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Accepted Manuscript

Antarctic and sub-Antarctic reveal novel evolutionary charac- teristics of mitochondrial genomes in

Juan D. Gaitán-Espitia, Claudio A. González-Wevar, Elie Poulin, Leyla Cardenas

PII: S1055-7903(17)30583-3 DOI: https://doi.org/10.1016/j.ympev.2018.10.036 Reference: YMPEV 6324

To appear in: Molecular Phylogenetics and Evolution

Received Date: 15 August 2017 Revised Date: 23 July 2018 Accepted Date: 30 October 2018

Please cite this article as: Gaitán-Espitia, J.D., González-Wevar, C.A., Poulin, E., Cardenas, L., Antarctic and sub- Antarctic Nacella limpets reveal novel evolutionary characteristics of mitochondrial genomes in Patellogastropoda, Molecular Phylogenetics and Evolution (2018), doi: https://doi.org/10.1016/j.ympev.2018.10.036

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Version: 23-07-2018

SHORT COMMUNICATION

Running head: mitogenomes Nacella limpets

Antarctic and sub-Antarctic Nacella limpets reveal novel evolutionary characteristics of mitochondrial genomes in Patellogastropoda

Juan D. Gaitán-Espitia1,2,3*; Claudio A. González-Wevar4,5; Elie Poulin5 & Leyla

Cardenas3

1 The Swire Institute of Marine Science and School of Biological Sciences, The University of Hong Kong, Pokfulam, Hong Kong, China

2 CSIRO Oceans and Atmosphere, GPO Box 1538, Hobart 7001, TAS, Australia.

3 Instituto de Ciencias Ambientales y Evolutivas, Facultad de Ciencias, Universidad Austral de Chile, Casilla 567 Valdivia, Chile.

4 Instituto de Ciencias Marinas y Limnológicas, Facultad de Ciencias, Universidad Austral de Chile, Casilla 567, Valdivia, Chile,

5 Laboratorio de Ecología Molecular, Instituto de Ecología y Biodiversidad, Departamento de Ciencias Ecológicas, Facultad de Ciencias, Universidad de Chile, Las Palmeras 3425,

Ñuñoa, Santiago, Chile

Corresponding author: [email protected]

Keywords: mitogenomes; Patellogastropoda; Nacella; ; gene order; phylogeny

1 Highlights

 We sequenced complete mitochondrial genome of three new patellogastropods  Novel gene rearrangements were detected for Gastropoda and Patellogastropoda  Two main translocations affect order of KARNI and MYCWQ clusters  Phylogenetic analyses support monophyly of Nacella and Patellogastropoda  New mitogenome-based phylogenetic hypothesis is proposed for Patellogastropoda

Abstract

Mitochondrial genomes (mitogenomes) provide valuable phylogenetic information and genome-level characters that are useful in resolving evolutionary relationships within major lineages of gastropods. However, for more than one decade, these relationships and the phylogenetic position of Patellogastropoda have been inferred based on the genomic architecture as well as the nucleotide and protein sequences of a single representative, the digitalis. This mitogenome exhibits extensive rearrangements and several repetitive units that may not represent universal features for Patellogastropoda. Here, we sequenced the complete mitogenomes of three Nacella limpets, providing new insights into the dynamics of gene order and phylogenetic relationships of Patellogastropoda.

Comparative analyses revealed novel gene rearrangements in Gastropoda, characterised by two main translocations that affect the KARNI and the MYCWQ clusters in Nacella limpets. Our phylogenetic reconstructions using combined sequence datasets of 13 mitochondrial protein-coding genes and two rRNAs, recovered Patellogastropoda, and

Gastropoda in general, as non-monophyletic. These findings could be related to the long- branch attraction tendency of these groups, and/or taxon sampling bias. In our novel mitogenome-based phylogenetic hypothesis, L. digitalis is placed in a sister position to

Bivalvia and , whereas Nacella limpets are placed sister to a clade

2 containing + and + Neomphalina.

1. Introduction

Patellogastropods or true limpets, are abundant and conspicuous keystone inhabitants of intertidal rocky shores throughout the world's oceans from the polar regions to the tropics

(Lindberg, 2008). Due to their particular morphological characteristics, these molluscs have been considered as the most external branch of living gastropods (Ponder and Lindberg,

1997). However, the phylogenetic position of the group is highly controversial. For example molecular analyses have placed patellogastropods as sister group of Gastropoda,

Vetigastropoda, Noemphalina, Heterobranchia or within Caenogastropoda (Colgan et al.,

2000; Harasewych and McArthur, 2000; Aktipis et al., 2008; Nakano and Sasaki, 2011;

Zapata et al., 2014).

The evolutionary relationships among families of true limpets are also controversial

(Harasewych and McArthur, 2000; Nakano and Ozawa, 2007; Lindberg, 2008; Nakano and

Sasaki, 2011). Molecular studies based on nuclear genes (18S and 28S rRNAs) have recovered the family as paraphyletic at the base of Patellogastropoda

(Harasewych and McArthur, 2000; Nakano and Ozawa, 2007), whereas analyses based on mitochondrial genes (rrnS, rrnL and cox1) recovered the monophyly of this group in a close relationship with and clearly distinguished from (Nakano and

Ozawa, 2007; González-Wevar et al., 2017a). The general consensus is that mitochondrial

3 genes are more informative for relationships within Patellogastropoda due to their faster and more uniform evolutionary rates among of this order, compared to nuclear genes (Harasewych and McArthur, 2000; Nakano and Ozawa, 2007; Nakano and Sasaki,

2011). Nevertheless, although more than seven thousand mitochondrial sequences of 253 species of patellogastropods have been deposited in GenBank (30/03/2018), most of these are partial sequences of rrnS, rrnL, cox1 and cob, and only one of them represents a complete mitochondrial genome (mitogenome) of the limpet . The genomic architecture as well as the nucleotide and protein sequences of this mitogenome have been widely used as references of Patellogastropoda to reconstruct the evolutionary history of different molluscan lineages (e.g. Grande et al., 2008; Stöger and Schrödl, 2013; Uribe et al., 2016b). Nonetheless, L. digitalis mitogenome exhibits extensive rearrangements and several repeating units (Simison et al., 2006; Grande et al., 2008) that may not represent universal features for Patellogastropoda. The aims of this study were thus: (a) to sequence the complete mitogenome of three Antarctic and sub-Antarctic Nacella limpets, a that is distantly related to Lottia (Nakano and Sasaki, 2011); (b) to investigate the dynamics of gene order arrangements within patellogastropods and in comparison with other gastropods; and (c) to understand the contribution of these newly sequenced mitogenomes to the phylogenetic relationships of Patellogastropoda.

2. Materials and Methods

2.1. Species collection and mitogenome sequencing

Adult limpets of Nacella concinna, N. magellanica and N. clypeater were collected from the intertidal and subtidal zones in the King George Island (62°10′S,

58°51′W), the Strait of Magellan (53° 37′S, 70° 54′W) and La Misión, Chile (39° 46′ S,

4 73° 23′W), respectively. For each species, pieces of foot muscle were isolated from 15 individuals for RNA and DNA sequencing. Total RNA was isolated by homogenization of each sample in a TRIzol (Invitrogen, Carlsbad, CA)/chloroform mixture, followed by a

DNase treatment and cleaning with the RNeasy mini kit (Qiagen, Chatsworth, CA).

Purified mRNA samples were pooled for cDNA library preparation (paired-end) and sequenced. DNA was extracted by the isolation of intact mitochondria from approximately 150 mg of tissue from single individuals using the Mitochondrial Isolation

Kit (Thermo Scientific). The isolated mitochondrial pellet of each limpet was used for the mtDNA extraction by mean of the Mitochondrial-DNA Isolation kit (BioVision). RNA and DNA quality and quantity were analysed using an Agilent 2100 Bioanalyzer (Agilent

Technologies, Palo Alto, CA). RNA and DNA library preparations and sequencing were done in the AUSTRAL-omics Core Facility (www.australomics.cl) on an Illumina Hiseq

2500 and a 454 GS Junior Titanium platform (Roche), for RNA and DNA respectively.

2.2. Assembly and annotation

Quality control of the raw data was performed using the CLC Genomics Workbench software v.8.5 (CLC bio, Denmark) by removing adapters and reads containing more than

5% of ambiguous nucleotides (‘N’). Additionally, sequences were trimmed with a Phred quality score ≤ 40; and reads shorter than 30 bp were removed after trimming. For the 454 raw reads we also used the condensation tool found in NextGENe v2.3.3 (Softgenetics,

USA) for correction of homopolymer errors and other base call errors produced by the pyrosequencing process. Then, DNA reads were assembled using the software Geneious

Pro 8.1(Biomatters), resulting in a single contig with uniform coverage distribution for each species. Assembly of high quality reads from Illumina sequencing (RNA-cDNA) was

5 carried out using the Trinity software (Grabherr et al., 2013) with default settings and a minimum contig length of 200 nt.

Mitochondrial DNA sequences were identified by local BLAST searches (BLASTn and BLASTx) using Geneious Pro 8.1(Biomatters). In addition, protein-coding genes

(PCGs) were identified with the ORF Finder at NCBI using the invertebrate mitochondrial genetic code. The limits of both protein-coding and ribosomal RNA genes were adjusted manually based on the location of adjacent genes and the presence of start and stop codons.

Transfer RNA genes were located and folded by their proposed cloverleaf to confirm their secondary structures using ARWEN v.1.2 (Laslett and Canbäck, 2008) following the generalized invertebrate mitochondrial tRNA settings. Finally, our annotations were double-checked using MITOS standalone version (Bernt et al., 2013b) under the invertebrate mitochondrial code. Mitogenome sequences obtained in this work were deposited in GenBank under the accession numbers KT990124 (N. clypeater), KT990125

(N. magellanica) and KT990126 (N. concinna).

2.3. Concatenated alignments

Nucleotide sequences of the 13 mitochondrial protein-coding genes (PCGs) and two rRNAs from gastropods used in this study (Table 1) were aligned separately using the

MAFFT platform implemented in TranslatorX (Abascal et al., 2010). Alignments were done using the L-INS-i option (accurate for alignment of ≤ 200 sequences) and default settings. For the PCGs, the alignments were translated into amino acid sequences using the invertebrate mitochondrial genetic code. Ambiguously-aligned sites were removed using

Gblocks v.0.19b implemented in TranslatorX (Abascal et al., 2010) with default settings.

6 Phylogenetic reconstructions were conducted based on the concatenation of the deduced amino acid sequences of the 13 PCGs and the nucleotide sequences of the two rRNAs.

2.4. Phylogenetic reconstructions

Best Partition Scheme (BPS) analyses were conducted with the program

PartitionFinder2 and PartitionFinder2 Protein (Lanfear et al., 2016), using the Akaike information criterion (AIC) and a heuristic search algorithm. Following Uribe et al.

(2016b), the MtZoa model (Rota-Stabelli et al., 2009) was also tested to examine if it could provide a better fit than the models selected by PartitionFinder2. Based on the likelihood values (Supplementary Information S1), we selected the models of molecular evolution used for both Bayesian (BI) and Maximum Likelihood (ML) analyses. ML inference were performed with RAXML v.7.2.6 (Stamatakis, 2006), invoking the GTRGAMMA (less than

50 taxa) and the rapid bootstrap option with 10000 replicates. In addition, a Bayesian inference (BI) MCMC analysis was conducted using MrBayes v.3.2 (Ronquist and

Huelsenbeck, 2003), running six Markov chains in two simultaneous runs of 10 million generations. Trees were sampled every 1000 generations and those inferred prior to stationarity were discarded as burn-in (first 25% of the sampled trees), and the remaining trees were used to construct a 50% majority-rule consensus tree.

Finally, an additional BI analysis was conducted in PhyloBayes MPI v.1.5a.

(Lartillot et al., 2013) using the CAT-GTR model of evolution. This method is described as a good strategy to alleviate the long-branch attraction biases that exist between

Heterobranchia and Patellogastropoda (Uribe et al., 2016b). The performance of the CAT-

GTR+G model was assessed using a 10-fold cross-validation performed on subsamples of

6000 non-constant positions randomly drawn from the original matrices. Convergence was

7 assessed using tools implemented in PhyloBayes. Posterior probabilities provided branch support for BI analyses.

3. Results and discussion

3.1. Mitogenomic architectures

Combined DNA and RNA sequencing approaches were able to recover the complete mitogenomes of Antarctic (N. concinna) and sub-Antarctic (N. magellanica and

N. clypeater) limpets. The size of these mitogenomes (~16 kb) falls within the range of other gastropods but is considerably smaller than in L. digitalis (Table 1), for which several repeated units are distributed across two regions of the genome (rrnS-cob and nad1-nad6) (Simison et al., 2006). As for most molluscs, Nacella mitogenomes consist of a single circular chromosome with the typical set of 13 protein-coding genes (PCGs), two ribosomal RNA genes (rRNA), 22 transfer RNA genes (tRNA), and a large AT-rich non- coding region (control region; CR) (Bernt et al., 2013a) that is implicated in the initiation of transcription and replication processes (Gaitán-Espitia et al., 2013). In these mitogenomes, genes are divided between the two strands about evenly, with the heavy strand (H) having 10 tRNAs and seven PCGs, and the light (L) strand having 12 tRNAs, six PCGs and the two rRNAs (Fig. 1). Although both strands are AT rich (65-68%), there is a compositional asymmetry favouring GT in the H-strand and AT in the L-strand

(Supplementary Information S2). The cause of this asymmetry is accounted to an asymmetric mutation process that favours transitions over transversions on the H-strand, which is prone to mutations when it is single stranded (i.e., during replication and transcription) (Bernt et al., 2013a).

8 In the 13 PCGs, three types of start codons (ATG, ATT and ATA) were present, of which ATG started 8 genes while ATA and ATT initiated two and three genes respectively. Nine open-reading frames ended with TAA whereas four ended with TAG.

The highest AT content was observed in nad4L (71.3%), whilst the lowest one was detected in cox3 (61.9%) (Supplementary Information S2). For the tRNAs, our findings are consistent with those documented for other metazoans (Bernt et al., 2013a), in which most of the tRNAs have the potential to fold into the typically cloverleaf secondary structure, with the exception of tRNA-S1 that lacks the dihydrouracil (DHU) arm

(Supplementary Information S3).

Gene arrangements in Antarctic and sub-Antarctic Nacella mitogenomes were highly conserved within the genus but exhibited clearly distinctive genomic architectures compared to L. digitalis (Fig. 1). Contrasting to this patellogastropod, as well as to other gastropods, no duplications and/or translocations+inversions were detected in Nacella limpets (Fig. 1). However, our newly sequenced mitogenomes showed similar arrangements to the hypothetical ancestral gene order of Gastropoda (Uribe et al., 2016b), and to some Neritomorpha and Vetigastropoda species (Fig. 1). Major differences with these groups could be explained by two main translocations that affect the KARNI (H- strand) and the MYCWQ (L-strand) clusters (Fig. 1). These translocations represent novel gene rearrangements in Gastropoda (Grande et al., 2008). With the exception of nad3, these findings are consistent with the idea that in Gastropoda, tRNA genes are more prone to switch their position than larger PCGs and rRNAs (Grande et al., 2008; Gaitán-Espitia et al., 2013).

3.2. Phylogenetic analysis

9 Best Partition Scheme (BPS) analyses for combined amino acid sequences of 13 PCGs and nucleotide sequences of the two rRNAs, revealed slightly better fit using the MtZoa model than the models selected by PartitionFinder2 (Supplementary Information S1).

Similar results has been documented in phylogenetic studies using mitochondrial genomes in other gastropod lineages (Stöger and Schrödl, 2013; Lee et al., 2016; Uribe et al.,

2016b). The MtZoa BPS was used for both Bayesian (BI) and Maximum Likelihood (ML) analyses, which produced nearly identical topologies with similar branch lengths and strong support of bootstraps (ML analysis) and posterior probabilities (Bayesian inference) for most of the nodes (Fig. 2 and Supplementary Information S4). Both, the ML (-LnL = -

143247.86) and the BI (-LnL = -137981.4) reconstructions, recovered with high statistical support the monophyly of the genus Nacella (Fig. 2). The evolutionary relationships within this group of Antarctic and sub-Antarctic limpets were consistent with previous molecular studies using partial sequences of mtDNA (cox1 and cob) and nDNA (28S rRNA) genes, supporting the division of Nacella according to the biogeographic regions described for the

Southern Ocean (González-Wevar et al., 2010, 2017b). Here, our results suggest that the

Antarctic limpet N. concinna branched off early in this monophyletic lineage (Fig. 2). This split is estimated to have occurred during late (~8 – 5.5 Mya), parallel to climatic transitions and intense thermal zonations between Antarctic and sub-Antarctic provinces

(González-Wevar et al., 2010, 2017b). Additionally, our results suggest that the sub-

Antarctic limpet N. magellanica shares a most recent common ancestor with N. clypeater

(Fig. 2), and their divergence time is estimated to be approximately 6-5 Mya in the transition between late Miocene and early Pliocene (González-Wevar et al., 2010).

In our study, the evolutionary relationships among gastropods and the phylogenetic position of Patellogastropoda were based on a taxon sampling that included representative

10 species of Cephalopoda, Scaphopoda, Polyplacophora Aplacophora, and

Monoplacophora as outgroups. The selection of these groups had important implications on our phylogenetic reconstructions. For instance, phylogenetic analyses using Octopus vulgaris (Cephalopoda) as a single outgroup recovered Heterobranchia as the most external branch of Gastropoda, and a monophyletic Patellogastropoda in a sister relationship to a clade containing Caenogastropoda + Neritimorpha and Vetigastropoda + Neomphalina

(Supplementary Information S5). However, a larger sampling of representative species from other molluscan groups (Table 1) recovered a different phylogenetic hypothesis with higher statistical support (Fig. 2). In this reconstruction, Gastropoda and Patellogastropoda were recovered as non-monophyletic (Fig. 2), probably as a result of the long-branch attraction tendency of these groups (Stöger and Schrödl, 2013; Osca et al., 2014), and/or taxon sampling bias (e.g., low representation in Patellogastropoda compared to other groups). The general topology for was highly consistent with other studies based on mitochondrial genome architectures (Stöger et al., 2016) and mitogenome sequences

(Stöger and Schrödl, 2013; Osca et al., 2014; Li et al., 2018). Within each Gastropoda order, our results are congruent with previous mitogenomic phylogenies of Vetigastropoda

(Uribe et al., 2016b) and Neritimorpha (Uribe et al., 2016a), with some discrepancies regarding the position of Patellogastropoda, which was previously based only on L. digitalis. Evolutionary reconstructions using L. digitalis as a reference for

Patellogastropoda may have led to misleading phylogenetic hypotheses in the past, not only due to the single-exemplar sampling effect (Spinks et al., 2013), but also because the particular features of its mitochondrial genome. This limpet exhibits extensive rearrangements and several repeating units (Simison et al., 2006; Grande et al., 2008) that are not universal features for Patellogastropoda, as evidenced by our newly sequenced

11 mitogenomes of Nacella limpets.

Former phylogenetic reconstructions of evolutionary relationships among gastropods have used a variety of different datasets, ranging from morphology (e.g., shell structure) to low number of molecular sequences (e.g., partial genes 18S, 28S, rrnS, rrnL and cox1), supermatrices of genes derived from transcriptomes, and the gene order of mitogenomes

(Ponder and Lindberg, 1997; Colgan et al., 2000; Harasewych and McArthur, 2000; Aktipis et al., 2008; Grande et al., 2008; Lindberg, 2008; Zapata et al., 2014). These studies have shown contrasting results placing patellogastropods as the most external branch of

Gastropoda or in a sister group relationship with Vetigastropoda, Noemphalina,

Heterobranchia or within Caenogastropoda (Colgan et al., 2000; Harasewych and

McArthur, 2000; Aktipis et al., 2008; Nakano and Sasaki, 2011; Zapata et al., 2014). In this study, patellogastropods were split in two groups with L. digitalis placed in a sister position to Bivalvia and Heterobranchia, whereas Nacella limpets were placed sister to a clade containing Caenogastropoda + Neritimorpha and Vetigastropoda + Neomphalina (Fig. 2).

Our novel phylogenetic hypothesis highlights the importance of increasing taxon sampling in patellogastropods in order to ameliorate the impact of long-branch attraction artefacts

(Felsenstein zone) and to improve our understanding of their phylogenetic relationships with other molluscan lineages. Moreover, considering that Patellogastropoda contains at least seven families (Nakano and Ozawa, 2007), a denser taxon sampling of mitogenomes of representative species from other groups such as , , Patellidae and

Nacellidae (e.g., , a sister genus of Nacella), will provide a better framework of the evolutionary relationships within patellogastropods and the dynamics of genome-level characters (e.g., rearranged structures) for this order.

12 Conflict of interest

The authors declare no conflicts of interest.

Acknowledgments

We would like to thank A/Prof Jan Strugnell, Prof Derek Wildman and two anonymous reviewers for their valuable comments on a previous version of the manuscript. This study was supported by the Instituto Antártico Chileno (INACH) with the grant No RT_27-14.

JDGE was supported by a CSIRO-OCE postdoctoral fellowship. LC and CAGW were supported by the FONDAP Program, Project No 15150003. EP and CAGW were supported by the Project GAB PIA CONICYT ACT172065. CAGW was also supported by Fondecyt

Initiation Project 11140087.

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15 Figure Legends

Figure 1. Mitochondrial gene order of patellogastropods in comparison with other

gastropod lineages. Protein coding and ribosomal genes are labelled with gene names

and tRNAs are denoted by single-letter codes according to the amino acid they

represent: A, Ala; G, Gly; P, Pro; T, Thr; V, Val; S, Ser; R, Arg; L, Leu; F, Phe; N,

Asn; K, Lys; D, Asp; E, Glu; H, His; Q, Gln; I, Ile; M, Met; Y, Tyr; C, Cys; W, Trp.

Transcripts in the light strand (reverse) are represented in red and dashed boxes.

Figure 2. Maximum likelihood (ML) tree of concatenated amino acid sequences of the 13

PCGs and the nucleotide sequences of the two rRNAs describing phylogenetic

relationships among Gastropoda. The ML bootstrap and Bayesian posterior probability

values for each node are indicated (black circles: bootstrap value ≥ 90% and posterior

probability of 1; grey circles: bootstrap value < 90% and posterior probability of 1;

white circles: bootstrap value < 90% and posterior probability < 1). The scale bar

represents the number of nucleotide substitutions per site.

16 Table 1. List of species used in this study. Length Order Superfamily Species %AT GenBank (bp) Patellogastropoda

Nacella clypeater 16742 66.1 KT990124

Nacella magellanica 16663 66.2 KT990125

Nacella concinna 16761 66.0 KT990126

Lottioidea Lottia digitalis 26835 66.4 DQ235899

Neritimorpha

Hydrocenoidea Georissa bangueyensis 15267 68.8 KU342664

Helicinoidea Pleuropoma jana 15851 74.6 GQ527172

Neritoidea melanotragus* 15261 62.0 GU810158

Neritopsoidea limacina* 15046 61.8 KU342669

Caenogastropoda maximum 15578 58.2 HM174253

Truncatelloidea Oncomelania hupensis 15216 72.1 JF284686

Tricula hortensis 15179 75.5 EU440735

Muricoidea Rapana venosa 15271 72.8 KM213962

Thais clavigera 15285 66.8 DQ159954

Conoidea Conus borgesi 15536 69.7 EU827198

Buccinoidea Ilyanassa obsoleta 15263 72.4 DQ238598

Vetigastropoda Neomphaloidea Chrysomallon squamiferum 15388 68.0 AP013032

Fissurelloidea Diodora graeca 17209 58.6 KT207825 volcano 17575 64.7 JN790612 Variegemarginula punctata* 14640 70.8 KX298889 Phasianelloidea Phasianella solida 16698 65.7 KR297251

Lepetodriloidea Lepetodrilus schrolli* 15579 64.6 KR297250

Haliotoidea Haliotis rubra 16907 62.8 AY588938

Seguenzioidea Granata lyrata 17632 62.2 KR297249 Stomatella planulata 17151 71.4 KX298894 Angaria neglecta 19470 68.7 KR297248 brunnea 17690 68.4 JN790613 Omphalius nigerrimus 17755 68.7 KX298895 argyrostomum 17780 69.4 KX298892 Astralium haematragum 16310 71.5 KX298891 rugosa 17432 71.2 KX207824 Lunella aff. cinerea 17670 70.8 KF700096 Heterobranchia Albinaria coerulea 14130 73.1 X83390

17 Helicoidea Cornu aspersum 14050 71.8 JQ417194

Ellobioidea 14246 57.7 AY345053

Lymnaeoidea Galba pervia 13768 74.9 JN564796

Planorboidea Biompjalaria glabrata 13670 77.4 AY380531

Acteonoidea Pupa strigosa 14189 64.7 AB028237

Anadoridoidea Roboastra europea 14472 70.3 AY083457

Aplysioidea Aplysia californica 14117 67.6 AY569552

Onchidioidea Onchidella celtica 14150 63.7 AY345048 Peronia pernii 13968 68.5 JN619346

Outgroups Class Cephalopoda Octopus vulgaris 15744 74.9 NC_006353 Nautilus macromphalus 16258 59.6 DQ472026 Scaphopoda Graptacme eborea 14492 74.1 AY484748 Siphonodentalium lobatum 13932 68.3 AY342055 Polyplacophora Katharina tunicata 15532 69.5 KTU09810 Sypharochiton sinclairi 15028 72.1 KJ534306 Aplacophora Scutopus ventrolineatus 14662 69.0 KC757645 Chaetoderma nitidulum 21008 68.0 EF211990 Bivalvia Mytilus chilensis 16765 61.8 KP100300 Paphia textile 18561 64.3 JF969277 Monoplacophora Vema ewingi 17910 63.3 KY244019 Laevipilina antarctica 18642 64.5 KY244020 * Nearly complete mitogenomes

18 tRNA-Trp tRNA-Gly tRNA-Glu Cox3 tRNA-Asn tRNA-Met tRNA-Cys tRNA-Arg tRNA-Phe Nad3 tRNA-Tyr tRNA-Ala 12S tRNA-Ile tRNA-Ile tRNA-Val tRNA-Val Nacella clypeater Nad2

16S Nacella clypeater tRNA-Leu tRNA-Leu KT990124

Nacella Nad1 Cox1 KT990125 magellanica tRNA-Pro Cox2 Nad6 Nacella concinna Cytb KT990126 tRNA-Asp

Atp8 Atp6 Nacella concinna tRNA-Ser tRNA-Thr Nad4L D-Loop

Nad4 Nad5 tRNA-Gln tRNA-His Hypothetical Ancestral Gastropoda

cox1 cox2 D atp8 atp6 F nad5 H nad4 nad4L T S cob nad6 P nad1 L L rrnL V rrnS M Y C W Q G E cox3 K A R N I nad3 S nad2

Patellogastropoda Nacella cox1 cox2 D atp8 atp6 T nad5 H Q nad4 nad4L S cob nad6 P nad1 L L rrnL V rrnS Y M F W C G E cox3 R N nad3 A K I S nad2

Lottia cox1 N cox3 F V cox2 E T P rrnL M rrnS Y cob K atp8 Q nad4 nad4L L I R nad2 W nad3 atp6 A G D nad5 S L H nad1 C nad6

Neritomorpha Nerita, Georissa cox1 cox2 D atp8 atp6 F nad5 H nad4 nad4L T S cob nad6 P nad1 L L rrnL V rrnS M Y C W Q G E cox3 K A R N I nad3 S nad2

Pleuropoma cox1 cox2 G atp8 atp6 M Y M rrnS V rrnL L nad1 P nad6 cob S T nad4L nad4 H nad5 F L W E C Q cox3 K A R N I nad3 S nad2

Neomphalina

Chrysomallon cox1 F T atp6 atp8 D cox2 nad5 H nad4 nad4L S cob E nad6 P nad1 L L rrnL V rrnS M Q W C Y G cox3 K A R N I nad3 S nad2

Vetigastropoda

Granata, Tegula, Stomatella, E D E G Chlorostoma, Phasieanella, cox1 cox2 D atp8 atp6 F nad5 H nad4 nad4L T S cob nad6 P nad1 L L rrnL V rrnS M Y C W Q G E cox3 K A R N I nad3 S nad2 Angaria, Lunella, Astralium, Omphalius, Bolma, Haliotis E G D T N

Diodora, Fisurela cox1 cox2 atp8 E G D Q W Y C M rrnS V rrnL L L nad1 P nad6 cob S S atp6 nad5 H nad4 nad4L T S R cox3 A N I nad3 K nad2 M K S I

Variegemarginula cox1 cox2 D atp8 E G Q W Y C M rrnS V cob nad6 P nad1 L L rrnL nad5 H nad4 nad4L T S R cox3 A I nad3 N K nad2

Lepetodrilus cox1 cox2 L L nad1 P nad6 cob S T nad4L nad4 H nad5 F atp6 atp8 D rrnL V rrnS cox3 K A R N I nad3 S nad2

Caenogastropoda T Rapana, Tricula, Thais, Oncomelania, Ilyanassa, cox1 cox2 D atp8 atp6 M Y C W Q G E rrnS V rrnL L L nad1 P nad6 cob S T nad4L nad4 H nad5 F cox3 K A R N I nad3 S nad2 Conus Heterobranchia

Peronia, Galba, Albinaria, P A P Y W C C Biomphalaria, Onchidella, cox1 V rrnL L A P nad6 nad5 nad1 nad4L cob D F cox2 Y W G H Q L atp8 N atp6 R E rrnS M nad3 S S nad4 T cox3 I nad2 K Myosotella, Cornu Q F nad4L S nad4

C C

Roboastra, Aplysia, Pupa cox1 V rrnL L A P nad6 nad5 nad1 Y W nad4L cob D F cox2 G H Q L atp8 N atp6 R E rrnS M nad3 S S nad4 T cox3 I nad2 K

Translocation and Translocation Inversion Duplication No information inversion Albinaria Cornu Onchidella Peronia Platevindex Aplysia HETEROBRANCHIA Pupa Roboastra Biomphalaria Galba Myosotella Biomphalaria BIVALVIA Galba Lottia PATELLOGASTROPODA Graptacme Siphonodentalium SCAPHOPODA Scutopus Chaetoderma APLACOPHORA Vema MONOPLACOPHORA Laevipilina Octopus CEPHALOPODA Nautilus Nacella clypeater Nacella magellanica PATELLOGASTROPODA Nacella concinna Dendropoma Oncomelania Tricula Rapana CAENOGASTROPODA Concholepas Thais Conus Ilyanassa Titiscania Pleuropoma NERITIMORPHA Nerita Georissa Variegemarginula Fissurella Diodora Lepetodrilus Haliotis Granata Stomatella Angaria VETIGASTROPODA Tegula Omphalius Chlorostoma Lunella Bolma Astralium Phasianella Chrysomallon NEOMPHALINA Katharina POLYPLACOPHORA Sypharochiton 0.4