DOI: 10.5061/dryad.31zcrjdxm
title: Data from: Phylogenetic history of the acquisition of molluscan hosts in acotylean flatworms

description: The question of how symbionts acquired hosts and diversified
 phylogenetically during their evolutionary history is a focus of attention
 in many symbiotic taxa. Marine polyclad flatworms are usually free-living,
 but some are symbiotic, using animals as hosts. However, the history of
 their acquisition of symbiotic systems is not well understood. Therefore,
 we focused on mollusc symbiotic flatworms in the suborder Acotylea and
 investigated the host specificity and phylogenetic history of symbiotic
 acquisition. Field surveys revealed that symbiotic flatworms utilized
 certain molluscs as hosts. In particular, Stylochoplana pusilla and
 Stylochoplana parasitica utilized different molluscan species as hosts
 sympatrically. Also, the phylogenetic analysis and the ancestral state
 reconstruction indicate that the mollusc symbiotic flatworms formed a
 monophyletic group and that their common ancestor shifted from free-living
 to mollusc symbiosis. These results suggest that each of the flatworms did
 not independently acquire a symbiotic system with molluscan hosts during
 its phylogenetic history, but that their common ancestor acquired a
 mollusc symbiotic system, which then underwent acquisition of host
 specificity and speciation. This study emphasises that multiple host use
 can be a driving force for niche advancement and speciation in the
 symbionts.
description: To investigate the phylogenetic relationships of the symbiotic
 flatworms, the whole mitochondrial genome and the nuclear ribosomal
 sequences of <em>S. pusilla</em>, <em>S.
 parasitica</em> and <em>Stylochoplana</em> sp. were
 determined. DNA extraction was conducted using the DNeasy Blood and Tissue
 Kit (Qiagen, Hilden, Germany). Partial sequences for CO1 and 28S were
 PCR-amplified following the PCR conditions described by the previous study
 <sup>1</sup>, and were obtained through sequencing on ABI
 3130xl automated DNA sequencer at Atmosphere and Ocean Research Institute
 (AORI), The University of Tokyo (Kashiwa, Chiba). The extracted DNA was
 quantified using a Nanodrop One (Thermo Scientific, Wilmington, DEL) and
 used for the library preparation for next-generation sequencing analysis.
 The DNA shotgun sequencing was conducted using a Miseq System (Illumina,
 San Diego, CA) with paired-end 300 bp reads at the National Institute for
 Environmental Studies (Tsukuba, Japan) for <em>S.
 parasitica</em>, and using DNBSEQ-T7 system (MGI Tech, Shenzhen,
 China) with pair-end 150 bp reads at Bioengineering Lab. Co., Ltd.
 (Sagamihara, Japan) for <em>S. pusilla</em> and
 <em>Stylochoplana</em> sp., respectively. The raw reads (Table
 S2) for each species were assembled into mitogenome and nuclear ribosomal
 DNA sequences using the NovoPlasty ver. 4.3.4 <sup>2</sup>.
 Partial CO1 and 28S sequences obtained from the same specimens were used
 as seed sequences for the NovoPlasty assembly of the mitogenome and
 nuclear ribosomal DNA sequences for three <em>Stylochoplana
 </em>species. The annotation of the mitochondrial genomes was
 initially performed using the MITOS 2 web server <sup>3</sup>,
 followed by manual verification of the annotation in Mesquite 3.11
 <sup>4</sup>. Due to the difficulty in automatic annotating of
 the ATP8 gene of Platyhelminthes species with MITOS
 <sup>5</sup>, ATP8 was re-annotated through manual inspection
 methods (e.g. <sup>5–7</sup>). The putative open reading
 frames (ORFs) of ATP8 were expected to be located in gap regions without
 annotations. To identify the ATP8 gene, ORFfinder
 (https://www.ncbi.nlm.nih.gov/orffinder/) was used to analyze long gap
 regions. The gap regions lacking annotations, which contained putative
 ORFs, were re-annotated using the MITOS 2 web server, resulting in the
 prediction of the ATP8 gene. The predicted ATP8 sequences were validated
 through BLAST searches <sup>8</sup> and manually compared
 among the three <em>Stylochoplana</em> species analysed.
 Circular mitogenome maps were constructed using Proksee
 <sup>9</sup>. The 28S and 18S sequences of nuclear ribosomal
 DNA were also annotated using BLAST. We constructed phylogenetic trees
 using 18S, 28S, 16S and CO1, which are commonly used in previous studies,
 and the full length of the mitochondrial genome, respectively. The
 mitochondrial and nuclear genome sequences newly determined in this study
 have been deposited in the DDBJ (DNA Data Bank of Japan) (Table S2). The
 sequences of 18S, 28S, 16S, and CO1 and the mitogenome sequences used for
 phylogenetic analysis have been summarized (Table S3 and Table S4). All
 sequences containing gaps were trimmed using trimAL after alignment
 <sup>10</sup>, with the -gt option set to 0.8. The final
 trimmed alignments resulted in 2,657 bp for the 18S, 28S, 16S and CO1
 dataset and 11,710 bp for the mitochondrial genome. Phylogenetic trees
 were constructed using Bayesian inference (BI) in MrBayes and maximum
 likelihood (ML) methods in IQ-TREE. <sup>11,12</sup>.
 Evolutionary models were selected using Kakusan and ModelFinder
 respectively, and the phylogenetic trees were finally constructed using
 the best model based on BIC (Table S5 and Table S6)
 <sup>13,14</sup>. Two Markov chains were run for Bayesian
 estimation. The BI tree was constructed using 18S, 28S, 16S, and CO1 with
 the following settings: ngen = 15,000,000, samplefreq = 1000, burn-in =
 375,000. The construction of a BI tree of the whole mitochondrial genome
 was constructed with the following settings: ngen = 60,000,000, samplefreq
 = 1,000, burn-in = 15,000,000. Convergence of the model was verified using
 Tracer v1.7.1 <sup>15</sup>. The model was considered to have
 converged if the ESS (MCMC effective sample size) of all statistics from
 the MCMC simulation was greater than 100. For ML tree, ultrafast
 bootstrapping was performed with 1,000 replicates
 <sup>16</sup>.               Ancestral
 trait reconstruction was performed using BayesTraitsV3 to estimate whether
 the common ancestor was a free-living or a symbiont
 <sup>17</sup>. Branch lengths were scaled to an average of 0.1
 with reference to the BayesTraitsV3 manual. The simulation was conducted
 using reversible-jump MCMC with the following settings: ngen = 15,000,000,
 samplefreq = 1000, burn-in = 5,000,000. The exponential reversible jump
 prior distribution (RevJump exp) was set to 10. Marginal likelihood
 estimation was performed using the stepping-stone sampler, with the number
 of stones set at 100 and the number of iterations at 1000. Ancestral trait
 reconstruction was based on the BI tree, considering nodes with high
 support (Bayesian posterior probability &gt; 0.9 or ultrafast
 bootstrapping &gt; 90%). Acotylean flatworms are typically free-living
 that attach to rocks and algae, but they also interact with other
 organisms in various ecological contexts, such as predator-prey
 relationships <sup>18–20</sup>. Therefore, based on clearer
 information, we defined only those species for which a symbiotic
 relationship (symbiosis or parasitism) was mentioned in previous studies
 as symbiotic species, and incorporated their host organisms as traits in
 the model. Specifically, <em>I. zebra,</em> which utilises
 hermit crabs <sup>21</sup>, and <em>E.
 lysiosquillae</em>, which utilises the mantis shrimp
 <em>Lysiosquilla maculata</em> <sup>22</sup>, were
 defined as symbionts, in addition to the mollusc-utilising flatworms,
 <em>S. pusilla</em>, <em>S. parasitic,</em> and
 <em>Stylochoplana</em> sp.. Their respective host organisms
 (molluscs, hermit crabs and the mantis shrimp) were considered as traits
 (Table S1). Species that were shown in previous studies to be free-living,
 or to have been attached to rocky or boulder sites, or that were not
 mentioned as being symbiotic, were classified as free-living.
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description: # Phylogenetic history of the acquisition of molluscan hosts in acotylean
 flatworms
 [https://doi.org/10.5061/dryad.31zcrjdxm](https://doi.org/10.5061/dryad.31zcrjdxm) ## Description of the data and file structure This dataset was used in the paper ‘Phylogenetic history of the acquisition of molluscan hosts in acotylean flatworms’ submitted to Biology Letters. ”`AncestralStateEstimation.zip`” is the file used for ancestral state estimation, which was based on the obtained phylogenetic tree and host-use information.  \&gt;"bayes.tree": Phylogenetic tree file used. \&gt;"habitat.txt": Text file showing current traits. The first column is the species name and the second column indicates the trait. Trait A indicates free-living, B indicates mantis shrimp host, C indicates hermit crab host, and D indicates mollusc host. \&gt;"run.txt": Run file showing detailed settings for ancestral trait estimation. ”`PhylogenyMtGenome.zip`” is the file used in the construction of the phylogenetic tree of the mitochondrial genome. The files include those used in maximum likelihood and Bayesian estimation respectively. \&gt;"MtGenome_IQtree_P.nex": Nexus file showing the partitions used in IQ-TREE. \&gt;"MtGenome_IQtree.fasta": Sequence information used in IQ-TREE. \&gt;"MtGenome_MrBayes.nex": Nexus file used in MrBayes. ”`PhylogenySangerMethods.zip`” is the file used in the construction of the phylogenetic tree of partial sequences obtained by the Sanger method. The files include those used in maximum likelihood and Bayesian estimation respectively. \&gt;"Sanger_IQtree_P.nex": Nexus file showing partitions used in IQ-TREE \&gt;"Sanger_IQtree.fasta": Sequence information used in IQ-TREE \&gt;"Sanger_MrBayes.nex": Nexus file used in MrBayes