1 Supplementary Material 2 Carbon and nitrogen to volume relationships for marine protist of the Rhizaria 3 lineage (Radiolaria and Phaeodaria) 4 Joost Samir Mansour, Andreas Norlin, Natalia Llopis Monferrer, Stéphane L’Helguen, and Fabrice Not 5 6 Supplementary Methodology 1 7 Collodaria transcriptome 18S extraction 8 RNA extraction, cDNA synthesis and sequencing were performed by Genoscope, Paris. RNA reads 9 obtained from Illumina HiSeq2500 sequencing of three Collodaria specimens, were trimmed using 10 SortMeRna to retrieve ribosomal sequences, at GENOSCOPE, Paris. The reads were further cleaned using 11 PrinSeQ (SLIDINGWINDOW:4:5; MINQUALITY:5; TRIM-POLYA/T:40; MINLEN:25), and assembled using 12 Trinity. Contigs were matched using NHMMER against a custom HMMR profile for Collodaria. The best 13 NHMMER contig hits were extracted and verified using NCBI blast to be of Collodaria origin. Contig 14 sequences were deposited under accession numbers MT985517-MT985527. 15 Acantharia / Collodaria DNA extraction, amplification and sequencing 16 Acantharia and Collodaria specimen for DNA extraction were collected in 96% EtOH. DNA extraction 17 purification were done using a Masterpure DNA purification kit (Epicentre-Illumina) following the 18 manufacturers protocol. Single-cell Polymerase Chain Reaction (PCR) was done to amplify the 18S 19 ribosomal DNA gene with specific primers for Acantharia, SA ad S879 as described in 20 Decelle et al., 2012, and for Collodaria S81Col and V9R as i Biad et al., . The 18S gene for 21 Acantharia was amplified in a total volume of 25 µL with: 0,35 µM of each primer, MgCL2 1.5 mM, 22 dNTPs mix 0.4 µM, buffer GoTaq 1x and 0.625u of GoTaq Flexi polymerase G2 (Promega). The reaction 23 proceeded with the following PCR parameters: 5 min at 95 °C, followed by 35 cycles of 30 s denaturation 24 at 95 °C, 30 s annealing at 53-55 °C, 2 min extension at 72 °C, with a final elongation step of 10 minutes 25 at 72 °C. For the Collodaria sample the reaction mix was as follows in a total volume of 25 µL: 0.35 µM of 1 26 each primer, 0.35 µM of each primer, DMSO 3%, and Mastermix Phusion GC (ref F532L, Finnzymes). PCR 27 parameters for Collodaria were: 30 sec at 98 °C, followed by 35 cycles of 10 s denaturation at 98 °C, 30 s 28 annealing at 55 °C and 30 s extension at 72 °C, with a final elongation step of 10 min at 72 °C. All positive 29 amplification products have been purified using the kit Nucleospin PCR Clean-Up (Macherey-Nagel) and 30 eluted with 22 µL of NE buffer. Sequencing was done using Big Dye Terminator Cycle Sequencing Kit (Life 31 Technologies) by Macrogen. Sequence were cleaned using ChromasPro software, contig construction 32 was only possible for one Acantharia sample (i.e. Ac-16). Sequences were deposited under Bioproject 33 PRJNA658429. 34 Phylogenetic analyses 35 Sequences from published and single cell identified Acantharia (Decelle et al. 2012b; a) and Collodaria 36 (Biard et al. 2015) were retrieved from NCBI Genbank for reference tree construction. Reference trees 37 were built with both 18S and 28S rRNA sequences for a concatenated alignment tree representing the 38 phylogenetic diversity as depicted in Decelle et al. 2012b (Acantharia) or Biard et al., 2015 (Collodaria). 39 For Acantharia phylogenetic identification 12 Spumellaria sequence were used as outgroup and for 40 Collodaria 6 Nassellaria sequences. 41 Sequences were aligned using MAFFT (Katoh et al. 2005) in AliView (Larsson 2014). Positions with ≥20% 42 gaps in Acantharia ad ≥% gaps i Collodaia seueces were removed using TrimAl v1.2 (Capella- 43 Gutiérrez et al. 2009). The Acantharia concatenated sequence alignment consisted of 1629 nucleotides 44 of the 18S region, and 561 nucleotides of the 28S region. For Collodaria the alignment was 1614 and 528 45 nucleotides of respectively the 18S and 28S regions. The concatenated alignments were analysed using 46 the Maxiu Likelihood ML ethods ude the GTR+Γ odel ad 4 rate categories) with RaxML 47 v8.2.4 (Stamatakis 2014). Node support was computed with 1000 bootstraps. Final tree was visualized 48 and edited with FigTree version 1.4.4. 2 49 Supplementary Methodology 2 50 Equations from Hillebrand et al. (1999) were used for volume calculation of Rhizarian samples. Rhizaria 51 were assigned the standardized shapes as follows: truncated cone for Nassellaria; prolate spheroid for 52 Protocystis, Challengeria and Spumellaria, and sphere for Aulacantha. Collodaria were assigned either 53 sphere, spheroid or cylinder with two half spheres depending on the colony shape. The equations used 54 are given below. 55 Truncated cone: 56 Supplemental Eq 1 휋 × × × 57 Sphere: ℎ + 푅 + 푅 = 푉 58 Supplemental Eq 2 × × 59 Supplemental Eq 3 휋 = 푉 60 Prolate Spheroid: 4 × 휋 × = 퐴 61 Supplemental Eq 4 × × × 62 Supplemental Eq 5 휋 푅 = 푉 2 2 2 휋×푟 푅 √푅 −푟 2 2 × 2 + √푅 −푟 푎푖푛 푅 = 퐴 63 Cylinder + two half spheres: 64 Supplemental Eq 6 65 Supplemental Eq 7 × 휋 × + (휋 × × 퐿 − 2) = 푉 66 r = short radius (or top radius of a4 cone) × 휋 × + (2휋 × 퐿 − 2) = 퐴 67 h = cone height 68 R = long radius (or base radius of a cone) 69 L = length of colony (used for cylinder + two half spheres, L = R) 70 V = Volume 71 A = Surface area 3 72 73 74 75 Supplemental Figure 1. Selection of light microscopy photographs of Collodaria to illustrate the 76 methodology for colonial volume measurements from images. The measurements done for use with the 77 equations of supplemental text 2 are illustrated with a red arrowed line. These lines are examples for 78 illustrative purpose and do not consist of actual measurements, actual measurements were done of 79 multiple lines to acquire an average and thus more accurate estimate. For Collodaria colonies these 80 measurements thus omit the extracellular matrix, and will allow calculation of the colonial volume as 81 within the green outline (top pictures). The extracellular matrix of the Collodaria is indicated between 82 red and green outlines (top photos). Note that the extracellular matrix can be variable with physiological 83 state, and is also not captured using in-situ photography like UVP5 (Biard and Ohman 2020). 4 100 100 98 Nassellaria 75 Pac12 naked_Collosphaeridae 72 Pac17 naked Collosphaeridae Type D 18S1 A2 KR058198 Disolenia quadrata Sat12 86 Sat21 Disolenia tenuissima 100 100 KR058199 Disolenia tenuissima Sat17 99 Sat26 Disolenia zanguebarica A3 54 KR058203 Disolenia zanguebarica Sat27 100 KR058200 Disolenia zanguebarica Sat20 90 KR058205 Thalassicolla melacapsa Pac3 100 KR058206 Siphonosphaera abyssi Pac7 A5 100 KR058204 Collosphaeridae sp. 79 100 KR058208 Collosphaera tuberosa Ind48 100 KR058210 Collosphaera_tuberosa Vil346 Collosphaeridae 100KR058209 Collosphaera tuberosa Pac2 A6 KR058207 Collosphaera tuberosa Ind44 100 KR058214 Collophidium ovatum Sat4 100 KR058216 Collophidium sp. Pac9 B1 KR058215 Procyttarium prototypus Pac1 93 Ind46 Collophidium serpentinuml 92 KR058211 Collophidium serpentinum Sat15 72 Sat25 Collophidium aff. ovatum KR058217 Collophidium cf. serpentinum Ind20 100 59 KR058220 Collophidium cf. serpentinum Pan8 Pan10 Collophidium serpentinum B2 Pan11 Collophidium serpentinum 100 KR058221 Collophidium cf. serpentinum Sat10 Collophidiida 100 Nat6 Sphaerozoum strigulosum KR058222 Sphaerozoum strigulosum Ind29 C1 5070 Ses47 Sphaerozoum fuscum 59 KR058223 Sphaerozoum fuscumInd38 57 56 AB613248 Sphaerozoum punctatum C2 KR058228 Sphaerozoum fuscum isolate Sat24 61 KR058227 Sphaerozoum sp. Sat13 86 KR058226 Sphaerozoum brandtii Pan3 C3 Pac26 Sphaerozoum brandtii KR058253 Procyttarium primordialis Vil366 C10 98 KR058232 Sphaerozoum trigenimum Sat18 100 KR058234 Sphaerozoum trigenimum Vil349 51 KR058233 Sphaerozoum trigenimum Sat23 C4 72 90 100 KR058230 Sphaerozoum trigenimum Pan18 KR058231 Sphaerozoum trigenimum Pan19 KR058236 Sphaerozoum punctatum Ind30 100 100 95 KR058237 Thalassicolla caerulea Pac15 92 KR058238 Sphaerozoum punctatum Pac16 C6 33 KR058240 Sphaerozoum punctatum Pac22 KR058239 Sphaerozoum punctatum Pac2 KR058235 Sphaerozoum armatum Pac24 C5 AY266295 Collozoum inerme 68 KR058244 Collozoum sp. Vil334 59 KR058243 Collozoum pelagicum Sat6 96 73 KR058298 Collozoum pelagicum Pac37 KR058242 Collozoum pelagicum Pan17 Sphaerozoidae 75 KR058241 Collozoum pelagicum Pac20 TypeA_s2_contig_c1_g1_i1 100 C7 55 66 TypeA s1 contig_DN0_c1_g3_i30 0.05 TypeA_s1_contig_DN0_c1_g3_i39 95 TypeA_s1_contig_DN0_c1_g3_i34 81 TypeB_contig_DN0_c0_g1_i19 100 TypeB_contig_DN0_c0_g1_i24 TypeB_contig_c0_g1_i1 KR058308 Collozoum inerme Vil339 85 Vil338 Collozoum inerme KR058245 Collozoum inerme Pan13 9282 KR058250 Collozoum inerme Vil345 C8 69 KR058246 Collozoum inerme Pan14 80 KR058247 Collozoum inerme Vil335 KR058248 Collozoum inerme Vil336 100 KR058251 Rhaphidozoum acuferum Ind47 KR058252 Rhaphidozoum acuferum Pac8 C9 100 KR058315 Collozoum pelagicum Ses46 100KR058256 Collozoum pelagicum Sat2 KR058257 Collozoum pelagicum Sat5 C11 91 92 93 Supplemental Figure 2. Phylogenetic tree for Collodaria identification. The molecular phylogeny of 94 Collodaria was inferred by concatenation of 18S and 28S rRNA genes, and obtained by a Maximum 95 Likelihood ecostuctio ethod usig the GTR + Γ odel of seuece eolutio o a aliget of 96 sequences and 2140 aligned nucleotide positions. Only RAxML bootstrap values (1000 replicates) higher 97 than 50% are shown. The new barcode and contig sequences extracted from our transcriptome data are 98 highlighted in bold and indicated by red lines on the right of them. The clade and sub-clade naming and 99 collaring follow the nomenclature