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 a d S879 as described in
20 Decelle et al., 2012, and for Collodaria S81Col and V9R as i Bia d 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 a d ≥ % gaps i Colloda ia se ue ces 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 Maxi u Likelihood ML ethods u de the GTR+Γ odel a d 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 푎 푖푛 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
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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).
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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 eco st uctio ethod usi g the GTR + Γ odel of se ue ce e olutio o a alig e t 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 system as in Biard et al. 2015. The tree has been rooted using
100 Nassellaria sequences.
101
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100 100 Spummellaria
100 AF290072 Uncultured marine acantharean 100 GU246591 Uncultured marine eukaryote clone Clade A 100 Ei59 Acanthoplegma sp. 100 KF130152 Uncultured eukaryote clone 71 KF129879 Uncultured eukaryote clone Clade Acanth1 100 KF129749 Uncultured eukaryote clone EF172802 Uncultured eukaryote clone 100 GU822163 Uncultured rhizarian clone 99 GU821070 Uncultured rhizarian clone Clade Acanth3 100 KF129913 Uncultured eukaryote clone 59 JX188361 Uncultured eukaryote clone 99 GU246582 Uncultured marine eukaryote clone 100 GU822868 Uncultured rhizarian clone 100 Clade Acanth2 44 EF172909 Unculture eukaryote clone EF172929 Uncultured eukaryote clone 100 Ei68 Phyllostaurus cuspidatus 70 KJ760026 Phyllostaurus cuspidatus 98 70 KJ761157 Phyllostaurus cuspidatus GU825535 Phyllostaurus cuspidatus Clade B 100 KJ758551 Acanthocyrta haeckeli 100 Vil64 Acanthocyrta haeckeli 93 100Vil39 Acanthocyrta haeckeli Vil51 Acanthocyrta haeckeli Oki33 Gigartacon fragilis 100 94 Ei71 Litholophus sp. 95 Oki79 Litholophus sp. Ei48 Acanthocyrta haeckeli 72 Clade C 96 Oki23 Gigartacon muelleri 82 JN811223 Heteracon biformis Vil126 83 JN811210 Gigartacon muelleri Vil61 100 JN811219 Gigartacon muelleri Vil105 100 Ros6 Trizoma brandti 93 Oki47 Staurolithium sp. 98 Oki51 Acanthocolla solidissima Clade D 100 Oki28 Acanthocolla cruciata JN811195 Acanthocolla cruciata Oki77 63 Oki73 Larcidium dodecanthum 98 84 JN811177 Lychnaspis giltschi Oki36 Oki45 Phractopelta sarmentosa 100 Oki54 Lychnaspis giltschii Clade E 100 Vil25 Coleaspis vaginata Oki4 Dorataspis loricata 100 JN811170 Amphibelone heteracanthum Oki21 100 JQ697716 Acanthometron sp. Ant24 51 99 JQ697715 Acanthometron sp. Ant23 JQ697714 Acanthometron sp. Ant20 JN811152 Amphilonche elongata_Ei27 60 51 JQ697703 Phyllostaurus cuspidatus Ind14 95 87 JQ697700 Xiphacantha alata Ind7 75 JN811169 Xiphacantha quadridentata Oki17 81 JQ697707 Xiphacantha alata Ind10 98 Ei53 Phyllostaurus cuspidatus 5560 Ei51 Amphiastrus tetrapterus 60 JQ697699 Phyllostaurus cuspidatus Ind5 61 JQ697701 Phyllostaurus cuspidatus Ind9 78 JQ697702 Phyllostaurus cuspidatus Ind11 JQ697698 Phyllostaurus cuspidatus Ind3 JQ697697 Xiphacantha alata Ind1 JQ697704 Phyllostaurus cuspidatus Ind6 66 Vil96 Xiphacantha alata JQ697706 Xiphacantha alata_Ind4 JN811196 Acanthometra pellucida Oki78 64 JN811190 Acanthometra pellucida Oki67 Clade F Vil100 Acanthostaurus purpurescens 59 Ei23 Lonchostaurus rhombicus 5359 Ac-16_18S1 53 Ac-6_18S1 Ac-17_18S1 0.04 Oki71 Amphistaurus complanatus 82 JN811224 Acanthostaurus purpurascens Vil127 90 JQ697708 Acanthometron sp. Ant2 77 JQ697713 Acanthometron sp. Ant14 JQ697710 Acanthometron sp. Ant7 JN811153 Phyllostaurus siculus Ei39 45 JQ697705 Acanthostaurus conacanthus Ind12 46 JN811158 Lonchostaurus rombicus Ei49 JN811172 Acanthostaurus conacanthus Oki24 JN811173 Phyllostaurus siculus Oki27 JQ697717 Phyllostaurus claparedi Seb1B GU246573 Acanthometra sp. 100 KC172857 Lithoptera JQ706068 Lithoptera sp.
107
108
109 Supplemental Figure 3. Phylogenetic tree for the Acantharia identification. The molecular phylogeny of
110 Acantharia was inferred by concatenation of 18S and 28S rRNA genes, and constructed by a Maximum
111 Likelihood reconstruction method using the GTR + Γ odel o a alig e t of se ue ces ith
112 positions. Only RAxML bootstrap values (1000 replicates) higher than 40% are shown. Our new 18S
113 barcode sequences are highlighted in bold and indicated with a red line on the right of them. The clade
114 and sub-clade naming and colouring are as in Decelle et al. 2012. The tree has been rooted using
115 Spumellaria sequences. Branches with a double barred symbol have been reduced in length for clarity.
116
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117
118 Supplemental Figure 4. Light microscopy photographs of Acantharia and Collodaria samples (left),
119 compared to photographs of phylogenetically closely related and previously identified specimens (right)
120 with location number as in the original study and available at http://renkan.sb-roscoff.fr.
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121
122
123
124 Supplemental Figure 5. Collodaria nitrogen and carbon density to colony volume. Graphs show log10
3 -3 3 125 transformed data, Log10 (volume) in mm to log10 (biomass) in µg N mm (A) or µg C mm- (B). The
126 different Collodaria genera investigated in this study are indicated by colour, Collosphaeridae (blue),
127 Collozoum (red), and Sphaerozoum (green). The line of best fit is shown as a solid black/blue line with
128 the 95% confidence interval of the fit in dark grey/blue shading and 95% prediction interval in lighter
129 grey/blue shading. Furthermore, the regression statistics are shown for each graph. The conversion of
130 the in log10 expressed regression equation is outlined in the Methods.
131
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132
133
134 Supplemental Figure 6. Nassellaria (A), Aulacantha (B) and Protocystis (C) nitrogen and carbon content
135 to biovolume. The line of best fit is shown as a solid black/blue line with the 95% confidence interval of
136 the fit in dark grey/blue shading and 95% prediction interval in lighter grey/blue shading. The regression
137 statistics are shown for each graph.
138
139
11
140
141
142
143
144 Supplemental Figure 7. Aulacantha nitrogen and carbon density to biovolume (A), and to number of
145 cells per sample (B). The line of best fit is shown as a solid black/blue line with the 95% confidence
146 interval of the fit in dark grey/blue shading and 95% prediction interval in lighter grey/blue shading. The
147 regression statistics are shown for each graph.
12
References
Biard, T., and M. . Ohman. 2020. Vertical niche definition of test-bearing protists (Rhizaria) into the twilight zone revealed by in situ imaging. Limnol. Oceanogr. 1–42. doi:10.1002/lno.11472 Biard, T., L. Pillet, J. Decelle, C. Poirier, N. Suzuki, and F. Not. 2015. Towards an Integrative Morpho- molecular Classification of the Collodaria (Polycystinea, Radiolaria). Protist 166: 374–388. doi:10.1016/j.protis.2015.05.002 Capella-Gutiérrez, S., J. M. Silla-Martínez, and T. Gabaldón. 2009. trimAl: A tool for automated alignment trimming in large-scale phylogenetic analyses. Bioinformatics 25: 1972–1973. doi:10.1093/bioinformatics/btp348 Decelle, J., I. Probert, L. Bittner, and others. 2012a. An original mode of symbiosis in open ocean plankton. Proc. Natl. Acad. Sci. 109: 18000–18005. doi:10.1073/pnas.1212303109 Decelle, J., N. Suzuki, F. Mahé, C. de Vargas, and F. Not. 2012b. Molecular Phylogeny and Morphological Evolution of the Acantharia (Radiolaria). Protist 163: 435–450. doi:10.1016/j.protis.2011.10.002 Hillebrand, H., C. D. Dürselen, D. Kirschtel, U. Pollingher, and T. Zohary. 1999. Biovolume calculation for pelagic and benthic microalgae. J. Phycol. 35: 403–424. doi:10.1046/j.1529-8817.1999.3520403.x Katoh, K., K. I. Kuma, H. Toh, and T. Miyata. 2005. MAFFT version 5: Improvement in accuracy of multiple sequence alignment. Nucleic Acids Res. 33: 511–518. doi:10.1093/nar/gki198 Larsson, A. 2014. AliView: A fast and lightweight alignment viewer and editor for large datasets. Bioinformatics 30: 3276–3278. doi:10.1093/bioinformatics/btu531 Stamatakis, A. 2014. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30: 1312–1313.
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