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1 RUNNING HEAD: Phylogenomics of Tunicata

2

3 Phylogenomics offers resolution of major tunicate relationships

4

5 Kevin M. Kocot1*, Michael G. Tassia2, Kenneth M. Halanych2, and Billie J. Swalla3,4

6

7 1Department of Biological Sciences and Alabama Museum of Natural History, University of

8 Alabama, Tuscaloosa, AL, 35487, U.S.A.

9 2Department of Biological Sciences, Auburn University, Auburn, AL, 36830, U.S.A.

10 3Department of Biology, University of Washington, 24 Kincaid Hall, Seattle, WA 98195, USA

11 4Friday Harbor Laboratories, University of Washington, Friday Harbor, WA 98250, USA

12 *Corresponding author

13

14 Contact: [email protected], 205-348-4052 (phone), 205-348-4039 (fax)

15

16 Keywords: Chordata; Tunicata; Urochordata; Thaliacea; phylogenomic

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18 Summary

19 Tunicata, a diverse clade of approximately 3,000 described species of marine, filter-feeding

20 chordates, is of great interest to researchers because tunicates are the closest living relatives of

21 vertebrates and they facilitate comparative studies of our own biology. The group also includes

22 numerous invasive species that cause considerable economic damage and some species of

23 tunicates are edible. Despite their diversity and importance, relationships among major lineages

24 of Tunicata are not completely resolved. Here, we supplemented public data with transcriptomes

25 from seven species spanning the diversity of Tunicata and conducted phylogenomic analyses on

26 data sets of up to 798 genes. Sensitivity analyses were employed to examine the influences of

27 reducing compositional heterogeneity and branch-length heterogeneity. All analyses maximally

28 supported a monophyletic Tunicata within Olfactores (Vertebrata + Tunicata). Within Tunicata,

29 all analyses recovered Appendicularia sister to the rest of Tunicata and confirmed (with maximal

30 support) that Thaliacea is nested within Ascidiacea. Stolidobranchia is the sister taxon to all

31 other tunicates except Appendicularia. In most analyses, phlebobranch tunicates were recovered

32 paraphyletic with respect to Aplousobranchia. Support for this topology varied but was strong in

33 some cases. However, when only the 50 best genes based on compositional heterogeneity were

34 analysed, we recovered Phlebobranchia and Aplousobranchia reciprocally monophyletic with

35 strong support, consistent with most traditional morphology-based hypotheses. Examination of

36 internode certainty also cast doubt on results of phlebobranch paraphyly, which may be due to

37 limited taxon sampling. Taken together, these results provide a higher-level phylogenetic

38 framework for our closest living invertebrate relatives.

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40 1. Introduction

41 Tunicata (Lamarck, 1816) is a diverse clade of approximately 3,000 described species of marine,

42 filter-feeding chordates (Shenkar and Swalla, 2011). This morphologically plastic group, which

43 may be benthic or pelagic and solitary or colonial, has intrigued researchers for a number of

44 reasons. Its position as the sister lineage of vertebrates (e.g., Bourlat et al., 2006; Delsuc et al.,

45 2005, 2006) makes it an import resource for studying the evolution of developmental

46 mechanisms (e.g., Racioppi et al., 2017; Suzuki et al., 2016; Swalla and Jeffery, 1996; Taketa

47 and De Tomaso, 2015). Additionally, Tunicata includes some of the most invasive benthic

48 marine animals known (e.g., Kaplan et al., 2017; Reem et al., 2013), which can cause

49 catastrophic damage if left unchecked. Lastly, tunicates are eaten in some parts of the world, thus

50 supporting fishery industries (Lambert et al., 2016). Despite their importance, evolutionary

51 relationships within Tunicata remain uncertain, hindering understanding of the evolutionary

52 history and ancestral character states of its constituent lineages and Chordata as a whole.

53

54 Tunicates date back to the Early Cambrian (Chen et al., 2003; Shu et al., 2001). Lamarck (1816)

55 first described modern tunicates for their hard, leathery outer covering, the “tunic,” and included

56 ascidians, pyrosomes and salps within the group. Tunicates were subsequently divided into three

57 recognized classes: Ascidiacea (sea squirts), Thaliacea (pelagic salps, doliolids, and pyrosomes;

58 Berrill, 1936), and Appendicularia (larvaceans), which have been associated with a suite of gross

59 morphological and life history features (Table 1). Of these classes, Ascidiacea, comprised of

60 sessile tunicates, is the most species rich (~3,000 species), but a growing body of evidence

61 indicates that the pelagic Thaliacea (~100 species) is nested within Ascidiacea (Swalla et al.,

62 2000; Stach et al., 2002; Tsagkogeorga et al., 2009; Winchell et al., 2002; Zeng et al., 2006).

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63 Appendicularia is a group of small, pelagic tunicates characterized by a putatively paedomorphic

64 adult body plan that resembles the tadpole larva, very short generation times, and secretion of a

65 unique mucous-like “house” used in feeding (Stach et al., 2008). Most appendicularian genes

66 studied to date exhibit high rates of nucleotide substitution leading to long branches in molecular

67 phylogenetic analyses (e.g., Delsuc et al., 2006, 2008; Govindarajan et al., 2011; Stach and

68 Turbeville, 2002; Swalla et al., 2000; Wada, 1998). Previous studies have recovered

69 appendicularians as either sister to the rest of Tunicata or within Ascidiacea (reviewed by

70 Tsagkogeorga et al., 2009).

71

72 Higher-level taxonomy of Ascidiacea is based primarily on morphological characters of the

73 branchial sac, the organ used to collect particles from the water column, and as such they are

74 divided into three traditionally recognized orders: Aplousobranchia (simple branchial sac),

75 Phlebobranchia (vascular branchial sac), and Stolidobranchia (folded branchial sac; Lahille,

76 1886). However, relationships among these clades remain uncertain and, as noted above,

77 Thaliacea and Appendicularia may be nested within this clade of otherwise benthic tunicates.

78 Stolidobranchia is a large and morphologically heterogeneous clade of solitary, social, and

79 colonial tunicates (Zeng and Swalla, 2005; Zeng et al., 2006). Despite this, molecular

80 phylogenetic analyses based on 18S rDNA and mitochondrial genes have shown convincingly

81 that Molgulidae, a group of solitary ascidians with various larval morphologies, is sister to all

82 other stolidobranchs whereas relationships within Pyuridae and Styelidae, the two other major

83 lineages of Stolidobranchia, have generally not been well-resolved (e.g., Stach et al., 2002;

84 Swalla et al., 2000; Tsagkogeorga et al., 2009; Winchell et al., 2002; Zeng and Swalla, 2005;

85 Zeng et al. 2006). Aplousobranchia has been recovered as unambiguously monophyletic in most

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86 molecular analyses to date (e.g., Stach and Turbeville, 2002; Tsagkogeorga et al., 2009; Turon

87 and López-Legentil, 2004), but these colonial tunicates tend to have high rates of nucleotide

88 substitution and support for their position relative to other tunicates has generally been weak.

89 Phlebobranchia is a traditionally recognized group of mostly solitary tunicates, but the

90 composition of this clade has been debated. For example, Cionidae, which includes the widely-

91 studied species Ciona intestinalis, C. robusta and C. savignyi, was originally included within

92 Phlebobranchia (Berrill, 1936). However, this genus of important model tunicate species has also

93 been viewed as a subclade of Aplousobranchia (e.g., Kott, 1990, 1969).

94

95 Although molecular phylogenetic studies of tunicates conducted to date (Govindarajan et al.,

96 2011; Shenkar et al., 2016; Singh et al., 2009; Stach et al., 2010; Stach and Turbeville, 2002;

97 Swalla et al., 2000; Tsagkogeorga et al., 2009; Turon and López-Legentil, 2004; Zeng et al.,

98 2006; Zeng and Swalla, 2005) have greatly advanced understanding of relationships within some

99 clades, tunicate higher-level phylogeny has been difficult to reconstruct. Evolutionary history of

100 Tunicata has likely been a particularly challenging question in invertebrate systematics because

101 several tunicate lineages (Appendicularia, Thaliacea, and many species within Aplousobranchia)

102 exhibit long branch lengths for 18S and at least some other genes (Stach and Turbeville, 2002;

103 Swalla et al., 2000; Tsagkogeorga et al., 2009; Winchell et al., 2002; Yokobori et al., 2006; Zeng

104 et al., 2006).

105

106 Phylogenomic analyses have been important to our understanding of chordate evolutionary

107 history by showing that tunicates and not cephalochordates are the sister taxon of the vertebrates

108 (Bourlat et al., 2006; Delsuc et al. 2006, 2008; Dunn et al. 2008; Putnam et al. 2008), but no

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109 study to date has had the necessary taxon sampling to address long-standing questions about

110 evolutionary relationships among the major lineages of tunicates. To this end, we supplemented

111 publicly available tunicate and outgroup genome and transcriptome data with transcriptomes

112 from taxa spanning the diversity of Tunicata and re-evaluated the higher-level evolutionary

113 history of this important group.

114

115 2. Methods

116 We sampled publicly available and newly generated transcriptome and/or genome data from all

117 extant tunicate orders with the exception of Doliolida, which was previously shown to be nested

118 within the otherwise well-sampled taxon Thaliacea (Govindarajan et al., 2011). Available

119 tunicate data were augmented with new transcriptomes from specimens collected from

120 Antarctica, the Northeastern Pacific, and the Northwestern Atlantic (Table 2). With the exception

121 of the unidentified Ascidia sp. (?) from Antarctica, all of the newly sequenced species are well-

122 known species from their respective collection localities that were easily identified based on

123 habitus and structure of the branchial basket and gut. RNA was extracted and purified from

124 RNAlater-preserved or frozen tissue samples using the Omega Bio-Tek Mollusc RNA kit or the

125 Qiagen RNeasy kit. In either case, an on-column DNAse digestion was performed. RNA

126 concentration was measured using a Qubit (Thermo Fisher) with the RNA High Sensitivity kit,

127 RNA purity was assessed by measuring the 260/280 nm absorbance ratio using a Nanodrop Lite

128 (Thermo Fisher), and RNA integrity was evaluated using a 1% SB agarose gel or a TapeStation

129 (Agilent). At least 1 µg of total RNA for each specimen was sent to Macrogen (Cambridge, MA,

130 USA) for Illumina TruSeq RNA v2 library preparation (polyA enrichment) and sequencing on

131 the Illumina HiSeq 2500 system using the HiSeq SBS V4 chemistry with 100 bp paired-end

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132 sequencing. For Styela gibbsii, two separate libraries from material from the same individual

133 were prepared and sequenced separately, but the raw reads were combined prior to transcriptome

134 assembly.

135

136 Dataset processing followed the general approach of Kocot et al. (2017). Publicly available

137 genomic data were downloaded as predicted proteins if available (Table 3). Otherwise, predicted

138 transcripts from genomes or assembled transcriptomes were downloaded. Publicly available

139 transcriptomes available only as raw read data and our new transcriptome data were assembled

140 using Trinity 2.2.0 with the --trimmomatic and --normalize_reads flags (Grabherr et al., 2011).

141 Transcripts were translated with TransDecoder 2.0.0 or 2.0.1 (Haas et al., 2014) using the

142 UniProt SwissProt database (accessed on September 20th, 2016; The Uniprot Consortium, 2014)

143 and PFAM (Pfam-A.hmm) version 27 (Finn et al., 2015).

144

145 For orthology inference, we employed HaMStR 13 (Ebersberger et al., 2009) with the “model

146 organsisms” core-ortholog set. Translated transcripts for all taxa except Ciona intestinalis and

147 human were searched against the 1,031 profile hidden Markov models (pHMMs) using the

148 default options. The “representative” flag was not used because it is not compatible with

149 PhyloTreePruner (Kocot et al., 2013; see below). Sequences matching a pHMM were compared

150 to the proteome of Ciona using BLASTP with the default search settings of HaMStR. If the

151 Ciona amino acid sequence contributing to the pHMM was the best BLASTP hit in each of

152 these back-BLASTs, the sequence was then assigned to that putative orthology group (simply

153 referred to as “gene” henceforth). Redundant sequences that were identical (including partial

154 sequences that were identical at least where they overlapped) were then removed with

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155 UniqHaplo (http://raven.iab.alaska.edu/~ntakebay/), leaving only unique sequences for each

156 taxon. Each gene was then aligned with MAFFT 7.273 using the automatic alignment strategy

157 with a “maxiterate” value of 1,000 (Katoh and Standley, 2013). Alignments were then trimmed

158 with Aliscore (Misof and Misof, 2009) and Alicut (Kück et al., 2010) with the default options to

159 remove ambiguously aligned regions. Lastly, we deleted sequences that did not overlap with all

160 other sequences in the alignment by at least 20 amino acids, starting with the shortest sequences

161 not meeting this criterion. This step was necessary for downstream single-gene tree

162 reconstruction. Finally, genes sampled for fewer than half of the 28 taxa after these steps were

163 discarded.

164

165 In some cases, a taxon was represented in an alignment by two or more sequences (splice

166 variants, lineage-specific gene duplications [=inparalogs], overlooked paralogs, or exogenous

167 contamination). To screen for evidence of paralogy or contamination and select just one

168 sequence for each taxon, an approximately maximum likelihood tree was inferred for each

169 remaining alignment using FastTree 2 (Price et al., 2010) using the -slow and -gamma options.

170 PhyloTreePruner (Kocot and Citarella et al., 2013) was then employed to use a tree-based

171 approach to screen each single-gene alignment for evidence of paralogy or contamination. First,

172 nodes with support values below 0.95 were collapsed into polytomies. Next, the maximally

173 inclusive subtree was selected where each taxon was represented by no more than one sequence

174 or, in cases where more than one sequence was present for any taxon, all sequences from that

175 taxon formed a clade or were part of the same polytomy. Putative paralogs and contaminants

176 (sequences falling outside of this maximally inclusive subtree) were then deleted from the input

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177 alignment. In cases where multiple sequences from the same taxon formed a clade or were part

178 of the same polytomy, all sequences except the longest were deleted.

179

180 In order to further screen for genes or taxa with paralogy or contamination issues, genes that

181 passed PhyloTreePruner screening and were still sampled for at least 15 of the 28 taxa were

182 retained and used for single-gene tree building in RAxML 8.2.8 (Stamatakis, 2014) with the

183 PROTGAMMAAUTOF model. The tree with the best likelihood score after 10 random

184 addition sequence replicates was retained and topological robustness (i.e., nodal support) was

185 assessed with rapid bootstrapping with the number of replicates determined by the autoMRE

186 criterion. Concatenation of remaining sequences to assemble the data matrix henceforth

187 referred to as the “original full dataset” was performed using FASconCAT-G (Kück and Longo,

188 2014).

189

190 Because compositional heterogeneity (Delsuc et al., 2005; Jermiin et al., 2004; Kocot et al.,

191 2017; Nesnidal et al., 2010; Rodríguez-Ezpeleta et al., 2007) and branch-length heterogeneity

192 (Kocot et al., 2017; Struck et al., 2014) have been shown to be potential sources of systematic

193 error in phylogenomics, we calculated relative composition frequency variability (RCFV;

194 Zhong et al., 2011) and branch-length heterogeneity score (LB; Struck et al., 2014) for each

195 gene in the original full dataset and assembled data matrices corresponding to the best 50, 100,

196 200, and 500 genes according to RCFV and LB. This allowed us to examine effects of

197 excluding genes with relatively high compositional heterogeneity or branch-length

198 heterogeneity. Average RCFV was calculated for each gene based on per-taxon RCFV scores

199 calculated in BaCoCa 1.104.r with a subclade definition file that divided the taxa into

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200 Ambulacraria (Hemichordata + Echinodermata), Vertebrata, Cephalochordata, and Tunicata.

201 LB was calculated for each gene with TreSpEx 1.1 (Struck, 2014) using RAxML single-gene

202 trees generated as described above. As above, concatenation was performed using

203 FASconCAT-G (Kück and Longo, 2014). For brevity, these matrices are referred to using

204 abbreviated names such as LB_50, which represents the best 50 genes according to branch-

205 length heterogeneity or RCFV_200, which represents the best 200 genes according to RCFV.

206

207 Maximum likelihood (ML) analyses were conducted for all data matrices in RAxML 8.2.8

208 (Stamatakis 2014). Matrices were partitioned by gene and the PROTGAMMAAUTOF model

209 was specified for all partitions. The tree with the best likelihood score after 10 random addition

210 sequence replicates was retained and nodal support was assessed with rapid bootstrapping with

211 the number of replicates determined by the autoMRE criterion.

212

213 We also conducted Bayesian inference (BI) analyses in Phylobayes MPI 1.6j (Lartillot et al.,

214 2013) using a site-heterogeneous mixture model. Specifically, the CAT+GTR+Γ4 model

215 (Lartillot and Philippe, 2004) was used to account for site-specific rate heterogeneity (-cat -gtr -

216 dgam 4). Because of the computationally intensive nature of Phylobayes analyses using this

217 model, BI was only conducted for the RCFV_50 and LB_50 data sets. For the BI analysis of

218 RCFV_50, five parallel chains were run for roughly 15,000-18,000 cycles with the first 10,000

219 discarded as burn-in. For the BI analysis of LB_50, four parallel chains were run for roughly

220 17,000-28,000 cycles with the first 10,000 discarded as burn-in. The bpcomp maxdiff values

221 (0.0039 for RCFV_50 and 0.0697 for LB_50) were used to assess convergence of chains.

222

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223 Because the CAT mixture model implemented in Phylobayes could not be feasibly applied to

224 all datasets, we also conducted ML analyses in IQ-TREE using the posterior mean site

225 frequency (PMSF) model (Wang et al. 2017). PMSF is a rapid approximation of the time- and

226 memory-intensive profile mixture model of Lee et al. (2008), which is a variant of the

227 Phylobayes CAT model. Specifically, the LG+C60+G+F model was specified. Because this

228 approach requires a guide tree to infer the site frequency model, we used the previously

229 generated RAxML tree for each PMSF analysis. For the PMSF analyses of RCFV_50 and

230 LB_50, we additionally tested the effect of using the consensus trees recovered by Phylobayes

231 as the guide tree. Nodal support was assessed with 1000 replicates of ultrafast bootstrapping (-

232 bb 1000).

233

234 To screen for outlier genes and taxa (genes or taxa that have contamination and/or paralogy

235 issues), single-gene RAxML trees were analyzed in Phylo-MCOA (De Vienne et al., 2012)

236 finding successive decomposition axes from individual ordinations (derived from distance

237 matrices) that maximize a covariance function. For detection of “complete outliers” (genes or

238 taxa most likely to have contamination and/or paralogy issues) we used values of k=1.5 and

239 thres=0.5.

240

241 After the above analyses were conducted, transcriptome data became available for two

242 additional tunicate species: Clavelina lepadiformis (Aplousobranchia) and Salpa thompsoni

243 (Thaliacea). We assembled an additional dataset (Full dataset+2) following an identical

244 approach to that described above to produce the original full dataset except for the addition of

245 these two taxa. The minimum number of taxa required to keep a gene was kept at fifteen (>50%

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246 of the original 28 sampled taxa). We also assembled datasets corresponding to the best 50 genes

247 according to RCFV (RCFV_50+2) and LB (LB_50+2) identified as described above that were

248 retained by our pipeline after the addition of these two taxa (47 and 48 genes, respectively).

249 Maximum likelihood analyses were conducted on these datasets in RAxML as described above.

250

251 We examined tree certainty (TC), relative tree certainty (RTC), and internode certainty (IC)

252 using the approaches of Salichos and Rokas (2013) and Salichos et al. (2014) as calculated in

253 RAxML 8.2.4. We calculated TC and RTC for the original full dataset and each of the sub-

254 matrices based on this dataset. Trees resulting from the RAxML analysis of each dataset

255 (provided to RAxML with “-t”) and trees based on the corresponding RAxML single-gene trees

256 (provided with "-z") were used. IC was calculated for the original full dataset based on the

257 corresponding RAxML single-gene trees (provided with "-z"). TC, RTC, and IC were calculated

258 under stochastic bipartition adjustment (excluding conflicting bipartitions).

259

260 Finally, we sought to confirm species identification by extracting cytochrome c oxidase subunit

261 I (COI) sequences from all of our newly generated transcriptomes analyzed herein (when

262 present) and conducting a phylogenetic analysis with publicly available tunicate COI

263 sequences. Sequences were manually put in the proper open reading from and were translated

264 to amino acids using the ascidian mitochondrial code in MEGA 7.0.14 (Kumar et al., 2016).

265 Amino acids were aligned with MUSCLE (Edgar, 2004) as implemented in MEGA 7.0.14. A

266 phylogenetic analysis was conducted in RAxML using the approach described above for our

267 concatenated data matrices except the alignment was not partitioned by gene.

268

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269 3. Results

270 Our bioinformatic pipeline resulted in a matrix (“original full dataset”) of 798 genes totaling

271 254,865 amino acid (AA) positions in length (Table 4). The average Alicut-trimmed alignment

272 length was 319 AAs with the longest being 1,326 AAs and the shortest being 55 AAs. All genes

273 were sampled for at least 15 taxa but some were sampled for all 28 taxa with an average of 24

274 taxa sampled per gene. Missing data in the original full dataset was 22.57% (i.e., 77.43% matrix

275 occupancy).

276

277 We conducted ML analyses on all twelve data sets (Figure 1, Supplementary Figures 1-23) and

278 BI analyses on the two smallest data sets: RCFV_50 and LB_50 (Figure 1). All ML and BI

279 analyses recovered Olfactores (Vertebrata + Tunicata) and Tunicata with maximal support

280 (bootstrap support, bs = 100 and posterior probability, pp = 1.00). Within Tunicata, all analyses

281 recovered the appendicularian Oikopleura dioica as the sister taxon to the rest of Tunicata with

282 maximal bootstrap support. Of significance, all ML and BI analyses recovered Thaliacea within

283 “Ascidiacea” with maximal support. Specifically, we consistently recovered Stolidobranchia

284 sister to a clade in which Thaliacea was sister to Phlebobranchia and Aplousobranchia. However,

285 a monophyletic Phlebobranchia was only recovered in the analyses of RCFV_50 (Figure 1,

286 Supplementary Figures 1-3) and LB_100 (Supplementary Figures 9-10). Bootstrap support for

287 Phlebobranchia was weak to moderate in the ML analyses recovering it, but this group was

288 strongly supported by posterior probabilities in the BI analysis of RCFV_50 (pp = 0.99). In the

289 BI analysis of LB_50 and all other ML analyses, phlebobranch tunicates were recovered

290 paraphyletic with respect to Aplousobranchia. BI analysis of LB_50 recovered Corella sister to

291 Aplousobranchia with strong support (pp = 0.99) but the ML analysis of this matrix weakly

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292 supported this relationship. Most other ML analyses recovered Ascidia + Corella sister to

293 Aplousobranchia. ML Bootstrap support for relationships among phlebobranchs and

294 Aplousobranchia varied, but as more genes (with increasingly poor average per-taxon RCFV or

295 LB scores) were sampled, support for phlebobranch paraphyly increased.

296

297 Aside from nodes dealing with placement of and relationships among phlebobranch tunicates, all

298 other major tunicate clades as well as relationships within them were consistently recovered and

299 maximally supported in all analyses. Molgulididae was recovered sister to the rest of

300 Stolidobranchia with Halocynthia (Pyuridae) sister to Styelidae. Within Styelidae, Styela was

301 recovered sister to Botryllus + Botrylloides. All analyses recovered Thaliacea monophyletic with

302 Pyrosomella sister to a clade consisting of the salps Iasis (=Weelia) and Salpa. Likewise, all

303 analyses recovered Aplousobranchia monophyletic.

304

305 ML analyses of a data matrix assembled in the same manner as the original full dataset but with

306 the addition of transcriptome data from Clavelina lepadiformis (Aplousobranchia) and Salpa

307 thompsoni (Thaliacea; “full dataset+2”; Supplementary Figure 21) resulted in the same general

308 branching order as the analysis of the original full dataset. C. lepadiformis was recovered sister

309 to the remaining Aplousobranchia with maximal support, and Aplousobranchia was recovered

310 within Phlebobranchia. S. thompsoni was recovered sister to S. fusiformis with maximal

311 support. Likewise, analyses of datasets with reduced compositional heterogeneity

312 (RCFV_50+2; Supplementary Figure 22) and branch-length heterogeneity (LB_50+2;

313 Supplementary Figure 23) including these taxa also reflected the topologies recovered in our

314 original analyses without the addition of these data.

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315

316 ML analyses based on reduced subsets of genes had higher TC and RTC scores than the analysis

317 of the original full dataset (Table 4), which had a TC of 8.76 and an RTC of 0.35. The dataset

318 with the highest tree certainty and relative tree certainty, LB_100, had a TC of 16.14 an RTC of

319 0.65. Among the smallest two data sets, RCFV_50 (TC = 13.81, RC = 0.55) had slightly lower

320 values than LB_50 (TC = 13.95 and RC = 0.56). We also examined internode certainty (IC)

321 values for the original full dataset (Supplementary Figure 24). IC values were generally low to

322 very low. Notably, the node nesting Aplousobranchia within Phlebobranchia received zero

323 support.

324

325 To search for evidence of overlooked paralogy or contamination that could explain the

326 inconsistencies observed among analyses, we screened the original full dataset (without the

327 addition of Clavelina lepadiformis or Salpa thompsoni) with Phylo-MCOA. This software did

328 not identify any taxa or genes as “complete outliers” (i.e., taxa with contamination or genes with

329 overlooked paralogs) and no individual sequences were identified as outliers (i.e., paralogs;

330 Supplementary Figures 25-26).

331

332 Our taxonomic identifications were generally confirmed to at least the genus-level by comparing

333 COI sequences derived from our transcriptomes to publicly available data. Although COI was

334 not recovered in all of our transcriptomes, placement of COI sequences in the tree

335 (Supplementary Figure 27; also see Supplementary Data on Dryad) was consistent with our

336 identifications and the current taxonomy of the sampled taxa in most cases. The only exception

337 was for the Antarctic tunicate we identified as Ascidia sp. in the field. The COI sequence derived

15 bioRxiv preprint doi: https://doi.org/10.1101/236505; this version posted December 19, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

338 from from this tunicate clustered with Phallusia, which is in the same family as Ascidia, but it is

339 unclear if we misidentified the sampled specimen or if there is a taxonomic problem with these

340 genera. Unfortunately the specimen was destroyed in the field to sample internal tissues for

341 molecular work.

342

343 4. Discussion

344 4.1 Higher-level tunicate phylogeny

345 Our phylogenomic analyses recovered Appendicularia sister to all other tunicates and confirm

346 that thaliaceans are derived ascidians. Stolidobranchia is monophyletic and sister to a clade that

347 encompasses all other ascidians and thaliaceans. However, monophyly of Phlebobranchia is

348 ambiguous in our analyses. Based on these results, the traditional groupings of higher level

349 tunicate taxa should be revisited as it is not surprising that features such as benthic versus pelagic

350 lifestyle, solitary versus colonial habit, or structure of feeding apparatuses are evolutionarily

351 plastic. Importantly, our results show that “Ascidiacea” is not monophyletic as traditionally

352 defined. Thus, this term should be abandoned as a formal taxonomic name as it represents a

353 paraphyletic group of benthic tunicates, or it should be redefined to also include Thaliacea.

354

355 Of particular interest, the taxonomic composition of Phlebobranchia has not been well

356 circumscribed. Cionidae, which includes the widely-studied model species Ciona intestinalis,

357 Ciona robusta and Ciona savignyi, was originally included within Phlebobranchia (Berrill,

358 1936), but this family is viewed as a subclade of Aplousobranchia by some taxonomists (e.g.,

359 Kott, 1969, 1990). This family was recovered sister to all other Aplousobranchia in an analysis

360 of mitochondrial cytochrome c oxidase subunit I (COI) by Turon and López-Legentil (2004) and

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361 biochemical analyses of vanadium oxidation state by Hawkins et al. (1983) also suggest a close

362 relationship of Cionidae to Aplousobranchia. In contrast, none of our analyses recovered

363 Cionidae within or sister to Aplousobranchia, even though Phlebobranchia was recovered

364 paraphyletic with respect to Aplousobranchia in some analyses. Conclusions about the

365 monophyly of Phlebobranchia are difficult to make based on the analyses presented herein as

366 some trees strongly support the paraphyly of this group and others recover it monophyletic.

367 Interestingly, the dataset with the highest TC and RTC, LB_100, was one of just two data sets

368 that resulted in trees recovering Phlebobranchia monophyletic.

369

370 One challenge of previous molecular phylogenetic studies of ascidians has been that

371 aplousobranchs show elevated rates of nucleotide substitution in ribosomal and mitochondrial

372 genes when compared to most other tunicates, leading to concerns about long branch artifacts

373 (e.g., Turon and López-Legentil, 2004; Tsagkogeorga et al., 2009). Many species of

374 aplousobranchs have rapidly-evolving 18S genes with large insertions in multiple parts of the

375 molecule when compared to other tunicates. However, in all of our reconstructed trees based on

376 nuclear protein-coding genes, the sampled aplousobranchs have comparable branch lengths to

377 most other tunicates. Notably, species of Clavelina and Distaplia sampled in the analysis of 18S

378 by Tsagkogeorga et al. (2009) were among the shortest-branched aplousobranch ascidians in that

379 study. However, they also sampled a species of Cystodites, which was a rather long-branched

380 taxon in that analysis, suggesting that evolutionary rates of 18S and nuclear protein-coding genes

381 differ in this lineage.

382

383 4.2 Thaliaceans evolved from a benthic ancestor

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384 Traditionally, tunicates were classified among three classes with the pelagic Thaliacea

385 (pyrosomes, salps, and doliolids) considered a distinct clade from Ascidiacea (e.g., Ruppert et al.

386 2004, Brusca et al., 2016). All of our results strongly support placement of Thaliacea within the

387 traditional class Ascidiacea as the sister group of Phlebobranchia + Aplousobranchia as

388 recovered by Swalla et al. (2000) and Stach and Turbeville (2002) with 18S rDNA and as hinted

389 at by analyses of 18S by Tsagkogeorga et al. (2009). These results suggest a greater degree of

390 lability in the evolution of benthic versus pelagic lifestyles than traditionally recognized. Given

391 that early cephalochordates, early vertebrates, and larvaceans were swimming organisms (Mallatt

392 and Chen, 2003), a benthic lifestyle must have evolved in the last common ancestor of the

393 ascidian-thaliacean clade. Subsequent to this change in lifestyle, Thaliacea, which is nested well-

394 within a clade of otherwise benthic ascidians, reacquired a pelagic lifestyle, supporting previous

395 assertions that these pelagic tunicates evolved from a benthic ancestor (Swalla et al. 2000).

396

397 Historically, Ascidiacea was classified on the basis of the relative position of the gonads with

398 Enterogona including Aplousobranchia and Phlebobranchia, who have gonads closely associated

399 with the gut, and Pleurogona consisting of Stolidobranchia, who generally have gonads distinct

400 from the gut (Garstang, 1928; Perrier, 1898). Thaliacea have gonads associated with the gut, like

401 Aplousobranchia and Phelobobranchia, reinforces the utility of this morphological character that

402 defined Enterogona as noted by Tsagkogeorga et al. (2009).

403

404 4.3 The phylogenetic position of Appendicularia

405 Our results are consistent with previous studies recovering Appendicularia outside of

406 “Ascidiacea” (Govindarajan et al., 2011; Stach and Turbeville, 2002; Swalla et al., 2000; Wada,

18 bioRxiv preprint doi: https://doi.org/10.1101/236505; this version posted December 19, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

407 1998). In studies based on markers such as 18S rDNA (e.g., Govindarajan et al., 2011; Swalla et

408 al., 2000; Tsagkogeorga et al., 2009) these unusual pelagic tunicates tend to have somewhat

409 elevated nucleotide substitution rates and in, phylogenomic analyses (e.g., Delsuc et al., 2006,

410 2008; this study), Oikopleura, the only larvacean from which genomic or transcriptomic data are

411 available, is an extremely long-branched taxon. Thus, recovery of Appendicularia as the sister

412 group of all other tunicates has been questioned as a possible result of long-branch attraction

413 (Swalla et al. 2000). The most recent study examining tunicate phylogeny with the broadest

414 taxon sampling of 18S to date recovered this unusual group within Ascidiacea as the sister group

415 of Stolidobranchia (Tsagkogeorga et al., 2009). However, to further complicate the issue,

416 Appendicularia and Molgulidae tend to have AT-rich 18S sequences, and this shared

417 compositional heterogeneity could be causing an artefactual attraction of these two taxa

418 (Tsagkogeorga et al., 2009).

419

420 The branch leading to Oikopleura was noticeably longer from root to tip than all other taxa in

421 our analyses of all genes retained by our pipeline and data sets with reduced compositional

422 heterogeneity. However, as data sets with fewer but ‘better’ genes according to branch-length

423 heterogeneity were analyzed, the branch leading to Oikopleura decreased in length relative to

424 other sampled taxa, and was even shorter than the branches leading to Salpa and Iasis in both the

425 ML and BI analyses of LB_50. Given our consistent recovery of Appendicularia as sister to the

426 rest of Tunicata with maximal support even when compositional heterogeneity and long-branch

427 attraction are reduced, we consider Appendicularia to be an early-branching tunicate lineage and

428 not a derived ascidian clade as previously hypothesized.

429

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430 4.4 Future directions

431 This study represents a first step towards resolving tunicate phylogeny using genomic data, but

432 greatly improved taxon sampling will be needed to begin to gain a full picture of the

433 evolutionary history of this diverse and important group. For example, Oikopleuridae is one of

434 three families of Appendicularia but the only family included in any molecular phylogenetic

435 investigation to date. Likewise, Sorberacea is an enigmatic clade of benthic, deep-sea tunicates

436 that has been considered to be a separate class from other tunicates (Monniot et al., 1975).

437 Whether this clade is indeed a distinct lineage of Tunicata or yet another derived ascidian lineage

438 has never been tested with any source of molecular data, let alone phylogenomics. Moreover,

439 many questions about tunicate evolutionary history at family level have been challenging to

440 address, particularly within Aplousobranchia, which exhibits extreme branch-length

441 heterogeneity for 18S rDNA. As most tunicates in the present study exhibit more-or-less

442 comparable branch lengths, especially when steps are taken to exclude genes with exceptional

443 branch-length heterogeneity scores, phylogenomics appears well-suited to address these

444 evolutionary questions.

445

446 Acknowledgements

447 This study was supported by University of Alabama start-up funds to KMK. We thank Alison

448 Sweeney and James Townsend for facilitating collection of specimens of Thaliacea and the

449 faculty and staff of Friday Harbor Labs for facilitating collection of Pacific tunicates. The crew

450 of the R/V Nathaniel B. Palmer and R/V Lawrence M. Gould facilitated collection of Antarctic

451 tunicates through NSF funds to KMH (ANT-1043745 and OPP-0132032). This study is based in

452 part upon work by BJS supported by the National Science Foundation under Cooperative

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453 Agreement No. DBI-0939454. Any opinions, findings, and conclusions or recommendations

454 expressed in this material are those of the author(s) and do not necessarily reflect the views of

455 the National Science Foundation. We thank Justin Cox and Emily Pabst for help with RNA

456 extractions. We thank Deb Crocker and Robert Griffin for assistance with the University of

457 Alabama High Performance Computing (UAHPC) system. This is Auburn University Marine

458 Biology Program contribution #169 and Molette Lab contribution #73.

459

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624 Yokobori, S. ichi, Kurabayashi, A., Neilan, B.A., Maruyama, T., Hirose, E., 2006. Multiple

625 origins of the ascidian-Prochloron symbiosis: Molecular phylogeny of photosymbiotic and

626 non-symbiotic colonial ascidians inferred from 18S rDNA sequences. Mol. Phylogenet.

627 Evol. 40, 8–19. doi:10.1016/j.ympev.2005.11.025

628 Zeng, L., Jacobs, M.W., Swalla, B.J., 2006. Coloniality has evolved once in Stolidobranch

629 Ascidians. Integr. Comp. Biol. 46, 255–268. doi:10.1093/icb/icj035

630 Zeng, L., Swalla, B.J., 2005. Molecular phylogeny of the protochordates: chordate evolution.

631 Can. J. Zool. 83, 24–33. doi:10.1139/z05-010

632 Zhong, B., Deusch, O., Goremykin, V. V., Penny, D., Biggs, P.J., Atherton, R.A., Nikiforova, S.

633 V., Lockhart, P.J., 2011. Systematic error in seed plant phylogenomics. Genome Biol. Evol.

634 3, 1340–1348. doi:10.1093/gbe/evr105

635

28 bioRxiv preprint doi: https://doi.org/10.1101/236505; this version posted December 19, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

637 Figure legends

638 Figure 1. Phylogeny of Tunicata as inferred in the present study. A. Consensus phylogram from

639 the Bayesian inference analysis of RCFV_50 with bootstrap support values from RAxML and

640 IQ-TREE ML analyses of RCFV_50, RCFV_100, RCFV_200, RCFV_500, and the original full

641 dataset shown. B. Consensus phylogram from the Bayesian inference analysis of LB_50 with

642 bootstrap support values from RAxML and IQ-TREE ML analyses of LB_50, LB_100, LB_200,

643 LB_500, and the original full dataset shown. Nodes without support matrices received maximal

644 support in all BI and ML analyses. For the IQ-TREE analyses of RCFV_50 and LB_50, both the

645 Phylobayes (PB) and RAxML (ML) topologies were tested as guide trees and bootstrap support

646 values resulting from these analyses are presented above and below the diagonal line in the

647 bottom left cells of the support matrices, respectively. Dashes in support matrices indicate that a

648 relationship was not recovered. Scale bars represent 0.1 substitutions per site. Corresponding ML

649 tree topologies are presented in Supplementary Figures 1-22.

650

651 Supplementary Figure 1. Phylogeny of Tunicata based on the RAxML analysis of the best 50

652 genes according to RCFV. Bootstrap support values are presented at each node. Scale bar

653 represents 0.1 substitutions per site.

654

655 Supplementary Figure 2. Phylogeny of Tunicata based on the IQ-TREE analysis of the best 50

656 genes according to RCFV with the RAxML tree used as the guide tree. Bootstrap support values

657 are presented at each node. Scale bar represents 0.1 substitutions per site.

658

29 bioRxiv preprint doi: https://doi.org/10.1101/236505; this version posted December 19, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

659 Supplementary Figure 3. Phylogeny of Tunicata based on the IQ-TREE analysis of the best 50

660 genes according to RCFV with the Phylobayes tree used as the guide tree. Bootstrap support

661 values are presented at each node. Scale bar represents 0.1 substitutions per site.

662

663 Supplementary Figure 4. Phylogeny of Tunicata based on the RAxML analysis of the best 50

664 genes according to LB. Bootstrap support values are presented at each node. Scale bar represents

665 0.1 substitutions per site.

666

667 Supplementary Figure 5. Phylogeny of Tunicata based on the IQ-TREE analysis of the best 50

668 genes according to LB with the RAxML tree used as the guide tree. Bootstrap support values are

669 presented at each node. Scale bar represents 0.1 substitutions per site.

670

671 Supplementary Figure 6. Phylogeny of Tunicata based on the IQ-TREE analysis of the best 50

672 genes according to LB with the Phylobayes tree used as the guide tree. Bootstrap support values

673 are presented at each node. Scale bar represents 0.1 substitutions per site.

674 Supplementary Figure 7. Phylogeny of Tunicata based on the RAxML analysis of the best 100

675 genes according to RCFV. Bootstrap support values are presented at each node. Scale bar

676 represents 0.1 substitutions per site.

677

678 Supplementary Figure 8. Phylogeny of Tunicata based on the IQ-TREE analysis of the best

679 100 genes according to RCFV. Bootstrap support values are presented at each node. Scale bar

680 represents 0.1 substitutions per site.

681

30 bioRxiv preprint doi: https://doi.org/10.1101/236505; this version posted December 19, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

682 Supplementary Figure 9. Phylogeny of Tunicata based on the RAxML analysis of the best 100

683 genes according to LB. Bootstrap support values are presented at each node. Scale bar represents

684 0.1 substitutions per site.

685

686 Supplementary Figure 10. Phylogeny of Tunicata based on the IQ-TREE analysis of the best

687 100 genes according to LB. Bootstrap support values are presented at each node. Scale bar

688 represents 0.1 substitutions per site.

689

690 Supplementary Figure 11. Phylogeny of Tunicata based on the RAxML analysis of the best

691 200 genes according to RCFV. Bootstrap support values are presented at each node. Scale bar

692 represents 0.1 substitutions per site.

693

694 Supplementary Figure 12. Phylogeny of Tunicata based on the IQ-TREE analysis of the best

695 200 genes according to RCFV. Bootstrap support values are presented at each node. Scale bar

696 represents 0.1 substitutions per site.

697

698 Supplementary Figure 13. Phylogeny of Tunicata based on the RAxML analysis of the best

699 200 genes according to LB. Bootstrap support values are presented at each node. Scale bar

700 represents 0.1 substitutions per site.

701

702 Supplementary Figure 14. Phylogeny of Tunicata based on the IQ-TREE analysis of the best

703 200 genes according to LB. Bootstrap support values are presented at each node. Scale bar

704 represents 0.1 substitutions per site.

31 bioRxiv preprint doi: https://doi.org/10.1101/236505; this version posted December 19, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

705

706 Supplementary Figure 15. Phylogeny of Tunicata based on the RAxML analysis of the best

707 500 genes according to RCFV. Bootstrap support values are presented at each node. Scale bar

708 represents 0.1 substitutions per site.

709

710 Supplementary Figure 16. Phylogeny of Tunicata based on the IQ-TREE analysis of the best

711 500 genes according to RCFV. Bootstrap support values are presented at each node. Scale bar

712 represents 0.1 substitutions per site.

713

714 Supplementary Figure 17. Phylogeny of Tunicata based on the RAxML analysis of the best

715 500 genes according to LB. Bootstrap support values are presented at each node. Scale bar

716 represents 0.1 substitutions per site.

717

718 Supplementary Figure 18. Phylogeny of Tunicata based on the IQ-TREE analysis of the best

719 500 genes according to LB. Bootstrap support values are presented at each node. Scale bar

720 represents 0.1 substitutions per site.

721

722 Supplementary Figure 19. Phylogeny of Tunicata based on the RAxML analysis of the original

723 full dataset (798 genes). Bootstrap support values are presented at each node. Scale bar

724 represents 0.1 substitutions per site.

725

32 bioRxiv preprint doi: https://doi.org/10.1101/236505; this version posted December 19, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

726 Supplementary Figure 20. Phylogeny of Tunicata based on the IQ-TREE analysis of the

727 original full dataset (798 genes). Bootstrap support values are presented at each node. Scale bar

728 represents 0.1 substitutions per site.

729

730 Supplementary Figure 21. Phylogeny of Tunicata based on the RAxML analysis of the subset

731 of the best 50 genes in the original full dataset according to RCFV retained by our pipeline after

732 the addition of Clavelina lepadiformis and Salpa thompsoni (47 genes). Bootstrap support values

733 are presented at each node. Scale bar represents 0.1 substitutions per site.

734

735 Supplementary Figure 22. Phylogeny of Tunicata based on the RAxML analysis of the subset

736 of the best 50 genes in the original full dataset according to LB retained by our pipeline after the

737 addition of Clavelina lepadiformis and Salpa thompsoni (48 genes). Bootstrap support values are

738 presented at each node. Scale bar represents 0.1 substitutions per site.

739

740 Supplementary Figure 23. Phylogeny of Tunicata based on the RAxML analysis of all genes

741 retained by our pipeline (788) after the addition of Clavelina lepadiformis and Salpa thompsoni.

742 Bootstrap support values are presented at each node. Scale bar represents 0.1 substitutions per

743 site.

744

745 Supplementary Figure 24. Phylogeny of Tunicata based on the RAxML analysis of all 798

746 genes with internode certainty scores presented at each node.

747

748 Supplementary Figure 25. Phylo-MCOA matrix.

33 bioRxiv preprint doi: https://doi.org/10.1101/236505; this version posted December 19, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

749

750 Supplementary Figure 26. Phylo-MCOA charts.

751

752 Supplementary Figure 27. Phylogenetic analysis of COI. Bootstrap support values are

753 presented at each node. Scale bar represents 0.1 substitutions per site. Tree presented as rooted

754 by RAxML but should be interpreted as unrooted.

755

756

757

758

759

760

761

762

763

764

765

766

767

768

769

770

771

34 bioRxiv preprint doi: https://doi.org/10.1101/236505; this version posted December 19, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

772 Table 1. Gross morphology and life history features of major tunicate clades

Taxon Benthic/Pelagic Solitary/Colonial Branchial sac Gonad position

Appendicularia Pelagic Solitary None Dorsal Phlebobranchia Benthic Usually solitary Vascular Enterogona1 Aplousobranchia Benthic Colonial Simple Enterogona1 Stolidobranchia Benthic Solitary or Colonial Folded Pleurogona2 Thaliacea Pelagic Solitary or Colonial Simple Enterogona1 773 1Unpaired gonads are situated on one side or behind the intestinal loop. 2Gonads are in the lateral mantle wall on both sides.

774

775 Table 2. Specimen collection data.

Species Collection locality Latitude Longitude Tissue extracted

Ascidia sp. Ross Sea, Antarctica 78°03'47.7"S 169°59'28.1"W Gonad Corella willmeriana Roche Harbor, San Juan Island, WA 48°36'34.6"N 123°09'18.8"W Gonad and branchial sac Distaplia occidentalis Roche Harbor, San Juan Island, WA 48°36'34.6"N 123°09'18.8"W Entire small colony Pyrosomella verticillata Northwestern Atlantic 37°45'46.8"N 73°38'37.8"W Several entire zooids Salpa fusiformis Northwestern Atlantic 39°15'07"N 71°56'24"W Gonad and branchial sac Styela gibbsii Friday Harbor, San Juan Island, WA 48°32'43.2"N 123°00'43.2"W Gonad Iasis (=Weelia) cylindrica Northwestern Atlantic 37°41'05"N 73°37'07"W Gonad and branchial sac 776

777 Table 3. Taxon sampling, number of HaMStR orthologous groups (OGs) recovered for each taxon (out of 1,031), and sources of data

778 used in phylogenomic analyses.

HaMStR Species Clade Abbrev. OGs Data source (Accession/URL/etc.) Acanthaster planci Echinodermata AAUS 991 http://marinegenomics.oist.jp/cots/download/gbr-

35 bioRxiv preprint doi: https://doi.org/10.1101/236505; this version posted December 19, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

cotsv1.0.EVM2.prot.gz Ascidia sp. Tunicata ASCI 961 SRR6363557 Asymmetron lucayanum Cephalochordata ALUC 995 NCBI TSA GESY00000000.1 Botrylloides leachi Tunicata BLEA 941 NCBI SRA SRR2641167, SRR2729871, & SRR2729872 http://botryllus.stanford.edu/botryllusgenome/download/start_st Botryllus schlosseri Tunicata BSCH 824 op_transcripts30.fa Branchiostoma belcheri Cephalochordata BBEL 1008 http://genome.bucm.edu.cn/lancelet/download_data.php Branchiostoma floridae Cephalochordata BFLO 1006 http://genome.jgi.doe.gov/Brafl1/Brafl1.home.html Ciona intestinalis Tunicata CINT 1031 HaMStR model organisms core ortholog set Ciona robusta Tunicata CROB 987 NCBI SRA SRR3953074, SRR3953075, & SRR3953076 http://www.aniseed.cnrs.fr/aniseed/download/?file=data%2Fcs Ciona savignyi Tunicata CSAV 933 %2Fciona_savignyi_transcripts_gff3_fasta.zip Corella willmeriana Tunicata CWIL 958 SRR6363558 Cystodites dellechiajei Tunicata CDEL 996 NCBI SRA SRR1324903 Distaplia occidentalis Tunicata DIST 993 SRR6363555 http://www.aniseed.cnrs.fr/aniseed/download/?file=data%2Fhr Halocynthia roretzi Tunicata HROR 853 %2Fhalocynthia_roretzi_est_fasta.zip Homo sapiens Vertebrata HSAP 1031 HaMStR model organisms core ortholog set ftp://ftp.ensembl.org/pub/release- Latimeria chalumnae Vertebrata LCHA 995 87/fasta/latimeria_chalumnae/pep/ Molgula occidentalis Tunicata OCCI 383 http://www.aniseed.cnrs.fr/aniseed/download/download_data Molgula occulta Tunicata OCCU 876 http://www.aniseed.cnrs.fr/aniseed/download/download_data http://www.aniseed.cnrs.fr/aniseed/download/?file=data%2Fmo Molgula oculata Tunicata OCUL 915 ocul%2Fmolgula_oculata_transcripts_gff3_fasta.zip Oikopleura dioica Tunicata ODIO 924 http://oikoarrays.biology.uiowa.edu/Oiko/Downloads.html ftp://ftp.ensembl.org/pub/release- Petromyzon marinus Vertebrata PMAR 739 87/fasta/petromyzon_marinus/pep/ Ptychodera flava Hemichordata PFLA 870 http://octopus.unit.oist.jp/HEMIDATA/pfl.prot Pyrosomella Tunicata PVER 979 SRR6363556 verticillata Saccoglossus Hemichordata SKOW 999 http://octopus.unit.oist.jp/HEMIDATA/sko.prot kowalevskii Salpa fusiformis Tunicata SFUS 980 SRR6363561

36 bioRxiv preprint doi: https://doi.org/10.1101/236505; this version posted December 19, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Strongylocentrotus http://www.echinobase.org/Echinobase/SpDownloads/SPU_pep Echinodermata SPUR 1016 purpuratus tide.fasta.zip Styela gibbsii Tunicata SGIB 1010 SRR6363562 Iasis (=Weelia) Tunicata WCYL 984 SRR6363560 cylindrica

37 bioRxiv preprint doi: https://doi.org/10.1101/236505; this version posted December 19, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

780 Table 4. Length, percent missing data, tree certainty (TC), and relative tree certainty (RTC) for

781 each data matrix analyzed.

Genes Taxa Length Missing TC RTC Data matrix (AAs) data Original full dataset 798 28 256,609 22.6% 8.76 0.35 RCFV_50 50 28 31,260 23.0% 13.81 0.55 RCFV_100 100 28 54,689 22.0% 14.22 0.57 RCFV_200 200 28 99,576 21.9% 10.14 0.41 RCFV_500 500 28 196,200 21.9% 9.78 0.39 LB_50 50 28 15,203 24.6% 13.95 0.56 LB_100 100 28 32,776 23.4% 16.14 0.65 LB_200 200 28 66,032 22.6% 16.02 0.64 LB_500 500 28 166,734 22.4% 11.73 0.47 Full dataset+2 788 30 258,910 22.6% RCFV_50+2 47 30 29,831 22.6% LB_50+2 48 30 14,990 24.4% 782

38 A 0.96 66 - - - - RCFV Ciona robusta Phylobayes 76 - - - - 50 76 Ciona intestinalis 0.96 Ciona savignyi Phlebobranchia RCFV RCFV All 88 82 86 100 100 Corella willmeriana RAxML RCFV RCFV 87 Ascidia sp. 50 100 200 500 798 86 83 97 100 100 Distaplia occidentalis Cystodites dellechiajei Aplousobranchia RCFV 50 (PB) RCFV RCFV RCFV All Pyrosomella verticillata IQ-TREE Weelia cylindrica Thaliacea RCFV 100 200 500 798 Salpa fusiformis 50 (ML) Molgula oculata Molgula occulta Molgula occidentalis bioRxiv preprint doi: https://doi.org/10.1101/236505; this version posted DecemberHalocynthia 19, 2017. The copyrightroretzi holder for this preprintStolidobranchia (which was not certified by peer review) is the author/funder. All rights reserved.Styela Nogibbsii reuse allowed without permission. Botryllus schlosseri Botrylloides leachi Oikopleura dioica Petromyzon marinus Latimeria chalumnae Appendicularia Homo sapiens Asymmetron lucayanum Branchiostoma floridae Branchiostoma belcheri Acanthaster planci Strongylocentrotus purpuratus Saccoglossus kowalevskii Ptychodera flava

0.95 B 47 - - - - 86 - - - - 87 Ciona robusta Ciona intestinalis 0.99 Ciona savignyi Phlebobranchs 40 -- -- Ascidia sp. Corella willmeriana 84 - - - - 85 Distaplia occidentalis Cystodites dellechiajei Aplousobranchia Pyrosomella verticillata Weelia cylindrica Thaliacea Salpa fusiformis Molgula oculata Molgula occulta Molgula occidentalis Halocynthia roretzi Styela gibbsii Stolidobranchia Botryllus schlosseri Botrylloides leachi Oikopleura dioica Petromyzon marinus Latimeria chalumnae Appendicularia Homo sapiens Asymmetron lucayanum Branchiostoma floridae Phylobayes LB Branchiostoma belcheri 50 Acanthaster planci Strongylocentrotus purpuratus LB LB All Saccoglossus kowalevskii RAxML LB LB Ptychodera flava 50 100 200 500 798

LB 50 (PB) LB LB LB All IQ-TREE LB 100 200 500 798 50 (ML) bioRxiv preprint doi: https://doi.org/10.1101/236505; this version posted December 19, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

100 Ciona robusta 100 Ciona intestinalis 66 Ciona savignyi Ascidia sp. 100 88 Corella willmeriana Cystodites dellechiajei 100 100 Distaplia occidentalis Pyrosomella verticillata

100 Salpa fusiformis 100 100 Weelia cylindrica

100 Molgula oculata 100 Molgula occulta Molgula occidentalis 100 100 Halocynthia roretzi Styela gibbsii 100 100 Botrylloides leachi 100 100 Botryllus schlosseri Oikopleura dioica Petromyzon marinus

100 Latimeria chalumnae 100 Homo sapiens Asymmetron lucayanum

100 Branchiostoma belcheri 100 Branchiostoma floridae

100 Strongylocentrotus purpuratus Acanthaster planci 100 Ptychodera flava 100 Saccoglossus kowalevskii

0.050 bioRxiv preprint doi: https://doi.org/10.1101/236505; this version posted December 19, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

100 Ciona robusta

100 Ciona intestinalis

76 Ciona savignyi

Corella willmeriana 100 87 Ascidia sp.

Cystodites dellechiajei 100 100 Distaplia occidentalis

Pyrosomella verticillata

100 Salpa fusiformis

100 100 Weelia cylindrica

100 Molgula oculata

100 Molgula occulta

Molgula occidentalis

100 100 Halocynthia roretzi

100 Styela gibbsii

100 Botryllus schlosseri 100 100 Botrylloides leachi

Oikopleura dioica

Petromyzon marinus

100 Homo sapiens

100 Latimeria chalumnae

Asymmetron lucayanum

100 Branchiostoma floridae

100 Branchiostoma belcheri

100 Strongylocentrotus purpuratus

Acanthaster planci

100 Ptychodera flava

100 Saccoglossus kowalevskii

0.050 bioRxiv preprint doi: https://doi.org/10.1101/236505; this version posted December 19, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

100 Ciona robusta

100 Ciona intestinalis

76 Ciona savignyi

Corella willmeriana 100 86 Ascidia sp.

Cystodites dellechiajei 100 100 Distaplia occidentalis

Pyrosomella verticillata

100 Salpa fusiformis

100 100 Weelia cylindrica

100 Molgula oculata

100 Molgula occulta

Molgula occidentalis

100 100 Halocynthia roretzi

100 Styela gibbsii

100 Botryllus schlosseri 100 100 Botrylloides leachi

Oikopleura dioica

Petromyzon marinus

100 Homo sapiens

100 Latimeria chalumnae

Asymmetron lucayanum

100 Branchiostoma floridae

100 Branchiostoma belcheri

100 Strongylocentrotus purpuratus

Acanthaster planci

100 Ptychodera flava

100 Saccoglossus kowalevskii

0.050 bioRxiv preprint doi: https://doi.org/10.1101/236505; this version posted December 19, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

100 Ciona robusta 100 Ciona intestinalis 47 Ciona savignyi

100 Ascidia sp. Corella willmeriana

100 40 Cystodites dellechiajei 100 Distaplia occidentalis Pyrosomella verticillata

100 Salpa fusiformis 100 100 Weelia cylindrica

100 Molgula oculata 100 Molgula occulta Molgula occidentalis 100 100 Halocynthia roretzi Styela gibbsii 100 100 Botrylloides leachi 100 100 Botryllus schlosseri Oikopleura dioica Petromyzon marinus

100 Homo sapiens 100 Latimeria chalumnae Asymmetron lucayanum

100 Branchiostoma floridae 100 Branchiostoma belcheri

100 Saccoglossus kowalevskii Ptychodera flava 100 Acanthaster planci 100 Strongylocentrotus purpuratus

0.10 bioRxiv preprint doi: https://doi.org/10.1101/236505; this version posted December 19, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

100 Ciona intestinalis

100 Ciona robusta

87 Ciona savignyi

100 Ascidia sp.

Corella willmeriana

85 Distaplia occidentalis 100 100 Cystodites dellechiajei

Pyrosomella verticillata

100 Salpa fusiformis

100 100 Weelia cylindrica

100 Molgula oculata

100 Molgula occulta

Molgula occidentalis

100 100 Halocynthia roretzi

100 Styela gibbsii

100 Botryllus schlosseri 100 100 Botrylloides leachi

Oikopleura dioica

Petromyzon marinus

100 Homo sapiens

100 Latimeria chalumnae

Asymmetron lucayanum

100 Branchiostoma floridae

100 Branchiostoma belcheri

100 Acanthaster planci

Strongylocentrotus purpuratus

100 Saccoglossus kowalevskii

100 Ptychodera flava

0.10 bioRxiv preprint doi: https://doi.org/10.1101/236505; this version posted December 19, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

100 Ciona intestinalis

100 Ciona robusta

86 Ciona savignyi

100 Ascidia sp.

Corella willmeriana

84 Distaplia occidentalis 100 100 Cystodites dellechiajei

Pyrosomella verticillata

100 Salpa fusiformis

100 100 Weelia cylindrica

100 Molgula oculata

100 Molgula occulta

Molgula occidentalis

100 100 Halocynthia roretzi

100 Styela gibbsii

100 Botryllus schlosseri 100 100 Botrylloides leachi

Oikopleura dioica

Petromyzon marinus

100 Homo sapiens

100 Latimeria chalumnae

Asymmetron lucayanum

100 Branchiostoma floridae

100 Branchiostoma belcheri

100 Acanthaster planci

Strongylocentrotus purpuratus

100 Saccoglossus kowalevskii

100 Ptychodera flava

0.10 bioRxiv preprint doi: https://doi.org/10.1101/236505; this version posted December 19, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

100 Distaplia occidentalis

52 Cystodites dellechiajei Ascidia sp. 100 82 Corella willmeriana Ciona savignyi

100 100 Ciona robusta 100 Ciona intestinalis Pyrosomella verticillata

100 Salpa fusiformis 100 100 Weelia cylindrica

100 Molgula occulta 100 Molgula oculata Molgula occidentalis 100 100 Halocynthia roretzi Styela gibbsii 100 100 Botrylloides leachi 100 100 Botryllus schlosseri Oikopleura dioica Petromyzon marinus

100 Homo sapiens 100 Latimeria chalumnae Asymmetron lucayanum

100 Branchiostoma belcheri 100 Branchiostoma floridae

100 Saccoglossus kowalevskii Ptychodera flava 100 Strongylocentrotus purpuratus 100 Acanthaster planci

0.050 bioRxiv preprint doi: https://doi.org/10.1101/236505; this version posted December 19, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

100 Cystodites dellechiajei

78 Distaplia occidentalis

Ascidia sp.

10083 Corella willmeriana

Ciona savignyi

100 100 Ciona robusta

100 Ciona intestinalis

Pyrosomella verticillata

100 Salpa fusiformis 100 100 Weelia cylindrica

100 Molgula oculata

100 Molgula occulta

Molgula occidentalis 100 100 Halocynthia roretzi

100 Styela gibbsii

100 Botrylloides leachi 100 100 Botryllus schlosseri

Oikopleura dioica

Petromyzon marinus

100 Latimeria chalumnae

100 Homo sapiens

Asymmetron lucayanum

100 Branchiostoma floridae

100 Branchiostoma belcheri

100 Acanthaster planci

Strongylocentrotus purpuratus

100 Saccoglossus kowalevskii

100 Ptychodera flava

0.050 bioRxiv preprint doi: https://doi.org/10.1101/236505; this version posted December 19, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

100 Ciona robusta 100 Ciona intestinalis 50 Ciona savignyi Corella willmeriana 100 67 Ascidia sp. Distaplia occidentalis 100 100 Cystodites dellechiajei Pyrosomella verticillata

100 Salpa fusiformis 100 100 Weelia cylindrica

100 Molgula occulta 100 Molgula oculata Molgula occidentalis 100 100 Halocynthia roretzi Styela gibbsii 100 100 Botryllus schlosseri 100 100 Botrylloides leachi Oikopleura dioica Petromyzon marinus

100 Homo sapiens 100 Latimeria chalumnae Asymmetron lucayanum

100 Branchiostoma floridae 100 Branchiostoma belcheri

100 Ptychodera flava Saccoglossus kowalevskii 100 Acanthaster planci 100 Strongylocentrotus purpuratus

0.10 bioRxiv preprint doi: https://doi.org/10.1101/236505; this version posted December 19, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

100 Ciona robusta

100 Ciona intestinalis

82 Ciona savignyi

Corella willmeriana 100 78 Ascidia sp.

Cystodites dellechiajei 100 100 Distaplia occidentalis

Pyrosomella verticillata

100 Weelia cylindrica

100 100 Salpa fusiformis

100 Molgula occulta

100 Molgula oculata

Molgula occidentalis

100 100 Halocynthia roretzi

100 Styela gibbsii

100 Botrylloides leachi 100 100 Botryllus schlosseri

Oikopleura dioica

Petromyzon marinus

100 Homo sapiens

100 Latimeria chalumnae

Asymmetron lucayanum

100 Branchiostoma floridae

100 Branchiostoma belcheri

100 Ptychodera flava

Saccoglossus kowalevskii

100 Acanthaster planci

100 Strongylocentrotus purpuratus

0.10 bioRxiv preprint doi: https://doi.org/10.1101/236505; this version posted December 19, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

100 Cystodites dellechiajei

86 Distaplia occidentalis Ascidia sp. 100 86 Corella willmeriana Ciona savignyi

100 100 Ciona robusta 100 Ciona intestinalis Pyrosomella verticillata

100 Salpa fusiformis 100 100 Weelia cylindrica

100 Molgula occulta 100 Molgula oculata Molgula occidentalis 100 100 Halocynthia roretzi Styela gibbsii 100 100 Botryllus schlosseri 100 100 Botrylloides leachi Oikopleura dioica Petromyzon marinus

100 Latimeria chalumnae 100 Homo sapiens Asymmetron lucayanum

100 Branchiostoma floridae 100 Branchiostoma belcheri

100 Acanthaster planci Strongylocentrotus purpuratus 100 Saccoglossus kowalevskii 100 Ptychodera flava

0.050 bioRxiv preprint doi: https://doi.org/10.1101/236505; this version posted December 19, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

100 Distaplia occidentalis

100 Cystodites dellechiajei

Ascidia sp. 97 100 Corella willmeriana

Ciona savignyi

100 100 Ciona robusta

100 Ciona intestinalis

Pyrosomella verticillata

100 Weelia cylindrica 100 100 Salpa fusiformis

100 Molgula occulta

100 Molgula oculata

Molgula occidentalis 100 100 Halocynthia roretzi

100 Styela gibbsii

100 Botryllus schlosseri 100 100 Botrylloides leachi

Oikopleura dioica

Petromyzon marinus

100 Homo sapiens

100 Latimeria chalumnae

Asymmetron lucayanum

100 Branchiostoma belcheri

100 Branchiostoma floridae

100 Acanthaster planci

Strongylocentrotus purpuratus

100 Saccoglossus kowalevskii

100 Ptychodera flava

0.050 bioRxiv preprint doi: https://doi.org/10.1101/236505; this version posted December 19, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

70 Ascidia sp.

66 Corella willmeriana Cystodites dellechiajei 100 100 Distaplia occidentalis Ciona savignyi

100 100 Ciona robusta 100 Ciona intestinalis Pyrosomella verticillata

100 Salpa fusiformis 100 100 Weelia cylindrica

100 Molgula oculata 100 Molgula occulta Molgula occidentalis 100 100 Halocynthia roretzi Styela gibbsii 100 100 Botryllus schlosseri 100 100 Botrylloides leachi Oikopleura dioica Petromyzon marinus

100 Latimeria chalumnae 100 Homo sapiens Asymmetron lucayanum

100 Branchiostoma floridae 100 Branchiostoma belcheri

100 Saccoglossus kowalevskii Ptychodera flava 100 Acanthaster planci 100 Strongylocentrotus purpuratus

0.10 bioRxiv preprint doi: https://doi.org/10.1101/236505; this version posted December 19, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

86 Ascidia sp.

98 Corella willmeriana

Cystodites dellechiajei

100 100 Distaplia occidentalis

Ciona savignyi

100 100 Ciona robusta

100 Ciona intestinalis

Pyrosomella verticillata

100 Salpa fusiformis 100 100 Weelia cylindrica

100 Molgula occulta

100 Molgula oculata

Molgula occidentalis 100 100 Halocynthia roretzi

100 Styela gibbsii

100 Botryllus schlosseri 100 100 Botrylloides leachi

Oikopleura dioica

Petromyzon marinus

100 Homo sapiens

100 Latimeria chalumnae

Asymmetron lucayanum

100 Branchiostoma floridae

100 Branchiostoma belcheri

100 Ptychodera flava

Saccoglossus kowalevskii

100 Acanthaster planci

100 Strongylocentrotus purpuratus

0.10 bioRxiv preprint doi: https://doi.org/10.1101/236505; this version posted December 19, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

100 Cystodites dellechiajei

100 Distaplia occidentalis Corella willmeriana 100 100 Ascidia sp. Ciona savignyi

100 100 Ciona intestinalis 100 Ciona robusta Pyrosomella verticillata

100 Salpa fusiformis 100 100 Weelia cylindrica

100 Molgula oculata 100 Molgula occulta Molgula occidentalis 100 100 Halocynthia roretzi Styela gibbsii 100 100 Botrylloides leachi 100 100 Botryllus schlosseri Oikopleura dioica Petromyzon marinus

100 Homo sapiens 100 Latimeria chalumnae Asymmetron lucayanum

100 Branchiostoma belcheri 100 Branchiostoma floridae

100 Ptychodera flava Saccoglossus kowalevskii 100 Acanthaster planci 100 Strongylocentrotus purpuratus

0.10 bioRxiv preprint doi: https://doi.org/10.1101/236505; this version posted December 19, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

100 Corella willmeriana

100 Ascidia sp.

Distaplia occidentalis

100 100 Cystodites dellechiajei

Ciona savignyi

100 100 Ciona robusta

100 Ciona intestinalis

Pyrosomella verticillata

100 Salpa fusiformis 100 100 Weelia cylindrica

100 Molgula oculata

100 Molgula occulta

Molgula occidentalis 100 100 Halocynthia roretzi

100 Styela gibbsii

100 Botrylloides leachi 100 100 Botryllus schlosseri

Oikopleura dioica

Petromyzon marinus

100 Latimeria chalumnae

100 Homo sapiens

Asymmetron lucayanum

100 Branchiostoma belcheri

100 Branchiostoma floridae

100 Ptychodera flava

Saccoglossus kowalevskii

100 Strongylocentrotus purpuratus

100 Acanthaster planci

0.050 bioRxiv preprint doi: https://doi.org/10.1101/236505; this version posted December 19, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

100 Ascidia sp.

100 Corella willmeriana Cystodites dellechiajei 100 100 Distaplia occidentalis Ciona savignyi

100 100 Ciona intestinalis 100 Ciona robusta Pyrosomella verticillata

100 Salpa fusiformis 100 100 Weelia cylindrica

100 Molgula occulta 100 Molgula oculata Molgula occidentalis 100 100 Halocynthia roretzi Styela gibbsii 100 100 Botryllus schlosseri 100 100 Botrylloides leachi Oikopleura dioica Petromyzon marinus

100 Homo sapiens 100 Latimeria chalumnae Asymmetron lucayanum

100 Branchiostoma belcheri 100 Branchiostoma floridae

100 Acanthaster planci Strongylocentrotus purpuratus 100 Ptychodera flava 100 Saccoglossus kowalevskii

0.10 bioRxiv preprint doi: https://doi.org/10.1101/236505; this version posted December 19, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

100 Ascidia sp.

100 Corella willmeriana

Distaplia occidentalis

100 100 Cystodites dellechiajei

Ciona savignyi

100 100 Ciona robusta

100 Ciona intestinalis

Pyrosomella verticillata

100 Salpa fusiformis 100 100 Weelia cylindrica

100 Molgula occulta

100 Molgula oculata

Molgula occidentalis 100 100 Halocynthia roretzi

100 Styela gibbsii

100 Botrylloides leachi 100 100 Botryllus schlosseri

Oikopleura dioica

Petromyzon marinus

100 Latimeria chalumnae

100 Homo sapiens

Asymmetron lucayanum

100 Branchiostoma floridae

100 Branchiostoma belcheri

100 Ptychodera flava

Saccoglossus kowalevskii

100 Strongylocentrotus purpuratus

100 Acanthaster planci

0.10 bioRxiv preprint doi: https://doi.org/10.1101/236505; this version posted December 19, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

100 Distaplia occidentalis

100 Cystodites dellechiajei Corella willmeriana 100 100 Ascidia sp. Ciona savignyi

100 100 Ciona robusta 100 Ciona intestinalis Pyrosomella verticillata

100 Weelia cylindrica 100 100 Salpa fusiformis

100 Molgula occulta 100 Molgula oculata Molgula occidentalis 100 100 Halocynthia roretzi Styela gibbsii 100 100 Botrylloides leachi 100 100 Botryllus schlosseri Oikopleura dioica Petromyzon marinus 100 100 Homo sapiens 100 Latimeria chalumnae Asymmetron lucayanum

100 Branchiostoma belcheri 100 Branchiostoma floridae

100 Acanthaster planci Strongylocentrotus purpuratus 100 Saccoglossus kowalevskii 100 Ptychodera flava

0.10 bioRxiv preprint doi: https://doi.org/10.1101/236505; this version posted December 19, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

100 Corella willmeriana

100 Ascidia sp.

Cystodites dellechiajei

100 100 Distaplia occidentalis

Ciona savignyi

100 100 Ciona robusta

100 Ciona intestinalis

Pyrosomella verticillata

100 Weelia cylindrica 100 100 Salpa fusiformis

100 Molgula oculata

100 Molgula occulta

Molgula occidentalis 100 100 Halocynthia roretzi

100 Styela gibbsii

100 Botryllus schlosseri 100 100 Botrylloides leachi

Oikopleura dioica

Petromyzon marinus

100 Homo sapiens

100 Latimeria chalumnae

Asymmetron lucayanum

100 Branchiostoma floridae

100 Branchiostoma belcheri

100 Strongylocentrotus purpuratus

Acanthaster planci

100 Saccoglossus kowalevskii

100 Ptychodera flava

0.10 bioRxiv preprint doi: https://doi.org/10.1101/236505; this version posted December 19, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

100 Distaplia occidentalis 100 Cystodites dellechiajei

34 Clavelina lepadiformis

Corella willmeriana 100 46 Ascidia sp.

Ciona savignyi

100 100 Ciona intestinalis

100 Ciona robusta

Pyrosomella verticillata

100 Weelia cylindrica

100 100 Salpa thompsoni 100 Salpa fusiformis

100 Molgula occulta 100 Molgula oculata

Molgula occidentalis 100 100 Halocynthia roretzi

100 Styela gibbsii

100 Botryllus schlosseri 100 100 Botrylloides leachi

Oikopleura dioica

Petromyzon marinus

100 Homo sapiens

100 Latimeria chalumnae

Asymmetron lucayanum

100 Branchiostoma floridae

100 Branchiostoma belcheri

100 Strongylocentrotus purpuratus Acanthaster planci

100 Saccoglossus kowalevskii

100 Ptychodera flava

0.10 bioRxiv preprint doi: https://doi.org/10.1101/236505; this version posted December 19, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

100 Ciona robusta 100 Ciona intestinalis

64 Ciona savignyi

Ascidia sp. 100 86 Corella willmeriana

Clavelina lepadiformis

100 100 Cystodites dellechiajei

100 Distaplia occidentalis

Pyrosomella verticillata

100 Weelia cylindrica

100 100 Salpa fusiformis 100 Salpa thompsoni

100 Molgula occulta 100 Molgula oculata

Molgula occidentalis 100 100 Halocynthia roretzi

100 Styela gibbsii

96 Botrylloides leachi 100 100 Botryllus schlosseri

Oikopleura dioica

Petromyzon marinus

100 Homo sapiens

100 Latimeria chalumnae

Asymmetron lucayanum

100 Branchiostoma floridae

100 Branchiostoma belcheri

100 Strongylocentrotus purpuratus Acanthaster planci

100 Saccoglossus kowalevskii

100 Ptychodera flava

0.050 bioRxiv preprint doi: https://doi.org/10.1101/236505; this version posted December 19, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

100 Distaplia occidentalis 100 Cystodites dellechiajei

34 Clavelina lepadiformis

Corella willmeriana 100 46 Ascidia sp.

Ciona savignyi

100 100 Ciona intestinalis

100 Ciona robusta

Pyrosomella verticillata

100 Weelia cylindrica

100 100 Salpa thompsoni 100 Salpa fusiformis

100 Molgula occulta 100 Molgula oculata

Molgula occidentalis 100 100 Halocynthia roretzi

100 Styela gibbsii

100 Botryllus schlosseri 100 100 Botrylloides leachi

Oikopleura dioica

Petromyzon marinus

100 Homo sapiens

100 Latimeria chalumnae

Asymmetron lucayanum

100 Branchiostoma floridae

100 Branchiostoma belcheri

100 Strongylocentrotus purpuratus Acanthaster planci

100 Saccoglossus kowalevskii

100 Ptychodera flava

0.10 bioRxiv preprint doi: https://doi.org/10.1101/236505; this version posted December 19, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

53 Cystodites dellechiajei

0 Distaplia occidentalis Corella willmeriana 6 0 Ascidia sp. Ciona savignyi

29 41 Ciona robusta 34 Ciona intestinalis Pyrosomella verticillata

36 Weelia cylindrica 25 84 Salpa fusiformis

69 Molgula occulta 9 Molgula oculata Molgula occidentalis 30 3 Halocynthia roretzi Styela gibbsii 7 6 Botrylloides leachi 22 48 Botryllus schlosseri Oikopleura dioica Petromyzon marinus

50 Homo sapiens 63 Latimeria chalumnae Asymmetron lucayanum

74 Branchiostoma belcheri 42 Branchiostoma floridae

55 Acanthaster planci Strongylocentrotus purpuratus 36 Saccoglossus kowalevskii 45 Ptychodera flava bioRxiv preprint doi: https://doi.org/10.1101/236505; this version posted December 19, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

CSAV HROR SPUR BFLO BBEL ALUC PVER WCYL SFUS CROB CINT ASCI CDEL DIST CWIL OCCI Taxon OCUL OCCU BLEA BSCH SGIB ODIO PMAR HSAP LCHA PFLA SKOW AAUS 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706 707 708 709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726 727 728 729 730 731 732 733 734 735 736 737 738 739 740 741 742 743 744 745 746 747 748 749 750 751 752 753 754 755 756 757 758 759 760

Gene outlier detection proportion Outlier detection proportion 0.00 0.05 0.10 0.15 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 bioRxiv preprint doi: not certifiedbypeerreview)istheauthor/funder.Allrightsreserved.Noreuseallowedwithoutpermission. https://doi.org/10.1101/236505 ; this versionpostedDecember19,2017.

GEN1 GEN2 GEN3 GEN4 GEN5 GEN6 GEN7 GEN8 GEN9 GEN10 GEN11 GEN12 AAUS GEN13 GEN14 GEN15 GEN16 GEN17 GEN18 GEN19 GEN20 GEN21 The copyrightholderforthispreprint(whichwas GEN22 GEN23 GEN24 GEN25 GEN26 GEN27 GEN28 GEN29 GEN30 GEN31 GEN32 GEN33 GEN34 GEN35 GEN36 GEN37 GEN38 GEN39 GEN40 GEN41 SKOW GEN42 GEN43 GEN44 GEN45 GEN46 GEN47 GEN48 GEN49 GEN50 GEN51 GEN52 GEN53 GEN54 GEN55 GEN56 GEN57 GEN58 GEN59 GEN60 GEN61 GEN62 GEN63 GEN64 GEN65 GEN66 GEN67 GEN68 GEN69 PFLA GEN70 GEN71 GEN72 GEN73 GEN74 GEN75 GEN76 GEN77 GEN78 GEN79 GEN80 GEN81 GEN82 GEN83 GEN84 GEN85 GEN86 GEN87 GEN88 GEN89 GEN90 GEN91 GEN92 GEN93 GEN94 GEN95 GEN96 GEN97 GEN98 LCHA GEN99 GEN100 GEN101 GEN102 GEN103 GEN104 GEN105 GEN106 GEN107 GEN108 GEN109 GEN110 GEN111 GEN112 GEN113 GEN114 GEN115 GEN116 GEN117 GEN118 GEN119 GEN120 GEN121 GEN122 GEN123 GEN124 GEN125 GEN126 GEN127 HSAP GEN128 GEN129 GEN130 GEN131 GEN132 GEN133 GEN134 GEN135 GEN136 GEN137 GEN138 GEN139 GEN140 GEN141 GEN142 GEN143 GEN144 GEN145 GEN146 GEN147 GEN148 GEN149 GEN150 GEN151 GEN152 GEN153 GEN154 GEN155 PMAR GEN156 GEN157 GEN158 GEN159 GEN160 GEN161 GEN162 GEN163 GEN164 GEN165 GEN166 GEN167 GEN168 GEN169 GEN170 GEN171 GEN172 GEN173 GEN174 GEN175 GEN176 GEN177 GEN178 GEN179 GEN180 GEN181 GEN182 GEN183 GEN184 ODIO GEN185 GEN186 GEN187 GEN188 GEN189 GEN190 GEN191 GEN192 GEN193 GEN194 GEN195 GEN196 GEN197 GEN198 GEN199 GEN200 GEN201 GEN202 GEN203 GEN204 GEN205 GEN206 GEN207 GEN208 GEN209 GEN210 GEN211 GEN212 GEN213 SGIB GEN214 GEN215 GEN216 GEN217 GEN218 GEN219 GEN220 GEN221 GEN222 GEN223 GEN224 GEN225 GEN226 GEN227 GEN228 GEN229 GEN230 GEN231 GEN232 GEN233 GEN234 GEN235 GEN236 GEN237 GEN238 GEN239 GEN240 GEN241 BSCH GEN242 GEN243 GEN244 GEN245 GEN246 GEN247 GEN248 GEN249 GEN250 GEN251 GEN252 GEN253 GEN254 GEN255 GEN256 GEN257 GEN258 GEN259 GEN260 GEN261 GEN262 GEN263 GEN264 GEN265 GEN266 GEN267 GEN268 GEN269 GEN270 BLEA GEN271 GEN272 GEN273 GEN274 GEN275 GEN276 GEN277 GEN278 GEN279 GEN280 GEN281 GEN282 GEN283 GEN284 GEN285 GEN286 GEN287 GEN288 GEN289 GEN290 GEN291 GEN292 GEN293 GEN294 GEN295 GEN296 GEN297 GEN298 GEN299 OCCU GEN300 GEN301 GEN302 GEN303 GEN304 GEN305 GEN306 GEN307 GEN308 GEN309 GEN310 GEN311 GEN312 GEN313 GEN314 GEN315 GEN316 GEN317 GEN318 GEN319 GEN320 GEN321 GEN322 GEN323 GEN324 GEN325 GEN326 GEN327 OCUL GEN328 GEN329 GEN330 GEN331 GEN332 GEN333 GEN334 GEN335 GEN336 GEN337 GEN338 GEN339 GEN340 GEN341 GEN342 GEN343 GEN344 GEN345 GEN346 GEN347 GEN348 GEN349 GEN350 GEN351 GEN352 GEN353 GEN354 GEN355 GEN356 OCCI GEN357 GEN358 GEN359 GEN360 GEN361 GEN362 GEN363 GEN364 GEN365 GEN366 GEN367 GEN368 GEN369 GEN370 GEN371 GEN372 GEN373 GEN374 GEN375 GEN376 GEN377 GEN378 GEN379 GEN380 GEN381 GEN382 GEN383 GEN384 GEN385 CWIL GEN386 GEN387 GEN388 GEN389 GEN390 GEN391 GEN392 GEN393 GEN394 Species

GEN395 Genes GEN396 GEN397 GEN398 GEN399 GEN400 GEN401 GEN402 GEN403 GEN404 GEN405 GEN406 GEN407 GEN408 GEN409 GEN410 GEN411 GEN412 GEN413 DIST GEN414 GEN415 GEN416 GEN417 GEN418 GEN419 GEN420 GEN421 GEN422 GEN423 GEN424 GEN425 GEN426 GEN427 GEN428 GEN429 GEN430 GEN431 GEN432 GEN433 GEN434 GEN435 GEN436 GEN437 GEN438 GEN439 GEN440 GEN441 GEN442 CDEL GEN443 GEN444 GEN445 GEN446 GEN447 GEN448 GEN449 GEN450 GEN451 GEN452 GEN453 GEN454 GEN455 GEN456 GEN457 GEN458 GEN459 GEN460 GEN461 GEN462 GEN463 GEN464 GEN465 GEN466 GEN467 GEN468 GEN469 GEN470 GEN471 ASCI GEN472 GEN473 GEN474 GEN475 GEN476 GEN477 GEN478 GEN479 GEN480 GEN481 GEN482 GEN483 GEN484 GEN485 GEN486 GEN487 GEN488 GEN489 GEN490 GEN491 GEN492 GEN493 GEN494 GEN495 GEN496 GEN497 GEN498 GEN499 CINT GEN500 GEN501 GEN502 GEN503 GEN504 GEN505 GEN506 GEN507 GEN508 GEN509 GEN510 GEN511 GEN512 GEN513 GEN514 GEN515 GEN516 GEN517 GEN518 GEN519 GEN520 GEN521 GEN522 GEN523 GEN524 GEN525 GEN526 GEN527 GEN528 CROB GEN529 GEN530 GEN531 GEN532 GEN533 GEN534 GEN535 GEN536 GEN537 GEN538 GEN539 GEN540 GEN541 GEN542 GEN543 GEN544 GEN545 GEN546 GEN547 GEN548 GEN549 GEN550 GEN551 GEN552 GEN553 GEN554 GEN555 GEN556 GEN557 SFUS GEN558 GEN559 GEN560 GEN561 GEN562 GEN563 GEN564 GEN565 GEN566 GEN567 GEN568 GEN569 GEN570 GEN571 GEN572 GEN573 GEN574 GEN575 GEN576 GEN577 GEN578 GEN579 GEN580 GEN581 GEN582 GEN583 GEN584 GEN585 WCYL GEN586 GEN587 GEN588 GEN589 GEN590 GEN591 GEN592 GEN593 GEN594 GEN595 GEN596 GEN597 GEN598 GEN599 GEN600 GEN601 GEN602 GEN603 GEN604 GEN605 GEN606 GEN607 GEN608 GEN609 GEN610 GEN611 GEN612 GEN613 GEN614 PVER GEN615 GEN616 GEN617 GEN618 GEN619 GEN620 GEN621 GEN622 GEN623 GEN624 GEN625 GEN626 GEN627 GEN628 GEN629 GEN630 GEN631 GEN632 GEN633 GEN634 GEN635 GEN636 GEN637 GEN638 GEN639 GEN640 GEN641 GEN642 GEN643 ALUC GEN644 GEN645 GEN646 GEN647 GEN648 GEN649 GEN650 GEN651 GEN652 GEN653 GEN654 GEN655 GEN656 GEN657 GEN658 GEN659 GEN660 GEN661 GEN662 GEN663 GEN664 GEN665 GEN666 GEN667 GEN668 GEN669 GEN670 GEN671 BBEL GEN672 GEN673 GEN674 GEN675 GEN676 GEN677 GEN678 GEN679 GEN680 GEN681 GEN682 GEN683 GEN684 GEN685 GEN686 GEN687 GEN688 GEN689 GEN690 GEN691 GEN692 GEN693 GEN694 GEN695 GEN696 GEN697 GEN698 GEN699 GEN700 BFLO GEN701 GEN702 GEN703 GEN704 GEN705 GEN706 GEN707 GEN708 GEN709 GEN710 GEN711 GEN712 GEN713 GEN714 GEN715 GEN716 GEN717 GEN718 GEN719 GEN720 GEN721 GEN722 GEN723 GEN724 GEN725 GEN726 GEN727 GEN728 GEN729 SPUR GEN730 GEN731 GEN732 GEN733 GEN734 GEN735 GEN736 GEN737 GEN738 GEN739 GEN740 GEN741 GEN742 GEN743 GEN744 GEN745 GEN746 GEN747 GEN748 GEN749 GEN750 GEN751 GEN752 GEN753 GEN754 GEN755 GEN756 GEN757 HROR GEN758 GEN759 GEN760 GEN761 GEN762 GEN763 GEN764 GEN765 GEN766 GEN767 GEN768 GEN769 GEN770 GEN771 GEN772 GEN773 GEN774 GEN775 GEN776 GEN777 GEN778 GEN779 GEN780 GEN781 GEN782 GEN783 GEN784 GEN785 GEN786 CSAV GEN787 GEN788 GEN789 GEN790 GEN791 GEN792 GEN793 GEN794 GEN795 GEN796 GEN797 GEN798 83 KX650801.1 Lissoclinum patella voucher DBTIC181 cytochrome oxidase subunit I COI gene partial cds mitochondrial 65 KX650800.1 Lissoclinum patella voucher DBTIC180 cytochrome oxidase subunit I COI gene partial cds mitochondrial KX650802.1 Lissoclinum patella voucher DBTIC182 cytochrome oxidase subunit I COI gene partial cds mitochondrial KU667258.1 Lissoclinum fragile voucher DBTIC68 cytochrome oxidase subunit I COI gene partial cds mitochondrial

18100 KM411611.1 Lissoclinum fragile voucher DBTIC26 cytochrome oxidase subunit I COI gene partial cds mitochondrial KX138473.1 Lissoclinum fragile voucher DBTIC100 cytochrome oxidase subunit I COI gene partial cds mitochondrial 57 KR822674.1 Lissoclinum fragile voucher DBTIC 34 cytochrome oxidase subunit I COI gene partial cds mitochondrial 80 KJ725150.1 Lissoclinum fragile voucher ICBT.ASC006 cytochrome oxidase subunit I COI gene partial cds mitochondrial KU667257.1 Lissoclinum fragile voucher DBTIC67 cytochrome oxidase subunit I COI gene partial cds mitochondrial 63 KX138474.1 Lissoclinum fragile voucher DBTIC101 cytochrome oxidase subunit I COI gene partial cds mitochondrial KU667256.1 Lissoclinum fragile voucher DBTIC66 cytochrome oxidase subunit I COI gene partial cds mitochondrial 91 29 KM411612.1 Lissoclinum fragile voucher DBTIC24 cytochrome oxidase subunit I COI gene partial cds mitochondrial 33 KX650799.1 Lissoclinum fragile voucher DBTIC179 cytochrome oxidase subunit I COI gene partial cds mitochondrial JF506185.1 Lissoclinum fragile clone 07Jun08-1-1 cytochrome c oxidase subunit I COI gene partial cds mitochondrial JF506180.1 Lissoclinum fragile clone 08Jun08-1-5 cytochrome c oxidase subunit I COI gene partial cds mitochondrial 93 JF506181.1 Lissoclinum fragile clone 08Jun08-1-2 cytochrome c oxidase subunit I COI gene partial cds mitochondrial 100 JF506184.1 Lissoclinum fragile clone 07Jun08-3-6 cytochrome c oxidase subunit I COI gene partial cds mitochondrial

92 JF506182.1 Lissoclinum aff. fragile SLL-2011 clone 09Jun081-2 cytochrome c oxidase subunit I COI gene partial cds mitochondrial JF506183.1 Lissoclinum aff. fragile SLL-2011 clone 04Jun08-1-7 cytochrome c oxidase subunit I COI gene partial cds mitochondrial 75 KU221205.1 Lissoclinum vareau voucher UF 606 cytochrome oxidase subunit I COI gene partial cds mitochondrial KU221214.1 Lissoclinum ravarava voucher UF 1398 cytochrome oxidase subunit I COI gene partial cds mitochondrial JX099361.1 Lissoclinum verrilli clone ZPLV cytochrome c oxidase subunit I COI gene partial cds mitochondrial 100 KU221194.1 Lissoclinum verrilli voucher UF 1244 cytochrome oxidase subunit I COI gene partial cds mitochondrial 96 18 KX650796.1 Diplosoma multifidum voucher DBTIC176 cytochrome oxidase subunit I COI gene partial cds mitochondrial 44 KU221221.1 Diplosoma listerianum voucher UF 649 cytochrome oxidase subunit I COI gene partial cds mitochondrial

70 KX650797.1 Diplosoma swamiensis voucher DBTIC177 cytochrome oxidase subunit I COI gene partial cds mitochondrial KX650792.1 Diplosoma gelatinosa voucher DBTIC172 cytochrome oxidase subunit I COI gene partial cds mitochondrial KU667283.1 Diplosoma virens voucher DBTIC94 cytochrome oxidase subunit I COI gene partial cds mitochondrial 67 KU221188.1 Diplosoma sp. AO-2016 voucher ColBIO TL 817 cytochrome oxidase subunit I COI gene partial cds mitochondrial 89 KF309633.1 Aplidium sp. PRE-AP cytochrome oxidase subunit I COI gene partial cds mitochondrial 100 KJ725153.1 Diplosoma simile isolate GAASC2 cytochrome c oxidase subunit 1 COI gene partial cds mitochondrial 42 KU221218.1 Diplosoma simile voucher UF 697 cytochrome oxidase subunit I COI gene partial cds mitochondrial KU221184.1 Diplosoma sp. 1 AO-2016 voucher ColBIO TL 729 cytochrome oxidase subunit I COI gene partial cds mitochondrial 48 99 KU221228.1 Diplosoma sp. 3 AO-2016 voucher UF 680 cytochrome oxidase subunit I COI gene partial cds mitochondrial 76 11 37 KU221226.1 Diplosoma sp. 3 AO-2016 voucher UF 759 cytochrome oxidase subunit I COI gene partial cds mitochondrial KU221220.1 Diplosoma sp. 2 AO-2016 voucher UF 644 cytochrome oxidase subunit I COI gene partial cds mitochondrial 75 KJ725166.1 Diplosoma spongiforme isolate GAASC15 cytochrome c oxidase subunit 1 COI gene partial cds mitochondrial KF309624.1 Diplosoma spongiforme isolate DS-DL cytochrome oxidase subunit I COI gene partial cds mitochondrial 97 AY600972.1 Diplosoma spongiforme cytochrome c oxidase subunit I COI gene partial cds mitochondrial KX650775.1 Polyclinum indicum voucher DBTIC155 cytochrome oxidase subunit I COI gene partial cds mitochondrial 63 KX650777.1 Polyclinum indicum voucher DBTIC157 cytochrome oxidase subunit I COI gene partial cds mitochondrial KT345963.1 Polyclinum indicum voucher DBTIC48 cytochrome oxidase subunit I COI gene partial cds mitochondrial KU220954.1 Polyclinum madrasensis voucher DBTIC15 cytochrome oxidase subunit I COI gene partial cds mitochondrial KT276223.1 Polyclinum indicum voucher DBTIC27 cytochrome c oxidase subunit I COI gene partial cds mitochondrial KP072781.1 Polyclinum indicum voucher DBTIC28 cytochrome oxidase subunit I COI gene partial cds mitochondrial 63 KX650778.1 Polyclinum indicum voucher DBTIC158 cytochrome oxidase subunit I COI gene partial cds mitochondrial 97 KX650774.1 Polyclinum indicum voucher DBTIC154 cytochrome oxidase subunit I COI gene partial cds mitochondrial 8 KX650780.1 Polyclinum indicum voucher DBTIC160 cytochrome oxidase subunit I COI gene partial cds mitochondrial KX650776.1 Polyclinum indicum voucher DBTIC156 cytochrome oxidase subunit I COI gene partial cds mitochondrial KJ944391.1 UNVERIFIED Polyclinum indicum voucher DBTIC 002 cytochrome oxidase subunit I-like COI gene partial sequence mitochondrial KX650786.1 Polyclinum indicum voucher DBTIC166 cytochrome oxidase subunit I COI gene partial cds mitochondrial KX650785.1 Polyclinum indicum voucher DBTIC165 cytochrome oxidase subunit I COI gene partial cds mitochondrial 33 KX650781.1 Polyclinum indicum voucher DBTIC161 cytochrome oxidase subunit I COI gene partial cds mitochondrial 32 KX650783.1 Polyclinum indicum voucher DBTIC163 cytochrome oxidase subunit I COI gene partial cds mitochondrial 34 40 KX650784.1 Polyclinum indicum voucher DBTIC164 cytochrome oxidase subunit I COI gene partial cds mitochondrial 71 KX650782.1 Polyclinum indicum voucher DBTIC162 cytochrome oxidase subunit I COI gene partial cds mitochondrial 89 KU667271.1 Diplosoma sp. JAHA-2016 voucher DBTIC81 cytochrome oxidase subunit I COI gene partial cds mitochondrial 87 KX650798.1 Diplosoma translucidum voucher DBTIC178 cytochrome oxidase subunit I COI gene partial cds mitochondrial 100 5 KU221224.1 Trididemnum pigmentatum voucher UF 696 cytochrome oxidase subunit I COI gene partial cds mitochondrial KU221203.1 Trididemnum tomarahi voucher UF 1424 cytochrome oxidase subunit I COI gene partial cds mitochondrial KF309632.1 Trididemnum cereum isolate PRE-AA cytochrome oxidase subunit I COI gene partial cds mitochondrial 81 84 KU221185.1 Trididemnum sp. AO-2016 voucher ColBIO TL 538 cytochrome oxidase subunit I COI gene partial cds mitochondrial

32 KU221206.1 Trididemnum fetia voucher UF 629 cytochrome oxidase subunit I COI gene partial cds mitochondrial KJ725158.1 Trididemnum cyanophorum isolate GAASC7 cytochrome c oxidase subunit 1 COI gene partial cds mitochondrial 91 3 77 KJ632947.1 Trididemnum cyanophorum strain MH-JMC3 cytochrome c oxidase subunit I COI gene partial cds mitochondrial JF506187.1 Trididemnum cyanophorum clone 30May08-2-1 cytochrome c oxidase subunit I COI gene partial cds mitochondrial 97 KU221183.1 Trididemnum maragogi voucher ColBIO TL 234 cytochrome oxidase subunit I COI gene partial cds mitochondrial JF506186.1 Trididemnum solidum clone 9Jun08-2-1 cytochrome c oxidase subunit I COI gene partial cds mitochondrial 79 KR604728.1 Trididemnum maragogi voucher ColBIO TL438 cytochrome oxidase subunit I COI gene partial cds mitochondrial AY600982.1 Polycitor adriaticum cytochrome c oxidase subunit I COI gene partial cds mitochondrial 100 AY523042.1 Cystodytes dellechiajei haplotype S1 cytochrome oxidase subunit I COI gene partial cds mitochondrial AM709504.1 Polycitorella coronaria mitochondrial partial COI gene for cytochrome-c oxidase 100 KR703628.1 Eudistoma malum voucher DBTIC 35 cytochrome oxidase subunit I COI gene partial cds mitochondrial 100 KU360793.1 Eudistoma malum isolate ASC08 cytochrome oxidase subunit I COI gene partial cds mitochondrial

99 KR703627.1 Eudistoma tumidum voucher DBTIC 33 cytochrome oxidase subunit I COI gene partial cds mitochondrial 100 KU360797.1 Eudistoma tumidum isolate ASC12 cytochrome oxidase subunit I COI gene partial cds mitochondrial 11 85 KR815818.1 Symplegma brakenhielmi voucher DBTIC 08 cytochrome oxidase subunit I COI gene partial cds mitochondrial

96 KJ944392.1 Eudistoma viride voucher DBTIC 003 cytochrome oxidase subunit I COI gene partial cds mitochondrial KJ944393.1 Eudistoma viride voucher DBTIC 004 cytochrome oxidase subunit I COI gene partial cds mitochondrial 40 KJ710709.1 Eudistoma viride voucher ICBT005 cytochrome oxidase subunit I COI gene partial cds mitochondrial KX650769.1 Eudistoma gilboviride voucher DBTIC149 cytochrome oxidase subunit I COI gene partial cds mitochondrial

72 HG005369.1 Eudistoma elongatum mitochondrial partial COI gene for cytochrome oxidase subunit 1 99 EF619346.1 Eudistoma elongatum isolate 3Houhora cytochrome oxidase subunit I COI gene partial cds mitochondrial 100 AY600979.1 Pseudodistoma crucigaster cytochrome c oxidase subunit I COI gene partial cds mitochondrial 60 AJ748708.1 Pseudodistoma crucigaster mitochondrial partial coi gene for cytochrome oxidase subunit 1 haplotype IV 4 AY600970.1 Pseudodistoma cyrnusense cytochrome c oxidase subunit I COI gene partial cds mitochondrial AY600977.1 Eudistoma planum cytochrome c oxidase subunit I COI gene partial cds mitochondrial 4 100 JQ403421.1 Eudistoma viride cytochrome c oxidase subunit I COI gene partial cds mitochondrial AY600978.1 Eudistoma plumbeum cytochrome c oxidase subunit I COI gene partial cds mitochondrial 72 KY111422.1 Eudistoma capsulatum isolate 24Jun15 Ep1 Ep2 Ep4 Ep5 cytochrome oxidase subunit I COI gene partial cds mitochondrial 1877 KY111423.1 Eudistoma capsulatum isolate 24Jun15 Ey3 cytochrome oxidase subunit I COI gene partial cds mitochondrial 44 KY111420.1 Eudistoma capsulatum isolate 24Jun15 Ey4 Ey5 cytochrome oxidase subunit I COI gene partial cds mitochondrial

12 KY111421.1 Eudistoma capsulatum isolate 22Jul14-1-1 cytochrome oxidase subunit I COI gene partial cds mitochondrial AY600974.1 Eudistoma posidoniarum cytochrome c oxidase subunit I COI gene partial cds mitochondrial 100 AY600973.1 Eudistoma banyulensis cytochrome c oxidase subunit I COI gene partial cds mitochondrial KX650768.1 Eudistoma angolanum voucher DBTIC148 cytochrome oxidase subunit I COI gene partial cds mitochondrial KM411614.1 Eudistoma microlarvum voucher DBTIC18 cytochrome oxidase subunit I COI gene partial cds mitochondrial 76 KU360794.1 Eudistoma microlarvum isolate ASC09 cytochrome oxidase subunit I COI gene partial cds mitochondrial 18 KU667266.1 Eudistoma microlarvum voucher DBTIC76 cytochrome oxidase subunit I COI gene partial cds mitochondrial 58 KX138484.1 Eudistoma amplum voucher DBTIC111 cytochrome oxidase subunit I COI gene partial cds mitochondrial 52 KX138482.1 Eudistoma amplum voucher DBTIC109 cytochrome oxidase subunit I COI gene partial cds mitochondrial 43 KX138480.1 Eudistoma amplum voucher DBTIC107 cytochrome oxidase subunit I COI gene partial cds mitochondrial

2 56 KU667265.1 Eudistoma amplum voucher DBTIC75 cytochrome oxidase subunit I COI gene partial cds mitochondrial 62 KM411609.1 Eudistoma amplum voucher DBTIC17 cytochrome oxidase subunit I COI gene partial cds mitochondrial

49 KX138483.1 Eudistoma amplum voucher DBTIC110 cytochrome oxidase subunit I COI gene partial cds mitochondrial 91 KX138481.1 Eudistoma amplum voucher DBTIC108 cytochrome oxidase subunit I COI gene partial cds mitochondrial 91 KM411615.1 Eudistoma laysani voucher DBTIC19 cytochrome oxidase subunit I COI gene partial cds mitochondrial 81 KU667263.1 Eudistoma laysani voucher DBTIC73 cytochrome oxidase subunit I COI gene partial cds mitochondrial 59 27 KU667264.1 Eudistoma laysani voucher DBTIC74 cytochrome oxidase subunit I COI gene partial cds mitochondrial KX138476.1 Eudistoma ovatum voucher DBTIC103 cytochrome oxidase subunit I COI gene partial cds mitochondrial

75 KX650771.1 Eudistoma ovatum voucher DBTIC151 cytochrome oxidase subunit I COI gene partial cds mitochondrial 60 KU667259.1 Eudistoma ovatum voucher DBTIC69 cytochrome oxidase subunit I COI gene partial cds mitochondrial KU667260.1 Eudistoma ovatum voucher DBTIC70 cytochrome oxidase subunit I COI gene partial cds mitochondrial 65 KU667262.1 Eudistoma ovatum voucher DBTIC72 cytochrome oxidase subunit I COI gene partial cds mitochondrial KM411610.1 Eudistoma ovatum voucher DBTIC20 cytochrome oxidase subunit I COI gene partial cds mitochondrial 56 KU667261.1 Eudistoma ovatum voucher DBTIC71 cytochrome oxidase subunit I COI gene partial cds mitochondrial 44 KR867634.1 Eudistoma ovatum voucher DBTIC41 cytochrome oxidase subunit I COI gene partial cds mitochondrial 65 KU715280.1 Eudistoma ovatum voucher DBTIC96 cytochrome oxidase subunit I COI gene partial cds mitochondrial

45 KX138475.1 Eudistoma ovatum voucher DBTIC102 cytochrome oxidase subunit I COI gene partial cds mitochondrial 63 KX138477.1 Eudistoma ovatum voucher DBTIC104 cytochrome oxidase subunit I COI gene partial cds mitochondrial 100 KU697732.1 Morchellium argus isolate A50 cytochrome c oxidase subunit I COI gene partial cds mitochondrial 29 KF309621.1 Morchellium argus isolate DS-3 cytochrome oxidase subunit I COI gene partial cds mitochondrial 61 KF309620.1 Morchellium argus isolate DS-2 cytochrome oxidase subunit I COI gene partial cds mitochondrial AY600971.1 Aplidium elegans cytochrome c oxidase subunit I COI gene partial cds mitochondrial KF309663.1 Aplidium cf. accarense TO-AP cytochrome oxidase subunit I COI gene partial cds mitochondrial KF309654.1 Aplidium cf. accarense SIT-1 cytochrome oxidase subunit I COI gene partial cds mitochondrial 3 KF309657.1 Aplidium cf. accarense TA-1 cytochrome oxidase subunit I COI gene partial cds mitochondrial KF309571.1 Aplidium cf. accarense ESC-6 cytochrome oxidase subunit I COI gene partial cds mitochondrial KF309558.1 Aplidium cf. accarense CA-4 cytochrome oxidase subunit I COI gene partial cds mitochondrial

48 KF309655.1 Aplidium cf. accarense SIT-2 cytochrome oxidase subunit I COI gene partial cds mitochondrial KF309584.1 Aplidium cf. accarense FOR-AP cytochrome oxidase subunit I COI gene partial cds mitochondrial KF309601.1 Aplidium cf. accarense MAT-AA cytochrome oxidase subunit I COI gene partial cds mitochondrial KF309599.1 Aplidium cf. accarense MAT-4 cytochrome oxidase subunit I COI gene partial cds mitochondrial KF309618.1 Aplidium cf. accarense PB-7 cytochrome oxidase subunit I COI gene partial cds mitochondrial KF309625.1 Aplidium cf. accarense POG-1 cytochrome oxidase subunit I COI gene partial cds mitochondrial KF309598.1 Aplidium cf. accarense MAS-AB cytochrome oxidase subunit I COI gene partial cds mitochondrial 97 KF309629.1 Aplidium cf. accarense POM-AA cytochrome oxidase subunit I COI gene partial cds mitochondrial KF309646.1 Aplidium cf. accarense SCR-6 cytochrome oxidase subunit I COI gene partial cds mitochondrial KF309656.1 Aplidium cf. accarense SIT-AA cytochrome oxidase subunit I COI gene partial cds mitochondrial KF309640.1 Aplidium cf. accarense SA-AA cytochrome oxidase subunit I COI gene partial cds mitochondrial KF309626.1 Aplidium cf. accarense POG-2 cytochrome oxidase subunit I COI gene partial cds mitochondrial 6 KF309585.1 Aplidium cf. accarense GAR-AA cytochrome oxidase subunit I COI gene partial cds mitochondrial KF309597.1 Aplidium cf. accarense MAS-AA cytochrome oxidase subunit I COI gene partial cds mitochondrial KF309586.1 Aplidium cf. accarense GAR-AB cytochrome oxidase subunit I COI gene partial cds mitochondrial KF309574.1 Aplidium cf. accarense ESC-AA cytochrome oxidase subunit I COI gene partial cds mitochondrial KF309553.1 Aplidium cf. accarense BLA-AA cytochrome oxidase subunit I COI gene partial cds mitochondrial KF309630.1 Aplidium cf. accarense POM-AB cytochrome oxidase subunit I COI gene partial cds mitochondrial KF309627.1 Aplidium cf. accarense POG-AA cytochrome oxidase subunit I COI gene partial cds mitochondrial KJ725161.1 Synoicum castellatum isolate GAASC10 cytochrome c oxidase subunit 1 COI gene partial cds mitochondrial KJ632946.1 Aplidium conicum strain MH-JMC2 cytochrome c oxidase subunit I COI gene partial cds mitochondrial 93 14 AY600969.1 Aplidium conicum cytochrome c oxidase subunit I COI gene partial cds mitochondrial 53 Aplidium conicum NC 013584.1 cds YP 003331401.1 9

97 KY111412.1 Aplidium stellatum isolate 21Jul14-3-1 cytochrome oxidase subunit I COI gene partial cds mitochondrial KY111414.1 Aplidium stellatum isolate 22Jul14-1-3 cytochrome oxidase subunit I COI gene partial cds mitochondrial 91 KY111413.1 Aplidium stellatum isolate 21Jul14-3-5 Grey cytochrome oxidase subunit I COI gene partial cds mitochondrial KY111411.1 Aplidium stellatum isolate 17Jul14-1-3 White cytochrome oxidase subunit I COI gene partial cds mitochondrial AY600967.1 Aplidium pseudolobatum cytochrome c oxidase subunit I COI gene partial cds mitochondrial

50 KJ725160.1 Aplidium fuscum isolate GAASC9 cytochrome c oxidase subunit 1 COI gene partial cds mitochondrial 100 AY600975.1 Aplidium fuscum cytochrome c oxidase subunit I COI gene partial cds mitochondrial 10 AY600983.1 Rophalaea neapolitana cytochrome c oxidase subunit I COI gene partial cds mitochondrial AM706464.1 Clavelina australis coi gene for cytochrome c oxidase AM706465.1 Clavelina breve coi gene for cytochrome c oxidasehaplotype Cbv1 KF309535.1 Clavelina sp. AM-4 cytochrome oxidase subunit I COI gene partial cds mitochondrial KF309645.1 Clavelina sp. SCR-5 cytochrome oxidase subunit I COI gene partial cds mitochondrial KF309638.1 Clavelina lepadiformis isolate ROS-CLY cytochrome oxidase subunit I COI gene partial cds mitochondrial 63 AJ884573.1 Clavelina gemmae mitochondrial partial COI gene for cytochrome oxidase 1 79 AJ866716.1 Clavelina gemmae mitochondrial partial coi gene for cytochrome oxidase subunit 1

30 Clavelina lepadiformis FJ839918.1 cds ACO40300.1 1 AY603104.1 Clavelina lepadiformis cytochrome c oxidase subunit I COI gene partial cds mitochondrial 77 63 KF309563.1 Clavelina lepadiformis isolate CF-4 cytochrome oxidase subunit I COI gene partial cds mitochondrial AM706468.1 Clavelina meridionalis coi gene for cytochrome c oxidasehaplotype Cme1 29 AY603105.1 Clavelina delavallei cytochrome c oxidase subunit I COI gene partial cds mitochondrial JN703738.1 Clavelina oblonga haplotype 1 cytochrome c oxidase subunit I gene partial cds mitochondrial Clavelina phlegraea NC 024105.1 cds YP 009029840.1 4 JN703739.1 Clavelina oblonga haplotype 2 cytochrome c oxidase subunit I gene partial cds mitochondrial

70 KJ725165.1 Clavelina oblonga isolate GAASC14 cytochrome c oxidase subunit 1 COI gene partial cds mitochondrial 29 KY111417.1 Clavelina oblonga isolate 22Jul14-1-4 cytochrome oxidase subunit I COI gene partial cds mitochondrial AY603106.1 Clavelina oblonga cytochrome c oxidase subunit I COI gene partial cds mitochondrial KF309648.1 Clavelina oblonga isolate SCR-CO cytochrome oxidase subunit I COI gene partial cds mitochondrial JN859182.1 Clavelina oblonga isolate FL2 cytochrome c oxidase subunit I gene partial cds mitochondrial 59 AM706472.1 Clavelina moluccensis coi gene for cytochrome c oxidase 7 JN703740.1 Clavelina picta cytochrome c oxidase subunit I gene partial cds mitochondrial AM706489.1 Nephtheis fascicularis coi gene for cytochrome c oxidase AM706462.1 Clavelina arafurensis coi gene for cytochrome c oxidase haplotype Car1 26 AM403684.1 Pycnoclavella atlantica mitochondrial coi gene for cytochrome c oxidase subunit I haplotype H1 99 AM706474.1 Pycnoclavella detorta coi gene for cytochrome c oxidase haplotype Pde2 48 36 AM706473.1 Pycnoclavella detorta coi gene for cytochrome c oxidase haplotype Pde1 AM706484.1 Pycnoclavella tabella coi gene for cytochrome c oxidase haplotype Ptb1 AM706479.1 Pycnoclavella martae coi gene for cytochrome c oxidase haplotype Pma1 82 44 AM706481.1 Pycnoclavella martae coi gene for cytochrome c oxidase haplotype Pma3 35 AM706480.1 Pycnoclavella martae coi gene for cytochrome c oxidase haplotype Pma2 KJ725156.1 Pycnoclavella diminuta isolate GAASC5 cytochrome c oxidase subunit 1 COI gene partial cds mitochondrial KJ632945.1 Pycnoclavella diminuta strain MH-JMC1 cytochrome c oxidase subunit I COI gene partial cds mitochondrial

29 AM746343.1 Pycnoclavella communis mitochondrial partial coi gene for cytochrome c oxidase subunit I haplotype H1 7 AM403696.1 Pycnoclavella nana mitochondrial coi gene for cytochrome c oxidase subunit I haplotype H26 35 AM403690.1 Pycnoclavella aurilucens mitochondrial coi gene for cytochrome c oxidase subunit I haplotype H10 34 AY600988.1 Pycnoclavella sp. SLL-2004 cytochrome c oxidase subunit I COI gene partial cds mitochondrial AM706475.1 Clavelina flava coi gene for cytochrome c oxidase haplotype Pfl1 17 AM403686.1 Pycnoclavella brava mitochondrial coi gene for cytochrome c oxidase subunit I haplotype H2 AM403703.1 Pycnoclavella sp. RPP-2006 mitochondrial coi gene for cytochrome c oxidase subunit I haplotype H6 32 AM706483.1 Clavelina producta coi gene for cytochrome c oxidase haplotype Ppd2 AM706482.1 Clavelina producta coi gene for cytochrome c oxidase haplotype Ppd1 69 AY600966.1 Archidistoma aggregatum cytochrome c oxidase subunit I COI gene partial cds mitochondrial Distaplia occidentalis|TRINITY DN13342 c0 g1 i1

57 KU221227.1 Distaplia sp. AO-2016 voucher TL 02 cytochrome oxidase subunit I COI gene partial cds mitochondrial 97 KY111419.1 Distaplia bermudensis isolate 1Mar16 3B-1 cytochrome oxidase subunit I COI gene partial cds mitochondrial 100 KU221217.1 Polysyncraton poro voucher UF 664 cytochrome oxidase subunit I COI gene partial cds mitochondrial KU221222.1 Polysyncraton poro voucher UF 682 cytochrome oxidase subunit I COI gene partial cds mitochondrial 18 84 AY600986.1 Polysyncraton lacazei cytochrome c oxidase subunit I COI gene partial cds mitochondrial

79 KU221187.1 Polysyncraton sp. AO-2016 voucher ColBIO TL 432 cytochrome oxidase subunit I COI gene partial cds mitochondrial 99 KU221186.1 Polysyncraton sp. AO-2016 voucher ColBIO TL 810 cytochrome oxidase subunit I COI gene partial cds mitochondrial KU906100.1 Molgula manhattensis voucher SERCINVERT0184 cytochrome oxidase subunit 1 COI gene partial cds mitochondrial KU905891.1 Molgula manhattensis voucher SERCINVERT0432 cytochrome oxidase subunit 1 COI gene partial cds mitochondrial KU905755.1 Molgula manhattensis voucher SERCINVERT0185 cytochrome oxidase subunit 1 COI gene partial cds mitochondrial 96 JQ742950.1 Molgula manhattensis clone SY011703 cytochrome c oxidase subunit 1 COI gene partial cds mitochondrial JQ742955.1 UNVERIFIED Molgula manhattensis clone SY011708 cytochrome c oxidase subunit 1-like COI gene partial sequence mitochondrial KY111424.1 Molgula manhattensis isolate 19Jul14-1-1 cytochrome oxidase subunit I COI gene partial cds mitochondrial 100 HM574376.1 Molgula provisionalis cytochrome oxidase subunit I COI gene partial cds mitochondrial HM574345.1 Molgula manhattensis haplotype 1 cytochrome oxidase subunit I COI gene partial cds mitochondrial HM574378.1 Molgula socialis haplotype HB cytochrome oxidase subunit I COI gene partial cds mitochondrial HM574379.1 Molgula socialis haplotype HC cytochrome oxidase subunit I COI gene partial cds mitochondrial 100 HM574377.1 Molgula socialis haplotype HA cytochrome oxidase subunit I COI gene partial cds mitochondrial KY111418.1 Didemnum lutarium isolate 17Jul14-1-4 cytochrome oxidase subunit I COI gene partial cds mitochondrial 42 KU221202.1 Didemnum ligulum voucher UF 1387 cytochrome oxidase subunit I COI gene partial cds mitochondrial 100 KU221198.1 Didemnum ligulum voucher UF 613 cytochrome oxidase subunit I COI gene partial cds mitochondrial 94 KU221200.1 Didemnum ligulum voucher UF 694 cytochrome oxidase subunit I COI gene partial cds mitochondrial 100 KU221210.1 Didemnum ligulum voucher ColBIO TL796 cytochrome oxidase subunit I COI gene partial cds mitochondrial KJ725159.1 Didemnum albidum isolate GAASC8 cytochrome c oxidase subunit 1 COI gene partial cds mitochondrial KJ725155.1 Didemnum granulatum isolate GAASC4 cytochrome c oxidase subunit 1 COI gene partial cds mitochondrial 79 93 JQ013198.1 Didemnum granulatum strain Sri2 cytochrome oxidase subunit I COI gene partial cds mitochondrial 55 KP779901.1 Didemnum granulatum cytochrome oxidase subunit I COI gene partial cds mitochondrial KR612209.1 Didemnum vexillum isolate Fairlie11 cytochrome c oxidase CoI gene partial cds mitochondrial

87 Didemnum vexillum NC 026107.1 cds YP 009114980.1 11 KU221189.1 Didemnum psammatodes voucher ColBIO TL 27 cytochrome oxidase subunit I COI gene partial cds mitochondrial

66 96 JN624758.1 Didemnum psammatode strain sri1 cytochrome C oxidase subunit I COI gene partial cds mitochondrial

78 KP779902.1 Didemnum psammatodes cytochrome oxidase subunit I COI gene partial cds mitochondrial KU667268.1 Didemnum spadix voucher DBTIC78 cytochrome oxidase subunit I COI gene partial cds mitochondrial 69 KU667267.1 Didemnum spadix voucher DBTIC77 cytochrome oxidase subunit I COI gene partial cds mitochondrial KR604727.1 Polysyncraton maurizeliae voucher ColBIO TL61 cytochrome oxidase subunit I COI gene partial cds mitochondrial 100 KU221211.1 Didemnum apuroto voucher UF 813 cytochrome oxidase subunit I COI gene partial cds mitochondrial 21 KU221212.1 Didemnum apuroto voucher UF 814 cytochrome oxidase subunit I COI gene partial cds mitochondrial KU221192.1 Didemnum cineraceum voucher ColBIO TL 839 cytochrome oxidase subunit I COI gene partial cds mitochondrial 13 91 94 KU221193.1 Didemnum cineraceum voucher ColBIO TL 230 cytochrome oxidase subunit I COI gene partial cds mitochondrial

3 KF309573.1 Didemnum sp. ESC-10 cytochrome oxidase subunit I COI gene partial cds mitochondrial 93 KF309622.1 Didemnum sp. DS-4 cytochrome oxidase subunit I COI gene partial cds mitochondrial KM411613.1 Didemnum sp. JAHA-2014 voucher DBTIC25 cytochrome oxidase subunit I COI gene partial cds mitochondrial 2 5 87 KU667269.1 Didemnum sp. JAHA-2014 voucher DBTIC79 cytochrome oxidase subunit I COI gene partial cds mitochondrial 100 KU221201.1 Didemnum fragile voucher UF 839 cytochrome oxidase subunit I COI gene partial cds mitochondrial KU221209.1 Didemnum fragile voucher UF 746 cytochrome oxidase subunit I COI gene partial cds mitochondrial 1 57 KU221207.1 Didemnum mutabile voucher UF 689 cytochrome oxidase subunit I COI gene partial cds mitochondrial 100 KU221223.1 Didemnum mutabile voucher UF 691 cytochrome oxidase subunit I COI gene partial cds mitochondrial 44 KU221208.1 Didemnum sordidum voucher UF 734 cytochrome oxidase subunit I COI gene partial cds mitochondrial 1 KU221195.1 Didemnum sordidum voucher UF 597 cytochrome oxidase subunit I COI gene partial cds mitochondrial 89 KU221197.1 Didemnum sordidum voucher UF 612 cytochrome oxidase subunit I COI gene partial cds mitochondrial

2 KF309576.1 Didemnum fulgens isolate ESC-DF cytochrome oxidase subunit I COI gene partial cds mitochondrial 100 KJ725152.1 Didemnum fulgens isolate GAASC1 cytochrome c oxidase subunit 1 COI gene partial cds mitochondrial KU221199.1 Didemnum cuculliferum voucher UF 676 cytochrome oxidase subunit I COI gene partial cds mitochondrial KU221219.1 Didemnum cuculliferum voucher UF 724 cytochrome oxidase subunit I COI gene partial cds mitochondrial 98 KU221191.1 Didemnum granulatum voucher ColBIO TL 686 cytochrome oxidase subunit I COI gene partial cds mitochondrial 65 KU221190.1 Didemnum granulatum voucher ColBIO TL 671 cytochrome oxidase subunit I COI gene partial cds mitochondrial 43 JQ780668.1 Didemnum granulatum haplotype HAP01 cytochrome oxidase subunit I COI gene partial cds mitochondrial KU221196.1 Didemnum granulatum voucher UF 573 cytochrome oxidase subunit I COI gene partial cds mitochondrial

3738 JQ731741.1 Didemnum sp. BAOR-2012 haplotype 11 cytochrome oxidase subunit I COI gene partial cds mitochondrial JQ731742.1 Didemnum sp. BAOR-2012 haplotype 12 cytochrome oxidase subunit I COI gene partial cds mitochondrial KU221225.1 Didemnum membranaceum voucher UF 720 cytochrome oxidase subunit I COI gene partial cds mitochondrial KU221216.1 Didemnum membranaceum voucher UF 526 cytochrome oxidase subunit I COI gene partial cds mitochondrial 6371 KU221213.1 Didemnum membranaceum voucher UF 1389 cytochrome oxidase subunit I COI gene partial cds mitochondrial KU221215.1 Didemnum granulatum voucher UF 1408 cytochrome oxidase subunit I COI gene partial cds mitochondrial 77 KU221204.1 Didemnum granulatum voucher UF 601 cytochrome oxidase subunit I COI gene partial cds mitochondrial 2 JQ692626.1 Didemnum incanum haplotype 8 cytochrome oxidase subunit I COI gene partial cds mitochondrial 6 KJ725167.1 Didemnum sp. GA-2014 cytochrome c oxidase subunit 1 COI gene partial cds mitochondrial JQ731736.1 Didemnum patulum haplotype 1 cytochrome oxidase subunit I COI gene partial cds mitochondrial KX641024.1 Didemnum sp. IEAPMC1237 cytochrome oxidase subunit I COI gene partial cds mitochondrial 9 KX138486.1 Didemnum perlucidum voucher DBTIC113 cytochrome oxidase subunit I COI gene partial cds mitochondrial 100 KX138487.1 Didemnum perlucidum voucher DBTIC114 cytochrome oxidase subunit I COI gene partial cds mitochondrial 54 KX138490.1 Didemnum perlucidum voucher DBTIC117 cytochrome oxidase subunit I COI gene partial cds mitochondrial KU667270.1 Didemnum perlucidum voucher DBTIC80 cytochrome oxidase subunit I COI gene partial cds mitochondrial 98 KR537439.1 Didemnum perlucidum voucher DBTIC 30 cytochrome oxidase subunit I COI gene partial cds mitochondrial 51 KR303626.1 Didemnum perlucidum voucher DBTIC 29 cytochrome oxidase subunit I COI gene partial cds mitochondrial JQ731735.1 Didemnum perlucidum isolate 2 cytochrome oxidase subunit I COI gene partial cds mitochondrial KU883151.1 Didemnum perlucidum isolate JD781 cytochrome oxidase subunit I COI gene partial cds mitochondrial KX650790.1 Didemnum perlucidum voucher DBTIC170 cytochrome oxidase subunit I COI gene partial cds mitochondrial 98 KX650791.1 Didemnum perlucidum voucher DBTIC171 cytochrome oxidase subunit I COI gene partial cds mitochondrial 54 AB104866.1 Megalodicopia hians COI for cytochrome c oxidase subunit 1 partial cds AB104867.1 Chelyosoma siboja COI for cytochrome c oxidase subunit I partial cds 69 Corella willmeriana|TRINITY DN3184 c0 g1 i1 99 EU140801.1 Corella eumyota haplotype H1 cytochrome c oxidase subunit I COI gene partial cds mitochondrial 91 KX138509.1 Ecteinascidia thurstoni voucher DBTIC134 cytochrome oxidase subunit I COI gene partial cds mitochondrial

100 KX138508.1 Ecteinascidia venui voucher DBTIC133 cytochrome oxidase subunit I COI gene partial cds mitochondrial KT693190.1 Ecteinascidia venui voucher DBTIC50 cytochrome oxidase subunit I COI gene partial cds mitochondrial EF643374.1 Ecteinascidia turbinata cytochrome c oxidase subunit I COI gene partial cds mitochondrial KY111425.1 Perophora viridis isolate 22Jul14-3-1 cytochrome oxidase subunit I COI gene partial cds mitochondrial 95 95 JQ863390.1 Perophora japonica haplotype H 1 cytochrome c oxidase subunit I COI gene partial cds mitochondrial AY600968.1 Ecteinascidia herdmanni cytochrome c oxidase subunit I COI gene partial cds mitochondrial KU667277.1 Perophora multiclathrata voucher DBTIC88 cytochrome oxidase subunit I COI gene partial cds mitochondrial 88 KU667278.1 Perophora multiclathrata voucher DBTIC89 cytochrome oxidase subunit I COI gene partial cds mitochondrial KT693196.1 Perophora multiclathrata voucher DBTIC56 cytochrome oxidase subunit I COI gene partial cds mitochondrial KT276225.1 Perophora multiclathrata voucher DBTIC45 cytochrome c oxidase subunit I COI gene partial cds mitochondrial 96 KU360801.1 Perophora multiclathrata isolate ASC16 cytochrome oxidase subunit I COI gene partial cds mitochondrial KU667276.1 Perophora multiclathrata voucher DBTIC87 cytochrome oxidase subunit I COI gene partial cds mitochondrial 7 KT693197.1 Perophora multiclathrata voucher DBTIC 57 cytochrome oxidase subunit I COI gene partial cds mitochondrial Ciona intestinalis NC 004447.2 cds NP 758778.1 12 KF309570.1 Ciona robusta isolate ESC-5 cytochrome oxidase subunit I COI gene partial cds mitochondrial KF309580.1 Ciona robusta isolate EST-CI cytochrome oxidase subunit I COI gene partial cds mitochondrial KF309554.1 Ciona robusta isolate BLA-CI cytochrome oxidase subunit I COI gene partial cds mitochondrial KF309587.1 Ciona robusta isolate HI-1 cytochrome oxidase subunit I COI gene partial cds mitochondrial KF309575.1 Ciona robusta isolate ESC-AS cytochrome oxidase subunit I COI gene partial cds mitochondrial KF309578.1 Ciona robusta isolate EST-AS cytochrome oxidase subunit I COI gene partial cds mitochondrial KF309532.1 Ciona robusta isolate AM-1 cytochrome oxidase subunit I COI gene partial cds mitochondrial KF309651.1 Ciona robusta isolate SFG-AS cytochrome oxidase subunit I COI gene partial cds mitochondrial 87 KF309591.1 Ciona robusta isolate LLAN-AS cytochrome oxidase subunit I COI gene partial cds mitochondrial KF309662.1 Ciona robusta isolate TO-AA cytochrome oxidase subunit I COI gene partial cds mitochondrial KF309658.1 Ciona robusta isolate TA-2 cytochrome oxidase subunit I COI gene partial cds mitochondrial KF309613.1 Ciona robusta isolate MP-CIT cytochrome oxidase subunit I COI gene partial cds mitochondrial KF309628.1 Ciona robusta isolate POG-AS cytochrome oxidase subunit I COI gene partial cds mitochondrial

25 KF309604.1 Ciona robusta isolate MAT-CI cytochrome oxidase subunit I COI gene partial cds mitochondrial KF309614.1 Ciona robusta isolate PB-1 cytochrome oxidase subunit I COI gene partial cds mitochondrial KF309593.1 Ciona robusta isolate LLAN-CI cytochrome oxidase subunit I COI gene partial cds mitochondrial KF309603.1 Ciona robusta isolate MAT-AS cytochrome oxidase subunit I COI gene partial cds mitochondrial 74 KF309602.1 Ciona robusta isolate MAT-AP cytochrome oxidase subunit I COI gene partial cds mitochondrial Ciona robusta NC 034372.1 cds YP 009357192.1 12 82 Ciona intestinalis NC 017929.1 cds YP 006341036.1 12 KF309636.1 Ciona sp. ROS-5 cytochrome oxidase subunit I COI gene partial cds mitochondrial KF597196.1 Ciona savignyi isolate KCI00031 cytochrome c oxidase subunit I COI gene partial cds mitochondrial 100 Ciona savignyi NC 004570.1 cds NP 786952.1 1 KF414706.1 Phallusia arabica isolate ASP9 cytochrome c oxidase subunit I COI gene partial cds mitochondrial Phallusia mammillata NC 009833.1 cds YP 001481141.1 12 72 AY600980.1 Phallusia mammillata cytochrome c oxidase subunit I COI gene partial cds mitochondrial KF309607.1 Phallusia mammillata isolate MP-1 cytochrome oxidase subunit I COI gene partial cds mitochondrial KP779903.1 Phallusia arabica cytochrome oxidase subunit I COI gene partial cds mitochondrial 46 KP779905.1 Phallusia mammillata cytochrome oxidase subunit I COI gene partial cds mitochondrial KP779904.1 Phallusia fumigata cytochrome oxidase subunit I COI gene partial cds mitochondrial 10 46 KF309548.1 Phallusia fumigata isolate AR-1 cytochrome oxidase subunit I COI gene partial cds mitochondrial 85 KF414698.1 Phallusia mammillata isolate ASP1 cytochrome c oxidase subunit I COI gene partial cds mitochondrial 40 Phallusia fumigata NC 009834.1 cds YP 001481152.1 10 Ascidia sp|TRINITY DN8074 c1 g1 i1 KX650762.1 Phallusia nigra voucher DBTIC142 cytochrome oxidase subunit I COI gene partial cds mitochondrial 94 67 AY600976.1 Phallusia ingeria cytochrome c oxidase subunit I COI gene partial cds mitochondrial 96 KY111416.1 Ascidia interrupta isolate 21Jul14-2-1 cytochrome oxidase subunit I COI gene partial cds mitochondrial KY111415.1 Ascidia interrupta isolate 17Jul14-1-10 cytochrome oxidase subunit I COI gene partial cds mitochondrial 91 KR604726.1 Ascidia viridina voucher ColBIO TL524 cytochrome oxidase subunit I COI gene partial cds mitochondrial 12 31 KJ725162.1 Ascidia virginea isolate GAASC11 cytochrome c oxidase subunit 1 COI gene partial cds mitochondrial 93 KF309647.1 Ascidia virginea isolate SCR-AS cytochrome oxidase subunit I COI gene partial cds mitochondrial 46 KX650763.1 Ascidia gemmata voucher DBTIC143 cytochrome oxidase subunit I COI gene partial cds mitochondrial KF309529.1 Ascidiella scabra isolate AB-AS cytochrome oxidase subunit I COI gene partial cds mitochondrial KF309556.1 Ascidiella scabra isolate BU-AS cytochrome oxidase subunit I COI gene partial cds mitochondrial KF309572.1 Ascidiella scabra isolate ESC-9 cytochrome oxidase subunit I COI gene partial cds mitochondrial

72 KF309560.1 Ascidiella scabra isolate CF-1 cytochrome oxidase subunit I COI gene partial cds mitochondrial KF309650.1 Ascidiella scabra isolate SFG-3 cytochrome oxidase subunit I COI gene partial cds mitochondrial 88 KJ725163.1 Ascidiella aspersa isolate GAASC12 cytochrome c oxidase subunit 1 COI gene partial cds mitochondrial JQ742948.1 Ascidiella aspersa clone SY011701 cytochrome c oxidase subunit 1 COI gene partial cds mitochondrial 59 KF309559.1 Ascidiella aspersa isolate CA-AS cytochrome oxidase subunit I COI gene partial cds mitochondrial 59 KF309631.1 Ascidiella aspersa isolate POM-SC cytochrome oxidase subunit I COI gene partial cds mitochondrial KF309653.1 Ascidiella aspersa isolate SFG-SC cytochrome oxidase subunit I COI gene partial cds mitochondrial KU695283.1 Ascidiella aspersa isolate 91 cytochrome c oxidase subunit I COI gene partial cds mitochondrial KF309594.1 Ascidiella aspersa isolate LLAN-SC cytochrome oxidase subunit I COI gene partial cds mitochondrial KF886702.1 Ascidiella aspersa isolate DNAS-54-45089 cytochrome c oxidase subunit I COI gene partial cds mitochondrial KF309555.1 Ascidiella aspersa isolate BLA-SC cytochrome oxidase subunit I COI gene partial cds mitochondrial 33 KF309534.1 Ascidiella aspersa isolate AM-3 cytochrome oxidase subunit I COI gene partial cds mitochondrial KF309568.1 Ascidiella aspersa isolate EMB-AS cytochrome oxidase subunit I COI gene partial cds mitochondrial KF309533.1 Ascidiella aspersa isolate AM-2 cytochrome oxidase subunit I COI gene partial cds mitochondrial KF309562.1 Ascidiella aspersa isolate CF-3 cytochrome oxidase subunit I COI gene partial cds mitochondrial KF309661.1 Ascidiella aspersa isolate TO-3 cytochrome oxidase subunit I COI gene partial cds mitochondrial KF309606.1 Ascidiella aspersa isolate MAT-SC cytochrome oxidase subunit I COI gene partial cds mitochondrial KF309637.1 Ascidiella aspersa isolate ROS-AS cytochrome oxidase subunit I COI gene partial cds mitochondrial KF309617.1 Ascidiella aspersa isolate PB-6 cytochrome oxidase subunit I COI gene partial cds mitochondrial FJ528625.1 Pyura squamulosa isolate 60 cytochrome oxidase subunit I COI gene partial cds mitochondrial FJ528614.1 Pyura gibbosa isolate 20 cytochrome oxidase subunit I COI gene partial cds mitochondrial 80 FJ528613.1 Pyura gibbosa isolate 6 cytochrome oxidase subunit I COI gene partial cds mitochondrial 50 FJ528615.1 Pyura gibbosa isolate 21 cytochrome oxidase subunit I COI gene partial cds mitochondrial 78 FJ528617.1 Pyura australis isolate 22 cytochrome oxidase subunit I COI gene partial cds mitochondrial 73 FJ528616.1 Pyura australis isolate 13 cytochrome oxidase subunit I COI gene partial cds mitochondrial FJ528612.1 Pyura spinifera isolate 23 cytochrome oxidase subunit I COI gene partial cds mitochondrial 99 FJ528611.1 Pyura spinifera isolate 4 cytochrome oxidase subunit I COI gene partial cds mitochondrial DQ658850.1 Pyura praeputialis isolate cyp6 cytochrome oxidase subunit I COI gene partial cds mitochondrial 91 FJ528624.1 Pyura praeputialis isolate 64 cytochrome oxidase subunit I COI gene partial cds mitochondrial FJ528621.1 Pyura praeputialis isolate 55 cytochrome oxidase subunit I COI gene partial cds mitochondrial FJ528623.1 Pyura praeputialis isolate 58 cytochrome oxidase subunit I COI gene partial cds mitochondrial FJ528622.1 Pyura praeputialis isolate 57 cytochrome oxidase subunit I COI gene partial cds mitochondrial FJ528620.1 Pyura praeputialis isolate 14 cytochrome oxidase subunit I COI gene partial cds mitochondrial 71 GU065362.1 Boltenia echinata from Canada cytochrome c oxidase subunit I COI gene partial cds mitochondrial 96 GU065361.1 Boltenia echinata from Canada cytochrome c oxidase subunit I COI gene partial cds mitochondrial 94 89 GU065360.1 Boltenia echinata from Canada cytochrome c oxidase subunit I COI gene partial cds mitochondrial FJ528628.1 Boltenia ovifera isolate 18 cytochrome oxidase subunit I COI gene partial cds mitochondrial 4597 FJ528627.1 Boltenia ovifera isolate 16 cytochrome oxidase subunit I COI gene partial cds mitochondrial 100 KF309582.1 Styelidae EST-MI cytochrome oxidase subunit I COI gene partial cds mitochondrial KF309588.1 Polycarpa sp. HI-2 cytochrome oxidase subunit I COI gene partial cds mitochondrial

69 KF309596.1 Pyura dura isolate MAS-2 cytochrome oxidase subunit I COI gene partial cds mitochondrial FJ528619.1 Pyura dura isolate 25 cytochrome oxidase subunit I COI gene partial cds mitochondrial 100 FJ528618.1 Pyura dura isolate 24 cytochrome oxidase subunit I COI gene partial cds mitochondrial GU220387.1 Botrylloides violaceus haplotype Bv6 cytochrome oxidase subunit I COI gene partial cds mitochondrial 91 GQ365694.1 Botrylloides violaceus isolate Bv4 cytochrome oxidase subunit I COI gene partial cds mitochondrial GQ365695.1 Botrylloides violaceus isolate Bv5 cytochrome oxidase subunit I COI gene partial cds mitochondrial GQ365693.1 Botrylloides violaceus isolate Bv3 cytochrome oxidase subunit I COI gene partial cds mitochondrial GQ365691.1 Botrylloides violaceus isolate Bv1 cytochrome oxidase subunit I COI gene partial cds mitochondrial GU220388.1 Botrylloides violaceus haplotype Bv7 cytochrome oxidase subunit I COI gene partial cds mitochondrial 92 GQ365692.1 Botrylloides violaceus isolate Bv2 cytochrome oxidase subunit I COI gene partial cds mitochondrial FJ528644.2 Botrylloides violaceum isolate 67 cytochrome oxidase subunit I COI gene partial cds mitochondrial 33 GU065359.1 Botrylloides violaceus isolate 4 from USA cytochrome c oxidase subunit I COI gene partial cds mitochondrial GU065356.1 Botrylloides violaceus isolate 30 from Canada cytochrome c oxidase subunit I COI gene partial cds mitochondrial 11 GU065357.1 Botrylloides violaceus isolate 6 from Canada cytochrome c oxidase subunit I COI gene partial cds mitochondrial GU065355.1 Botrylloides violaceus isolate 27 from Canada cytochrome c oxidase subunit I COI gene partial cds mitochondrial GU065358.1 Botrylloides violaceus isolate 3 from USA cytochrome c oxidase subunit I COI gene partial cds mitochondrial 1 GQ365690.1 Botrylloides fuscus cytochrome oxidase subunit I COI gene partial cds mitochondrial KT693201.1 Botrylloides nigrum voucher DBTIC61 cytochrome oxidase subunit I COI gene partial cds mitochondrial Botrylloides nigrum NC 021467.1 cds YP 008083006.1 8 KT693200.1 Botrylloides nigrum voucher DBTIC60 cytochrome oxidase subunit I COI gene partial cds mitochondrial 85 KT693198.1 Botrylloides nigrum voucher DBTIC58 cytochrome oxidase subunit I COI gene partial cds mitochondrial KF309551.1 Botrylloides leachii isolate AR-5 cytochrome oxidase subunit I COI gene partial cds mitochondrial KF309549.1 Botrylloides leachii isolate AR-2 cytochrome oxidase subunit I COI gene partial cds mitochondrial KF309641.1 Botrylloides leachii isolate SCR-1 cytochrome oxidase subunit I COI gene partial cds mitochondrial

31 KF309608.1 Botrylloides leachii isolate MP-2 cytochrome oxidase subunit I COI gene partial cds mitochondrial KU711782.1 Botrylloides nigrum isolate IRAR2 cytochrome c oxidase subunit I COI gene partial cds mitochondrial KF309610.1 Botrylloides leachii isolate MP-5 cytochrome oxidase subunit I COI gene partial cds mitochondrial KP254541.1 Botrylloides nigrum voucher FTP 0791 cytochrome oxidase subunit 1 COI gene partial cds mitochondrial KF309642.1 Botrylloides leachii isolate SCR-2 cytochrome oxidase subunit I COI gene partial cds mitochondrial KF309611.1 Botrylloides leachii isolate MP-6 cytochrome oxidase subunit I COI gene partial cds mitochondrial 45 FJ528645.1 Botrylloides leachi isolate 53 cytochrome oxidase subunit I COI gene partial cds mitochondrial KF309609.1 Botrylloides leachii isolate MP-4 cytochrome oxidase subunit I COI gene partial cds mitochondrial KF309644.1 Botrylloides leachii isolate SCR-4 cytochrome oxidase subunit I COI gene partial cds mitochondrial Botrylloides leachii NC 024103.1 cds YP 009029811.1 1 20 LT908007.1 Botryllus schlosseri mitochondrial partial COI gene for cytochrome oxidase subunit 1 specimen voucher MSNVE-24197 isolate NeoA 100 KX650765.1 Botrylloides chevalense voucher DBTIC145 cytochrome oxidase subunit I COI gene partial cds mitochondrial 8730 KX650764.1 Botrylloides chevalense voucher DBTIC144 cytochrome oxidase subunit I COI gene partial cds mitochondrial KT276224.1 Symplegma brakenhielmi voucher DBTIC38 cytochrome c oxidase subunit I COI gene partial cds mitochondrial 49 FJ528648.1 Symplegma rubra isolate 74 cytochrome oxidase subunit I COI gene partial cds mitochondrial KU360802.1 Symplegma brakenhielmi isolate ASC17 cytochrome oxidase subunit I COI gene partial cds mitochondrial 58 36 FJ528651.1 Stolonica socialis isolate 69 cytochrome oxidase subunit I COI gene partial cds mitochondrial KX138510.1 Symplegma brakenhielmi voucher DBTIC136 cytochrome oxidase subunit I COI gene partial cds mitochondrial HF922625.1 Botrylloides giganteum mitochondrial partial COI gene for cytochrome oxidase subunit 1 isolate PE 7 94 Botrylloides pizoni NC 024104.1 cds YP 009029824.1 1 KF309623.1 Distomus variolosus isolate DS-5 cytochrome oxidase subunit I COI gene partial cds mitochondrial 14 FJ528652.1 Distomus variolosus isolate 84 cytochrome oxidase subunit I COI gene partial cds mitochondrial 100 FJ528653.1 Distomus variolosus isolate 85 cytochrome oxidase subunit I COI gene partial cds mitochondrial 100 FJ528650.1 Dendrodoa grossularia isolate 79 cytochrome oxidase subunit I COI gene partial cds mitochondrial FJ528649.1 Dendrodoa grossularia isolate 78 cytochrome oxidase subunit I COI gene partial cds mitochondrial 100 KY111429.1 Polyandrocarpa zorritensis isolate 17Jul14-2-1 cytochrome oxidase subunit I COI gene partial cds mitochondrial 70 KF309643.1 Polyandrocarpa zorritensis isolate SCR-3 cytochrome oxidase subunit I COI gene partial cds mitochondrial 33 FJ528646.1 Polycarpa aurata isolate 40 cytochrome oxidase subunit I COI gene partial cds mitochondrial

49 KF309583.1 Styelidae FOR-4 cytochrome oxidase subunit I COI gene partial cds mitochondrial 53 KF309577.1 Styelidae EST-2 cytochrome oxidase subunit I COI gene partial cds mitochondrial KJ725157.1 Styelidae sp. GA-2014 cytochrome c oxidase subunit 1 COI gene partial cds mitochondrial 90 AY600984.1 Polycarpa pomaria cytochrome c oxidase subunit I COI gene partial cds mitochondrial KF309569.1 Styelidae ESC-1 cytochrome oxidase subunit I COI gene partial cds mitochondrial FJ528655.1 Polycarpa tenera isolate 90 cytochrome oxidase subunit I COI gene partial cds mitochondrial FJ528647.1 Polyzoa opuntia isolate 73 cytochrome oxidase subunit I COI gene partial cds mitochondrial KY111427.1 Polyandrocarpa anguinea isolate 17Jul14-3-4 cytochrome oxidase subunit I COI gene partial cds mitochondrial KY111426.1 Polyandrocarpa anguinea isolate 17Jul14-1-1B cytochrome oxidase subunit I COI gene partial cds mitochondrial 97 KY111428.1 Polyandrocarpa anguinea isolate 22Jul14-1-5 cytochrome oxidase subunit I COI gene partial cds mitochondrial 55 KU697729.1 Microcosmus squamiger isolate Ll3I cytochrome c oxidase subunit I COI gene partial cds mitochondrial KX650804.1 Microcosmus helleri voucher DBTIC184 cytochrome oxidase subunit I COI gene partial cds mitochondrial 84 KF414709.1 Microcosmus sp. ASP-2013 cytochrome c oxidase subunit I COI gene partial cds mitochondrial KX650803.1 Microcosmus helleri voucher DBTIC183 cytochrome oxidase subunit I COI gene partial cds mitochondrial

53 98 KX138507.1 Microcosmus exasperatus voucher DBTIC132 cytochrome oxidase subunit I COI gene partial cds mitochondrial 100 KX138506.1 Microcosmus exasperatus voucher DBTIC131 cytochrome oxidase subunit I COI gene partial cds mitochondrial

60 KX650767.1 Cnemidocarpa sp. AHJ-2016 voucher DBTIC147 cytochrome oxidase subunit I COI gene partial cds mitochondrial 13 99 KX138513.1 Cnemidocarpa areolata voucher DBTIC137 cytochrome oxidase subunit I COI gene partial cds mitochondrial 75 39 KU667275.1 Cnemidocarpa sp. n. JAHA-2016 voucher DBTIC86 cytochrome oxidase subunit I COI gene partial cds mitochondrial FJ528605.1 Microcosmus claudicans isolate 59 cytochrome oxidase subunit I COI gene partial cds mitochondrial

22 Microcosmus sulcatus NC 013752.1 cds YP 003406728.1 13 KF297502.1 Halocynthia papillosa haplotype HP Hap 1 cytochrome oxidase subunit 1 COI gene partial cds mitochondrial S54796.1 cytochrome oxidase subunit I Halocynthia roretzi ascidian Mitochondrial 1263 nt 71 Halocynthia roretzi NC 002177.1 cds NP 038239.1 1 100 FJ528610.1 Halocynthia pyriformis isolate 30 cytochrome oxidase subunit I COI gene partial cds mitochondrial FJ528607.1 Halocynthia papillosa isolate 76 cytochrome oxidase subunit I COI gene partial cds mitochondrial 65 FJ528606.1 Halocynthia papillosa isolate 75 cytochrome oxidase subunit I COI gene partial cds mitochondrial FJ528608.1 Halocynthia papillosa isolate 77 cytochrome oxidase subunit I COI gene partial cds mitochondrial 87 FJ528609.1 Halocynthia papillosa isolate 62 cytochrome oxidase subunit I COI gene partial cds mitochondrial AY600981.1 Halocynthia papillosa cytochrome c oxidase subunit I COI gene partial cds mitochondrial 86 JX312279.1 Asterocarpa humilis haplotype H2 cytochrome c oxidase subunit I COI gene partial cds mitochondrial JX312278.1 Asterocarpa humilis haplotype H1 cytochrome c oxidase subunit I COI gene partial cds mitochondrial 76 14 FJ528634.1 Styela plicata isolate 34 cytochrome oxidase subunit I COI gene partial cds mitochondrial FJ528633.1 Styela plicata isolate 32 cytochrome oxidase subunit I COI gene partial cds mitochondrial 99 FJ528626.1 Boltenia ovifera isolate 1 cytochrome oxidase subunit I COI gene partial cds mitochondrial FJ528640.1 Styela sp. RPP-2008 isolate 91 cytochrome oxidase subunit I COI gene partial cds mitochondrial FJ528639.1 Styela sp. RPP-2008 isolate 51 cytochrome oxidase subunit I COI gene partial cds mitochondrial 23 100 FJ528604.1 Microcosmus polymorphus isolate 54 cytochrome oxidase subunit I COI gene partial cds mitochondrial Herdmania momus NC 013561.1 cds YP 003331094.1 1 HM490289.1 Herdmania momus haplotype 1 cytochrome oxidase subunit I COI gene partial cds mitochondrial 94 FJ528629.1 Herdmania grandis isolate 26 cytochrome oxidase subunit I COI gene partial cds mitochondrial 94 FJ528630.1 Herdmania grandis isolate 27 cytochrome oxidase subunit I COI gene partial cds mitochondrial

96 KU667272.1 Herdmania momus voucher DBTIC82 cytochrome oxidase subunit I COI gene partial cds mitochondrial 26 KR867633.1 Herdmania momus voucher DBTIC42 cytochrome oxidase subunit I COI gene partial cds mitochondrial 67 KM411616.1 Herdmania momus voucher DBTIC22 cytochrome oxidase subunit I COI gene partial cds mitochondrial KX138500.1 Styela canopus voucher DBTIC127 cytochrome oxidase subunit I COI gene partial cds mitochondrial KF309557.1 Styelidae CA-2 cytochrome oxidase subunit I COI gene partial cds mitochondrial KX138501.1 Styela canopus voucher DBTIC128 cytochrome oxidase subunit I COI gene partial cds mitochondrial 56 KF309589.1 Styelidae HI-5 cytochrome oxidase subunit I COI gene partial cds mitochondrial 97 KF309590.1 Styela canopus isolate HI-6 cytochrome oxidase subunit I COI gene partial cds mitochondrial KT693195.1 Styela canopus voucher DBTIC55 cytochrome oxidase subunit I COI gene partial cds mitochondrial KX138498.1 Styela canopus voucher DBTIC125 cytochrome oxidase subunit I COI gene partial cds mitochondrial 29 KT693194.1 Microcosmus curvus voucher DBTIC54 cytochrome oxidase subunit I COI gene partial cds mitochondrial FJ528637.1 Styela gibbsii isolate 47 cytochrome oxidase subunit I COI gene partial cds mitochondrial 100 Styela gibbsii|TRINITY DN32987 c0 g1 i1 13 HQ916447.1 Styela gibbsii cytochrome oxidase subunit I COI gene partial cds mitochondrial 60 FJ528638.1 Styela montereyensis isolate 48 cytochrome oxidase subunit I COI gene partial cds mitochondrial KU905887.1 Styela canopus voucher SERCINVERT0909 cytochrome oxidase subunit 1 COI gene partial cds mitochondrial 27 KU714784.1 Styela clava isolate L3C3 cytochrome c oxidase subunit I COI gene partial cds mitochondrial 16 KU714785.1 Styela plicata isolate G3B5 cytochrome c oxidase subunit I COI gene partial cds mitochondrial FJ528631.1 Styela plicata isolate 10 cytochrome oxidase subunit I COI gene partial cds mitochondrial 16 KY111432.1 Styela plicata isolate BIO585 S11 cytochrome oxidase subunit I COI gene partial cds mitochondrial 65 bioRxiv preprint doi: https://doi.org/10.1101/236505KY111430.1; this version posted December Styela 19, 2017.plicata The copyright isolate holder for BIO585 this preprint (which S2 wascytochrome oxidase subunit I COI gene partial cds mitochondrial not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. KY111431.1 Styela plicata isolate BIO585 S6 cytochrome oxidase subunit I COI gene partial cds mitochondrial 25 AY600985.1 Styela plicata cytochrome c oxidase subunit I COI gene partial cds mitochondrial JQ742957.1 Styela plicata clone SY011710 cytochrome c oxidase subunit 1 COI gene partial cds mitochondrial FJ528632.1 Styela plicata isolate 31 cytochrome oxidase subunit I COI gene partial cds mitochondrial Styela plicata NC 013565.1 cds YP 003331150.1 5 62 AB104868.1 Styela plicata COI for cytochrome c oxidase subunit I partial cds HQ916425.1 Styela plicata haplotype 1 cytochrome oxidase subunit I COI gene partial cds mitochondrial KT988335.1 Styela plicata cytochrome oxidase subunit I COI gene partial cds mitochondrial KX871693.1 Styela plicata isolate B1 20 jgLCO-1490 cytochrome oxidase subunit I COI gene partial cds mitochondrial

97 KX871692.1 Styela plicata isolate B5 Asc1 jgLCO-1490 cytochrome oxidase subunit I COI gene partial cds mitochondrial 79 KX871694.1 Styela plicata isolate B5 Asc4 jgLCO-1490 cytochrome oxidase subunit I COI gene partial cds mitochondrial 100 KT693192.1 Eudistoma sp. AHJ-2015 voucher DBTIC52 cytochrome oxidase subunit I COI gene partial cds mitochondrial 11 KT693193.1 Eudistoma sp. AHJ-2015 voucher DBTIC53 cytochrome oxidase subunit I COI gene partial cds mitochondrial KR030569.1 Macropsis tunicata voucher CNC*HEM305577 cytochrome oxidase subunit 1 COI gene partial cds mitochondrial 18 KU667285.1 Ascidia sydneiensis voucher DBTIC85 cytochrome oxidase subunit I COI gene partial cds mitochondrial

100 KU667273.1 Ascidia sydneiensis voucher DBTIC83 cytochrome oxidase subunit I COI gene partial cds mitochondrial KU667274.1 Ascidia sydneiensis voucher DBTIC84 cytochrome oxidase subunit I COI gene partial cds mitochondrial 33 KR815822.1 Ascidia sydneiensis voucher DBTIC 13 cytochrome oxidase subunit I COI gene partial cds mitochondrial 52 KC974532.1 Amauronematus tunicatus voucher DEIGISHym18102 cytochrome oxidase subunit 1 COI gene partial cds mitochondrial 15 KC976922.1 Amauronematus tunicatus voucher DEIGISHym18903 cytochrome oxidase subunit 1 COI gene partial cds mitochondrial 100 KC974460.1 Amauronematus tunicatus voucher DEIGISHym18905 cytochrome oxidase subunit 1 COI gene partial cds mitochondrial KR630374.1 Eutrichota tunicata voucher BIOUG17280-B08 cytochrome oxidase subunit 1 COI gene partial cds mitochondrial 100 KJ725172.1 Ascidia ahodori isolate GAPB6 cytochrome c oxidase subunit 1 COI gene partial cds mitochondrial 19 77 AB104870.1 Ascidia ahodori COI for cytochrome c oxidase subunit I partial cds AB104869.1 Perophora sagamiensis COI for cytochrome c oxidase subunit I partial cds

40 AJ830012.1 Cnemidocarpa verrucosa mitochondrial partial COI gene for cytochrome c oxidase subunit I KJ725168.1 Didemnum candidum isolate GAPB2 cytochrome c oxidase subunit 1 COI gene partial cds mitochondrial 100 KJ725164.1 Didemnum candidum isolate GAASC13 cytochrome c oxidase subunit 1 COI gene partial cds mitochondrial 100 KJ725170.1 Didemnum candidum isolate GAPB4 cytochrome c oxidase subunit 1 COI gene partial cds mitochondrial 69 KJ725169.1 Didemnum candidum isolate GAPB3 cytochrome c oxidase subunit 1 COI gene partial cds mitochondrial KC429151.1 Corbula tunicata voucher BivAToL-85 cytochrome c oxidase subunit I COI gene partial cds mitochondrial KC706853.1 Salpa sp. BMOO-06976 cytochrome c oxidase subunit I COI gene partial cds mitochondrial 30 100 Salpa fusiformis|TRINITY DN12252 c0 g1 i1 100 Salpa fusiformis|TRINITY DN12252 c0 g2 i1 83 Iasia cylindrica|TRINITY DN5006 c0 g2 i1

49 KT818685.1 Brooksia lacromae dev-stage oozooid cytochrome oxidase subunit I COI gene partial cds mitochondrial 100 KT818686.1 Brooksia lacromae dev-stage blastozooid cytochrome oxidase subunit I COI gene partial cds mitochondrial KF977307.1 Oikopleura intermedia isolate DNZ117 cytochrome oxidase subunit I COI gene partial cds mitochondrial

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