1 Revised version: November 21st, 2016 2 3 4 Denser mitogenomic sampling improves resolution of the phylogeny of the 5 superfamily Trochoidea (Gastropoda: Vetigastropoda) 6 7 8 Juan E. Uribe1, Suzanne T. Williams2, José Templado1, Samuel 9 Abalde1, and Rafael Zardoya1* 10 11 1Museo Nacional de Ciencias Naturales (MNCN-CSIC), José Gutiérrez 12 Abascal 2, 28006, Madrid, Spain 13 2Department of Life Sciences, Natural History Museum, Cromwell Rd, 14 London SW7 5BD, UK 15 16 17 18 19 20 21 *Correspondence: R. Zardoya; email: [email protected] 22 23 ABSTRACT 24 The great morphological and ecological diversity within the superfamily Trochoidea s.l. 25 (Gastropoda: Vetigastropoda) has in the past hindered the reconstruction of a robust 26 phylogeny for the group based on morphology. Moreover, previous molecular 27 phylogenies disagreed on the monophyly and internal relationships of Trochoidea s.l., 28 as well as on its relative phylogenetic position within Vetigastropoda. In order to further 29 resolve the trochoidean and vetigastropod phylogenetic trees, we considerably increased 30 the representation of trochoidean families for which no previous mitochondrial (mt) 31 genomes were available: the complete mt genome of Cittarium pica (Tegulidae) and the 32 nearly complete mt genomes of Tectus virgatus (Tegulidae), Gibbula umbilicaris 33 (Trochidae), and Margarites vorticiferus (Margaritidae) were sequenced. In addition, 34 the nucleotide sequences of all protein coding and rRNA genes of Clanculus 35 margaritarius (Trochidae) and of Calliostoma zizyphinum (Calliostomatidae) were 36 derived from transcriptomic sequence data. The reconstructed phylogenetic trees using 37 probabilistic methods and Neomphalina as outgroup recovered with maximal support a 38 Trochoidea sensu Hickman & McLean, 1990 clade that included superfamilies 39 Angarioidea and Phasianelloidea deeply nested within superfamily Trochoidea sensu 40 Williams (2012). The families Trochidae and Calliostomatidae were the sister group to 41 the remaining trochoidean lineages. Of these, the family Margaritidae was sister to a 42 clade including Phasianelloidea + Angarioidea and Turbinidae + Tegulidae, this latter 43 family being paraphyletic (Cittarium and Tectus need to be assigned to a new family). 44 Gene order within newly determined mt genomes was very stable (with only few 45 rearrangements restricted to tRNA genes) and conformed to the vetigastropod and 46 gastropod consensus genome organizations. 47 48 Keys words: Mitogenomic phylogeny, rearrangement, Vetigastropoda, Trochoidea, 49 Trochidae, Calliostomatidae, Margaritidae, Cittarium, Tectus. 50 51 INTRODUCTION 52 Trochoidea s.l. Rafinesque, 1815 (top shells, turban shells, and allies) is one of the 53 most ecologically and morphologically diverse lineage of marine gastropods and by far 54 the largest superfamily belonging to the subclass Vetigastropoda, with more than 2,000 55 living species grouped into about 500 recognized genera (Hickman, 1996; Geiger, 56 Nützel & Sasaki, 2008). The clade is distributed worldwide and is present throughout 57 all seas and oceans, at all latitudes and bathymetric ranges (Hickman & McLean, 1990; 58 Williams, Karube & Ozawa, 2008). Trochoideans play an important ecological role as a 59 predominant element in different marine communities such as intertidal rocky shores, 60 seagrass beds, or coral reefs, and they are also found in many other marine habitats 61 (Williams et al., 2008). They have a long fossil record that goes back to the Middle 62 Triassic, 228-245 million years ago, but the time of the origin of the group is certainly 63 much older (Hickman & McLean, 1990; Williams et al., 2008). 64 The taxonomic internal classification of Trochoidea has a long history of 65 controversy and instability. In their comprehensive morphological monograph on 66 trochacean gastropods, Hickman & McLean (1990) maintained the three families 67 traditionally recognized within the superfamily i.e., Trochidae, Turbinidae and 68 Skeneidae, and organized the different genera into various subfamilies and tribes based 69 on suites of shared morphological characters. Later, in the taxonomic classification of 70 gastropods proposed by Bouchet et al. (2005), the family Turbinidae (including the 71 subfamily Skeneinae) was classified within the superfamily Turbinoidea. However, 72 major changes to the systematics of Trochoidea were based on recent molecular 73 phylogenies (Geiger & Thacker, 2005; Williams & Ozawa, 2006; Kano, 2008; Williams 74 et al., 2008; Williams, 2012), which challenged the monophyly of the superfamily as 75 well as of several of the internal groups as defined by Hickman & McLean (1990), and 76 prompted for important changes to the taxon composition and arrangement of families 77 (Williams, 2012). For instance, some taxa were transferred to the superfamily 78 Seguenzioidea (Verrill, 1884), newly redefined by Kano (2008), and a number of 79 minute skeneimorph genera were variously relocated either to Seguenzoidea (Kano, 80 Chikyu & Warén, 2009; Haszprunar et al., 2016), Neomphalina (Kunze et al., 2008), or 81 to the new family Crosseolidae of uncertain taxonomic position (Hickman, 2013). 82 Furthermore, several molecular studies redefined the family Turbinidae (Williams & 83 Ozawa, 2006), reinterpreted the superfamilies Angarioidea and Phasianelloidea 84 (Williams et al., 2008), and restricted Trochoidea to the families Trochidae, Turbinidae, 85 Solariellidae, Calliostomatidae, Liotiidae, Skeneidae, Margaritidae and Tegulidae 86 (Williams, 2012). 87 None of these taxonomic changes was definitive and the debates over the final 88 composition and internal phylogenetic relationships of Trochoidea remain more alive 89 than ever. Moreover, this question is directly related to resolving phylogenetic 90 relationships among the different superfamilies of Vetigastropoda. In this regard, some 91 studies recovered Phasianelloidea and/ or Angarioidea in early-branching positions of 92 the Vetigastropoda tree after the divergence of Pleurotomarioidea ((Williams & Ozawa, 93 2006; Kano, 2008; Williams et al., 2008; Aktipis & Giribet, 2012) whereas several 94 recent phylogenies grouped Phasianelloidea and/ or Angarioidea with Trochoidea 95 (Zapata et al., 2014; Uribe et al., 2016; Lee et al. 2017; Wort, Fenberg & Williams, 96 2017). While earlier studies were based on few partial mitochondrial and nuclear genes 97 and a rather extensive lineage representation, later ones were based on phylogenomic 98 data but with reduced taxon sampling. 99 Phylogenetic analysis of complete mitochondrial (mt) genomes resulted in good 100 resolution among vetigastropod superfamilies (e.g. Uribe et al., 2016) and therefore, 101 they are good candidates to resolve phylogenetic relationships within Trochoidea. Until 102 recently, there were available 22 complete or near-complete mt genomes of 103 Vetigastropoda, which represent the living superfamilies Fissurelloidea, 104 Lepetodriloidea, Seguenzioidea, Haliotoidea, Angarioidea, Phasianelloidea, and 105 Trochoidea (no mt genome has been sequenced for Pleurotomarioidea and 106 Lepetelloidea). However, the great diversity of Trochoidea was clearly 107 underrepresented, as mt genomes for only 12 species belonging to families Turbinidae, 108 Trochidae, and Tegulidae had been published (Uribe et al., 2016; Lee et al. 2017; Wort 109 et al. 2017). Here, we increased the number of complete mt genomes representing 110 different families within Trochoidea to test the monophyly and address internal 111 phylogenetic relationships of the superfamily (in particular the relative positions of 112 families Trochidae, Calliostomatidae, and Margaritidae plus of the genera Tectus and 113 Cittarium , of which onlyTrochidae was previously included in phylogenetic analyses), 114 as well as to resolve its relative phylogenetic position within Vetigastropoda. In 115 addition, the reconstructed phylogeny was used to determine whether trochoidean mt 116 genomes show rearrangements in their genes orders. During the review process of this 117 paper, Lee et al. (2016) published a related mitogenomic phylogenetic study, which 118 complemented our taxon sampling and enriched it at the family level. Therefore, the mt 119 genomes reported by Lee et al. (2016) were incorporated into our phylogenetic 120 analyses. 121 122 MATERIALS AND METHODS 123 Samples and DNA/ RNA extraction 124 One specimen each of Cittarium pica (Tegulidae), Tectus virgatus (Tegulidae), 125 Gibbula umbilicaris (Trochidae), Clanculus margaritarius (Trochidae), Calliostoma 126 zizyphinum (Calliostomatidae), and Margarites vorticiferus (Margaritidae) was used for 127 this study (See Table 1, for details on the locality, collector, and voucher ID of each 128 sample; family assignment was based on WoRMS: accessed October 2016, Gofas, 129 2009). Samples of C. pica, T. virgatus, G. umbilicaris, and M. vorticiferus, were stored 130 in 100% ethanol at -20 ºC, and total genomic DNA was isolated from up to 30 mg of 131 foot tissue following a standard phenol chloroform extraction. 132 Samples of C. margaritarius and C. zizyphinum were stored in RNALater at -80 133 ºC, and total RNA was isolated from mantle tissue using the RNeasy Fibrous Tissue 134 Mini Kit (Qiagen) according to the manufacturer’s instructions. Total RNA was 135 quantified and its integrity assessed using a Qubit® 2.0 Fluorometer RNA assay kit and 136 an Agilent 2200 Tapestation with a high sensitivity R6K Screen Tape, respectively. 137 Dynabeads® mRNA DIRECT™ Micro Kit (Ambion,