Title: Molecular Data From Orthonectid Worms Show They Are Highly Degenerate Members of Phylum Annelida Not Phylum Mesozoa

bioRxiv preprint first posted online Dec. 18, 2017; doi: http://dx.doi.org/10.1101/235549.
The copyright holder for this preprint (which was

not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.
1
2 Title: Molecular data from Orthonectid worms show they
3 are highly degenerate members of phylum Annelida not
4 phylum Mesozoa.
5 Authors: Philipp H. Schiffer1, Helen E. Robertson1, Maximilian J. Telford1*
7 Affiliations: 1 Centre for Life’s Origins and Evolution, Department of Genetics, Evolution
8 and Environment, University College London, Gower Street, London, WC1E 6BT, UK
9 *Correspondence to: m.telford@ucl.ac.uk
10
11
12 Summary:
13 The Mesozoa are a group of tiny, extremely simple, vermiform endoparasites of various
14 marine animals (Fig. 1). There are two recognised groups within the Mesozoa: the
15 Orthonectida (Fig. 1a,b; with a few hundred cells including a nervous system made up of just
16 10 cells [1]) and the Dicyemids (Fig. 1c; with at most 42 cells [2]). They are
17 classic ’Problematica’ [3] - the name Mesozoa suggests an evolutionary position
18 intermediate between Protozoa and Metazoa (animals) [4] and implies their simplicity is a
19 primitive state, but molecular data have shown they are members of Lophotrochozoa within
20 Bilateria [5-8] which would mean they derive from a more complex ancestor. Their precise
21 phylogenetic affinities remain uncertain, however, and ascertaining this is complicated by the
22 very fast evolution observed in genes from both groups, leading to the common systematic
23 error of Long Branch Attraction (LBA) [9]. Here we use mitochondrial and nuclear gene
bioRxiv preprint first posted online Dec. 18, 2017; doi: http://dx.doi.org/10.1101/235549. The copyright holder for this preprint (which was
24 sequence data, and show beyond doubt that both dicyemids and orthonectids are members of
25 the Lophotrochozoa. Carefully addressing the effects of systematic errors due to unequal
26 rates of evolution, we show that the phylum Mesozoa is polyphyletic. While the precise
27 position of dicyemids remains unresolved within Lophotrochozoa, we unequivocally identify
28 orthonectids as members of the phylum Annelida. This result reveals one of the most extreme
29 cases of body plan simplification in the animal kingdom; our finding makes sense of an
30 annelid-like cuticle in orthonectids [1] and suggests the circular muscle cells repeated along
31 their body [10] may be segmental in origin.
32
33 Results
34 Using a new assembly of available genomic and transcriptomic sequence data we identified an
35 almost complete mitochondrial genome from Intoshia linei (2 ribosomal RNAs, 20 transfer
36 RNAs and all protein coding genes apart from atp8) and recovered 9 individual mitochondrial
37 gene containing contigs from Dicyema japonicum and from a second unidentified species
38 (Dicyema sp.; cox1, 2, 3; cob; and nad1, 2, 3, 4, 5). Cob, nad3, nad4, and nad5 had not
39 previously been identified in any Dicyma species. All protostomes studied possess a unique,
40 derived combination of amino acid signatures and conserved deletions in their mitochondrial
41 NAD5 genes. Comparing the NAD5 protein coding regions of Intoshia and Dicyema to those
42 of other Metazoa shows that both share almost all of the conserved protostome signatures [11]
43 (Fig. 2a). This signature is significantly more complex than the two amino acids of the
44 Lox5/DoxC signature from Dicyema previously published [11-13] and shows beyond doubt
45 that both groups are protostomes.
46 It has been suggested that mesozoans are derived from the parasitic neodermatan flatworms. If
47 this were correct mesozoans would be expected to share two changes in mitochondrial genetic
48 code that unite all rhabditophoran flatworms, where the triplet AAA codes for Asparagine (N)
49 rather than the normal Lysine (K) and ATA codes for Isoleucine (I) rather than the usual
50 Methionine (M) [14]. We inferred the mitochondrial genetic codes for Dicyema and Intoshia.
51 Both groups have the standard invertebrate mitochondrial code arguing against a relationship
52 with the parasitic rhabditophoran platyhelminths (table S1).
53 We next aligned the mitochondrial genes of Intoshia and three species of Dicyema with
54 orthologs from a diversity of other Metazoans and concatenated these to produce a matrix of
55 2,969 reliably aligned amino acids from 69 species. Phylogenetic analyses of this
56 comparatively small data set is not expected to be as reliable as a much larger set of nuclear
57 genes and aspects of the topology and observed branch lengths suggest it was affected by LBA
58 (Fig. 2b). To reduce the effects of LBA on the inference of the affinities of the mesozoans we
59 removed the taxa with the longest branches and considered the position of the dicyemids and
60 orthonectid separately (as both are very long branched). We were unable to resolve the position
61 of the dicyemids (although they are clearly lophotrochozoans), but found some support for
62 placing the orthonectid Intoshia linei with the annelids (Fig. 2c and figures S1, 2). Intoshia
63 linei has a unique mitochondrial gene order although the order of the genes nad1, nad6, and
64 cob match that seen in the Lophotrochozoan ground plan and the early branching annelid
65 Owenia (Fig. 2d).
66 We next assembled a data set of 469 orthologous genes, 227,187 reliably aligned amino acids,
67 from 45 species of animals including Intoshia linei and two species of Dicyema. After
68 removing positions in the concatenated alignment with less than 50% occupancy we had an
69 alignment length of 190,027 amino acids and average completeness of ~68%. Intoshia linei
70 was 65% complete, while Dicyema japonicum and Dicyema sp. were 77% and 43% complete
71 respectively (table S2). We conducted a bayesian phylogenetic analyses of these data with the
72 site heterogeneous CAT+G4 model in Phylobayes [15]. To provide an additional, conservative

73 estimate of clade support and to enable further analyses in a practical time frame, we also used
74 jackknife subsampling. For each jackknife analysis we took 50 random subsamples of 30,000
75 amino acids each and ran 2,000 cycles (phylobayes CAT+G4) per sample. All 50 subsamples
76 were summarised into a single tree with the first 1800 trees from each excluded as ‘burnin’
77 [16].
78 We observed strong support for a clade of Lophotrochozoa (excluding Rotifers) including both
79 dicyemids and the orthonectid (Bayesian Posterior Probability (PP) = 1.0; Jackknife Proportion
80 (JP) = 0.97) (Fig. 3a). The dicyemids and orthonectids were not each other’s closest relatives;
81 the position of the dicyemids within the Lophotrochozoa was not resolved; they were not the
82 sister group of the platyhelminths nor of the gastrotrichs in our analysis. The position of the
83 orthonectid Intoshia, in contrast, was resolved as being within the clade of annelids (Fig. 2a PP
84 = 0.97; JP = 0.74).
85 We next asked whether there was any effect from long branched dicyemids on the strength of
86 support for inclusion of Intoshia within the Annelida - Intoshia also being a long-branched
87 taxon. Repeating our jackknife analyses with dicyemids excluded increased the support for
88 Intoshia as an annelid from JP = 0.74 to JP = 0.86 (Fig. 3b) showing that when the expected
89 LBA between Dicyema spp and Intoshia is prevented, there is stronger support for including
90 the orthonectid in Annelida. An equivalent analysis omitting Intoshia did not help to resolve
91 the position of dicyemids (figure S3).
92 To test further the support for Intoshia being a member of Annelida, we reasoned that an
93 analysis restricted to genes showing the strongest signal supporting monophyletic Annelida
94 should give stronger support to Intoshia within Annelida but only if it is indeed a member of
95 the clade; if not, support should decrease when using this subset of genes. We first removed all
96 mesozoan sequences from each individual gene alignment and reconstructed a tree for each
97 gene. We ranked these gene trees according to the proportion of all annelids present in a given
98 gene data set that were observed united in a clade. We concatenated the genes (now including
99 mesozoans) from strongest supporters of monophyletic Annelida to weakest. We repeated our
100 jackknife analyses using the best quarter of genes. An analysis of the genes that most strongly
101 support monophyletic Annelida results in an increase support for inclusion of Intoshia within
102 Annelida from JP = 0.74 to JP = 0.94 (Fig. 3c).
103 Our results suggest that recent findings of a close relationship between Intoshia and Dicyema
104 and the linking of both these taxa to rapidly evolving gastrotrichs and platyhelminths [7,8] is
105 due to long branch attraction. To test this prediction we exaggerated the expected effects of
106 LBA on our own data set by using less well fitting models. We first conducted cross validation
107 comparing the site heterogeneous CAT+G4 model we have used to the site homogenous
108 LG+G4 and show that LG+G4 is a significantly less good fit to our data (CAT+G4 is better
109 than LG+G4: ΔlnL = 9787 +/- 249.265). We used the less well fitting LG+G4 model to
110 reanalyse the jackknife replicates of a data set including our four most complete annelids. We
111 observed a topology clearly influenced by LBA in which long branched taxa including
112 flatworms, annelids, rotifers and nematodes were grouped. We also observed within this ‘LBA
113 assemblage’ the two longest branched clades, dicyemids and the orthonectid as each other’s
114 closest relatives. As a further test we reanalysed the published data set [8] which had linked
115 orthonectid and dicyemid with platyhelminths and gastrotrichs. When we removed the most
116 obvious source of LBA - the long branched dicyemid - we found that the orthonectid Intoshia
117 was, as expected, found not with platyhelminths or gastrotrichs but with the two annelids
118 present in this data set, again providing evidence of the effects of long branch attraction (Fig
119 4).
120 Discussion
121 We have analysed the first, almost complete mitochondrial genome sequence of an orthonectid
122 mesozoan and added to the known mitochondrial genes of Dicyemida to provide two powerful
123 rare genomic changes. Our analyses of mitochondrial NAD5 gene sequences show
124 unequivocally that both Dicyemida and Orthonectida are members of the protostomes and the
125 absence of rhabditophoran flatworm mitochondrial genetic code changes rejects existing ideas
126 that either group might be derived from parasitic flatworms. Both groups show unusually high
127 rates of evolution and this required steps to test for and avoid the possible effects of long branch
128 attraction, not least between the orthonectids and dicyemids.
129 Our mitochondrial data set and our large, taxonomically broad set of nuclear genes with a low
130 percentage of missing data, analysed with well fitting, site heterogeneous models of sequence
131 evolution, do not support the close relationship between orthonectids and dicyemids.
132 Orthonectids are annelids and not members of the Mesozoa and the phylum Mesozoa sensu
133 lato is an unnatural polyphyletic assemblage. We were unable to place the dicyemids more
134 precisely and they may be considered a phylum in their own right. Experiments manipulating
135 the expected effects of LBA strongly suggest previous phylogenies were affected by this
136 important source of systematic error. Finding the orthonectids and dicyemids not closely
137 associated demonstrates a remarkable instance of convergent evolution in two unrelated,
138 miniaturised parasites.
139 The finding that the orthonectid Intoshia is a member of the Annelida shows that it has evolved
140 its extraordinary simplicity by drastic simplification from a much more complex annelid
141 common ancestor. Our phylogenetic analyses could not more precisely place Intoshia within
142 the annelids, however, a short stretch of mitochondrial genes (nad1, nad6, cob) that are found
143 in the same order as in the lophotrochozoan ancestor and in the early branching annelid Owenia
144 fusiformis but not in the pleistoannelid ground plan argues for a position outside of the
145 Pleistoannelida [17] (Fig 2d). Possible evidence of an ancestral segmented body plan is still
146 apparent in the series of circular muscles regularly spaced along the antero-posterior axis of
147 Intoshia (Fig 1b), along with similarly repeated bands of cilia (Fig 1 and ref [18]). Further
148 analysis of the genome, embryology and morphology of Intoshia or other orthonectids are
149 predicted to show additional clues as to their cryptic annelidan ancestry.
150 Methods
151
152 Genome and transcriptome assemblies.
153 We downloaded genomic (Intoshia linei: SRR4418796, SRR4418797) and transcriptomic
154 (Dicyema sp.: SRR827581; Dicyema japonicum: DRR057371) data from the NCBI Short
155 Read Archives and DDBJ, and used Trimmomatic [19] to clean residual adapter sequences
156 from the sequencing reads and to remove low quality bases. We used the clc assembly cell
157 (clcBIO/Qiagen; v.5.0) to re-assemble the I. linei genome and the Trinity pipeline [20]
158 (v.2.3.2) to assemble the Dicyema. sp. and D. japonicum transcriptomes using default
159 settings. We additionally assembled transcriptomes for Phascolopsis gouldii,
160 Spiochaetopterus sp., Arenicola marina, Sabella pavonina, Magelona pitelkai,
161 Pharyngocirrus tridentiger and Bonellia viridis from SRA datasets (SRR1654498,
162 SRR1224605, SRR2005653, SRR2005708, SRR2015609, SRR2016714, SRR2017645)
163 using the same approach.
164
165 Identifying mitochondrial genome fragments.
166 Using mitochondrial gene protein coding sequences from flatworms as queries [21] we used
167 tblastn [22] and blastp to search for Dicyema sp. and D. japonicum mitochondrial fragments
168 in the Trinity RNA-Seq assemblies, and screened the I. linei genome re-assembly in a similar
169 way. Positively identified ORFs were then blasted against NCBI nr to detect possible
170 contamination from host species in the RNA-Seq data. For each Dicyema sp. gene-bearing
171 contig, we also found additional contigs which had strongly matching blast hits to Octopus or
172 other cephalopods (or in some cases to the gastropod mollusc Aplysia) and we discarded
173 these as likely contaminations.
174
175 Annotating mitochondrial genomes.
176 Using blast we identified a 14.2kb mitochondrial contig in the assembled I. linei genome,
177 which we annotated using MITOS [23]. The location of protein-coding genes were manually
178 verified from MITOS prediction, and inferred to start from the first in-frame start codon
179 (ATN, GTG, TTG, or GTT). The C-terminal of the protein-coding genes was inferred to be
180 the first in-frame stop codon (TAA, TAG or TGA). We aligned the Intoshia and Dicyema
181 NAD5 genes with those from 5 protostomes, 4 deuterostomes, and 2 non-bilaterian species in
182 the Geneious software to visualise Protostome specific signatures in the sequence.
183
184 Mitochondrial Phylogenetics
185 We grouped the mesozoan mitochondrial protein coding genes with their orthologs from 65
186 other species selected to cover the diversity of the Metazoa including diploblasts,
187 deuterostomes and ecdysozoans but with an emphasis on the diversity of Lophotrochozoa.
188 We aligned each set of orthologs using Muscle [24] v3.8.31 using default parameters and
189 trimmed these alignments to exclude unreliably aligned positions using TrimAl [25] (version
190 1.2 rev 59 using default settings). Finally, we concatenated the trimmed alignments of all
191 genes into a supermatrix of 2969 positions. We inferred a phylogeny with phylobayes (4.1b)
192 under the CAT+G4 model. We ran 10 independent chains for 10,000 cycles each. We
193 summarised all ten chains (bpcomp) discarding the first 8,000 trees from each as burnin. We
194 reconstructed additional mitochondrial phylogenies omitting (i) the long branching flatworm
195 species, (ii) all long branch taxa and also Intoshia, and (iii) long branch taxa and the Dicyema
196 species. Here and elsewhere we visualised and edited phylogenetic trees with FigTree
197 (v1.4.3; http://tree.bio.ed.ac.uk/software/figtree/).
198
199 Nuclear gene orthology determination
200 We chose to add the mesozoan data to sets of orthologous genes that were previously
201 successfully used to infer lophotrochozoan phylogeny [26,27]. We first used Orthofinder [28]
202 (v.1.0.8) to calculate orthologous relationships between the genes predicted for I. linei in the
203 recent genome paper [8] and our Dicyema sp. gene predictions. To ensure robustness of the
204 analysis we included several outgroup species (Supplementary table 2) In particular, as we
205 were concerned about potential contamination by the hosts of the parasitic Dicyema we
206 included the Octopus bimaculoides proteome. Since the published phylogenomic studies
207 included few annelid species we added our own Trinity assemblies of several additional
208 species (see above). We then extracted all orthologous groups containing the Octopus and the
209 two mesozoan taxa from the Orthofinder output and inserted these sequences into the original
210 alignments. This resulted in 590 orthologous groups. With the aid of OMA [29] and custom
211 Perl scripts we filtered these groups to contain single copy orthologs of all species. We re-
212 aligned each set of orthologs using clustal-omega [30]; we removed unreliably aligned
213 positions from each alignment using TrimAl; finally we constructed individual gene trees
214 from these trimmed alignments using phyml [31] (v20160207). Using Python code and the
215 ETE3 toolkit we checked each tree for instances where sequences from Octopus and Dicyema
216 sp. were each other’s closest relatives (suggesting the sequence is an Octopus contaminant)
217 and removed the 5 alignments where the trees had this topology from our set. We
218 concatenated all single trimmed alignments of 45 taxa into a supermatrix of 227,646
219 positions. We used a custom script to eliminate all positions in the alignment with less than
220 50% occupancy.
221
222 Nuclear Gene Phylogenomic analyses
223 Using the mpi version of phylobayes (in v.1.7) run over four independent chains for 5000
224 cycles and discarding the first 4500 trees as burnin we reconstructed a phylogeny using this
225 alignment under both the CAT+G4 model of molecular evolution. To provide a conservative
226 measure of clade support and to test different data samples in a reasonable time we also
227 reconstructed trees using 50 jackknife sub-samples of 30,000 positions each from the
228 supermatrix. We used phylobayes 4.1c with the aid of the gnu-parallel command line tool
229 [32] and the UCL HPC cluster. We used the CAT+G4 model, and also compared results
230 from LG+G4. We ran phylobayes for 2000 cycles per jackknife sample which consistently
231 resulted in a plateauing of the likelihood score. We summarised all 50 of these phylobayes
232 analyses per model (using bpcomp) discarding the first 1800 sampled trees per jackknife as
233 burnin. We also tested the effect of different species compositions in our dataset by
234 performing phylobayes jackknife sampling with different subsets of taxa.
235
236 Cross validation
237 We compared the fit of CAT+G4 and LG+G4 models to our data using cross validation as
238 described in the phylobayes user manual. We ran 10 replicates and for each replicate we used
239 a randomly selected 30,000 positions of the data as a training set and 10,000 randomly
240 selected positions as the test set. Log likelihood scores were averaged over the ten replicates
241 using the sumcv command.
242
243 Ranking genes according to support for monophyletic Annelida.
244 We first removed all Intoshia and Dicyema sequences from each individual gene alignment.
245 For each individual gene, we reconstructed a tree from the aligned protein coding sequences
246 using Ninja [33]. Each tree was parsed using a custom script to find the proportion of
247 annelids in the data set present in the largest clade of annelids found. The tree was given a
248 score which was calculated as the number of annelids in the largest clade/total number of
249 annelids on the tree. Trees with larger monophyletic annelid clades scored highest. The
250 genes were then concatenated in order of their score. We took the first 25% of positions from
251 this concatenation (those genes with the strongest signal supporting monophyletic annelids)
252 and analysed jackknife replicates as before.
253
254 Acknowledgements:
255
256 The authors are grateful to Prof George Slyusarev (Saint Petersburg State University, Russia)
257 for providing images of Intoshia linei and for sharing an unpublished book chapter and to Dr
258 Tsai-Ming Lu and colleagues (Okinawa Institute of Science and Technology, Japan) for
259 sharing data. The authors also thank Fraser Simpson (UCL) for help in editing Figure 1b. The
260 research was funded by ERC grant (ERC-2012-AdG 322790) to MJT. Alignments have been
261 deposited with Zenodo (XXX) and phylogenetic trees are available through treebase.org
262 (XXX).
263
264
265 Author Contributions
266 Conceived the study: MJT. Planned the study: MJT and PHS. Assembled the data sets: PHS.
267 Analysed the data: PHS and MJT. Drafted the manuscript: MJT and PHS. Analysed
268 mitochondrial data: HER.
269
270 Declaration of Interests:
271 The authors declare no competing financial interests.

272
273
274 Figure Legends
275
276 Fig. 1: The mesozoans Intoshia variabili and Dicyema typus
277
278 A. Differential Interference contrast micrograph of an Intoshia variabili female showing
279 repeated bands of ciliated cells. Picture G. Slyusarev (St Petersburg State University,
280 Russia).
281 B. Confocal image of a phalloidin stained female specimen of Intoshia linei reveals repeated
282 set of circular muscles. Picture G. Slyusarev (St Petersburg State Univ.).
283 C. Rhombogen stage of a dicyemid (Dicyema typus from the Octopus) adapted from Hyman
284 L.H. The Invertebrates: Protozoa through Ctenophora McGraw-Hill, New York 1940(19).
285 Anterior to right in all images.
286
287 Fig. 2: Analyses of the phylogenetic positions of Dicyema and Intoshia based on
288 mitochondrial gene sequences.
289
290 A. Alignment of the mitochondrial NAD5 gene from selected protostomes, deuterostomes,
291 and outgroups, highlighting derived substitutions and amino acid deletions shared by the
292 orthonectids, dicyemids, and other protostomes.
293 B. A mitochondrial bayesian phylogeny based on 2969 positions places orthonectids and
294 dicyemids inside Lophotrochozoa, but the unlikely assemblage of Intoshia linei and
295 flatworms with annelids suggest this is affected by systematic error.
296 C. Mitochondrial bayesian phylogeny omitting the long branching taxa including Dicyema
297 gives some support for a position of Intoshia within Annelida.D. Order of the Intoshia nad1,
298 nad6, and cob mitochondrial genes in comparison to the early branching annelid Owenia
299 fusiformis, the pleistoannelid ground plan and the lophotrochozoan ground plan (see ref [17]).
300
301 Fig. 3: Analyses of the phylogenetic positions of Dicyema and Intoshia based on nuclear
302 gene sequences.
303 A. A bayesian phylogeny reconstructed from 190,027 aligned amino acid positions analysed
304 under the CAT+G4 model. Support values are from bayesian posterior probabilities (PP) and
305 from 50 jackknifed sub-samples of 30,000 residues (JP support values in brackets). Both
306 analyses reveal Mesozoa to be polyphyletic and place Intoshia linei in Annelida (see Supp
307 Fig 4a for support values).
308 B. A repeat of the jackknife analysis omitting the long-branching Dicyema species eliminates
309 the potential for LBA between Intoshia and Dicyema. This leads to an increase in the support
310 for a position of Intoshia within Annelida from JP 0.74 to JP 0.86 JP. (Only lophotrochozoan
311 part of the tree shown, see Supplementary Fig 4c for full tree).
312 C. Bayesian jackknife using CAT+G4 model using the best quarter of genes supporting
313 monophyletic annelids leads to increased support for Intoshia within Annelida to JP 0.94
314 even with the inclusion of the Dicyema species. (Only lophotrochozoan part of the tree
315 shown, see Supplementary Fig 4d for full tree).
316
317 Fig. 4: Reanalysis of a published data set addressing potential LBA between mesozoans
318 supports annelid affinity for Intoshia.
319 Repeating the analyses on a previously published data set [8] excluding the long branching
320 Dicyema leads to Intoshia being placed with the annelids, showing the likely effect of LBA
321 on the original analysis. Support values are bayesian posterior probabilities (PP).
A B
C
Fig. 1: The mesozoans Intoshia variabili and Dicyema typus

A. Differential Interference contrast micrograph of an Intoshia variabili female showing repeated bands of ciliated cells. Picture G. Slyusarev
(St Petersburg State University, Russia).
B. Confocal image of a phalloidin stained female specimen of Intoshia linei reveals repeated set of circular muscles. Picture G. Slyusarev (St
Petersburg State Univ.).
C. Rhombogen stage of a dicyemid (Dicyema typus from the Octopus) adapted from Hyman L.H. The Invertebrates: Protozoa through
Ctenophora McGraw-Hill, New York 1940(19).
Anterior to right in all images.
180 190 260 270 280 290
Lophotrochozoa
A D
Dicyema
Intoshia
Octopus
rrnS rrnL nad1 nad6 cob nad4L nad4
Phonoris
Taenia Intoshia linei
Brugia
Drosophila -nad4 -cox1 nad1 nad6 cob nad3 rrnL
Xenopus
Pongo
Xenoturbella Owenia fusiformis
Hydra
Paramecium nad5 nad2 nad1 nad6 cob nad4L nad4
Pleistoannelida
B Acropora_tenuis
C cox2 atp8 cox3 nad6 cob atp6 nad5
Acropora_tenuis
Nematostella_sp Nematostella_sp
Aurelia_aurita Trichoplax_adhaerens
Trichoplax_adhaerens Aurelia_aurita
Xenoturbella_bocki
0.62 Strongylocentrotus_pallidus
Saccoglossus_kowalevskii
Xenoturbella_bocki
Strongylocentrotus_pallidus
Balanoglossus_carnosus 1 0.83 Saccoglossus_kowalevskii
0.98 Myxine_glutinosa
Lampetra_fluviatilis
Balanoglossus_carnosus
Doliolum_nationalis
0.96 Salmo_salar
Ornithorhynchus_anatinus 0.74 Ciona_intestinalis
Homo_sapiens Myxine_glutinosa
Doliolum_nationalis Lampetra_fluviatilis
Ciona_intestinalis Salmo_salar
Branchiostoma_floridae
1 Asymmetron_inferum Ornithorhynchus_anatinus
Homo_sapiens
Priapulus_caudatus
Tribolium_castaneum 1 Branchiostoma_floridae
Locusta_migratoria Asymmetron_inferum
Limulus_polyphemus Priapulus_caudatus
Daphnia_pulex Tribolium_castaneum
Trichinella_spiralis Locusta_migratoria
Strongyloides_stercoralis
1 Steinernema_carpocapsae
Schmidtea_mediterranea
Limulus_polyphemus
Daphnia_pulex
Spadella_cephaloptera Spadella_cephaloptera
Sagitta_inflata
Decipisagitta_decipiens 1 Sagitta_inflata
Decipisagitta_decipiens
Siphonodentalium_lobatum
Siphonodentalium_lobatum
0.87 Mytilus_trossulus
Flustrellidra_hispida Mytilus_trossulus
Bugula_neritina Thais_clavigera
Pupa_strigosa Lineus_alborostratus
Biomphalaria_tenagophila
Aplysia_californica 0.92 Nautilus_macromphalus
0.68 Thais_clavigera
Lineus_alborostratus
Octopus_vulgaris
Octopus_ocellatus
Nautilus_macromphalus Sepia_officinalis
Octopus_vulgaris Sepia_esculenta
Octopus_ocellatus Sepioteuthis_lessoniana
Sepia_officinalis Loligo_bleekeri
Sepia_esculenta
Sepioteuthis_lessoniana 0.94 Dosidicus_gigas
0.3 Loligo_bleekeri Pupa_strigosa
Dosidicus_gigas Biomphalaria_tenagophila
Terebratulina_retusa Aplysia_californica
Laqueus_rubellus Terebratulina_retusa
Sipunculus_nudus Laqueus_rubellus
Orbinia_latreillii
0.41 Urechis_caupo Flustrellidra_hispida
Platynereis_dumerilii
Myzostoma_seymourcollegiorum 0.8 Sipunculus_nudusBugula_neritina
Marphysa_sanguinea
Intoshia_linei 0.69 Intoshia_linei
0.57 Microstomum_lineare
Schistosoma_mansoni 0.39
Orbinia_latreillii
Clymenella_torquata
Fasciola_hepatica Platynereis_dumerilii
0.33 Taenia_crassiceps
Echinococcus_shiquicus
Myzostoma_seymourcollegiorum
Urechis_caupo
Echinococcus_multilocularis
Annelida
Dicyema_sp Marphysa_sanguinea
1 Dicyema_misakiense Escarpia_spicata
Lumbricus_terrestris
Dicyema_japonicum
Clymenella_torquata Perionyx_excavatus
Escarpia_spicata Amynthas_jiriensis
Perionyx_excavatus
Amynthas_jiriensis
0.1
0.1
Fig. 2: Analyses of the phylogenetic positions of Dicyema and Intoshia based on mitochondrial gene sequences.
A. Alignment of the mitochondrial NAD5 gene from selected protostomes, deuterostomes, and outgroups, highlighting derived substitutions and amino
acid deletions shared by the orthonectids, dicyemids, and other protostomes.
B. A mitochondrial bayesian phylogeny based on 2969 positions places orthonectids and dicyemids inside Lophotrochozoa, but the unlikely assemblage
of Intoshia linei and flatworms with annelids suggest this is affected by systematic error.
C. Mitochondrial bayesian phylogeny omitting the long branching taxa including Dicyema gives some support for a position of Intoshia within Annelida.
D. Order of the Intoshia nad1, nad6, and cob mitochondrial genes in comparison to the early branching annelid Owenia fusiformis, the pleistoannelid
ground plan and the lophotrochozoan ground plan (see ref [17]).
Amphimedon_queenslandica
Sycon_coactum
Trichoplax_adhaerens
Nematostella_vectensis
Hydra_vulgaris
A
Rhabdopleura_sp
Strongylocentrotus_purpuratus
Patiria_miniata
Homo_sapiens
Branchiostoma_floridae Fig. 3: Analyses of the phylogenetic positions of
Priapulus_caudatus
Tribolium_castaneum
Trichinella_spiralis Dicyema and Intoshia based on nuclear gene
Pediculus_humanus
Daphnia_pulex
sequences.
Macracanthorhynchus_hirudinaceus
Adineta_ricciae
Neomenia_megatrapezata
A. A bayesian phylogeny reconstructed from
190,027 aligned amino acid positions analysed
1
Octopus_bimaculoides
Lymnea_stagnalis
Lottia_gigantea under the CAT+G4 model. Support values are
(0.97) Dicyema_sp
Dicyema_japonicum from bayesian posterior probabilities (PP) and
Cerebratulus_sp
Tubulanus_polymorphus
Cephalothrix_linearis
from 50 jackknifed sub-samples of 30,000
Mesodasys_laticaudatus
Macrodasys_sp
residues (JP support values in brackets). Both
Lepidodermella_squamata
Macrostomum_lignano
analyses reveal Mesozoa to be polyphyletic and
Maritigrella_crozieri
Monocelis_sp
place Intoshia linei in Annelida (see Supp Fig 4a
Schistosoma_mansoni
Dendrocoelum_lacteum
for support values).
Stenostomum_sp
Magelona_pitelkai
Golfingia_vulgaris
B. A repeat of the jackknife analysis omitting the
long-branching Dicyema species eliminates the
0.97
Pharyngocirrus_tridentiger
Intoshia_linei
Sabella_pavonina potential for LBA between Intoshia and Dicyema.
(0.74)
Annelida
Capitella_tellata
Bonellia_viridis
Helobdella_robusta
This leads to an increase in the support for a
0.1
Alvinella_pompejana position of Intoshia within Annelida from JP 0.74
to JP 0.86 JP. (Only lophotrochozoan part of the
B C
0.1
Adineta_ricciae
Adineta_ricciae tree shown, see Supplementary Fig 4c for full
tree).
Macrodasys_sp
Macrodasys_sp
C. Bayesian jackknife using CAT+G4 model using
Lymnea_stagnalis
Lymnea_stagnalis the best quarter of genes supporting monophyletic
Lottia_gigantea
Lottia_gigantea Dicyema_sp annelids leads to increased support for Intoshia
Cerebratulus_sp
Tubulanus_polymorphus Cerebratulus_sp
Dicyema_japonicum
within Annelida to JP 0.94 even with the inclusion
Macrostomum_lignano
of the Dicyema species. (Only lophotrochozoan
Macrostomum_lignano
part of the tree shown, see Supplementary Fig 4d
Monocelis_sp
Schistosoma_mansoni
Monocelis_sp for full tree).
Schistosoma_mansoni
Stenostomum_sp Stenostomum_sp
Magelona_pitelkai Magelona_pitelkai
0.86 Golfingia_vulgaris
0.94 Golfingia_vulgaris
Intoshia_linei Sabella_pavonina
Sabella_pavonina Intoshia_linei
Helobdella_robusta Helobdella_robusta
Annelida
Capitella_tellata Capitella_tellata
Bonellia_viridis Bonellia_viridis
Alvinella_pompejana
Alvinella_pompejana
0.5
0.5
0.5
0.5
Daphnia_pulex
1
1
Tribolium_castaneum
Drosophila_melanogaster
Limnognathia_maerski
1
1
Brachionus_koreanus
1
0.91
Macrodasys_sp
1
1
0.96 Diuronotus_aspetos
0.98
1
Stenostomum_sthenum
Stenostomum_leucops
1 Microstomum_lineare
1 1
Prostheceraeus_vittatus
Geocentrophora_applanata
1 1
Monocelis_fusca
1
0.99 1
Schistosoma_mansoni
Bothrioplana_semperi
1
Octopus_bimaculatus
Lottia_gigantea
0.94
Annelida
Intoshia_linei
0.93
0.98
Helobdella_robusta
Capitella_teleta
1
Gnathostomula_paradoxa
Austrognathia_sp
1
Homo_sapiens
1 Ciona_intestinalis
0.1
Fig. 4: Reanalysis of a published data set addressing potential LBA between mesozoans supports annelid affinity for Intoshia.
Repeating the analyses on a previously published data set [7] excluding the long branching Dicyema leads to Intoshia being placed with the
annelids, showing the likely effect of LBA on the original analysis. Support values are bayesian posterior probabilities (PP).
322 Supplementary Tables

323
324 Supplementary Table 1
325 Predicted correspondence of nucleotide triplets to amino acids in Intoshia and three Dicyema
326 species. For each triplet, the amino acid corresponding to the triplet in the standard
327 invertebrate mitochondrial code is shown, the number of observations of the triplet to
328 prediction is based on, the predicted amino acid and its score and finally the second highest
329 scoring amino acid prediction. The triplets AAA and ATA are highlighted in green and likely
330 errors highlighted in blue. Likely errors are mostly associated with very low numbers of
331 observed GC rich triplets in these very AT rich mitochondrial genomes.
332
333 Supplementary Table 2
334 List of species used in the final phylogenetic analysis, data sources, and representation in the
335 final alignment.
336
337
338 Supplementary Figures:
339
340 Figure S1. Related to Figure 2.
341 Phylogram and corresponding cladogram of a Bayesian analysis of our mitochondrial data set
342 omitting the long-branching flatworm species. Phylobayes CAT+G4 model was run in 10
343 independent runs for 10,000 cycles each on an alignment with 2969 positions and 8000 trees
344 were discarded as burnin.
345
347 Phylogram and corresponding cladogram of a Bayesian analysis of our mitochondrial data set
348 omitting Intoshia linei. Phylobayes CAT+G4 model was run in 10 independent runs for
349 10,000 cycles each on an alignment with 2969 positions and 8000 trees were discarded as
350 burnin.
351
353 A phylogram based on our analysis of the jackknifed dataset omitting Intoshia linei. Contrary
354 to the improvement in placing I. linei observed when excluding the Dicyema species, the
355 exclusion of I. linei does not lead to a better resolution of the Dicyema species’ position. This
356 can be seen as further evidence for the non-affiliation of orthonectids and dicyemids and the
357 correct inference that orthonectids are part of Annelida.

359 A. Cladogram corresponding to Fig 3a showing all PP support values for the CAT+G4
360 phylogeny based on the full alignment of 190,027 amino acid positions.
361
362 B. A cladogram including JP support values based on 50 jackknife subsamples of 30,000
363 amino acid positions each independently analysed for 2000 cycles under the CAT+G4 model
364 in phylobayes and summarised with the bpcomp command setting 1800 as burnin. As in the
365 analysis of the full dataset I. linei is found within the annelids and phylum Mesozoa is found
366 as an unnatural assemblage.
367
368 C. Cladogram corresponding to Fig 3b showing all support values.
369
370 D. Cladogram corresponding to Fig 3c showing all support values.
371
372 References:
373
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~50µm
Figure S1. Related to Figure 2.
Acropora_tenuis
Nematostella_sp
Aurelia_aurita
Xenoturbella_bocki
1 Strongylocentrotus_pallidus
0.94 1 Saccoglossus_kowalevskii
1
0.55 1 Doliolum_nationalis
0.69 Ciona_intestinalis
1 Myxine_glutinosa
0.74
1
Lampetra_fluviatilis
Salmo_salar
Acropora_tenuis 1 Ornithorhynchus_anatinus
Nematostella_sp 0.64 1
Homo_sapiens
Aurelia_aurita 1 1 Branchiostoma_floridae
Trichoplax_adhaerens Asymmetron_inferum
Xenoturbella_bocki Priapulus_caudatus
Strongylocentrotus_pallidus Tribolium_castaneum
Saccoglossus_kowalevskii 0.96 0.97
Balanoglossus_carnosus 0.97 Locusta_migratoria
Doliolum_nationalis 0.96 Limulus_polyphemus
Ciona_intestinalis Daphnia_pulex
Myxine_glutinosa Spadella_cephaloptera
0.99 Sagitta_inflata
Lampetra_fluviatilis 0.78
Salmo_salar 1 Decipisagitta_decipiens
Ornithorhynchus_anatinus 0.74 Siphonodentalium_lobatum
Homo_sapiens Mytilus_trossulus
Branchiostoma_floridae 0.39 Thais_clavigera
Asymmetron_inferum Lineus_alborostratus
Priapulus_caudatus 0.41 0.58 Nautilus_macromphalus
Tribolium_castaneum 0.94 Octopus_vulgaris
Locusta_migratoria 0.83 0.99
Limulus_polyphemus Octopus_ocellatus
Daphnia_pulex 0.85 0.94 Sepia_officinalis
Spadella_cephaloptera 0.38 Sepia_esculenta
0.87 Sepioteuthis_lessoniana
Sagitta_inflata 0.99
Decipisagitta_decipiens 0.97 Loligo_bleekeri
Siphonodentalium_lobatum Dosidicus_gigas
Mytilus_trossulus 0.87 Pupa_strigosa
Thais_clavigera 0.99 Biomphalaria_tenagophila
0.63
Lineus_alborostratus Aplysia_californica
Nautilus_macromphalus 0.99 Terebratulina_retusa
Octopus_vulgaris 0.73 Laqueus_rubellus
Octopus_ocellatus 1 Flustrellidra_hispida
Sepia_officinalis Bugula_neritina
Sepia_esculenta Sipunculus_nudus
Sepioteuthis_lessoniana 0.47 0.32
Loligo_bleekeri Dicyema_sp
1 Dicyema_misakiense
Dosidicus_gigas 0.98
Dicyema_japonicum
Pupa_strigosa 0.43
Biomphalaria_tenagophila Intoshia_linei
Aplysia_californica 0.32 Orbinia_latreillii
Terebratulina_retusa 0.39 Clymenella_torquata
Laqueus_rubellus Platynereis_dumerilii
Flustrellidra_hispida 0.42 0.63 Myzostoma_seymourcollegiorum
Bugula_neritina 0.57 Urechis_caupo
Sipunculus_nudus 0.44 Marphysa_sanguinea
Dicyema_sp Escarpia_spicata
Dicyema_misakiense 0.89 Lumbricus_terrestris
Dicyema_japonicum 0.92 Perionyx_excavatus
Intoshia_linei 0.94
Orbinia_latreillii Amynthas_jiriensis
Clymenella_torquata
Myzostoma_seymourcollegiorum 0.1
Urechis_caupo
Marphysa_sanguinea
Escarpia_spicata
Perionyx_excavatus
Amynthas_jiriensis
Figure S1:  0.1
Phylogram and corresponding cladogram of a Bayesian analysis of our mitochondrial data set omitting the long-branching flatworm species.
Phylobayes CAT+G4 model was run in 10 independent runs for 10,000 cycles each on an alignment with 2969 positions and 8000 trees were
discarded as burnin.
Acropora_tenuis
Nematostella_sp
Aurelia_aurita
Xenoturbella_bocki
1 1
0.85 1 Saccoglossus_kowalevskii
0.57 1 Doliolum_nationalis
0.72 Ciona_intestinalis
1 Myxine_glutinosa
0.66
1 Lampetra_fluviatilis
0.99
Salmo_salar
0.64 1 Ornithorhynchus_anatinus
Homo_sapiens
1 1 Branchiostoma_floridae
Asymmetron_inferum
Acropora_tenuis
Priapulus_caudatus
Nematostella_sp 0.95 0.97 Tribolium_castaneum
Trichoplax_adhaerens 0.97 Locusta_migratoria
Aurelia_aurita 0.97 Limulus_polyphemus
Xenoturbella_bocki Daphnia_pulex
1
Spadella_cephaloptera
Saccoglossus_kowalevskii 0.76 Sagitta_inflata
Balanoglossus_carnosus Decipisagitta_decipiens
Doliolum_nationalis 1 Thais_clavigera
Ciona_intestinalis Siphonodentalium_lobatum
Myxine_glutinosa 0.88
Lampetra_fluviatilis Mytilus_trossulus
Salmo_salar Lineus_alborostratus
Ornithorhynchus_anatinus 0.56 Nautilus_macromphalus
Homo_sapiens Octopus_vulgaris
Branchiostoma_floridae 0.89 0.99
Asymmetron_inferum
0.92 Octopus_ocellatus
Priapulus_caudatus
0.59 0.91 0.97 Sepia_officinalis
Tribolium_castaneum Sepia_esculenta
Locusta_migratoria 0.93 Sepioteuthis_lessoniana
0.98
Limulus_polyphemus 0.98 Loligo_bleekeri
Daphnia_pulex Dosidicus_gigas
Spadella_cephaloptera Dicyema_sp
Sagitta_inflata 1 Dicyema_misakiense
Decipisagitta_decipiens 0.99
Thais_clavigera 0.91 Dicyema_japonicum
Siphonodentalium_lobatum Pupa_strigosa
Mytilus_trossulus 0.71 Biomphalaria_tenagophila
Lineus_alborostratus Aplysia_californica
Nautilus_macromphalus 0.99 Terebratulina_retusa
Octopus_vulgaris Laqueus_rubellus
Octopus_ocellatus 0.76
Sepia_officinalis 1 Flustrellidra_hispida
Sepia_esculenta 0.74 Bugula_neritina
Sepioteuthis_lessoniana Sipunculus_nudus
Loligo_bleekeri 0.81 Orbinia_latreillii
Dosidicus_gigas Clymenella_torquata
Dicyema_sp 0.8 Urechis_caupo
Dicyema_misakiense Platynereis_dumerilii
Dicyema_japonicum 0.68
Pupa_strigosa Myzostoma_seymourcollegiorum
Biomphalaria_tenagophila 0.56 Marphysa_sanguinea
Aplysia_californica Escarpia_spicata
Terebratulina_retusa 0.96 Lumbricus_terrestris
Laqueus_rubellus 0.96 Perionyx_excavatus
Flustrellidra_hispida 0.98
Amynthas_jiriensis
Bugula_neritina
Sipunculus_nudus
Orbinia_latreillii
Clymenella_torquata 0.1
Urechis_caupo
Myzostoma_seymourcollegiorum
Marphysa_sanguinea
Escarpia_spicata
Perionyx_excavatus
Amynthas_jiriensis
Figure S2: 0.1
Phylogram and corresponding cladogram of a Bayesian analysis of our mitochondrial data set omitting Intoshia linei. Phylobayes CAT+G4
model was run in 10 independent runs for 10,000 cycles each on an alignment with 2969 positions and 8000 trees were discarded as burnin.
Amphimedon_queenslandica
Sycon_coactum
1 Nematostella_vectensis
0.97
Hydra_vulgaris
0.96 Saccoglossus_kowalevskii
1 Rhabdopleura_sp
0.87 1 Strongylocentrotus_purpuratus
Patiria_miniata
1 Homo_sapiens
1 Priapulus_caudatus
0.9 Trichinella_spiralis
0.82 1 Tribolium_castaneum
1 Pediculus_humanus
Daphnia_pulex
1 1 Macracanthorhynchus_hirudinaceus
Adineta_ricciae
Amphimedon_queenslandica 1 Mesodasys_laticaudatus
Sycon_coactum Macrodasys_sp
Trichoplax_adhaerens Neomenia_megatrapezata
Nematostella_vectensis 0.84 0.98 Octopus_bimaculoides
Hydra_vulgaris 0.94
Saccoglossus_kowalevskii 1 Lymnea_stagnalis
Rhabdopleura_sp Lottia_gigantea
Strongylocentrotus_purpuratus 1 Dicyema_sp
Patiria_miniata Dicyema_japonicum
Homo_sapiens 0.98 Cerebratulus_sp
Branchiostoma_floridae 1 Tubulanus_polymorphus
Priapulus_caudatus 0.98
Trichinella_spiralis Macrostomum_lignano
Tribolium_castaneum 0.53
Pediculus_humanus 1 Maritigrella_crozieri
1 Monocelis_sp
Daphnia_pulex 1
Macracanthorhynchus_hirudinaceus 0.99
1 Schistosoma_mansoni
Adineta_ricciae Dendrocoelum_lacteum
Mesodasys_laticaudatus Stenostomum_sp
Macrodasys_sp Magelona_pitelkai
Neomenia_megatrapezata 0.79 Golfingia_vulgaris
Octopus_bimaculoides 0.93 Pharyngocirrus_tridentiger
Lymnea_stagnalis
Lottia_gigantea 0.98 Sabella_pavonina
Dicyema_sp 0.98 Helobdella_robusta
Dicyema_japonicum 0.51 1 Capitella_tellata
Cerebratulus_sp Bonellia_viridis
Tubulanus_polymorphus Alvinella_pompejana
Macrostomum_lignano
Maritigrella_crozieri 0.1
Monocelis_sp
Schistosoma_mansoni
Stenostomum_sp Figure S3:
Magelona_pitelkai
Golfingia_vulgaris A phylogram based on our analysis of the jackknifed dataset omitting Intoshia linei. Contrary to the
Sabella_pavonina
Helobdella_robusta
improvement in placing I. linei observed when excluding the Dicyema species, the exclusion of I. linei does
Capitella_tellata not lead to a better resolution of the Dicyema species’ position. This can be seen as further evidence for the
Bonellia_viridis
Alvinella_pompejana non-affiliation of orthonectids and dicyemids and the correct inference that orthonectids are part of
Annelida.
0.1
A Amphimedon_queenslandica
Sycon_coactum
B Amphimedon_queenslandica
Sycon_coactum
Trichoplax_adhaerens Trichoplax_adhaerens
1
Nematostella_vectensis Nematostella_vectensis
1 Hydra_vulgaris 1
Hydra_vulgaris
1 0.96 Saccoglossus_kowalevskii
1
Rhabdopleura_sp 0.96
1 Rhabdopleura_sp
1
Strongylocentrotus_purpuratus 1
Patiria_miniata 0.86 Strongylocentrotus_purpuratus
1
1
Homo_sapiens Patiria_miniata
1
Branchiostoma_floridae Homo_sapiens
Priapulus_caudatus 1
0.75 Trichinella_spiralis
0.75 Tribolium_castaneum
1 Priapulus_caudatus
0.75
1
Pediculus_humanus 0.92 Trichinella_spiralis
1
Daphnia_pulex 0.82 Tribolium_castaneum
1
1
1
Macracanthorhynchus_hirudinaceus 1 Pediculus_humanus
Adineta_ricciae Daphnia_pulex
Neomenia_megatrapezata Macracanthorhynchus_hirudinaceus
1 Octopus_bimaculoides 1
1 1 Adineta_ricciae
0.75 1
Lymnea_stagnalis Mesodasys_laticaudatus
Lottia_gigantea 1
Dicyema_sp 0.5 Macrodasys_sp
1 Lepidodermella_squamata
Dicyema_japonicum
1 Cerebratulus_sp Neomenia_megatrapezata
1 0.88 0.98
1
Tubulanus_polymorphus Octopus_bimaculoides
Cephalothrix_linearis 0.93 Lymnea_stagnalis
0.75 Mesodasys_laticaudatus 1
1 Lottia_gigantea
Macrodasys_sp Dicyema_sp
0.58 Lepidodermella_squamata 1
1
Macrostomum_lignano Dicyema_japonicum
0.75 1 Maritigrella_crozieri 0.97 Cerebratulus_sp
1 Monocelis_sp 0.99 Tubulanus_polymorphus
1 0.98
1
1
Schistosoma_mansoni Cephalothrix_linearis
Dendrocoelum_lacteum Macrostomum_lignano
Stenostomum_sp 1 Maritigrella_crozieri
Magelona_pitelkai 1 Monocelis_sp
0.97 Golfingia_vulgaris 1
Pharyngocirrus_tridentiger 0.94 Schistosoma_mansoni
1 0.98
Intoshia_linei Dendrocoelum_lacteum
1
Sabella_pavonina Stenostomum_sp
1 1
Capitella_tellata Magelona_pitelkai
1
Bonellia_viridis 0.74 Golfingia_vulgaris
1
Helobdella_robusta Pharyngocirrus_tridentiger
Alvinella_pompejana 0.75
Intoshia_linei
0.6 Sabella_pavonina
0.1
Helobdella_robusta
0.77 Capitella_tellata
0.99
Bonellia_viridis
Alvinella_pompejana
0.1
C D
Amphimedon_queenslandica Amphimedon_queenslandica
Sycon_coactum Sycon_coactum
Trichoplax_adhaerens Trichoplax_adhaerens
1
Nematostella_vectensis Nematostella_vectensis
Hydra_vulgaris 1
0.97 0.97 Hydra_vulgaris
0.97
Saccoglossus_kowalevskii Homo_sapiens
Rhabdopleura_sp 0.97
1 0.67 Branchiostoma_floridae
0.9 1
Strongylocentrotus_purpuratus Saccoglossus_kowalevskii
Patiria_miniata 0.99
1
Rhabdopleura_sp
0.99
Homo_sapiens 1 Strongylocentrotus_purpuratus
1
Branchiostoma_floridae Patiria_miniata
1 Priapulus_caudatus Priapulus_caudatus
0.91 Trichinella_spiralis 0.68 1 Trichinella_spiralis
0.8
1
Tribolium_castaneum 0.94 Tribolium_castaneum
1
1 Pediculus_humanus 1 Pediculus_humanus
Daphnia_pulex Daphnia_pulex
1
Macracanthorhynchus_hirudinaceus 1
1 1
Adineta_ricciae Adineta_ricciae
1
Mesodasys_laticaudatus Mesodasys_laticaudatus
1
0.55 Macrodasys_sp 1 0.82 Macrodasys_sp
Lepidodermella_squamata Lepidodermella_squamata
0.86 Neomenia_megatrapezata Neomenia_megatrapezata
1 Octopus_bimaculoides 0.99
0.99 Octopus_bimaculoides
0.92 0.99
1
Lymnea_stagnalis 1
Lymnea_stagnalis
Lottia_gigantea Lottia_gigantea
Cerebratulus_sp 1
Dicyema_sp
0.99 0.59
0.99
0.97
Tubulanus_polymorphus Dicyema_japonicum
Cephalothrix_linearis Cerebratulus_sp
1
0.51 Macrostomum_lignano 1
1 Maritigrella_crozieri 0.54
1 Monocelis_sp 0.8 Macrostomum_lignano
0.95 1
Schistosoma_mansoni 1 Maritigrella_crozieri
1
Dendrocoelum_lacteum 1 Monocelis_sp
1
Stenostomum_sp 0.96
1
Schistosoma_mansoni
0.52
Magelona_pitelkai Dendrocoelum_lacteum
0.86 Golfingia_vulgaris Stenostomum_sp
0.81 Pharyngocirrus_tridentiger Magelona_pitelkai
Intoshia_linei 0.94 Golfingia_vulgaris
0.65
Sabella_pavonina 0.99 Pharyngocirrus_tridentiger
Helobdella_robusta 0.88 Sabella_pavonina
0.72 Capitella_tellata 0.7 Intoshia_linei
0.99
Bonellia_viridis 0.43 Helobdella_robusta
Alvinella_pompejana 0.66
0.99
Capitella_tellata
0.54 Bonellia_viridis
Alvinella_pompejana
0.1
0.1
Figure S4:
A. Cladogram corresponding to Fig 3a showing all PP support values for the CAT+G4 phylogeny based on the full alignment of 190,027 amino acid positions.
B. A cladogram including JP support values based on 50 jackknife subsamples of 30,000 amino acid positions each independently analysed for 2000 cycles under
the CAT+G4 model in phylobayes and summarised with the bpcomp command setting 1800 as burnin. As in the analysis of the full dataset I. linei is found
within the annelids and phylum Mesozoa is found as an unnatural assemblage.
C. Cladogram corresponding to Fig 3b showing all support values.
D. Cladogram corresponding to Fig 3c showing all support values.

Title: Molecular Data From Orthonectid Worms Show They Are Highly Degenerate Members of Phylum Annelida Not Phylum Mesozoa

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Title: Molecular Data From Orthonectid Worms Show They Are Highly Degenerate Members of Phylum Annelida Not Phylum Mesozoa

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bioRxiv preprint first posted online Dec. 18, 2017; doi: http://dx.doi.org/10.1101/235549.

The copyright holder for this preprint (which was

3 are highly degenerate members of phylum Annelida not

5 Authors: Philipp H. Schiffer1, Helen E. Robertson1, Maximilian J. Telford1*

9 *Correspondence to: m.telford@ucl.ac.uk

17 classic ’Problematica’ [3] - the name Mesozoa suggests an evolutionary position

27 position of dicyemids remains unresolved within Lophotrochozoa, we unequivocally identify

31 their body [10] may be segmental in origin.

45 that both groups are protostomes.

52 with the parasitic rhabditophoran platyhelminths (table S1).

65 Owenia (Fig. 2d).

72 site heterogeneous CAT+G4 model in Phylobayes [15]. To provide an additional, conservative

91 the position of dicyemids (figure S3).

99 mesozoans) from strongest supporters of monophyletic Annelida to weakest. We repeated our

102 Annelida from JP = 0.74 to JP = 0.94 (Fig. 3c).

128 attraction, not least between the orthonectids and dicyemids.

137 associated demonstrates a remarkable instance of convergent evolution in two unrelated,

138 miniaturised parasites.

149 predicted to show additional clues as to their cryptic annelidan ancestry.

153 We downloaded genomic (Intoshia linei: SRR4418796, SRR4418797) and transcriptomic

159 settings. We additionally assembled transcriptomes for Phascolopsis gouldii,

160 Spiochaetopterus sp., Arenicola marina, Sabella pavonina, Magelona pitelkai,

162 SRR1224605, SRR2005653, SRR2005708, SRR2015609, SRR2016714, SRR2017645)

163 using the same approach.

165 Identifying mitochondrial genome fragments.

173 these as likely contaminations.

175 Annotating mitochondrial genomes.

184 Mitochondrial Phylogenetics

197 (v1.4.3; http://tree.bio.ed.ac.uk/software/figtree/).

199 Nuclear gene orthology determination

204 analysis we included several outgroup species (Supplementary table 2) In particular, as we

220 50% occupancy.

222 Nuclear Gene Phylogenomic analyses

234 performing phylobayes jackknife sampling with different subsets of taxa.

236 Cross validation

241 using the sumcv command.

243 Ranking genes according to support for monophyletic Annelida.

252 and analysed jackknife replicates as before.

265 Author Contributions

268 mitochondrial data: HER.

270 Declaration of Interests:

271 The authors declare no competing financial interests.

Fig. 1: The mesozoans Intoshia variabili and Dicyema typus

322 Supplementary Tables

358 Figure S4. Related to Figure 3.

Figure S1: 0.1

Figure S2: 0.1

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Figure S1:  0.1