopylidae Spongopyle AB860151 L L2 Spongopyloidea Spongopylidae Spongopyle AB246689 L L2 Spongopyloidea Spongopylidae Un-identifiable AB617589 L L2 Spongopyloidea Spongopylidae Un-identifiable HQ651803 M M1 Pylonioidea Tholoniidae Sphaeropylolena Toy110 M M1 Pylonioidea Tholoniidae Tholomura Vil200 M M1 Pylonioidea Tholoniidae Tholomura Osh16 M M2 Pylonioidea Pyloniidae Larcopyle LC049343 M M2 Pylonioidea Pyloniidae Larcopyle LC049344 M M2 Pylonioidea Pyloniidae Larcopyle AB246688 M M2 Pylonioidea Pyloniidae Sphaerolarnacillium Mge17-20 M M2 Pylonioidea Pyloniidae Tetrapyle Ses18 M M2 Pylonioidea Pyloniidae Tetrapyle Vil231 M M2 Pylonioidea Pyloniidae Tetrapyle Vil298 M M2 Pylonioidea Pyloniidae Tetrapyle Vil432 M M2 Pylonioidea Pyloniidae Tetrapyle Vil480 M M2 Pylonioidea Pyloniidae Tetrapyle Vil506 M M2 Pylonioidea Pyloniidae Tetrapyle AB246680 M M2 Pylonioidea Pyloniidae Tetrapyle AB617585 M M2 Pylonioidea Pyloniidae Un-identifiable Vil437 M M3 Pylonioidea Pyloniidae Cryptolarnaciinae Ses13 M M3 Pylonioidea Pyloniidae Pylodiscus Ses53 M M3 Pylonioidea Pyloniidae Pyloniidae HQ651783 M M3 Pylonioidea Pyloniidae Un-identifiable Species Authority Hexacontella_sp NA Hexastylus_nobilis Cleve Hexastylus_nobilis Cleve Hexacontella?_sp NA Hexacontium_cf_hexagonum (Ehrenberg) Hexacontium_sp_A NA Hexacontium_sp_A NA Hexacontium_sp_A NA Hexacontium_sp_A NA Stigmostylus_ferrusi Hollande and Enjument Hollandosphaera_hexagonia (Hollande and Enjument) Hollandosphaera_hexagonia (Hollande and Enjument) Hollandosphaera_hexagonia (Hollande and Enjument) Hollandosphaera_hexagonia (Hollande and Enjument) Hollandosphaera_hexagonia (Hollande and Enjument) Spongosphaera_streptacantha Haeckel Spongosphaera_streptacantha Haeckel Spongosphaera_streptacantha Haeckel Spongosphaera_streptacantha Haeckel Cypassis_irregularis Nigrini Cypassis_irregularis Nigrini Cypassis_oblongus Takahashi Didymocyrtis_sp Didymocyrtis_tetrathalamus (Haeckel) Didymocyrtis_tetrathalamus (Haeckel) Didymocyrtis_tetrathalamus (Haeckel) Didymocyrtis_tetrathalamus_coronatus (Haeckel) Didymocyrtis_tetrathalamus_coronatus (Haeckel) gen_et_sp_indet gen_et_sp_indet gen_et_sp_indet gen_et_sp_indet gen_et_sp_indet Spongasteriscus_toxon (Tan and Su) Spongobracium_ellipticum (Haeckel) Dictyocoryne_gegenbauri (Haeckel) Dictyocoryne_koellikeri (Haeckel) Dictyocoryne_koellikeri (Haeckel) Dictyocoryne_profunda Ehrenberg Dictyocoryne_sp Dictyocoryne_truncata (Ehrenberg) Dictyocoryne_truncata (Ehrenberg) Dictyocoryne_truncata (Ehrenberg) Myelastrum_aurivillii (Cleve) Myelastrum_elegans (Ehrenberg) Myelastrum_furcatum (Haeckel) Myelastrum_lobum (Swanberg, Anderson and Bennett Spongaster_tetras_tetras (Ehrenberg) Spongodiscus_resurgens Ehrenberg Heliosphaera_aff_actinota (Haeckel) Heliosphaera_aff_actinota (Haeckel) Heliosphaera_aff_actinota (Haeckel) Heliosphaera_bifurcus (Haeckel) Arachnospongus_varians Mast Astrosphaera_hexagonalis Haeckel Astrosphaera_hexagonalis Haeckel Cladococcus_irregularis Popofsky Cladococcus_scoparius Haeckel Cladococcus_sp_A Cladococcus_sp_A Cladococcus_viminalis Haeckel Cladococcus_viminalis Haeckel Haliommilla_capillacea (Haeckel) Haliommilla_capillacea (Haeckel) Haliommilla_capillacea (Haeckel) Rhizosphaera_sp Rhizosphaera_sp Rhizosphaera_trigonacantha Haeckel Rhizosphaera_trigonacantha Haeckel Rhizosphaera_trigonacantha Haeckel Centrocubus_cladostylus Haeckel Uncultured_rhirarian_clone Uncultured_rhirarian_clone Plegmosphaeromma?_sp Plegmosphaeromma?_sp Uncultured_eukaryote Uncultured_marine_eukaryote Uncultured_marine_eukaryote Uncultured_Rhizaria Uncultured_Rhizaria Uncultured_Rhizaria Perichlamydium_venustum Stylodictya_arachnea Müller Stylodictya_sp Stylodictya_arachnea Müller Stylodictya_arachnea Müller Stylodictya_stellata Bailey Stylodictya_stellata Bailey Stylodictya_subtilis (Ehrenberg) Actinomma_boreale Cleve Actinomma_boreale Cleve Actinomma_boreale Cleve Actinomma_boreale Cleve Actinomma_trinacrium (Haeckel) Lithelius_cf_alveolina Haeckel Spongurus?_pylomaticus Trilobatum_aff_acuferum Popofsky Calcaromma_morum Müller Calcaromma_morum Müller Calcaromma_morum Müller Calcaromma_morum Müller Schizodiscus_asymmetricus Dogiel in Dogiel and Reshetny Schizodiscus_stylotrochoides Dogiel in Dogiel and Reshetny Schizodiscus?_spp Schizodiscus?_spp Schizodiscus?_spp Schizodiscus?_spp Schizodiscus?_spp Spongopyle_osculosa Dreyer Spongopyle_osculosa Dreyer Spongopyle_osculosa Dreyer Un-identifiable_specimen Un-identifiable_specimen Sphaeropylolena_circumtexta (Joergensen) Tholomura_pilula Zhang and Suzuki Tholomura_transversara (Chen) Larcopyle_aff_buetschlii Dreyer Larcopyle_buetschlii Dreyer Larcopyle_buetschlii Dreyer Sphaerolarnacillium_tanzhiyuani Zhang and Suzuki Tetrapyle_octacantha Müller Tetrapyle_octacantha Müller Tetrapyle_octacantha Müller Tetrapyle_octacantha Müller Tetrapyle_octacantha Müller Tetrapyle_octacantha Müller Tetrapyle_octacantha Müller Tetrapyle_sp Un-identifiable_specimen Cryptolarnaciinae_gen_et_sp_indet Pylodiscus_spinulosus (Chen and Tan) Pyloniidae_gen_et_sp_indet Un-identifiable_specimen Location Depth Reference Accession_number_18S Villefranche-sur-mer NA This study MT670526 Sogndalsfjorden (Norway) NA Krabberod et al. (2011) HQ651795 Sogndalsfjorden (Norway) NA Krabberod et al. (2011) HQ651798 Westearn mediterranean sea 500-0 This study MT670484 Sogndalsfjord, Norway 250-25 Yuasa et al. (2009) AB284519 Sogndalsfjorden (Norway) NA Krabberod et al. (2011) HQ651784 Sogndalsfjorden (Norway) NA Krabberod et al. (2011) HQ651794 Sogndalsfjorden (Norway) NA Krabberod et al. (2011) HQ651796 Sogndalsfjorden (Norway) NA Krabberod et al. (2011) HQ651797 Villefranche-sur-mer 30 This study MT670544 Villefranche-sur-mer Euphotic This study MT670512 Villefranche-sur-mer Euphotic This study MT670515 Villefranche-sur-mer Euphotic This study MT670517 Villefranche-sur-mer 450-0 This study MT670535 Villefranche-sur-mer 450-0 This study MT670540 Westearn mediterranean sea 500-0 This study MT670490 Okinawa, Japan NA This study MT670492 East_off_Japan 25-0 This study MT670496 East_off_Japan NA This study MT670499 Sagami Bay, Japan Surface Kimoto et al. (2011) AB439010 Villefranche-sur-mer 450-0 This study MT670541 Villefranche-sur-mer 450-0 This study MT670537 Villefranche-sur-mer NA This study MT670513 Okinawa, Japan NA Yuasa et al. (2004) AB193605 Sesoko Okinawa Japan NA This study MT670505 Sesoko Okinawa Japan NA This study MT670509 Okinawa, Japan Surface Yuasa et al. (2016) LC093106 Sesoko Okinawa Japan NA This study MT670508 Shimoda, Izu peninsula, Japan Surface Kunitomo et al. (2006) AB246695 Shimoda, Izu peninsula, Japan NA Ando et al. (2009) AB430760 East_off_Japan 25-0 This study MT670495 East_off_Japan NA This study MT670498 East_off_Japan 25-0 This study MT670500 Villefranche-sur-mer 450-0 This study MT670536 Westearn mediterranean sea 500-0 This study MT670485 Westearn mediterranean sea 500-0 This study MT670488 Villefranche-sur-mer Euphotic This study MT670524 Villefranche-sur-mer NA This study MT670527 Okinawa, Japan Surface Takahashi et al. (2004) AB101540 Westearn mediterranean sea 500-0 This study MT670491 Okinawa, Japan Surface Takahashi et al. (2004) AB101541 Shimoda, Izu peninsula, Japan NA Ando et al. (2009) AB430757 Shimoda, Izu peninsula, Japan NA Ando et al. (2009) AB430758 Okinawa, Japan Surface Yuasa et al. (2016) LC093107 Okinawa, Japan Surface Yuasa et al. (2005) AB179732 Okinawa, Japan Surface Yuasa et al. (2005) AB179733 Okinawa, Japan Surface Yuasa et al. (2005) AB179734 Okinawa, Japan Surface Takahashi et al. (2004) AB101542 Shimoda, Izu peninsula, Japan Surface Kunitomo et al. (2006) AB246696 Okinawa, Japan NA Yuasa et al. (2009) AB284518 Sogndalsfjorden (Norway) NA Krabberod et al. (2011) HQ651782 Sogndalsfjorden (Norway) NA Krabberod et al. (2011) HQ651792 East_off_Japan NA This study MT670497 Villefranche-sur-mer 30 This study MT670545 Okinawa, Japan NA Yuasa et al. (2009) AB490705 Okinawa, Japan NA Yuasa et al. (2009) AB490706 Villefranche-sur-mer NA This study MT670520 Villefranche-sur-mer NA This study MT670518 Villefranche-sur-mer NA This study MT670511 Villefranche-sur-mer 30 This study MT670546 Villefranche-sur-mer NA This study MT670522 Villefranche-sur-mer NA This study MT670523 Westearn mediterranean sea 500-0 This study MT670487 Sesoko Okinawa Japan NA This study MT670503 Villefranche-sur-mer NA This study MT670521 Shimoda, Izu peninsula, Japan Surface Kunitomo et al. (2006) AB246684 Shimoda, Izu peninsula, Japan Surface Kunitomo et al. (2006) AB246686 Akajima Island, Japan NA Decelle et al. Unpublished JQ706069 Westearn mediterranean sea 500-0 This study MT670489 Villefranche-sur-mer Surface This study MT670542 Sesoko Okinawa Japan NA This study MT670507 oxygenated_water_column_CariacNA Edgcomb et al. (2011) GU821394 oxygenated_water_column_CariacNA Edgcomb et al. (2011) GU822672 Villefranche-sur-mer NA This study MT670516 Villefranche-sur-mer 450-0 This study MT670538 East_Pacific_Rise 2500 Lie et al. (2014) KJ757677 seawater_sample 500 Kim et al. (2012) JX842018 ocean_water NA Xu et al. press KX532703 anoxic_water_column_Cariaco_BaNA Edgcomb et al. (2011) GU821097 anoxic_water_column_Cariaco_BaNA Edgcomb et al. (2011) GU822292 anoxic_water_column_Cariaco_BaNA Edgcomb et al. (2011) GU822590 Central Pacific NA Ishitani et al. (2014) AB860148 Villefranche-sur-mer 450-0 This study MT670534 Villefranche-sur-mer 450-0 This study MT670548 Villefranche-sur-mer 450-0 This study MT670539 Villefranche-sur-mer 30 This study MT670547 Central Pacific NA Ishitani et al. (2014) AB860147 East_off_Japan 250-150 This study MT670501 Shimoda, Izu peninsula, Japan Surface Kunitomo et al. (2006) AB246698 Sogndalsfjorden (Norway) NA Krabberod et al. (2011) HQ651780 Sogndalsfjorden (Norway) NA Krabberod et al. (2011) HQ651781 Sogndalsfjorden (Norway) NA Krabberod et al. (2011) HQ651788 Sogndalsfjorden (Norway) NA Krabberod et al. (2011) HQ651789 Villefranche-sur-mer 450-0 This study MT670533 North Pacific Ocean 1000-750 Ishitani et al. (2012) AB617584 North Pacific Ocean 500-250 Ishitani et al. (2012) AB617587 Westearn mediterranean sea 500-0 This study MT670483 East_off_Japan 25-0 This study MT670493 Villefranche-sur-mer Euphotic This study MT670525 Villefranche-sur-mer Euphotic This study MT670528 Villefranche-sur-mer Euphotic This study MT670529 North Pacific Ocean 150-0 Ishitani et al. (2012) AB617590 North Pacific Ocean 500-350 Ishitani et al. (2012) AB617588 Central Pacific NA Ishitani et al. (2014) AB860152 Central Pacific 180 Ishitani et al. (2014) AB860153 Central Pacific NA Ishitani et al. (2014) AB860154 Central Pacific NA Ishitani et al. (2014) AB860155 Central Pacific NA Ishitani et al. (2014) AB860156 Central Pacific NA Ishitani et al. (2014) AB860149 Central Pacific NA Ishitani et al. (2014) AB860150 Central Pacific NA Ishitani et al. (2014) AB860151 Shimoda, Izu peninsula, Japan Surface Kunitomo et al. (2006) AB246689 North Pacific Ocean 150-0 Ishitani et al. (2012) AB617589 Sogndalsfjorden (Norway) NA Krabberod et al. (2011) HQ651803 Okinawa Japan NA This study MT670510 Villefranche-sur-mer NA This study MT670514 East_off_Japan NA This study MT670494 Japan_sea NA Ishitani et al. (2015) LC049343 Japan_sea NA Ishitani et al. (2015) LC049344 Shimoda, Izu peninsula, Japan Surface Kunitomo et al. (2006) AB246688 Westearn mediterranean sea 500-0 This study MT670486 Sesoko Okinawa Japan NA This study MT670504 Villefranche-sur-mer Euphotic This study MT670519 Villefranche-sur-mer NA This study MT670530 Villefranche-sur-mer 450-0 This study MT670531 Villefranche-sur-mer Surface This study MT670543 Villefranche-sur-mer 450-0 This study MT670549 Shimoda, Izu peninsula, Japan Surface Kunitomo et al. (2006) AB246680 North Pacific Ocean 150-0 Ishitani et al. (2012) AB617585 Villefranche-sur-mer 450-0 This study MT670532 Sesoko Okinawa Japan NA This study MT670502 Sesoko Okinawa Japan NA This study MT670506 Sogndalsfjorden (Norway) NA Krabberod et al. (2011) HQ651783 Accession_number_28S NA NA NA NA NA HQ651784 NA NA NA MT670477 MT670460 NA MT670463 NA NA MT670451 NA NA NA NA NA NA MT670461 NA NA NA NA NA NA AB430760 MT670452 NA MT670453 NA MT670447 MT670449 NA MT670469 NA NA NA AB430757 AB430758 NA NA NA NA NA NA NA HQ651782 NA NA MT670478 NA NA NA MT670464 NA MT670479 MT670466 MT670467 MT670448 MT670456 MT670465 NA NA NA MT670450 MT670475 MT670459 NA NA NA NA NA NA NA NA NA NA AB860148 NA MT670481 MT670474 MT670480 AB860147 MT670454 NA HQ651780 HQ651781 NA NA NA NA NA MT670446 NA MT670468 MT670470 MT670471 NA NA AB860152 AB860153 AB860154 AB860155 AB860156 AB860149 AB860150 AB860151 NA NA NA NA MT670462 NA NA NA 18S NA MT670457 NA MT670472 MT670473 MT670476 MT670482 NA NA NA MT670455 MT670458 HQ651783 ID Clade Superfamily_skeleton Family_skeleton Genus_skeleton Osh82 Env1 Rhizosphaeroidea Rhizosphaeridae Rhizosphaera Osh87 Env1 Spongodiscoidea Spongobrachiidae gen_indet Osh97 Env1 Rhizosphaeroidea Rhizosphaeridae Rhizosphaera Osh108 Env1 Heliosaturnaloidea Saturnulidae Saturnulus Osh114 Env1 Rhizosphaeroidea Rhizosphaeridae Rhizosphaera Osh116 Env1 Lithelioidea Litheliidae Lithelius Osh156 Env1 Liosphaeroidea Astrosphaeridae Cladococcus Osh174 Env1 Spongodiscoidea Spongobrachiidae gen_indet Osh187 Env1 Excentroconchidae ExcentroconchidaePlegmosphaeromm Species_skeleton Location Depth Reference Accession_number_18S Accession_ Rhizosphaera haeckeli East_off_Japan 50-25 This study MT670437 NA gen_et_sp_indet East_off_Japan 750-500 This study MT670438 NA Rhizosphaera sp. East_off_Japan 50-25 This study MT670439 NA Saturnulus circularis East_off_Japan 2000-1000 This study MT670440 NA Rhizosphaera haeckeli East_off_Japan 50-25 This study MT670441 NA Lithelius spiralis East_off_Japan 700-500 This study MT670442 NA Cladococcus scoparius East_off_Japan 25-0 This study MT670443 NA gen_et_sp_indet East_off_Japan 25-0 This study MT670444 NA Plegmosphaeromma?_sp East_off_Japan 25-0 This study MT670445 NA _number_28S Ando,H., Kunitomo,Y., Sarashina,I., Iijima,M., Endo,K. and, Sashida,K. (2009). Intraspecific variations in the IT Decelle,J., Bittner,L. and Not,F., Unpublished. Symbiosis in Acantharia (Radiolaria) Edgcomb,V., Orsi,W., Bunge,J., Jeon,S., Christen,R., Leslin,C., (2011). Protistan microbial observatory in the C Ishitani,Y. and Takishita,K., (2015). Molecular evidence for wide vertical distribution of the marine planktoni Ishitani,Y., Ujiie,Y. and Takishita,K., (2014). Uncovering sibling species in Radiolaria: evidence for ecological p Ishitani,Y., Ujiie,Y., De Vargas,C., Not,F. and Takahashi,K., (2012). Two distinct lineages in the radiolarian ord Kim,D.Y., Countway,P.D., Yamashita,W. and Caron,D.A., (2012). A combined sequence-based and fragment- Kimoto,K., Yuasa,T. and Takahashi,O., (2011). Molecular identification of reproductive cells released from Cy Krabberod,A.K., Brate,J., Dolven,J.K., Ose,R.F., Klaveness,D., (2011). Radiolaria Divided into Polycystina and S Kunitomo,Y., Sarashina,I., Iijima,M., Endo,K. and Sashida,K., (2006). Molecular phylogeny of acantharian and Lie,A.A., Liu,Z., Hu,S.K., Jones,A.C., Kim,D.Y., Countway,P.D., (2014). Investigating Microbial Eukaryotic Diver Takahashi,O., Yuasa,T., Honda,D. and Mayama,S., (2004). Molecular phylogeny of solitary shell-bearing Poly Xu,D., Jiao,N., Ren,R. and Warren,A., (2017). Distribution and Diversity of Microbial Eukaryotes in Bathypela Yuasa,T. and Takahashi,O., (2016). Light and electron microscopic observations of the reproductive swarme Yuasa,T., Dolven,J.K., Bjorklund,K.R., Mayama,S. and Takahashi,O., (2009). Molecular phylogenetic position o Yuasa,T., Takahashi,O. and Mayama,S., (2004). PCR primers for the amplification of the nuclear small subuni Yuasa,T., Takahashi,O., Honda,D. and Mayama,S., (2005). Phylogenetic analyses of the polycystine Radiolaria TS region of Recent radiolarians JOURNAL Earth Evol. Sci. Univ. Tsukuba 3, 37-44 (2009).Earth Evol. Sci. Univ.

Cariaco Basin, Caribbean. I. Pyrosequencing vs Sanger insights into species richness.ISME J 5 (8), 1344-1356

der Spumellaria having different ecological preferences.Deep Sea Res. Part II Top. Stud. Oceanogr. 61-64, 172 based characterization of microbial eukaryote assemblages provides taxonomic context for the.J. Microbiol. d polycystine radiolarians based on ribosomal DNA sequences, and some comparisons with data.Eur. J. Protis rsity from a Global Census: Insights from a Comparison of Pyrotag and Full-Length Sequences of.Appl. Environ

it ribosomal DNA sequences from polycystine radiolarians.Gensei Dobutsugaku Zasshi 37, 133-137 a based on the 18s rDNA sequences of the Spumellarida and the Nassellarida.Eur. J. Protistol. 41, 287-298 n. Microbiol. 80 (14), 4363-4373 Table S2. List of morphological characters (traits) and their states (1 to 5) used for the ancestral state reconstruction analysis.

Traits Number of Number of Internal spines arisen Skeleton shape Symmetry Central structure distinctive cavity from central cortical shell structure

0 Spherical Lineb Empty Wide space 0 1

Entactinaria-type, Space 1 Spherical modifieda Spherical or plane fused fibers or soft 6 2 dividers spongye State Double medullary Reflection and/or More or less 2 Cubic-, box-, dice-shaped shell or highly dense Denseh 3 planec than 6 centref Dense with Significantly 3 Flat Circular or rotationald Complexg More than 3 pylomei more than 6 a Cylindrical or spherical to ellipsoidal. b Line symmetry for one, three or n axes. c Reflection symmetry for one axes and plane symmetry or reflection symmetry for three axes. d Circular, tria-rotational, tetra-rotational or n-fold rotational and plane symmetry. e Tetrapetaloid, cube or Rhizosphaerid-type microsphere, fibers fused at the centre or very delicate framed central structure. f Spherical or flatten double medullary shell, spherical microsphere with decussate 4 radial beams, margarita-type microsphere or spinose and homogeneous dense structure. g Pit-like microsphere with tunnel-like pylome, spinose with straight radial beams, tetrapyd system or phorticid system microsphere. h Dense to coarse homogeneous skeletons or dense concentric structure with or without a non-walled pylome. i Dense concentric structure with a tunnel-like pylome. Accession_number Organism Environment AJ829791 uncultured_marine_eukaryote Marine EU333030 uncultured_eukaryote South_China_Sea EU333051 uncultured_eukaryote South_China_Sea EU562088 uncultured_marine_eukaryote ocean_water EU562119 uncultured_marine_eukaryote ocean_water FJ000073 uncultured_eukaryote Bannock_Basin_brine GU820862 uncultured_Rhizaria oxygenated_water_column_Cariaco_Basin GU820872 uncultured_Rhizaria micro-oxic_water_column_Cariaco_Basin GU820920 uncultured_Rhizaria oxygenated_water_column_Cariaco_Basin GU820930 uncultured_Rhizaria oxygenated_water_column_Cariaco_Basin GU820941 uncultured_Rhizaria oxygenated_water_column_Cariaco_Basin GU820962 uncultured_Rhizaria anoxic_water_column_Cariaco_Basin GU821028 uncultured_Rhizaria oxygenated_water_column_Cariaco_Basin GU821081 uncultured_Rhizaria anoxic_water_column_Cariaco_Basin GU821092 uncultured_Rhizaria oxygenated_water_column_Cariaco_Basin GU821096 uncultured_Rhizaria oxygenated_water_column_Cariaco_Basin GU821129 uncultured_Rhizaria micro-oxic_water_column_Cariaco_Basin GU821131 uncultured_Rhizaria oxygenated_water_column_Cariaco_Basin GU821178 uncultured_Rhizaria micro-oxic_water_column_Cariaco_Basin GU821184 uncultured_Rhizaria oxygenated_water_column_Cariaco_Basin GU821188 uncultured_Rhizaria micro-oxic_water_column_Cariaco_Basin GU821211 uncultured_Rhizaria anoxic_water_column_Cariaco_Basin GU821243 uncultured_Rhizaria oxygenated_water_column_Cariaco_Basin GU821288 uncultured_Rhizaria micro-oxic_water_column_Cariaco_Basin GU821311 uncultured_Rhizaria micro-oxic_water_column_Cariaco_Basin GU821369 uncultured_Rhizaria oxygenated_water_column_Cariaco_Basin GU821445 uncultured_Rhizaria micro-oxic_water_column_Cariaco_Basin GU821466 uncultured_Rhizaria oxygenated_water_column_Cariaco_Basin GU821495 uncultured_Rhizaria oxygenated_water_column_Cariaco_Basin GU821507 uncultured_Rhizaria oxygenated_water_column_Cariaco_Basin GU821529 uncultured_Rhizaria anoxic_water_column_Cariaco_Basin GU821541 uncultured_Rhizaria oxygenated_water_column_Cariaco_Basin GU821557 uncultured_Rhizaria anoxic_water_column_Cariaco_Basin GU821574 uncultured_Rhizaria anoxic_water_column_Cariaco_Basin GU821583 uncultured_Rhizaria micro-oxic_water_column_Cariaco_Basin GU821597 uncultured_Rhizaria oxygenated_water_column_Cariaco_Basin GU821660 uncultured_Rhizaria micro-oxic_water_column_Cariaco_Basin GU821686 uncultured_Rhizaria oxygenated_water_column_Cariaco_Basin GU821706 uncultured_Rhizaria micro-oxic_water_column_Cariaco_Basin GU821741 uncultured_Rhizaria oxygenated_water_column_Cariaco_Basin GU821851 uncultured_Rhizaria anoxic_water_column_Cariaco_Basin GU821873 uncultured_Rhizaria oxygenated_water_column_Cariaco_Basin GU821884 uncultured_Rhizaria oxygenated_water_column_Cariaco_Basin GU821971 uncultured_Rhizaria oxygenated_water_column_Cariaco_Basin GU821993 uncultured_Rhizaria oxygenated_water_column_Cariaco_Basin GU822026 uncultured_Rhizaria oxygenated_water_column_Cariaco_Basin GU822040 uncultured_Rhizaria micro-oxic_water_column_Cariaco_Basin GU822082 uncultured_Rhizaria oxygenated_water_column_Cariaco_Basin GU822103 uncultured_Rhizaria oxygenated_water_column_Cariaco_Basin GU822114 uncultured_Rhizaria oxygenated_water_column_Cariaco_Basin GU822124 uncultured_Rhizaria anoxic_water_column_Cariaco_Basin GU822157 uncultured_Rhizaria micro-oxic_water_column_Cariaco_Basin GU822215 uncultured_Rhizaria micro-oxic_water_column_Cariaco_Basin GU822242 uncultured_Rhizaria oxygenated_water_column_Cariaco_Basin GU822263 uncultured_Rhizaria anoxic_water_column_Cariaco_Basin GU822280 uncultured_Rhizaria oxygenated_water_column_Cariaco_Basin GU822333 uncultured_Rhizaria oxygenated_water_column_Cariaco_Basin GU822349 uncultured_Rhizaria anoxic_water_column_Cariaco_Basin GU822398 uncultured_Rhizaria oxygenated_water_column_Cariaco_Basin GU822404 uncultured_Rhizaria oxygenated_water_column_Cariaco_Basin GU822487 uncultured_Rhizaria oxygenated_water_column_Cariaco_Basin GU822564 uncultured_Rhizaria anoxic_water_column_Cariaco_Basin GU822576 uncultured_Rhizaria oxygenated_water_column_Cariaco_Basin GU822592 uncultured_Rhizaria oxygenated_water_column_Cariaco_Basin GU822619 uncultured_Rhizaria oxygenated_water_column_Cariaco_Basin GU822677 uncultured_Rhizaria oxygenated_water_column_Cariaco_Basin GU822687 uncultured_Rhizaria oxygenated_water_column_Cariaco_Basin GU822713 uncultured_Rhizaria micro-oxic_water_column_Cariaco_Basin GU822724 uncultured_Rhizaria oxygenated_water_column_Cariaco_Basin GU822734 uncultured_Rhizaria oxygenated_water_column_Cariaco_Basin GU822780 uncultured_Rhizaria oxygenated_water_column_Cariaco_Basin GU822792 uncultured_Rhizaria oxygenated_water_column_Cariaco_Basin GU822828 uncultured_Rhizaria oxygenated_water_column_Cariaco_Basin GU822879 uncultured_Rhizaria oxygenated_water_column_Cariaco_Basin GU822935 uncultured_Rhizaria micro-oxic_water_column_Cariaco_Basin HQ688692 uncultured_eukaryote oceanic_East_Sea_of_South_Korea JX188292 uncultured_eukaryote South_China JX188300 uncultured_eukaryote South_China JX188364 uncultured_eukaryote South_China JX194695 Uncultured_organism Juan_de_Fuca_Ridge_flank JX842096 uncultured_marine_eukaryote seawater_sample JX842264 uncultured_marine_eukaryote seawater_sample JX842341 uncultured_marine_eukaryote seawater_sample JX842586 uncultured_marine_eukaryote seawater_sample KC488591 uncultured_Rhizaria Atlantic_Zone_Monitoring_Program_AZMP KC583133 uncultured_Rhizaria Red_Sea KF129781 uncultured_eukaryote South_China KF129897 uncultured_eukaryote South_China KF130096 uncultured_eukaryote South_China KF130443 uncultured_eukaryote South_China KF130486 uncultured_eukaryote South_China KJ757051 uncultured_eukaryote East_Pacific_Rise KJ757088 uncultured_eukaryote East_Pacific_Rise KJ757143 uncultured_eukaryote East_Pacific_Rise KJ757252 uncultured_eukaryote East_Pacific_Rise KJ757260 uncultured_eukaryote East_Pacific_Rise KJ757269 uncultured_eukaryote East_Pacific_Rise KJ757293 uncultured_eukaryote East_Pacific_Rise KJ757301 uncultured_eukaryote East_Pacific_Rise KJ757422 uncultured_eukaryote East_Pacific_Rise KJ757451 uncultured_eukaryote East_Pacific_Rise KJ757476 uncultured_eukaryote East_Pacific_Rise KJ757530 uncultured_eukaryote East_Pacific_Rise KJ757574 uncultured_eukaryote East_Pacific_Rise KJ757582 uncultured_eukaryote East_Pacific_Rise KJ757620 uncultured_eukaryote East_Pacific_Rise KJ757629 uncultured_eukaryote East_Pacific_Rise KJ757642 uncultured_eukaryote East_Pacific_Rise KJ757647 uncultured_eukaryote East_Pacific_Rise KJ757675 uncultured_eukaryote East_Pacific_Rise KJ759538 uncultured_eukaryote Gulf_Stream KJ759742 uncultured_eukaryote Gulf_Stream KJ759795 uncultured_eukaryote Gulf_Stream KJ759804 uncultured_eukaryote Gulf_Stream KJ759887 uncultured_eukaryote Gulf_Stream KJ759906 uncultured_eukaryote Gulf_Stream KJ759931 uncultured_eukaryote Gulf_Stream KJ759957 uncultured_eukaryote Gulf_Stream KJ759962 uncultured_eukaryote Gulf_Stream KJ759977 uncultured_eukaryote Gulf_Stream KJ760028 uncultured_eukaryote Gulf_Stream KJ760039 uncultured_eukaryote Gulf_Stream KJ760054 uncultured_eukaryote Gulf_Stream KJ760085 uncultured_eukaryote Gulf_Stream KJ760144 uncultured_eukaryote Gulf_Stream KJ760145 uncultured_eukaryote Gulf_Stream KJ760157 uncultured_eukaryote Gulf_Stream KJ760223 uncultured_eukaryote Gulf_Stream KJ760250 uncultured_eukaryote Gulf_Stream KJ760253 uncultured_eukaryote Gulf_Stream KJ760292 uncultured_eukaryote Gulf_Stream KJ760300 uncultured_eukaryote Gulf_Stream KJ760313 uncultured_eukaryote Gulf_Stream KJ760331 uncultured_eukaryote Gulf_Stream KJ760338 uncultured_eukaryote Gulf_Stream KJ760355 uncultured_eukaryote Gulf_Stream KJ760359 uncultured_eukaryote Gulf_Stream KJ760854 uncultured_eukaryote East_Pacific_Rise KJ760870 uncultured_eukaryote East_Pacific_Rise KJ760873 uncultured_eukaryote East_Pacific_Rise KJ760884 uncultured_eukaryote East_Pacific_Rise KJ760892 uncultured_eukaryote East_Pacific_Rise KJ760894 uncultured_eukaryote East_Pacific_Rise KJ760899 uncultured_eukaryote East_Pacific_Rise KJ760902 uncultured_eukaryote East_Pacific_Rise KJ760904 uncultured_eukaryote East_Pacific_Rise KJ760905 uncultured_eukaryote East_Pacific_Rise KJ760910 uncultured_eukaryote East_Pacific_Rise KJ760944 uncultured_eukaryote East_Pacific_Rise KJ760949 uncultured_eukaryote East_Pacific_Rise KJ760978 uncultured_eukaryote East_Pacific_Rise KJ760994 uncultured_eukaryote East_Pacific_Rise KJ760995 uncultured_eukaryote East_Pacific_Rise KJ761003 uncultured_eukaryote East_Pacific_Rise KJ761004 uncultured_eukaryote East_Pacific_Rise KJ761013 uncultured_eukaryote East_Pacific_Rise KJ761031 uncultured_eukaryote East_Pacific_Rise KJ761045 uncultured_eukaryote East_Pacific_Rise KJ761055 uncultured_eukaryote East_Pacific_Rise KJ761059 uncultured_eukaryote East_Pacific_Rise KJ761084 uncultured_eukaryote East_Pacific_Rise KJ761089 uncultured_eukaryote East_Pacific_Rise KJ761097 uncultured_eukaryote 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uncultured_eukaryote East_Pacific_Rise KJ761260 uncultured_eukaryote East_Pacific_Rise KJ761262 uncultured_eukaryote East_Pacific_Rise KJ761266 uncultured_eukaryote East_Pacific_Rise KJ761268 uncultured_eukaryote East_Pacific_Rise KJ761275 uncultured_eukaryote East_Pacific_Rise KJ761282 uncultured_eukaryote East_Pacific_Rise KJ761284 uncultured_eukaryote East_Pacific_Rise KJ761287 uncultured_eukaryote East_Pacific_Rise KJ761289 uncultured_eukaryote East_Pacific_Rise KJ761290 uncultured_eukaryote East_Pacific_Rise KJ761299 uncultured_eukaryote East_Pacific_Rise KJ761301 uncultured_eukaryote East_Pacific_Rise KJ761305 uncultured_eukaryote East_Pacific_Rise KJ761321 uncultured_eukaryote East_Pacific_Rise KJ761322 uncultured_eukaryote East_Pacific_Rise KJ761324 uncultured_eukaryote East_Pacific_Rise KJ761325 uncultured_eukaryote East_Pacific_Rise KJ761326 uncultured_eukaryote East_Pacific_Rise KJ761329 uncultured_eukaryote East_Pacific_Rise KJ761337 uncultured_eukaryote East_Pacific_Rise KJ761339 uncultured_eukaryote East_Pacific_Rise KJ761340 uncultured_eukaryote East_Pacific_Rise KJ761352 uncultured_eukaryote East_Pacific_Rise KJ761357 uncultured_eukaryote East_Pacific_Rise KJ761368 uncultured_eukaryote East_Pacific_Rise KJ761370 uncultured_eukaryote East_Pacific_Rise KJ761371 uncultured_eukaryote East_Pacific_Rise KJ761375 uncultured_eukaryote East_Pacific_Rise KJ761382 uncultured_eukaryote East_Pacific_Rise KJ761386 uncultured_eukaryote East_Pacific_Rise KJ761389 uncultured_eukaryote East_Pacific_Rise KJ761390 uncultured_eukaryote East_Pacific_Rise KJ761392 uncultured_eukaryote East_Pacific_Rise KJ761394 uncultured_eukaryote East_Pacific_Rise KJ761399 uncultured_eukaryote East_Pacific_Rise KJ761408 uncultured_eukaryote East_Pacific_Rise KJ761417 uncultured_eukaryote East_Pacific_Rise KJ761432 uncultured_eukaryote East_Pacific_Rise KJ761441 uncultured_eukaryote East_Pacific_Rise KJ761442 uncultured_eukaryote East_Pacific_Rise KJ761443 uncultured_eukaryote 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uncultured_eukaryote East_Pacific_Rise KJ761558 uncultured_eukaryote East_Pacific_Rise KJ761560 uncultured_eukaryote East_Pacific_Rise KJ761569 uncultured_eukaryote East_Pacific_Rise KJ761571 uncultured_eukaryote East_Pacific_Rise KJ761579 uncultured_eukaryote East_Pacific_Rise KJ761585 uncultured_eukaryote East_Pacific_Rise KJ761588 uncultured_eukaryote East_Pacific_Rise KJ761592 uncultured_eukaryote East_Pacific_Rise KJ761594 uncultured_eukaryote East_Pacific_Rise KJ761597 uncultured_eukaryote East_Pacific_Rise KJ761598 uncultured_eukaryote East_Pacific_Rise KJ761605 uncultured_eukaryote East_Pacific_Rise KJ761611 uncultured_eukaryote East_Pacific_Rise KJ761612 uncultured_eukaryote East_Pacific_Rise KJ761619 uncultured_eukaryote East_Pacific_Rise KJ761624 uncultured_eukaryote East_Pacific_Rise KJ761629 uncultured_eukaryote East_Pacific_Rise KJ761636 uncultured_eukaryote East_Pacific_Rise KJ761638 uncultured_eukaryote East_Pacific_Rise KJ761639 uncultured_eukaryote East_Pacific_Rise KJ761645 uncultured_eukaryote East_Pacific_Rise KJ761657 uncultured_eukaryote East_Pacific_Rise KJ761663 uncultured_eukaryote East_Pacific_Rise KJ761665 uncultured_eukaryote East_Pacific_Rise KJ761666 uncultured_eukaryote East_Pacific_Rise KJ761679 uncultured_eukaryote East_Pacific_Rise KJ761681 uncultured_eukaryote East_Pacific_Rise KJ761683 uncultured_eukaryote East_Pacific_Rise KJ761685 uncultured_eukaryote East_Pacific_Rise KJ761687 uncultured_eukaryote East_Pacific_Rise KJ761695 uncultured_eukaryote East_Pacific_Rise KJ761698 uncultured_eukaryote East_Pacific_Rise KJ761701 uncultured_eukaryote East_Pacific_Rise KJ761706 uncultured_eukaryote East_Pacific_Rise KJ761708 uncultured_eukaryote East_Pacific_Rise KJ761718 uncultured_eukaryote East_Pacific_Rise KJ761719 uncultured_eukaryote East_Pacific_Rise KJ762482 uncultured_eukaryote SPOTstation KJ762533 uncultured_eukaryote SPOTstation KJ762537 uncultured_eukaryote SPOTstation KJ762562 uncultured_eukaryote SPOTstation KJ762613 uncultured_eukaryote SPOTstation KJ762656 uncultured_eukaryote SPOTstation KJ762669 uncultured_eukaryote SPOTstation KJ762723 uncultured_eukaryote SPOTstation KJ762797 uncultured_eukaryote SPOTstation KJ762817 uncultured_eukaryote SPOTstation KJ763443 uncultured_eukaryote Gulf_Stream KJ763627 uncultured_eukaryote Gulf_Stream KJ763679 uncultured_eukaryote Gulf_Stream KJ763858 uncultured_eukaryote Gulf_Stream KJ763887 uncultured_eukaryote Gulf_Stream KP175029 uncultured_spumellarian central_Pacific_Ocean KP175032 uncultured_spumellarian central_Pacific_Ocean KX532536 uncultured_marine_eukaryote ocean_water KX532575 uncultured_marine_eukaryote ocean_water KX532598 uncultured_marine_eukaryote ocean_water KX532606 uncultured_marine_eukaryote ocean_water KX532622 uncultured_marine_eukaryote ocean_water KX532838 uncultured_marine_eukaryote ocean_water KX533015 uncultured_marine_eukaryote ocean_water KX533018 uncultured_marine_eukaryote ocean_water KX533054 uncultured_marine_eukaryote ocean_water KX533142 uncultured_marine_eukaryote ocean_water KX533182 uncultured_marine_eukaryote ocean_water KX533205 uncultured_marine_eukaryote ocean_water KX533292 uncultured_marine_eukaryote ocean_water KX533298 uncultured_marine_eukaryote ocean_water KX533305 uncultured_marine_eukaryote ocean_water KX533313 uncultured_marine_eukaryote ocean_water KX533370 uncultured_marine_eukaryote ocean_water Depth Others Affiliation_pplacer_Fig5 Pplacer_comment NA NA NA Basal_to_Clades_C_D_E NA NA NA Basal_to_Linneage_I NA NA NA Basal_to_Clade_B 75 NA NA Basal_to_Linneage_I 75 NA NA Basal_to_Linneage_I NA NA NA Basal_to_Clade_G NA NA NA Basal_to_Linneage_I NA NA NA Basal_to_Clade_F NA NA NA Basal_to_Linneage_I NA NA NA Basal_to_Clades_D_E NA NA NA Basal_to_Linneage_I NA NA NA Basal_to_Clade_G NA NA NA Basal_to_Linneage_I NA NA NA Basal_to_all NA NA NA Basal_to_Clades_C_D_E NA NA NA Basal_to_Clade_F NA NA NA Basal_to_Clades_D_E NA NA NA Basal_to_Clades_D_E NA NA NA Basal_to_Linneage_I NA NA NA Basal_to_Linneage_I NA NA NA Basal_to_Linneage_I NA NA NA Basal_to_Clade_J NA NA NA Basal_to_Clade_F NA NA NA Basal_to_Clades_C_D_E NA NA NA Basal_to_Linneage_I NA NA NA Basal_to_Clade_M NA NA NA Basal_to_Linneage_I NA NA NA Basal_to_Linneage_I NA NA NA Basal_to_Clades_D_E NA NA NA Basal_to_Linneage_I NA NA NA Basal_to_Clade_G NA NA NA Basal_to_Clade_G NA NA NA Basal_to_Clade_J NA NA NA Basal_to_Clade_J NA NA NA Basal_to_Clades_C_D_E NA NA NA Basal_to_Linneage_I NA NA NA Basal_to_Clades_D_E NA NA NA Basal_to_Linneage_I NA NA NA Basal_to_Lineages_II_III_IV NA NA NA Basal_to_Clade_B NA NA NA Basal_to_Clade_J NA NA NA Basal_to_Linneage_I NA NA NA Basal_to_Clade_B NA NA NA Basal_to_Clades_D_E NA NA NA Basal_to_Linneage_I NA NA NA Basal_to_Clades_D_E NA NA NA Basal_to_Clade_F NA NA NA Basal_to_Clades_C_D_E NA NA NA Basal_to_Clades_C_D_E NA NA NA Basal_to_Clades_D_E NA NA NA Basal_to_Clades_D_E NA NA NA Basal_to_Clade_F NA NA NA Basal_to_Linneage_I NA NA NA Basal_to_Clades_C_D_E NA NA NA Basal_to_Lineages_II_III_IV NA NA NA Basal_to_Clades_D_E NA NA NA Basal_to_Clades_D_E NA NA NA Basal_to_Clade_J NA NA NA Basal_to_Clades_C_D_E NA NA NA Basal_to_Clades_D_E NA NA NA Basal_to_Linneage_I NA NA NA Basal_to_Linneage_I NA NA NA Basal_to_Clade_F NA NA NA Basal_to_Linneage_I NA NA NA Basal_to_Clade_B NA NA NA Basal_to_Linneage_I NA NA NA Basal_to_Linneage_I NA NA NA Basal_to_Linneage_I NA NA NA Basal_to_Linneage_I NA NA NA Basal_to_Linneage_I NA NA NA Basal_to_Linneage_I NA NA NA Basal_to_Linneage_I NA NA NA Basal_to_Clades_C_D_E NA NA NA Basal_to_Clade_B NA NA NA Basal_to_Linneage_I 75 NA NA Basal_to_Clade_C 75 NA NA Basal_to_Clade_F 75 NA NA Basal_to_Linneage_I 75 NA NA Basal_to_Clades_C_D_E 2648 NA NA Basal_to_Linneage_I 500 NA NA Basal_to_Clade_F 500 NA NA Basal_to_Clades_C_D_E 500 NA NA Basal_to_Clade_M 500 NA NA Basal_to_Clade_M 20 NA NA Basal_to_Clade_F 10 NA NA Basal_to_Clade_F 60 NA NA Basal_to_Linneage_I 60 NA NA Basal_to_Clade_F 60 NA NA Basal_to_Linneage_I 60 NA NA Basal_to_Linneage_I 60 NA NA Basal_to_Linneage_I 2500 NA NA Basal_to_Clade_F 2500 NA NA Basal_to_Clade_F 2500 NA NA Basal_to_Clade_J 2500 NA NA Basal_to_Clade_F 2500 NA NA Basal_to_Clade_M 2500 NA NA Basal_to_Clade_F 2500 NA NA Basal_to_Clade_F 2500 NA NA Basal_to_Clade_F 2500 NA NA Basal_to_Clade_F 2500 NA NA Basal_to_Clade_F 2500 NA NA Basal_to_Clade_F 2500 NA NA Basal_to_Clade_F 2500 NA NA Basal_to_Clade_F 2500 NA NA Basal_to_Clade_F 2500 NA NA Basal_to_Clade_J 2500 NA NA Basal_to_all 2500 NA NA Basal_to_Clade_F 2500 NA NA Basal_to_Clade_F 2500 NA NA Basal_to_Lineage_IV 15 NA NA Basal_to_Linneage_I 2500 NA NA Basal_to_Clade_F 2500 NA NA Basal_to_Clade_F 2500 NA NA Basal_to_Clade_J 2500 NA NA Basal_to_Lineage_IV 2500 NA NA Basal_to_Clade_J 2500 NA NA Basal_to_Clade_F 2500 NA NA Basal_to_Clade_F 2500 NA NA Basal_to_Clade_F 2500 NA NA Basal_to_Clade_J 2500 NA NA Basal_to_Clade_F 2500 NA NA Basal_to_Lineage_IV 2500 NA NA Basal_to_Clade_F 2500 NA NA Basal_to_Clade_F 2500 NA NA Basal_to_Clade_J 2500 NA NA Basal_to_all 2500 NA NA Basal_to_Lineage_IV 2500 NA NA Basal_to_Clade_F 2500 NA NA Basal_to_all 2500 NA NA Basal_to_Clade_J 2500 NA NA Basal_to_Clade_J 2500 NA NA Basal_to_Clade_F 2500 NA NA Basal_to_all 2500 NA NA Basal_to_Clade_F 2500 NA NA Basal_to_Clade_F 2500 NA NA Basal_to_Clade_F 2500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 1500 NA NA Basal_to_Clade_F 500 NA NA Basal_to_all 500 NA NA Basal_to_Clade_F 500 NA NA Basal_to_Clade_F 500 NA NA Basal_to_Clade_F 500 NA NA Basal_to_Clade_M 500 NA NA Basal_to_all 500 NA NA Basal_to_Clade_F 500 NA NA Basal_to_Clade_F 500 NA NA Basal_to_Clade_F 500 NA NA Basal_to_Clade_F 105 NA NA Basal_to_Clade_F 105 NA NA Basal_to_all 105 NA NA Basal_to_Linneage_I 105 NA NA Basal_to_Clade_F 105 NA NA Basal_to_Linneage_I NA NA NA Basal_to_Clade_F NA NA NA Basal_to_Clade_F NA NA NA Basal_to_Linneage_I NA NA NA Basal_to_Linneage_I NA NA NA Basal_to_Clade_F NA NA NA Basal_to_Clade_B NA NA NA Basal_to_Lineage_IV NA NA NA Basal_to_Clade_J NA NA NA Basal_to_Clade_F NA NA NA Basal_to_Clade_F NA NA NA Basal_to_Clade_F NA NA NA Basal_to_Clade_F NA NA NA Basal_to_Clade_F NA NA NA Basal_to_Clade_F NA NA NA Basal_to_Clade_J NA NA NA Basal_to_Clade_F NA NA NA Basal_to_Clade_F NA NA NA Basal_to_Clade_J NA NA NA Basal_to_Clade_F pplacer_MCL_clu Affiliation_Fig6 Reference mcl9 Env_1 Yuan et al. (2004) mcl8 Env_1 Li et al. (2011) mcl9 Env_1 Li et al. (2011) mcl8 Env_1 Not et al. (2008) mcl8 Env_1 Not et al. (2008) mcl20 NA Edgcomb et al. Unpublished mcl8 Env_1 Edgcomb et al. (2011) mcl1 Env_6 Edgcomb et al. (2011) mcl8 Env_1 Edgcomb et al. (2011) mcl9 Env_1 Edgcomb et al. (2011) mcl8 Env_1 Edgcomb et al. (2011) mcl20 Chimeric Edgcomb et al. (2011) mcl8 Env_1 Edgcomb et al. (2011) mcl16 Env_1 Edgcomb et al. (2011) mcl9 Env_1 Edgcomb et al. (2011) mcl8 Chimeric Edgcomb et al. (2011) mcl8 Env_1 Edgcomb et al. (2011) mcl9 Env_1 Edgcomb et al. (2011) mcl8 Env_1 Edgcomb et al. (2011) mcl8 Env_1 Edgcomb et al. (2011) mcl8 Env_1 Edgcomb et al. (2011) mcl11 Env_4 Edgcomb et al. (2011) mcl1 Env_6 Edgcomb et al. (2011) mcl9 Env_1 Edgcomb et al. (2011) mcl8 Env_1 Edgcomb et al. (2011) mcl18 Chimeric Edgcomb et al. (2011) mcl8 Env_1 Edgcomb et al. (2011) mcl8 Env_1 Edgcomb et al. (2011) mcl9 Env_1 Edgcomb et al. (2011) mcl8 Env_1 Edgcomb et al. (2011) mcl20 NA Edgcomb et al. (2011) mcl20 Chimeric Edgcomb et al. (2011) mcl11 Env_4 Edgcomb et al. (2011) mcl11 Env_4 Edgcomb et al. (2011) mcl9 Env_1 Edgcomb et al. (2011) mcl8 Env_2 Edgcomb et al. (2011) mcl9 Env_1 Edgcomb et al. (2011) mcl8 Env_2 Edgcomb et al. (2011) mcl1 Env_6 Edgcomb et al. (2011) mcl8 Env_2 Edgcomb et al. (2011) mcl11 Env_4 Edgcomb et al. (2011) mcl8 Env_1 Edgcomb et al. (2011) mcl8 Env_2 Edgcomb et al. (2011) mcl9 Env_1 Edgcomb et al. (2011) mcl8 Env_1 Edgcomb et al. (2011) mcl9 Env_1 Edgcomb et al. (2011) mcl1 Env_5 Edgcomb et al. (2011) mcl9 Env_1 Edgcomb et al. (2011) mcl9 Env_1 Edgcomb et al. (2011) mcl9 Env_1 Edgcomb et al. (2011) mcl9 Env_1 Edgcomb et al. (2011) mcl1 Env_5 Edgcomb et al. (2011) mcl8 Env_1 Edgcomb et al. (2011) mcl9 Env_1 Edgcomb et al. (2011) mcl1 Suspicious Edgcomb et al. (2011) mcl9 Env_1 Edgcomb et al. (2011) mcl9 Env_1 Edgcomb et al. (2011) mcl11 Env_4 Edgcomb et al. (2011) mcl9 Env_1 Edgcomb et al. (2011) mcl9 Env_1 Edgcomb et al. (2011) mcl8 Env_1 Edgcomb et al. (2011) mcl8 Env_1 Edgcomb et al. (2011) mcl1 Env_5 Edgcomb et al. (2011) mcl8 Env_1 Edgcomb et al. (2011) mcl8 Env_2 Edgcomb et al. (2011) mcl8 Env_1 Edgcomb et al. (2011) mcl8 Env_1 Edgcomb et al. (2011) mcl8 Env_1 Edgcomb et al. (2011) mcl8 Env_2 Edgcomb et al. (2011) mcl8 Env_1 Edgcomb et al. (2011) mcl8 Env_1 Edgcomb et al. (2011) mcl8 Env_1 Edgcomb et al. (2011) mcl9 Env_1 Edgcomb et al. (2011) mcl8 Env_2 Edgcomb et al. (2011) mcl8 Env_1 Edgcomb et al. (2011) mcl9 Env_1 Lee et al. (2012) mcl1 Env_5 Wu et al. (2014a) mcl8 Env_1 Wu et al. (2014a) mcl9 Env_1 Wu et al. (2014a) mcl8 Env_1 Jungbluth et al. (2013) mcl1 Env_5 Kim et al. (2012) mcl9 Env_1 Kim et al. (2012) mcl18 Chimeric Kim et al. (2012) mcl18 Clade_M Kim et al. (2012) mcl1 Env_6 Dasilva et al. Unpublished mcl1 Chimeric Acosta et al. (2013) mcl8 Env_1 Wu et al. (2014b) mcl1 Suspicious Wu et al. (2014b) mcl8 Env_1 Wu et al. (2014b) mcl8 Env_1 Wu et al. (2014b) mcl8 Env_1 Wu et al. (2014b) mcl1 Env_5 Lie et al. (2014) mcl1 Chimeric Lie et al. (2014) mcl11 Env_4 Lie et al. (2014) mcl1 Chimeric Lie et al. (2014) mcl18 Clade_M Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl11 Env_4 Lie et al. (2014) mcl16 Suspicious Lie et al. (2014) mcl1 Chimeric Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl17 Env_3 Lie et al. (2014) mcl8 Env_1 Lie et al. (2014) mcl0 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl11 Env_4 Lie et al. (2014) mcl17 Chimeric Lie et al. (2014) mcl11 Env_4 Lie et al. (2014) mcl1 Chimeric Lie et al. (2014) mcl1 Chimeric Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl11 Env_4 Lie et al. (2014) mcl1 Chimeric Lie et al. (2014) mcl17 Env_3 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Chimeric Lie et al. (2014) mcl11 Env_4 Lie et al. (2014) mcl16 Suspicious Lie et al. (2014) mcl17 Chimeric Lie et al. (2014) mcl1 Chimeric Lie et al. (2014) mcl16 Suspicious Lie et al. (2014) mcl11 Env_4 Lie et al. (2014) mcl11 Env_4 Lie et al. (2014) mcl1 Suspicious Lie et al. (2014) mcl16 Chimeric Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Suspicious Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Chimeric Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Chimeric Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Chimeric Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Chimeric Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Chimeric Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Chimeric Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Chimeric Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Chimeric Lie et al. (2014) mcl1 Chimeric Lie et al. (2014) mcl1 Chimeric Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Chimeric Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Chimeric Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Chimeric Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Chimeric Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Chimeric Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Chimeric Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Chimeric Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Chimeric Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Chimeric Lie et al. (2014) mcl1 Chimeric Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Chimeric Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Chimeric Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Chimeric Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Chimeric Lie et al. (2014) mcl1 Chimeric Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Chimeric Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Chimeric Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl3 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Chimeric Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Chimeric Lie et al. (2014) mcl1 Chimeric Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl16 Suspicious Lie et al. (2014) mcl1 Env_6 Lie et al. (2014) mcl1 Env_6 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl18 Chimeric Lie et al. (2014) mcl16 Suspicious Lie et al. (2014) mcl1 Env_6 Lie et al. (2014) mcl1 Env_5 Lie et al. (2014) mcl1 Env_6 Lie et al. (2014) mcl1 Chimeric Lie et al. (2014) mcl1 Suspicious Lie et al. (2014) mcl16 Chimeric Lie et al. (2014) mcl8 Chimeric Lie et al. (2014) mcl1 Chimeric Lie et al. (2014) mcl8 Env_1 Lie et al. (2014) mcl2 Env_5 Li et al. Unpublished mcl1 Env_5 Li et al. Unpublished mcl8 Env_1 Xu et al. (2016) mcl8 Env_1 Xu et al. (2016) mcl1 Suspicious Xu et al. (2016) mcl9 NA Xu et al. (2016) mcl17 Env_3 Xu et al. (2016) mcl11 Env_4 Xu et al. (2016) mcl1 Env_5 Xu et al. (2016) mcl1 Env_5 Xu et al. (2016) mcl1 Env_5 Xu et al. (2016) mcl1 Env_5 Xu et al. (2016) mcl1 Env_5 Xu et al. (2016) mcl1 Env_5 Xu et al. (2016) mcl11 Env_4 Xu et al. (2016) mcl1 Env_5 Xu et al. (2016) mcl1 Env_5 Xu et al. (2016) mcl11 Env_4 Xu et al. (2016) mcl1 Env_5 Xu et al. (2016) Acosta,F., Ngugi,D.K. and Stingl,U., (2013). 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