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and Evolution 158 (2021) 107081

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Molecular Phylogenetics and Evolution

journal homepage: www.elsevier.com/locate/ympev

Eight new mitogenomes clarify the phylogenetic relationships of within the caenogastropod phylogenetic framework

Alison R. Irwin a,b,*, Ellen E. Strong c, Yasunori Kano d, Elizabeth M. Harper e, Suzanne T. Williams a a Department of Life Sciences, Natural History Museum, Cromwell Rd, London SW7 5BD, United Kingdom b School of Biological Sciences, University of Bristol, 24 Tyndall Ave, Bristol BS8 1TQ, United Kingdom c Department of , National Museum of Natural History, Smithsonian Institution, 10th St. & Constitution Ave. NW, Washington, D.C. 20560, United States d Department of Marine Ecosystems Dynamics, Atmosphere and Research Institute, University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8564, e Department of Earth Sciences, University of Cambridge, Downing Street, Cambridge CB2 3EQ, United Kingdom

ARTICLE INFO ABSTRACT

Keywords: Members of the gastropod superfamily Stromboidea () are characterised by their elaborate shell Stromboidea morphologies, distinctive mode of locomotion, and often large and colourful . This iconic group comprises over 130 , including many large and charismatic species. The family Strombidae is of particular interest, largely due to its commercial importance and wide distribution in tropical and subtropical waters. Although a Mitochondrial genome few strombid mitochondrial genomes have been sequenced, data for the other four Recent families in Strom­ Systematics Phylogenetics boidea are lacking. In this study we report seven new stromboid mitogenomes obtained from transcriptomic and genomic data, with taxonomic representation from each Recent stromboid family, including the first mitoge­ nomes for , , and . We also report a new mitogenome for the family . We use these data, along with published sequences, to investigate the relationships among these and other caenogastropod groups. All analyses undertaken in this study support of Stromboidea as redefined here to include Xenophoridae, a finding consistent with morphological and behav­ ioural data. Consistent with previous morphological and molecular analyses, including those based on mitoge­ nomes, monophyly of is confirmed but monophyly of Littorinimorpha is again rejected.

1. Introduction n/im/2007-36711). By comparison, the other four stromboidean fam­ ilies are represented in the Recent fauna by only a handful of species The superfamily Stromboidea is a highly diverse group within the with restricted distributions, although records show they were largest and most successful clade of living gastropods, the Caenogas­ more diverse and geographically widespread until the end- tropoda (Bouchet and Rocroi, 2005; Bouchet et al., 2017). Stromboidea (K/Pg) mass (Morton, 1951; Roy, 1996; Nielsen, 2005). is currently understood to comprise five Recent families: Aporrhaidae, Despite their diverse shell morphologies (Savazzi, 1991), extensive fossil Rostellariidae, Seraphsidae, Strombidae and Struthiolariidae (Bouchet record (Roy, 1996), and recognition as a commercially important group et al., 2017). By far the largest of these is the family Strombidae, which is (within the shell trade and fishing industry; Aiken et al., 2006; Dias widespread in tropical and subtropical seas, and comprises more than 90 et al., 2011; Stoner et al., 2019), strombids have been the focus of few species currently considered valid (Abbott, 1960, 1961; Kreipl and morphological or molecular systematic studies (Stone, 2001; Simone, Poppe, 1999; Liverani, 2013; WoRMS, 2020). Strombids inhabit mostly 2005; Latiolais et al., 2006; Maxwell et al., 2020). shallow waters, but some have been found in deeper ; for Extreme differences in shell morphology exist among stromboid example, labiosa has been collected at depths in excess of 780 families (Savazzi, 1991; Bandel, 2007), yet equally striking is the vari­ m (MNHN IM-2007-36711; http://coldb.mnhn.fr/catalognumber/mnh ation in morphology. Rostellariidae, Seraphsidae, and Strombidae

* Corresponding author at: Department of Life Sciences, Natural History Museum, Cromwell Rd, London SW7 5BD, United Kingdom. E-mail addresses: [email protected] (A.R. Irwin), [email protected] (E.E. Strong), [email protected] (Y. Kano), [email protected] (E.M. Harper), s. [email protected] (S.T. Williams). https://doi.org/10.1016/j.ympev.2021.107081 Received 5 October 2020; Received in revised form 18 December 2020; Accepted 12 January 2021 Available online 20 January 2021 1055-7903/Crown Copyright © 2021 Published by Elsevier Inc. All rights reserved. A.R. Irwin et al. Molecular Phylogenetics and Evolution 158 (2021) 107081 possess large and colourful -type eyes (which probably impart genome skimming). Type species were used for Strombidae and Stru­ excellent vision; Seyer, 1994) on the ends of long, mobile eyestalks thiolariidae; ( pugilis and papulosa; Table 1); how­ (ommatophores), and are characterised by a u-shaped “stromboid ever, appropriately preserved specimens of type species were not notch” near the anterior end of the shell outer lip which accommodates available for the other families. Therefore, specimens were chosen from the right ommatophore (Woodward, 1894; D.P. Abbott, 1962; Berg, the type ( serresiana, Aporrhaidae; japonica, 1974; Simone, 2005). By contrast, Aporrhaidae and Struthiolariidae Xenophoridae), or in Rostellariidae, where samples were limited, the have small, darkly pigmented eyes near the base of cephalic , species with the highest quality extracted DNA ( cancellata; and no (Simone, 2005). Despite this diversity, the Table 1). The type genus of Seraphsidae, Seraphs, is extinct, however the incorporation of these five families within Stromboidea is widely name Seraphsidae was introduced by Jung (1974) as a replacement for accepted due to a number of unreversed synapomorphies, including a the name Terebellidae (Bouchet and Rocroi, 2005; Bouchet et al., 2017) foot with a sub-terminal, projecting (Woodward, 1894; so we used the type species of () to Simone, 2005). represent the family (Table 1). Complete mitogenomes were also From cladistic analysis of morphological characters, Simone (2005) assembled from the transcriptomes of two additional strombid species also assigned Xenophoridae to Stromboidea, a decision not widely which have been sequenced for another project: Ministrombus variabilis accepted; Xenophoridae is currently assigned to a separate superfamily, (formerly Dolomena variabilis; Bandel, 2007; Dekkers and Maxwell, Xenophoroidea (WoRMS, 2020). The systematic relationship between 2020), and dentatus (Table 1). Stromboidea and Xenophoridae has long been problematic, with the latter variably included in Xenophoroidea (Boss, 1982; Ponder, 1983; 2.2. DNA extractions Ponder and De Keyzer, 1998; Bouchet et al., 2005, 2017; Ponder et al., 2020), Stromboidea (Wenz, 1940; Berg, 1974, 1975; Kiel and Perrilliat, Tissue samples taken from the foot were cut into small pieces and 2001; Simone, 2005, 2011; Kronenberg and Wieneke, 2020; also DNA was extracted according to the manufacturer’s instructions for the tentatively by Bandel, 2007), or Calyptraeoidea (Morton, 1958; Ponder, E.Z.N.A.® Mollusc DNA extraction kit (Omega Bio-tek; Terebellum ter­ 1983). Stromboidea differ from the Xenophoroidea principally in the ebellum, , Xenophora japonica and Struthiolaria papulosa), fusiform shell shape (as opposed to the flattened, conical xenophorid or High Pure PCR Template Preparation kit (Roche; Aporrhais serresiana shell, which is arbitrarily termed ‘trochiform’) and in the presence of a and Varicospira cancellata), with the minor modifications that tissue ◦ extending from the anterior edge of the (Ponder samples in proteinase K and tissue lysis buffer were incubated at 50 C and De Keyzer, 1998; Simone, 2005). Nevertheless, all studies suggest a overnight whilst rotated at 15 rpm. DNA was quantified with a Nano­ close relationship between Stromboidea and Xenophoroidea, which Drop™ 8000 spectrophotometer (Thermo Fisher Scientific),and further were united by Ponder et al. (2020) in the Strombida, and the two su­ purifiedwhen necessary by precipitation with absolute ethanol and 3 M perfamilies share numerous morphological characters, including the sodium acetate. Final DNA concentrations were within the range projecting operculum (Simone, 2005). However, to date, neither 14.3–41.7 ng/mL. morphological nor molecular phylogenetic analyses have fully tested the relationship between these two superfamilies, or among the currently 2.3. RNA extractions recognised stromboid families. Both Stromboidea and Xenophoroidea belong to the gastropod group Transcriptomic data were obtained from RNA extracted from Hypsogastropoda, which includes all living caenogastropods other than strombid eyes (Ministrombus variabilis and Tridentarius dentatus) using and Cerithioidea (Bouchet et al., 2017; Ponder et al., the E.Z.N.A.® Mollusc RNA Kit protocol (Omega Bio-tek) following the 2020). Within the hypsogastropods, these two superfamilies are manufacturer’s instructions, with modifications as follows: tissue was currently assigned to Littorinimorpha, a highly diverse group of fifteen homogenised in TRIzol® (Thermo Fisher Scientific) instead of the superfamilies (Ponder and Lindberg, 1997). Phylogenetic analyses of manufacturer’s MRL Buffer, all centrifugation steps were performed at ◦ ◦ caenogastropods based upon morphological characters (Ponder et al., 4 C, and samples were frozen at 20 C for 2 h after adding isopropanol 2008; Simone, 2011) found Littorinimorpha to be paraphyletic. This is and prior to precipitation. Subsequent mRNA purification was per­ now supported by molecular data (Hayashi, 2005; Colgan et al., 2007; formed with a Dynabeads® mRNA Purification Kit (Invitrogen) Takano and Kano, 2014; Williams et al., 2014; Cunha and Giribet, following the manufacturer’s protocol. 2019), including phylogenetic studies using complete mitochondrial genomes (mitogenomes). In recent years, the number of major caeno­ 2.4. Illumina sequencing with NextSeq 500 gastropod groups with available mitogenomes has increased, several of which have not yet been included in phylogenetic analyses that inves­ DNA libraries were prepared using the Nextera DNA Flex Library tigate relationships across the . In this study we report Prep Kit (Illumina, USA), and transcriptome libraries using the NEB­ new mitochondrial genome sequences for eight caenogastropod species, Next® UltraTM II RNA Library Prep Kit for Illumina (New England Bio­ with one representative each from Aporrhaidae, Rostellariidae, Ser­ labs, UK), by the NHMUK Sequencing Facility following manufacturers’ aphsidae, Struthiolariidae and Xenophoridae, and three from Strombi­ protocols. All libraries were sequenced in the same facility in a single run dae. These new sequences are combined with published mitogenome using the NextSeq 500 Desktop Sequencing System (Illumina), yielding sequences from selected caenogastropods in phylogenetic analyses with paired-end reads of 150 bp in length, in a high output run with 45% the following aims: 1) to examine relationships within Stromboidea for dedicated to the genomic DNA (gDNA) libraries and 25% for the the firsttime using molecular data, 2) to the systematic placement of transcriptomes. Stromboidea and Xenophoroidea within Caenogastropoda and their relationship to each other by inclusion of representatives of all available 2.5. Assembly of mitogenomes caenogastropod superfamilies. Raw Illumina reads (WGS) were assembled using Geneious Prime 2. Materials and methods v.2019.2.3 (https://www.geneious.com). Paired-end reads were trim­ med where there was more than 5% chance of error. All paired-end 2.1. Sample choice reads for each species were mapped to the previously published strom­ bid mitogenome for gigas (formerly Strombus and recently ; Representatives of each stromboid family were chosen for Whole Kronenberg and Lee, 2007; NC_024932), and, from the resulting contig, Genome Sequencing (WGS) (also known as shotgun sequencing or the consensus sequence from the largest area of good coverage (selected

2 A.R. Irwin et al. Molecular Phylogenetics and Evolution 158 (2021) 107081

Table 1 Sample details for specimens sequenced in this study. Family and species identification, voucher registration number, collection date, depth, and locality (with expedition, station number, longitude and latitude, where applicable). An asterisk (*) marks samples from which transcriptome data were collected to obtain mito­ genomes; for all other specimens, mitogenomes were recovered via Whole Genome Sequencing. Type species are indicated in bold font.

Family Species Registration no. Date Depth Sampling locality collected (m) ◦ ′ ◦ ′ Aporrhaidae Aporrhais serresiana (Michaud, NHMUK 20200347 11.05.2017 104–105 Malaga´ Bay, Alboran´ Sea; 36 37 N 04 22 W; BV61 1828) ◦ ′ ◦ ′ Rostellariidae Varicospira cancellata MNHN IM- 28.06.2014 10–15 Silver Sound, Kavieng Lagoon, Papua ; 02 40 S 150 41 E; (Lamarck, 1816) 2013–55759 MNHN KAVIENG 2014, KD91 ◦ ′ ◦ ′ Seraphsidae Terebellum terebellum MNHN IM- 13.11.2012 0–1 S.E. of Megas Islet, Alexishafen, Papua New Guinea; 05 05 S 145 49 E; (Linnaeus, 1758) 2013–12884 MNHN PAPUA NIUGINI, PM19 ◦ ′ Strombidae Ministrombus variabilis MNHN IM- 08.09.2018 0 Karemb´e Plateau (North Port), near Koumac, ; 20 38 S ◦ ′ (Swainson, 1820)* 2019–1736 164 17 E; MNHN KOUMAC 2.1, KM301 ◦ ′ ◦ ′ Strombus pugilis Linnaeus, MNHN IM- 25.09.2016 1–8 E. of Madame Island, Robert Bay, Martinique; 14 40 N 60 53 W; MNHN 1758 2013–71190 MADIBENTHOS, AR455 ◦ ′ ◦ ′ Tridentarius dentatus (Linnaeus, MNHN IM- 09.09.2018 0 ˆIle Kendec, near Koumac, New Caledonia; 20 40 S 164 15 E; MNHN 1758)* 2019–1738 KOUMAC 2.1, KM302 Struthiolariidae Struthiolaria papulosa AORI_YK#4036 20.01.2004 0–1 Orewa Beach, Whangaparaoa Bay, N. of Auckland, New Zealand; ◦ ′ ◦ ′ (Martyn, 1784) 36 35 S 174 42 E ◦ ′ Xenophoridae Xenophora japonica Kuroda and AORI_YK#4037 07.06.2012 133–141 Off Oosezaki, Suruga Bay, Honshu Island, Shizuoka, Japan; 35 03 N ◦ ′ Habe, 1971 138 47 E via the graphic produced in Geneious) was extracted to be used as a new 2019). Therefore, each stromboid family was represented by one species reference sequence. The paired-end reads were then iteratively mapped except for Strombidae and Xenophoridae (seven and two taxa included, back to the reference sequence until all mitochondrial genes could be respectively). Together, these sequences comprised the ‘Stromboidea’ ′ ′ identified in the consensus sequence. Where an overlap at the 5 and 3 dataset, with either 8 or 13 ingroup sequences (depending on whether ends of the final assembly was detected, the genomes were assumed to previously published sequences were included) and saxatilis be complete and the sequences were circularised. (NC_030595, Y11751, HE590811) as the outgroup (see Suppl. Mat. 1 for The quality assessment of all transcriptome reads was performed list of published sequences used in this study). The choice of Littorina with FastQC (Andrews, 2010). The raw reads were then cleaned using saxatilis as outgroup was based on analyses of the ‘Caenogastropoda’ Trimmomatic v.0.39 (Bolger et al., 2014) with the following settings: dataset (see below). ILLUMINACLIP:TruSeq3-PE.fa:2:30:10 LEADING:3 TRAILING:3 SLI­ In order to determine the relationship between Stromboidea and DINGWINDOW:4:28 MINLEN:36. All paired-end reads were imported Xenophoridae, a second taxon dataset (‘Caenogastropoda’), included into Geneious and the mitogenomes assembled using the same method sequences from the ‘Stromboidea’ dataset plus 1–4 representatives from as for the WGS reads. each of the remaining caenogastropod superfamilies for which mitoge­ nome data are available, including the littorinimorph groups Calyp­ 2.6. Genome annotation traeoidea, , , and . Note that taxa used to represent in previous mitogenome Mitogenomes were annotated using MITOS (Bernt et al., 2013) and analyses (Williams et al. 2014; Osca et al., 2015; Jiang et al., 2019) are MitoZ (Meng et al., 2019). Gene boundaries were most accurately now assigned to Truncatelloidea (WoRMS, 2020) and as such there are determined by MitoZ, which was better able to precisely locate the start no mitogenomes available for Rissooidea as presently defined. Taxa and stop codons in most cases. However, MitoZ was specifically devel­ were selected to ensure balanced representation of caenogastropod oped for annotation of mammal and mitogenomes (Meng groups, as unbalanced taxon sampling can result in artefactual topol­ et al., 2019), and some genes required manual editing. Modifications ogies as a consequence of long-branch attraction (Lartillot et al., 2007; were inferred by comparison with annotations for previously published Uribe et al., 2019). When selecting sequences, slower-evolving taxa gastropod sequences and supported by new transcriptome data, thereby were preferred (identified by comparing branch lengths during initial mitigating the potentially problematic method of defining gene analyses) and, when possible, type species were chosen. Only complete boundaries based solely on GenBank sequences (which relies on a cir­ mitogenomes were selected, with the exception of the partial mitoge­ cular argument; Williams et al., 2017). The ribosomal RNA (rRNA) gene nome for chinensis EU827193, which was the only available boundaries were also edited based on similarity with other reported sequence for Calyptraeoidea. The final ‘Caenogastropoda’ dataset gastropod genes. Putative transfer RNA (tRNA) genes were identified included 40 ingroup sequences, with a vetigastropod (Haliotis iris with ARWEN (Laslett and Canback,¨ 2008), which infers cloverleaf sec­ NC_031361) selected as a suitable outgroup, based on previous mito­ ondary structures that were subsequently drawn using mt-tRNA-Draw genome analyses of (Uribe et al., 2019). (BETA Version 0.5; Youngblood and Masta, unpublished). Nuclear ‘Stromboidea’ and ‘Caenogastropoda’ datasets were comprised of gene sequences were also obtained for 18S rRNA and 28S rRNA by nucleotide residues only, as amino acid matrices can result in lower mapping reads to a reference sequence. branch support than nucleotide datasets for shallower nodes (Regier et al., 2010; Zwick et al., 2012; Breinholt and Kawahara, 2013). Third 2.7. Phylogenetic analyses codon positions were removed from the ‘Caenogastropoda’ dataset in order to mitigate problems associated with saturated sites (Regier et al., The eight new mitogenomes (Aporrhais serresiana, Varicospira can­ 2010; Zwick et al., 2012). The ‘Caenogastropoda’ dataset included only cellata, Terebellum terebellum, Ministrombus variabilis, Strombus pugilis, protein-coding genes (PCGs). The ‘Stromboidea’ datasets were analysed Tridentarius dentatus, Struthiolaria papulosa and Xenophora japonica; including: (1) all 13 mitochondrial PCGs, as with the ‘Caenogastropoda’ Table 1) were included in phylogenetic analyses, along with four pub­ dataset (13 ingroup sequences); (2) 17 genes: all PCGs and rrnS, rrnL, lished mitogenomes for Stromboidea and one for Xenophoridae: Aliger 18S and 28S (13 or 8 ingroup sequences, see below); (3) nuclear genes: gigas NC_024932, luhuanus NC_035726, chiragra 18S and 28S only (8 ingroup sequences). These different datasets were MH122656, lambis MH115428, exutus MK32736 analysed to provide more phylogenetically informative sites and inde­ (Marquez´ et al., 2016; Zhao et al., 2018; Jiang et al., 2019; Xu et al., pendent loci, because different genes are often informative at different

3 A.R. Irwin et al. Molecular Phylogenetics and Evolution 158 (2021) 107081 hierarchical levels and because studies have shown that gene choice can 3. Results affect gastropod phylogenies (e.g. Wort et al., 2017). Sequences of nu­ clear genes 18S and 28S were not available for all previously published 3.1. Mitochondrial genome organization and structural features taxa used in the ‘Stromboidea’ dataset; 18S sequence data were un­ available for , and Onustus exutus, Complete mitogenomes were recovered for seven species: Aporrhais and 28S sequences were unavailable for Aliger gigas, Harpago chiragra, serresiana MW244817, Varicospira cancellata MW244822, Terebellum and Onustus exutus. Therefore, two 17-gene datasets were terebellum MW244821, Strombus pugilis MW244819, Tridentarius denta­ analysed: (1) 13 ingroup sequences, with all available published se­ tus MW244920, Struthiolaria papulosa MW244818 and Xenophora quences (some nuclear genes missing), and (2) 8 ingroup sequences from japonica MW244823 (Table 2). Circularity of the mitogenome was this study only (all nuclear genes available). confirmedby identificationof a region ranging from 37 to 410 bp which Genes were aligned using an iterative process as implemented in was repeated at the two ends of the consensus sequence, indicating that PASTA (Mirarab et al., 2014), which used MAFFT L-INS-i (Katoh et al., the ends could be overlapped to form a circular sequence. The raw, 2009) to align, OPAL (Wheeler and Kececioglu, 2007) to merge pairs of trimmed paired-end reads were mapped again to the final, annotated adjacent subset alignments, FASTTREE (Price et al., 2009) to estimate a circular mitogenome to obtain coverage data (Table 2). The mitogenome maximum likelihood tree, GTR + CAT as the nucleotide substitution could not be circularised for Ministrombus variabilis and was therefore model, 50% subproblem and centroid decomposition with 5 iterations incomplete, but all protein-coding genes (PCGs) were obtained and were and the best alignment retained as determined by likelihood value. included in the analyses. Low coverage in certain areas of the two Gblocks (Castresana, 2000) was used to remove ambiguously aligned mitogenomes recovered from transcriptomic data (Tridentarius dentatus regions with the following settings: allow gap positions within the final and Ministrombus variabilis) prevented the reliable recovery of some blocks, do not allow many contiguous non-conserved positions. IQ-TREE tRNAs and these require further work. All eight mitogenomes contained was used to test for homogeneity of character composition in the 13 protein-coding sequences, as with most mitogenomes (Boore alignment of each gene within datasets (Suppl. Mat. 2). Finally, genes and Brown, 1994), and 2 rRNA and 22 tRNA genes (except for Tri­ were concatenated for phylogenetic analysis. dentarius dentatus and Ministrombus variabilis with 21 and 18 tRNA se­ Site-homogenous nucleotide substitution models partitioned by gene quences recovered, respectively), as well as non-coding regions (except were used in analyses, inferred from empirical alignments (see Suppl. for Ministrombus variabilis; see Table 2 for genome lengths; Fig. 1 for Mat. 3 for model choice). The best fitmodels for each gene were selected Strombus pugilis gene order, applicable to all newly obtained mitoge­ using ModelFinder (Kalyaanamoorthy et al., 2017) with the Bayesian nomes; Fig. 2 and Suppl. Mat. 4 for putative tRNA structures; Suppl. Mat. Information Criterion (BIC) as implemented in IQ-TREE; “-m MF” option 5 for gene annotations). (Suppl. Mat. 3). Phylogenetic relationships were inferred using All protein-coding and rRNA genes were located on the plus (heavy) Maximum Likelihood (ML; Felsenstein, 1981) and Bayesian Inference strand, which also encoded for trnD, trnV, trnL(UAG), trnL(UAA), trnP, methods (BI; Huelsenbeck et al., 2001). trnS(UGA), trnS(GCU), trnH, trnF and the cluster KARNI (trnK, trnA, trnR, ML analyses were implemented in IQ-TREE, using the ultrafast trnN, trnI; Fig. 1). The minor strand encoded for the cluster MYCWQGE bootstrap (UFBoot) feature as it obtains less biased support values than (trnM, trnY, trnC, trnW, trnQ, trnG, trnE), and trnT (Fig. 1). In the Ter­ RAxML rapid bootstrap (Minh et al., 2013; Hoang et al., 2018) to assess ebellum terebellum mitogenome, genes nad2/cox1 overlapped by one the robustness of the inferred topology. Note that ultrafast bootstrap base; in all species, cox2/trnD overlapped by two bases, and nad4/nad4L approximation implemented by IQ-TREE has a different interpretation by 6 bases (Suppl. Mat. 5). In all species, genes rrnS/trnV, rrnL/trnV, and to the standard bootstrap; only nodes with support values exceeding rrnL/trnL also overlapped. There was no overlap between atp6/atp8 as 95% are considered ‘highly’ supported (Minh et al., 2013; Hoang et al., has been observed in some gastropod species (Rawlings et al., 2010; 2018). BI analyses were implemented in MrBayes v.3.2.5 (Huelsenbeck Osca et al., 2014, 2015), but not all (Williams et al., 2014; Xu et al., and Ronquist, 2001), using four MCMC chains run for 10,000,000 2016; Harasewych et al., 2019). Some PCG annotations in published generations (or until convergence was reached) with a sample frequency strombid and xenophorid mitogenome sequences were judged to be of 1000. Stationarity and convergence between the runs was determined inaccurate when compared to other caenogastropod sequences in by visual examination of .p files and confirming that effective sample alignments; either too long (Aliger gigas NC_024932, nd5; Marquez´ et al., sizes for all parameters >200 via Tracer v.1.7.1 (Rambaut et al., 2018). 2016) or missing one end of the sequence (Conomurex luhuanus Consensus trees were obtained after discarding the first 10% of gener­ NC_035726 and Onustus exutus MK327366, atp6, nad4L, nad4; also ations as burnin. In all analyses, nodes with support values under 50% Onustus exutus, nad6; Zhao et al., 2018, Xu et al., 2019). Annotations for were collapsed. the remaining published strombid sequences (Harpago chiragra MH122656 and Lambis lambis MH115428; Jiang et al., 2019) matched those recovered in this study, which are supported by transcriptome data.

Table 2 New mitochondrial genomes for Stromboidea, including Xenophoridae. Mitogenome lengths, completeness (as demonstrated by the presence of an overlap that allowed circularisation of the genome), coverage statistics (minimum number of reads, maximum number of reads and standard deviation) and GC content (base composition). Note that mitogenomes recovered from transcriptomes (Ministrombus variabilis and Tridentarius dentatus) show extremes in coverage values, compared to the remaining sequences obtained from genomic DNA.

Family Species GenBank Acc. No. Mitogenome length (bp) Complete Genome Min coverage Max coverage SD coverage GC%

Aporrhaidae Aporrhais serresiana MW244817 15,455 YES 13 210 34.7 30.3 Rostellariidae Varicospira cancellata MW244822 15,864 YES 439 990 81.5 30.5 Seraphsidae Terebellum terebellum MW244821 15,478 YES 247 609 52.5 29.4 Strombidae Ministrombus variabilis MW244824 15,292 NO 1 89,246 10,449 33.1 Strombidae Strombus pugilis MW244819 15,809 YES 141 367 36.4 34.7 Strombidae Tridentarius dentatus MW244820 15,500 YES 1 47,990 8,338 31.8 Struthiolariidae Struthiolaria papulosa MW244818 15,475 YES 265 652 58.7 28.5 Xenophoridae Xenophora japonica MW244823 15,684 YES 118 306 27.0 30.4

4 A.R. Irwin et al. Molecular Phylogenetics and Evolution 158 (2021) 107081

Fig. 1. Variation in mitochondrial gene orders within Caenogastropoda. Numbers to the left of species names indicate taxon groups: (1) Architaenioglossa, (2) currently unassigned caenogastropods, (3) Littorinimorpha (names shown in red) and (4) . The most common caenogastropod gene order is exem­ plified by Strombus pugilis (this study); see Suppl. Mat. 1 for all other GenBank accession numbers. Gene inversions (circles) and transpositions (lines) are shown in red, except for Vermetoidea and Cerithioidea due to the high number of differences in these two superfamilies. Gene lengths are to scale except for tRNAs and atp8 (all x3 scale), nad3 and nad4L (both x2 scale) for ease of reading. Protein-coding, rRNA, and tRNA genes are shown in blue, grey, and yellow, respectively. Genes are shown oriented either to the right or to the left depending on whether they are encoded by the major or minor strand. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Almost all PCGs started their open reading frame with ATG (Suppl. nuclear genes consisted of 16,339 (13 ingroup sequences) and 16,588 (8 Mat. 5), although some alternate start codons existed. For example, the ingroup sequences) nucleotide residues after ambiguously aligned sites start codon for nad5 in Aporrhais serresiana was TTG (Suppl. Mat. 5); this were removed; 93.3% and 94.8% of residues in the initial alignment start codon has also been found in some other molluscs (Cunha et al., (Table 3). The ‘Stromboidea’ alignment of nuclear genes only consisted 2009; Williams et al., 2017). The start codon for nad4 in Aporrhais ser­ of 3,320 nucleotide residues after ambiguously aligned sites were resiana, Struthiolaria papulosa and Xenophora japonica was ATT, which removed, which comprised 92.3% of nucleotide residues in the initial has also been found in some bivalves (Williams et al., 2017). The most alignment (Table 3). common stop codon was TAA; however, exceptions exist (Suppl. Mat. 5). ‘Caenogastropoda’ analyses showed maximal support for monophyly For example, the stop codon for Terebellum terebellum gene nad1 was of Stromboidea including Xenophoridae (Tree 1, PP = 1.0; Tree 2, B/S = truncated to TA (the full codon, TAC, is not an accepted stop codon). 100%; H1 in Table 4). In all analyses, two clades were recovered: Ros­ Incomplete stop codons have been identified in several mollusc species tellariidae + Seraphsidae + Strombidae, and Aporrhaidae + Struthio­ (Rawlings et al., 2010; Osca et al., 2015; Williams et al., 2017), which lariidae + Xenophoridae (H2 in Table 4). In analyses of mitochondrial presumably become functional stop codons by subsequent poly­ genes on their own and combined mitochondrial and nuclear gene an­ adenylation of transcribed mRNAs (Lopez Sanchez et al., 2011). alyses, Xenophoridae was resolved as sister to Struthiolariidae + Apor­ Secondary structures determined for putative tRNA genes were rhaidae with maximal support (Trees 1, 3, 5, 7, PP = 1.0; Trees 2, 4, 6, 8, largely consistent within Stromboidea, with variations in base pairing B/S = 100%), whereas in nuclear gene trees Xenophoridae + Apor­ mainly occurring in the D loop (Fig. 2; Suppl. Mat. 4). With the exception rhaidae were sister to Struthiolariidae (Tree 9, PP = 1.0; Tree 10, B/S = of Strombus pugilis, one or both of the two serine tRNAs had truncated D 77%). The phylogenetic positions of Rostellariidae and Seraphsidae also arms, which is also seen in some other molluscs (Sevigny et al., 2015; varied among analyses (Fig. 3). Most of the analyses with PCGs only Williams et al., 2017). Although sequences were identifiedfor all tRNAs recovered Rostellariidae as the sister of Strombidae with varied support for all species sequenced from gDNA, the structures of some serine (Tree 1, PP = 0.73–0.94; Trees 2, 4, B/S = 59–99%; H3 in Table 4; Figs. 3 tRNAs were not resolved with the conventional cloverleaf structure, and and 4; Suppl. Mat. 6). However, the ‘Stromboidea’ BI analysis with PCGs several putative cloverleaf structures involved non-canonical pairings only, in addition to all ML and BI analyses with mitochondrial and nu­ (Suppl. Mat. 4). Other tRNA sequences could not be fully recovered in clear genes, resolved Rostellariidae as sister to Seraphsidae with varied Ministrombus variabilis (methionine, phenylalanine, proline and valine support (Trees 3, 5, 7, PP = 0.98–1.0; Trees 6, 8, B/S = 70–90%; Figs. 3 tRNAs) and Tridentarius dentatus (valine tRNA), in regions of the mito­ and 5; Suppl. Mat. 6). Analyses that included only nuclear genes genome where the transcriptomic data were poor, leading to uncertain recovered Seraphsidae as sister to Strombidae (Tree 9, PP = 1.0; Tree 10, base calls (Suppl. Mat. 4). B/S = 83–100%; H3 in Table 4; Fig. 3; Suppl. Mat. 6). Trees also varied in topology with regard to the relationships among species in Strombidae, with several different clades resolved (H4 in 3.2. Phylogenetic analyses Table 4; Fig. 3); Clade A, Aliger gigas + Strombus pugilis; Clade B, Con­ omurex luhuanus + ((Harpago chiragra + Lambis lambis) + (Ministrombus The ‘Caenogastropoda’ and ‘Stromboidea’ alignments containing variabilis + Tridentarius dentatus))); Clade C, (Harpago chiragra + Lambis PCGs only consisted of 7,492 and 10,972 nucleotide residues, respec­ lambis) + (Ministrombus variabilis + Tridentarius dentatus)). Analyses with tively, after ambiguously aligned sites were removed, corresponding to PCGs only recovered A + B (Trees 1–4; Fig. 4; Suppl. Mat. 6), whereas 95.2% and 96.6% of nucleotide residues in the initial alignment analyses with mitochondrial and nuclear genes recovered Conomurex (Table 3). The ‘Stromboidea’ alignments containing mitochondrial and

5 A.R. Irwin et al. Molecular Phylogenetics and Evolution 158 (2021) 107081

Fig. 2. Putative secondary structures for all 22 mitochondrial tRNAs of Strombus pugilis, identified using MitoZ (Meng et al., 2019), corroborated with ARWEN (Laslett and Canback,¨ 2008) and drawn using mt-tRNA-Draw (BETA Version 0.5; Youngblood and Masta, unpublished). See Suppl. Mat. 4 for mitochondrial tRNAs for all eight species. Watson-Crick pairing is shown by lines and G-T pairing by dots. Red text indicates non-canonical pairings. Photo included is of specimen sequenced. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

6 A.R. Irwin et al. Molecular Phylogenetics and Evolution 158 (2021) 107081

Table 3 recovery of a fully resolved systematic framework for this large and The number of sites in alignments before and after (the latter in parentheses) highly diverse gastropod group. Until now, no phylogenetic analysis has removing ambiguous sites via Gblocks. Taxon dataset name, number of ingroup included representatives from all Recent families within Stromboidea taxa, sequence dataset (13, all 13 mitochondrial protein-coding genes (PCGs); and Xenophoroidea, leaving the affinities of Xenophoridae controver­ + + + 17, mitochondrial (PCGs rrnS rrnL) and nuclear (28S 18S) genes; 2, nu­ sial. Many major clades are still under sampled, however, new sequences clear genes only), residue types (1 & 2, nucleotides with the third codon posi­ produced for this study and available from GenBank afford the oppor­ tions removed; all, including all nucleotide bases). Note that for ‘Stromboidea’ tunity to revisit the relationship between these two superfamilies and the number of ingroup taxa varies as 18S and 28S sequence data were unavai­ lable for Aliger gigas, Conomurex luhuanus, Harpago chiragra, Lambis lambis and their position within Caenogastropoda, as well as phylogenetic re­ Onustus exutus. lationships among lower taxonomic levels within Stromboidea.

Dataset Caenogastropoda Stromboidea 4.1. Stromboid systematics Taxon 40 13 13 8 8 sampling (ingroup) The superfamily Stromboidea was recovered in all analyses with high support, and always included the two species representing the family No. genes 13 13 17 17 2 Xenophoridae. Inclusion of Xenophoridae in Stromboidea is strongly included Residue type 1 & 2 all all all all supported by molecular analyses and receives additional support from No. sites 7,870 11,359 17,511 17,504 3,597 behavioural traits (Berg, 1974, 1975) and morphological characters prior (Simone, 2005, 2011). Although all families exhibit unique differences No. sites 7,492 10,972 16,339 16,588 3,320 in and shell morphology (Fig. 5), morphological characters after % sites 4.8% 3.4% 6.7% 5.2% 7.7% shared between Stromboidea and Xenophoridae include a foot with a removed sub-terminal, projecting operculum (Woodward, 1894; Simone, 2005). cox1 1,034 (1,024) 1,536 1,536 1,536 n/a In both these groups, this particular characteristic enables a leaping (1,536) (1,536) (1,536) mode of locomotion, which is readily distinguishable from the smooth cox2 474 (458) 687 687 687 n/a creeping or gliding motion typical of most gastropods (Berg, 1974, (687) (687) (687) atp8 131 (106) 159 159 159 n/a 1975). (159) (159) (159) In this study two clades were recovered in Stromboidea with atp6 492 (464) 696 696 696 n/a maximal support in all analyses: (A) Xenophoridae + Aporrhaidae + (648) (648) (648) Struthiolariidae, and (B) Seraphsidae + Rostellariidae + Strombidae nad1 651 (628) 966 966 966 n/a (935) (935) (941) (Table 4). Cladistic analyses of morphological characters for twenty nad6 394 (334) 519 519 519 n/a species from Aporrhaidae, Seraphsidae, Strombidae, Struthiolariidae (481) (481) (497) and Xenophoridae (with outgroups from Architaenioglossa and Cer­ cob 771 (760) 1,140 1,140 1,140 n/a ithioidea), did not recover Clade A but did recover Clade B (Simone, (1,128) (1,128) (1,140) 2005). Clade B is supported by several morphological characters, nad4l 209 (198) 297 297 297 n/a “ ” (277) (277) (297) including the stromboid notch in the outer lip of the shell, and the nad4 951 (910) 1,377 1,377 1,377 n/a large eyes located at the end of long ommatophores (Woodward, 1894; (1,350) (1,350) (1,363) Abbott, 1962; Berg, 1974; Seyer, 1994; Simone, 2005). Previously, these nad5 1,201 (1,148) 1,759 1,759 1,759 n/a shared characters have resulted in Rostellariidae and Seraphsidae being (1,671) (1,671) (1,671) cox3 539 (520) 780 780 780 n/a included in Strombidae by several authors (Wells, 1998; Kreipl and (780) (780) (780) Poppe, 1999; Simone, 2005), although they are now generally consid­ nad3 236 (236) 354 354 354 n/a ered to be separate, closely related families (Jung, 1974; Kronenberg (353) (353) (353) and Burger, 2002; Maxwell et al., 2019). Simone (2005) did not include nad2 787 (706) 1,089 1,089 1,089 n/a Rostellariidae in morphological analyses but suggested that (Ros­ (1,007) (1,007) (1,046) rrnS n/a n/a 1,037 1,037 n/a tellariidae) would be closely related to Terebellum (Seraphsidae) due to (817) (817) the shared character of a flattened foot not seen in the strombids. rrnL n/a n/a 1,511 1,511 n/a Choice of phylogenetic method (BI or ML) resulted in few well- (1,225) (1,225) supported incongruences among trees for families within Stromboidea 18S n/a n/a 1,910 1,903 1,903 (1,818) (1,819) (1,819) (compare Trees 3 and 4; Suppl. Mat. 6). However, gene choice strongly 28S n/a n/a 1,694 1,694 1,694 affected phylogenetic relationships within Stromboidea. Analyses using (1,467) (1,501) (1,501) only nuclear genes recovered Xenophoridae + Aporrhaidae as sister to Struthiolariidae (Tree 9, PP = 1.0; Tree 10, B/S = 77%), whereas ana­ lyses of mitochondrial genes as well as combined mitochondrial and luhuanus as sister to A + C (Trees 5, 6; Fig. 5; Suppl. Mat. 6). nuclear gene analyses recovered Xenophoridae as sister to Struthiolar­ The monophyly of major clades Littorinimorpha and Neogastropoda iidae + Aporrhaidae with maximal support (Trees 1, 3, 5, 7, PP = 1.0; were not recovered in ‘Caenogastropoda’ analyses (Trees 1, 2; H in 5 Trees 2, 4, 6, 8, B/S = 100%). These topologies were not consistent with Table 4). The two consensus trees resulting from the Bayesian Inference the results of morphological analyses, which recovered Xenophoridae as and Maximum Likelihood analyses had similar topologies, with no well- sister to Seraphsidae + Strombidae (Simone, 2005). In this study, there supported branches in conflict, though some poorly-supported groups was also some variation in the phylogeny of Stromboidea regarding the were sensitive to changes in mode of inference. Several littorinimorph position of families Seraphsidae and Rostellariidae with respect to groups were unresolved in analyses, including Calyptraeoidea, the po­ Strombidae. Most analyses using only nuclear genes recovered Ser­ sition of which varied among trees recovered using different inference aphsidae as the sister of Strombidae (Tree 9, PP = 1.0; Tree 10, B/S = methods (Trees 1, 2; Fig. 4, Suppl. Mat. 6). 94%; Fig. 3; Suppl. Mat. 6). Analyses with protein-coding genes (PCGs) only recovered Rostellariidae as the sister of Strombidae (Tree 1, PP = 4. Discussion 0.97; Tree 2, 4, B/S = 59–75%; Figs. 3 and 4; Suppl. Mat. 6); however, the BI ‘Stromboidea’ analysis with PCGs only, as well as all analyses that Molecular phylogenetic studies of caenogastropods face the chal­ included mixtures of mitochondrial and nuclear genes, recovered lenge of sparse taxon sampling at all taxonomic levels, which prevents Strombidae + (Rostellariidae + Seraphsidae), with varied support

7 A.R. Irwin et al. Molecular Phylogenetics and Evolution 158 (2021) 107081

Table 4 Impact of taxon sampling, gene selection, residue type, and mode of inference on the phylogeny of Stromboidea and Littorinimorpha for every tree recovered in this study. Tree number, taxon dataset name (ingroup taxon sampling in parentheses), sequence dataset (13, all 13 mitochondrial protein-coding genes (PCGs); 17, mitochondrial (PCGs + rrnS + rrnL) and nuclear (28S + 18S) genes; 2, nuclear genes only), residue types (1 & 2, nucleotides with the third codon positions removed; all, including all nucleotide bases), mode of inference (BI, Bayesian Inference; ML, Maximum Likelihood). Hypotheses (support values given in parentheses where appropriate): H1, inclusion of Xenophoridae in Stromboidea; H2, Stromboidea split into two clades (A/B; A, Xenophoridae + Aporrhaidae + Struthiolariidae; B, Rostellariidae + Seraphsidae + Strombidae); H3, trees recover: (1) Seraphsidae + (Rostellariidae + Strombidae), (2) Strombidae + (Rostellariidae + Seraphsidae), or (3) Rostellariidae + (Seraphsidae + Strombidae); H4, trees recover (1) Strombidae split into two clades (A/B; A, Aliger gigas + Strombus pugilis; B, Conomurex luhuanus + ((Harpago chiragra + Lambis lambis) + (Ministrombus variabilis + Tridentarius dentatus))), or (2) Conomurex luhuanus sister to two clades (A/C; A, Aliger gigas + Strombus pugilis; C, (Harpago chiragra + Lambis lambis) + (Ministrombus variabilis + Tridentarius dentatus)), or (3) other topology; H5, inclusion of Tonnoidea in Neogastropoda., and Suppl. Mat. 6. An asterisk (*) marks inclusion of both Tonnoidea and Calyptraeoidea in Neogastropoda; H2 and H4 values are given as support for clades as described above (A/B, or A/C).

Tree no. Taxon-sampling Gene Residue type Method H1 H2 H3 H4 H5

1 Caenogastropoda (40) 13 1 & 2 BI YES (1.0) YES (1.0/1.0) 1 (0.97) 1 (1.0/1.0) YES* 2 Caenogastropoda (40) 13 1 & 2 BI YES (100) YES (100/100) 1 (75) 1 (100/93) YES (50) 3 Stromboidea (13) 13 all BI n/a YES (1.0/1.0) 2 (0.98) 1 (1.0/1.0) n/a 4 Stromboidea (13) 13 all ML n/a YES (100/86) 1 (59) 1 (100/84) n/a 5 Stromboidea (13) 17 all BI n/a YES (1.0/1.0) 2 (1.0) 2 (1.0/1.0) n/a 6 Stromboidea (13) 17 all ML n/a YES (100/100) 2 (90) 2 (100/75) n/a 7 Stromboidea (8) 17 all BI n/a YES (1.0/1.0) 2 (1.0) n/a n/a 8 Stromboidea (8) 17 all ML n/a YES (100/100) 2 (70) n/a n/a 9 Stromboidea (8) 2 all BI n/a YES (1.0/1.0) 3 (1.0) n/a n/a 10 Stromboidea (8) 2 all ML n/a YES (77/99) 3 (94) n/a n/a

(Trees 3, 5, 7, PP = 0.98–1.0; Trees 6, 8, B/S = 70–90%; Figs. 3, 5; Suppl. Williams et al., 2008). Discordances between mitochondrial and nuclear Mat. 6). Future analyses should include greater sampling of stromboi­ genes suggest that in order to fully resolve strombid phylogenetic re­ dean species to further resolve this section of the tree. The inclusion of lationships, future studies should include both nuclear and mitochon­ the type species of Rostellariidae would be particularly beneficial; drial loci, as well as broader taxon sampling, and datasets should be Kronenberg and Burger (2002) noted that Varicospira was potentially analysed with the effects of gene choice taken into account. closer to Strombidae than Rostellariidae, based on shared morphological characters (serrated operculum and open siphonal canal). 4.2. Caenogastropod systematics Gene choice also strongly affected phylogenetic relationships within Strombidae. In this study, all analyses with only mitochondrial PCGs Although not the focus of this study, the inclusion of a large number recovered two biogeographically circumscribed clades: Clade A from the of caenogastropod outgroups provided the opportunity to re-examine Eastern Pacific/Atlantic (Aliger gigas + Strombus pugilis) and Clade B higher level systematic relationships within Caenogastropoda, as well from the Indo-West Pacific (IWP) (Conomurex luhuanus + ((Harpago as to investigate the relationship of Stromboidea with other caenogas­ chiragra + Lambis lambis) + (Ministrombus variabilis + Tridentarius den­ tropods. Hypsogastropoda (all caenogastropods except Architaenio­ tatus)), with maximal support for Clade A in all trees and varying support glossa and Cerithioidea; Bouchet et al., 2017; Ponder et al., 2020) was for Clade B (Trees 1, 3, PP = 1.0; Trees 2, 4, B/S = 84–93%; Table 4; recovered in Trees 1 and 2 (Tree 1, PP = 1.0; Tree 2, B/S = 98%; Fig. 4; Figs. 3–5; Suppl. Mat. 6). However, analyses of both nuclear and mito­ Suppl. Mat. 6), consistent with previous morphological (Healy, 1990; chondrial genes together recovered Conomurex luhuanus as sister to two Ponder et al., 2008) and molecular analyses (Colgan et al., 2007; Zapata clades (Clade A; Clade C, (Harpago chiragra + Lambis lambis) + (Minis­ et al., 2014; Osca et al., 2015; but see Zou et al., 2011). trombus variabilis + Tridentarius dentatus)) with low to maximal support Relationships among the three traditional hypsogastropod subgroups across all branches (Tree 5, PP = 1.0; Tree 6, 75–100%). Differences in (Littorinimorpha, Neogastropoda, and ) are notoriously taxon sampling notwithstanding, the topologies of Trees 5 and 6 were difficultto resolve, likely due to an early radiation event (Colgan et al., consistent with the results of Simone (2005), but differed from Latiolais 2007). The name Ptenoglossa has recently been abandoned because the et al. (2006), wherein analyses with nuclear (histone H3) and mito­ grouping is polyphyletic, with its component superfamilies Epitonioidea chondrial (cox1) sequence data resolved Tridentarius dentatus + ((Har­ and currently unassigned to a caenogastropod order pago chiragra + Lambis lambis) + (Conomurex luhuanus + (Aliger gigas + (WoRMS, 2020), and Eulimoidea now assigned to littorinimorph group Strombus pugilis))). Nevertheless, analyses produced by both mitochon­ as (Takano and Kano, 2014; Bouchet et al., drial and nuclear genes (Trees 5 and 6) were consistent with the finding 2017). These groups previously assigned to Ptenoglossa lack mitoge­ from Latiolais et al. (2006) of an Eastern Pacific/Atlantic clade derived nomic representation; however, the other two hypsogastropod groups from the IWP clade, as suggested by Kronenberg and Vermeij (2002). are represented here. Comparisons to other morphological (Stone, 2001) and molecular Neogastropoda is well-supported by morphological characters, (Maxwell et al., 2020) work were difficult due to the fact that these mostly associated with digestive system anatomy (Taylor and Morris, studies each included only two of the species represented in this study. 1988; Kantor, 1996, 2002; Strong, 2003; Ponder et al., 2008). However, Other studies have also shown that gastropod phylogenies can vary this group was not recovered as monophyletic in the ‘Caenogastropoda’ depending on the choice of genes, as well as the use of either amino acids analysis due to the inclusion of Tonnoidea with good support (Tree 2, B/ or nucleotides in analyses (Uribe et al., 2017; Wort et al., 2017), and that S = 88%; Suppl. Mat. 6), and in the BI analyses also to the inclusion of trees based solely on mitochondrial genes can also result in erroneous Calyptraeoidea with good support (Tree 1, PP = 0.98; Fig. 4). The un­ deep relationships among molluscan taxa (Stoger¨ and Schrodl,¨ 2013). certain placement of Calyptraeoidea may stem from missing genes as the Curiously, the phylogenies here based on mitogenomic data alone are mitogenome is incomplete. The inclusion of Tonnoidea in Neo­ the ones that identify biogeographically sensible clusters of species. gastropoda is consistent with previous molecular analyses (Hayashi, Phylogenetic studies using only mitogenome data have proved to be 2005; Colgan et al., 2007; Cunha et al., 2009; Williams et al., 2014; Osca better at resolving morphologically predicted groupings in Trochoidea et al., 2015). (Uribe et al., 2016, 2017; Wort et al., 2017) than a mixture of nuclear The third group, Littorinimorpha, where Stromboidea has tradi­ and mitochondrial genes (Williams and Ozawa, 2006; Kano, 2008; tionally been assigned, was not recovered as a clade in any analysis

8 A.R. Irwin et al. Molecular Phylogenetics and Evolution 158 (2021) 107081

Fig. 3. A summary of all topologies recov­ ered in this study for stromboid relation­ ships. See Table 4 for details of each analysis method; Suppl. Mat. 3 for substitution model choice. Trees 5 and 6 (highlighted in grey) recovered the most relationships corrobo­ rated by morphological data. Branches incongruent with Trees 5 and 6 are marked in red. Stromboid families, including Xen­ ophoridae, are indicated by blocks of colour (see legend). Outgroups have been trimmed for ease of reading. (For interpretation of the references to colour in this figurelegend, the reader is referred to the web version of this article.)

(Trees 1, 2; Fig. 4; Suppl. Mat. 6), consistent with all molecular studies to The sister group of Stromboidea remains uncertain. It is recovered as date (e.g. Hayashi, 2005; Colgan et al., 2007; Cunha et al., 2009; Takano sister to Cypraeoidea + (Calyptraeoidea + Neogastropoda) with poor and Kano, 2014; Williams et al. 2014; Osca et al. 2014, 2015; Cunha and support in the BI analysis (Tree 1, PP = 0.7; Fig. 4) and to Cypraeoidea + Giribet, 2019). However, alternative taxon groups that include Strom­ Neogastropoda in the ML analysis, although lacking support (Tree 2, B/ boidea were recovered. Latrogastropoda (Neogastropoda plus littor­ S = 50%; Suppl. Mat. 6). Previous molecular analyses have instead inimorph superfamilies Calyptraeoidea, Capuloidea, Cypraeoidea, recovered Cypraeoidea + Stromboidea as the sister group of Neo­ Stromboidea, Tonnoidea and Xenophoroidea; Riedel, 2000; Ponder gastropoda + Tonnoidea (Colgan et al., 2007; Osca et al., 2015), and et al., 2020) is recovered by mitogenome analyses in Osca et al. (2015), additionally Ficoidea in Zou et al. (2011). morphological analyses (Ponder et al., 2008) and in Tree 1 within this study, though with poor support (Tree 1, PP = 0.7; Fig. 4). The group 5. Conclusions Strombogastropoda (Stromboidea, Calyptraeoidea, Naticoidea, Cypraeoidea, Tonnoidea, , Cancellariidae and Muricoidea; Mitogenomes are a rich source of data to underpin detailed in­ Simone, 2011) was also recovered in this study, but also lacks support vestigations of the phylogeny of the superfamily Stromboidea, and its (Tree 1, PP = 0.7; Fig. 4). position within Caenogastropoda. The datasets analysed here include

9 A.R. Irwin et al. Molecular Phylogenetics and Evolution 158 (2021) 107081

Fig. 4. Phylogeny of Caenogastropoda (Tree 1) inferred using Bayesian analysis of 13 protein-coding genes for 40 ingroup taxa, with third codon positions removed, under site-homogeneous models partitioned by gene (see Suppl. Mat. 3 for substitution model choice; Table 4 for details of analysis method). Branch support values are included as Tree 1 / Tree 2 (Tree 1 PP, posterior probabilities; Tree 2 (resulting from Maximum Likelihood analysis of the same alignment) B/S, ultrafast bootstrap); where the topology of Tree 2 is not congruent with Tree 1, due to the placement of Calyptraeoidea, a dash (–) is used. Major taxon groups are identifiedby blocks of colour, with support values indicated in the top right of the block where relevant. Stromboidea is here defined as including Xenophoridae (highlighted in red font). See Suppl. Mat. 1 for GenBank Accession numbers. For ease of reading, outgroup (vetigastropod Haliotis iris) has been trimmed. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5. Phylogeny of Stromboidea (Tree 5) resulting from Bayesian analysis of 17 genes (13 mitochondrial protein-coding genes + rrnS + rrnL and two nuclear genes, 18S + 28S) for 13 ingroup taxa, using site-homogeneous models partitioned by gene (see Suppl. Mat. 3 for substitution model choice; Table 4 for details of analysis method). Branch support values are included as Tree 5 / Tree 6 (Tree 5 PP, posterior probabilities; Tree 6 (resulting from Maximum Likelihood analysis of the same alignment) B/S, ultrafast bootstrap). Families are identified by blocks of colour; sup­ port values are indicated in the top right of the block where relevant. Stromboidea here includes Xenophoridae. See Suppl. Mat. 1 for GenBank Accession numbers. Outgroup is removed for ease of reading. Photos included are of specimens sequenced.

10 A.R. Irwin et al. Molecular Phylogenetics and Evolution 158 (2021) 107081 representatives of all stromboidean families and substantially increase Appendix A. Supplementary material the total number of mitogenomes available for Stromboidea to thirteen. In this study we show that some relationships within Stromboidea are Supplementary data to this article can be found online at https://doi. sensitive to gene selection, but only a few well-supported branches were org/10.1016/j.ympev.2021.107081. sensitive to mode of inference. However, all results strongly support the monophyly of Stromboidea as redefined here to include the References Xenophoridae. Abbott, D.P., 1962. Observations on the gastropod Terebellum terebellum (Linnaeus), with particular reference to the behavior of the eyes during burrowing. The 5, Credit authorship contribution statement 1–3. Abbott, R.T., 1960. The genus Strombus in the Indo-Pacific. 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