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Home , Allonautilus, Argonautoidea, Bathyteuthis abyssicola, Coleoidea, Incirrata, Neocoleoidea

Version: December 1st, 2016

Revisiting the phylogeny of Cephalopoda using complete

mitochondrial genomes

Juan E. Uribe and Rafael Zardoya*

1Museo Nacional de Ciencias Naturales (MNCN-CSIC), José Gutiérrez

Abascal 2, 28006, Madrid, Spain

*Corresponding author: [email protected]

ABSTRACT

Living of Cephalopoda are classified into two main lineages, Nautiloidea and

Coleoidea. Within the latter, two main groups are distinguished, and

Decapodiformes. Phylogenetic relationships within have been the subject of numerous studies and the relative phylogenetic position of several enigmatic groups such as Vampyromorpha, Idiosepioidea, and , among others, has been contentious. Here, we reconstruct the phylogeny of Cephalopoda using all (39) currently reported complete mitochondrial (mt) genomes; in addition, we concatenated available partial mt genes from five important lineages (represented by genera Opistoteuthis,

Argonauta, Japetella, Thaumeledone, and ) lacking complete mt genomes to include them into the phylogenetic analyses. The reconstructed trees recovered with high statistical support the monophyly of Octopodiformes (Octopoda +

Vampyromorpha), Octopoda, and Decapodiformes. Within Octopoda, the first split was between Cirrata and , and within the latter, between and

Octopodoidea. Within Octopodoidea, the , as traditionally defined, is non-monophyletic. Within Decapodiformes, Sepiida were recovered sister to a clade including Idiosepiida as first diverging lineage. Among the remaining lineages of this clade, Myopsina were recovered sister to a “pelagic” clade including Bathyteuthoidea,

Spirulida and , to the exclusion of Sepiolida. Based on this fully resolved phylogeny, the evolution of gene rearrangements among the studied mitochondrial genomes following a tandem duplication and random loss model was inferred. Finally, a chronogram was estimated under an uncorrelated relaxed molecular clock, which dated major cladogenetic events within Cephalopoda considerably younger than previous studies.

INTRODUCTION

Cephalopoda are one of the most striking and morphologically disparate classes of

Mollusca (Young, Vecchione & Donovan, 1998; Nishiguchi & Mapes, 2008). Since early taxonomic studies (Naef, 1921-23), living (~700 species; Strugnell,

Lindgren & Allcock, 2009) have been classified into two main subdivisions (Fig. 1),

Nautiloidea (two genera, and Allonautilus), and Coleoidea (Young et al.,

1998). The latter is further subdivided into two groups with distinct body plans,

Octopoda (e.g. argonauts and ) and Decapodiformes (e.g. and ) that have eight or ten (due to two additional long ) appendages, respectively (Berthold & Engeser, 1987; Boletzky, 2003). The placement of the enigmatic, monotypic Vampyrotheutis (vampire squids) has been controversial for many years (Fig. 1). Ascribed to its own , Vampyromorpha by Pickford (1939), it has been hypothesized to be either close to Octopoda (e.g. Allcock, Cooke &

Strugnell, 2011; Lindgren et al., 2012; Groth et al., 2015; Sutton, Perales-Raya &

Gilbert, 2016) forming the clade Octopodiformes (also known as Vampyropoda) or sister to Decapodiformes (Lindgren, Giribet & Nishiguchi, 2004; Strugnell &

Nishiguchi, 2007; Zhang et al., 2016). Within Octopoda, two main groups are distinguished: Cirrata and Incirrata, which either have or lack rows of fleshy spines named cirri, respectively (Fig. 1). The Cirrata or deep-sea finned octopods are about 40 poorly known and understudied species. The Incirrata includes two superfamilies,

Argonautoidea with four pelagic families (Alloposidae, , Ocythoidae, and

Tremoctopodidade), and the highly diverse Octopodoidea, which traditionally was divided into three pelagic families (Vitreledonellidae, Bolitaenidae, and ), and the benthic family Octopodidae (Voight, 1997), but recently has been reorganized into six families based on molecular evidence despite less than ideal taxon sampling (Strugnell et al., 2013). Within Decapodiformes, up to five orders are distinguished,

Spirulida (ram’s horn squids, monogeneric), Sepiida (), Sepiolida (bobtail and bottletail squids), Idiosepiida (pigmy squids) and Teuthida (Boletzky, 2003). The latter is the most diverse and is further subdivided into and Oegopsida, close- and open-eye squids, respectively (Fig. 1). In addition, some classifications consider the

Bathyteuthoidea as a valid separate order (Lindgren, 2010; Kawashima et al., 2013).

Several studies have focused on phylogenetic relationships among the main lineages of recent cephalopods (see most recent review in Allcock, Lindgren &

Strugnell, 2015). The most extensive morphological phylogenetic analysis of the group examined 50 morphological characters, of which half were disregarded prior to analysis due to lack of independence or instances of homoplasy (Young & Vecchione, 1996).

The reconstructed phylogeny (Fig. 1) placed Vampyromorpha as sister group to the

Octopoda (supporting the clade Octopodiformes) and distinguished the incirrate and cirrate octopods, but showed little resolution within Decapodiformes (Young &

Vecchione, 1996). Early molecular studies were based on one or two partial gene sequences (Bonnaud, Boucher-Rodoni & Monnerot, 1994; Bonnaud, Boucher-Rodoni

& Monnerot, 1997; Carlini & Graves, 1999; Carlini, Reece & Graves, 2000). These pioneer analyses were not conclusive regarding higher-level phylogenetic relationships within Coleoidea due to poor statistical support of internal nodes and often the results of the different studies were conflicting each other (reviewed in Strugnell & Nishiguchi,

2007; Nisiguchi & Mapes, 2008; Allcock, Lindgren & Strugnell, 2015). More recent studies were based on larger data sets including four to nine partial gene sequences or even complete mitochondrial (mt) genomes (Lindgren et al., 2004; Strugnell et al.,

2005; Strugnell & Nishiguchi, 2007; Lindgren, 2010; Allcock et al., 2011; Lindgren et al., 2012; Groth et al. 2015; Zhang et al., 2016). These studies strongly supported the monophyly of Octopoda and Decapodiformes (reviewed in Allcock et al., 2015).

However, Vampyromorpha was placed either sister to Decapodiformes (Lindgren et al.,

2004; Strugnell & Nishiguchi, 2007; Zhang et al., 2016) or to Octopoda (Allcock et al.,

2011; Lindgren et al., 2012; Groth et al. 2015). This controversy is perfectly exemplified by one recent study (Kawashima et al., 2013), which was based on complete mt genomes and recovered Vampyromorpha as sister to Octopoda with maximum likelihood (ML), although with a relatively poor 59% bootstrap support (BP), and to Decapodiformes with Bayesian inference (BI) with maximal Bayesian Posterior

Probability (PP). Moreover, these studies showed low resolution within

Decapodiformes, and conflicting results particularly with regards to the relative position of Spirulida, Idiosepiida, and Bathyteuthoidea (Lindgren et al., 2004; Strugnell et al.,

2005; Strugnell & Nishiguchi, 2007; Allcock et al., 2011; Lindgren et al., 2012;

Kawashima et al., 2013; Zhang et al., 2016). Additionally, the monophyly of several traditionally accepted groups, such as Octopodidae (Strugnell et al., 2013) or Sepioidea

(Lindgren et al., 2012) was seriously questioned.

Therefore, higher-level relationships of Cephalopoda are still not well understood

(Fig. 1) and phylogenomic studies are largely wanted (Allcock et al., 2015). Here, we compiled all complete mitochondrial (mt) genomes available in GenBank to date as well as multi-gene mt sequences for several important lineages for which no complete mt genome has yet been sequenced. In total, we included 42 taxa that represent major lineages within Cephalopoda to reconstruct a fully resolved phylogeny of higher relationships of cephalopods using probabilistic methods. This robust molecular phylogeny was used as framework to analyse rearrangements in mt genome organization, to revisit the evolution of some morphological characters, and to date major cladogenetic events within the group using a relaxed uncorrelated molecular clock calibrated with fossil data.

MATERIALS AND METHODS

Sequence alignment

A full list of species included in phylogenetic analyses is provided in Table 1. Two different data sets were constructed. One was aimed to resolve phylogenetic relationships among cephalopod main lineages (hereafter the cephalopod data set); it included all taxa, and was rooted with Nautiloidea following previous studies (Allcock et al., 2011; Lindgren et al., 2012). In this data set, all 13 protein-coding genes were analysed at the amino acid level and concatenated together with rRNA genes analysed at the nucleotide level. For the few important lineages with no complete mt genomes available, existing partial mt genes (rrnL, rrnS. cox1, cox3, cob; mitochondrial gene abbreviations follow Boore, 1999; see Table 1) were concatenated and analysed at the amino acid level (protein coding) or at the nucleotide level (encoding rRNAs). This first data set rendered low resolution within Decapodiformes (see results). Therefore, in order to maximize phylogenetic information at this taxonomic level, a second data set

(hereafter the decapodiform data set) was constructed including representatives of main lineages of Decapodiformes other than Sepiida, which was used as outgroup according to the topology of the reconstructed tree based on the cephalopod data set (see results).

In this data set, all 13 protein-coding and rRNA genes were analysed at the nucleotide level and concatenated. Available partial mt genes were concatenated at the nucleotide level for lineages without complete mt genomes.

To construct the two data sets, amino acid or nucleotide sequences for the 13 mt protein-coding genes were aligned separately using Translator X (Abascal, Zardoya &

Telford, 2010) and the nucleotide sequences of the two mt rRNA genes were aligned independently using MAFFT v7 (Katoh & Standley, 2013) with default parameters. Ambiguously aligned positions were removed using Gblocks, v.0.91b (Castresana,

2000) in the Phylogeny.fr server (Dereeper et al., 2008) with the following settings: minimum sequence for flanking positions: 85%; maximum contiguous non-conserved positions: 8; minimum block length: 10; gaps in final blocks: no. Finally, the different single alignments were concatenated into the two data matrices using Geneious® 8.0.3.

Phylogenetic analyses

Phylogenetic relationships among major lineages within Cephalopoda were reconstructed using ML (Felsenstein, 1981) and BI (Huelsenbeck & Ronquist, 2001).

ML analyses were conducted with RAxML v7.3.1 (Stamatakis, 2006) using the rapid hill-climbing algorithm and 10,000 BP. BI analyses were conducted using MrBayes v3.1.2 (Ronquist & Huelsenbeck, 2003) and running four simultaneous Monte Carlo

Markov chains (MCMC) for 10 million generations, sampling every 1000 generations, and discarding the first 25% generations as burn-in (as judged by plots of ML scores and low SD of split frequencies) to prevent sampling before reaching stationarity. Two independent BI runs were performed to increase the chance of adequate mixing by the

MCMC and to increase the chance of detecting failure to converge. Statistical support of internal nodes was assessed using PP. In this study, we considered nodes with BP>70% and PP >0.95 as well supported; nodes with lower values were considered as poorly supported i.e., non-significantly different from random.

The best partition schemes and best-fit models of substitution for the two data sets were identified using Partition Finder or Partition Finder Protein (Lanfear et al., 2012) with the Bayesian Information Criterion (BIC; Schwarz, 1978). For the protein-coding genes of the cephalopod data set (analysed at the amino acid level) the partitions tested were: all genes grouped; all genes separated (except nad4/ 4L and atp6/8); genes grouped by subunits. For the protein-coding genes of the decapodiform data set

(analysed at the nucleotide level), codon positions were taken into account separately in the above-mentioned three partition schemes. In addition, following Williams, Foster &

Littlewood (2014), we tested manually whether the mtZoa model (Rota-Stabelli, Yang

& Telford, 2009), which is not implemented in Partiton Finder Prtotein, could fit better than the selected amino acid replacement models for each partition. The rRNA genes

(analyzed at the nucleotide level) in both data sets were tested separately with two different schemes, as genes separated or combined.

We also performed a BI using the site-heterogeneous mixture CAT model

(Lartillot & Philippe, 2004) as implemented in PhyloBayes MPI v.1.5 (Lartillot et al.,

2013). The CAT model assumes that the different sites of a protein evolve under distinct substitution processes. BI was performed without constant sites (‘-dc’ option), running two independent MCMC chains until convergence, sampling every cycle. The cephalopod data set was analysed only at the amino acid level (protein coding genes) under the best-fit CAT-GTR model, with the discrete gamma approximation to model among-site rate heterogeneity. The performance of the CAT-GTR+G model was assessed using a 10-fold cross-validation performed on subsamples of 6,000 non- constant positions randomly drawn from the original matrices. Convergence of analyses was checked a posteriori using the convergence tools implemented in PhyloBayes

(maxdiff < 0.125, maximum discrepancy < 0.1 and effective size > 100; see

Supplementary Material). Tree node support was assessed using BPP.

Estimation of divergence times Although different fossils such as Nectocaris (Smith & Caron, 2010) have been attributed to the cephalopod stem lineage, there is general agreement in considering

Plectronoceras from the of China (circa 520 MYA), as the earliest known bona fide fossil of the group (Teichert, 1988). Cephalopods have a rich fossil record but normally only the (the chambered portion of the shell) is preserved.

Therefore, fossil remains of Nautiloidea, and are relatively abundant, and offer important insights on the early evolution of the group (Kröger,

Vinther & Fuchs, 2011). However, the general trend to internalization and loss of the shell in the Coloidea has left a fragmented fossil record for many lineages within this group (Fuchs, Bracchi & Weis, 2009; Tanabe, Misaki & Ubukata, 2015; Neige,

Lapierre & Merle, 2016). In this regard, the dating of major cladogenetic events during the evolution of Cephalopoda using molecular data has been proposed as a useful complementary approach (Strugnell et al., 2006; Kröger et al., 2011).

We used BEAST v.1.7 (Drummond & Rambaut, 2007) to perform a Bayesian estimation of divergence times among major cephalopod lineages based on the cephalopod data set (only protein-coding genes at the amino acid level). This software was used to infer branch lengths, and nodal ages using a constrained “supertree” topology that summarizes the ML trees reconstructed based on the two data sets. For the clock model, we selected the lognormal uncorrelated relaxed-clock model, which allows rates to vary among branches without any a priori assumption of autocorrelation between adjacent branches. For the tree prior, we employed a Yule process of speciation. We employed the partitions selected by Partition Finder (see above) and the

WAG model (mtZoa or the best-fit model obtained by PartitionFinder were not available in BEAST). We estimated the K-tree score (Soria-Carrasco et al., 2007), which quantifies differences in the relative branch length and topology of phylogenetic trees. A low k-tree score (0.084) between the trees inferred under the mtZoa and WAG models (which arrived at the same topology) supported the decision taken indicating that the using of the WAG model should not affect dramatically branch length inferences under the molecular clock model. The final Markov chain was run twice for

100 million generations, sampling every 10,000 generations and the first ten million were discarded as part of the burn-in process, according to the convergence of chains checked with Tracer v.1.6 (Rambaut & Drummond, 2007). The effective sample size of all the parameters was above 200.

The posterior distribution of the estimated divergence times was obtained by specifying four calibration points as priors for divergence times of the corresponding splits (Ho & Phillips, 2009). Fossils provided hard minimum bounds (offset) and mean and standard deviations were chosen so that the 95% probability limit corresponds to a soft maximum bound. For the split of Vampyromorpha and Octopoda, a calibration point was set at a minimum of 162 million years ago (Mya) with a 95% upper limit of

171.5 Mya (lognormal distribution, offset: 162; mean: 3; standard deviation: 1) based on the age of the fossil Vampyronassa rhodanica (Fischer & Riou, 2002; Strugnell et al., 2006). For the split of Dorytheutis and Loligo, a calibration point was set at a minimum of 44 Mya with a 95% upper limit of 50 Mya (lognormal distribution, offset:

44; mean: 2; standard deviation: 1) based on the earliest appearance of statoliths of

Loligo (Clarke & Maddock, 1988; Strugnell, et al., 2006). For the ages of diversification of Ommastrephidae and Sepiidae, both calibration points were set at a minimum of 43 million years ago (Mya) with a 95% upper limit of 46 Mya (normal distribution, offset: 43; mean: 1; standard deviation: 0.9) based on the ages of the fossils recently reported for these two groups (Neige et al., 2016).

RESULTS AND DISCUSSION

Phylogenetic relationships of main lineages of cephalopods

The alignment of the cephalopod data set was 5658 positions long. The selected best-fit model was mtZoa and the selected best partition scheme unlinked each gene as a separate partition. The ML (-lnL = 97595.2) and BI (-lnL = 111202.0 for run 1; -lnL =

111200.7 for run 2) phylogenetic analyses arrived at the same topology using Nautilus and Allonautilus as outgroup taxa (Fig. 2). Both reconstructed trees recovered with high statistical support the monophyly of Octopodiformes (Octopoda + Vampyromorpha),

Octopoda, and Decapodiformes (Fig. 2). The relative phylogenetic position of the

“living fossil” Vampyroteuthis have been much debated (see e.g. Strugnell &

Nishiguchi, 2007 versus Lindgren, et al., 2012). In particular, different recent phylogenetic studies based on complete mt genomes supported both hypotheses indistinctly (Allcock et al., 2011; Kawashima et al., 2013; Groth et al., 2015; Zhang et al., 2016). Here, our reconstructed tree favoured with high statistical support (BP =

86%, BPP = 1) a sister group relationship between Vampyromorpha and Octopoda (Fig.

2), in support for a monophyletic Octopodiformes (Berthold & Engeser, 1987), the existing leading hypothesis when considering altogether evidence based on fossils, morphology, and molecules (Young & Vecchione, 1996. Yokobori et al., 2007, Allcock et al., 2011; Lindgren et al., 2012; Groth et al., 2015; Sutton et al., 2016). Therefore, our results further support the validity of the proposed morphological synapomorphies for Octopodiformes such as the modification into filaments or loss of the second pair of arms, and the presence of an outer capsule, among others (Young &

Vecchione, 1996; Allcock et al., 2011).

Within Octopoda, the first split was between Cirrata and Incirrata (Fig.2). This relationship was well supported in ML and BI analyses despite the fact that neither the Cirrata representative nor the early branching Incirrata representatives contributed complete mt genomes to the phylogenetic analyses. The branch length of the node leading to Incirrata is relatively long indicating strong phylogenetic signal, and numerous synapomorphies, which certainly will increase when complete genomes in this part of the tree will be sequenced. The major division of Octopoda into Cirrata and

Incirrata has long been acknowledged (e.g. Naef, 1921-23) due to the distinct morphological characters of Cirrata, which retain an internal shell and two fins on their head, and have rows of fleshy spines or cirri (see Voight, 1997 and references therein).

The peculiar sperm packets, horizontal arm , and the position of salivary glands within the buccal mass have been described as synapomorphies of Cirrata (Young &

Vecchione, 1996). Moreover, ancestral state reconstruction analyses based on a molecular phylogeny suggested that the right oviduct was lost in the ancestral cirrate

(Lindgren et al., 2012). The genus Argonauta was recovered as the sister group of the remaining Incirrata with moderately high statistical support (BP=75%, BPP=1; Fig. 2).

This sister group relationship supports the currently accepted division of Incirrata into two superfamilies, the Argonautoidea and the Octopodoidea, the former characterized by rather small males transferring sperm to females in a detached arm (Strugnell et al.,

2013). Within the Octopodoidea, the genera Japetella and Thaumeledone, which are representatives of traditional families Bolitaenidae and Octopodidae, respectively, were identified with moderately high support as sister to conispadiceus (family

Octopodidae). This clade was sister group to another clade including several species of the genera Octopus and Cistopus, of the traditional family Octopodidae (Fig. 2).

Therefore, the family Octopodidae, as formerly defined and despite being represented by only 10 taxa, is non-monophyletic in the reconstructed tree. This was already stated in phylogenetic studies based on three-four mt and three nuclear partial genes of more limited number of taxa (Strugnell et al., 2005; Strugnell et al., 2013), but was not supported by a morphological study which had better taxon sampling (Voight, 1997).

The genus Octopus is polyphyletic in our reconstructed tree. Alternatively,

Octopus minor and Octopus conispadiceus may belong to different genera,

Callistoctopus and Polypus, respectively (Risso, 1826; Sasaki, 1917; Sasaki, 1920).

This latter hypothesis agrees with a previous molecular phylogeny of Incirrata using a wider representation of Octopodoidea families and genera, which recovered

Callistoctopus ornatus as sister group of Octopus + Cistopus (Strugnell et al., 2013).

The monophyly of Decapodiformes is supported by several morphological synapomorphies, the most conspicuous being the modification of the fourth arms into tentacles (Young & Vecchione, 1996). However, resolving phylogenetic relationships within Decapodiformes has been particularly challenging, and for many years they were depicted as a polytomy due to poor support for internal nodes (Young & Vecchione,

1996; Bonnaud et al., 1997; Carlini & Graves, 1999; Boletzky, 2003; Lindgren et al.,

2004; Kröger et al., 2011). In our phylogenetic analyses, the family Sepiidae (Sepiida) was recovered sister to the remaining decapodiforms with high statistical support (Fig.

2). This result is in agreement with previous phylogenetic analyses using complete mt genomes (Allcock et al., 2011; Kawashima et al., 2013; Zhang et al., 2016). Groth et al.

(2015) combining cox and atp genes also obtained this result under maximum parsimony. Allcock et al. (2011) performed a second phylogenetic analysis including partial mt sequences of Idiosepiida and Spirulida, and placed with low statistical support the latter taxon as sister to all remaining Decapodiformes and the former taxon deeply nested together with Oegopsida and Bathyteuthoidea (i.e., the opposite to our results; see below). Our results also do not support studies based on three-four mt and six nuclear partial genes that recovered Idiosepiida as sister group of remaining Decapodiformes with Sepiida being either sister group of all other taxa but Idiosepiida

(Lindgren et al., 2012) or deeply nested (Strugnell & Nishiguchi, 2007). A phylogeny including extant as well as fossil taxa also recovered Idiosepiida as sister to Sepiida and other Decapodiformes (Sutton et al., 2016).

Within Sepiidae, phylogenetic relationships received high statistical support with two species of Sepia from the West Pacific Ocean species as sister group to a clade including two lineages, one formed by species of Sepia from the Indian Ocean and the other including Sepia officinalis from the Mediterranean Sea sister to species of Sepiella from the Indian Ocean (Fig. 2). The relative phylogenetic position of Sepiella made

Sepia paraphyletic. These results, although preliminary given the limited taxon sampling, are in full agreement with previous phylogenies based on complete mt genomes (Kawashima et al., 2013; Zhang et al., 2016), partial mt genomes (Groth et al.

2015), or mt and nuclear partial gene sequences (Lindgren et al., 2012). Phylogenetic relationships within a second clade comprising the remaining Decapodiformes were largely unresolved (Fig. 2). Additionally, a BI analysis using the site-heterogeneous mixture CAT model that ameliorates potential long-branch attraction biases recovered the same above-mentioned phylogenetic relationships (Supplementary Material).

Analysis & Phylogenetic relationships of the Decapod dataset

The alignment was 12,908 positions long. The selected best-fit model was GTR+I+G and the best partition, all genes and codon positions separated. The ML (-lnL =

111658.1) and BI (-lnL = 111822.2 for run 1; -lnL = 111825.4 for run) phylogenetic analyses arrived at the same fully resolved topology (Fig. 3). The pygmy , genus

Idiosepius, was recovered as sister to the remaining analysed taxa (Sepiolida, Myopsida,

Bathyteuthoidea, Spirulida and Oegopsida), as did Zhang et al. (2016) based on complete mt genomes. Other studies placed Idiosepiida close to Sepiida (e.g. Lindgren et al., 2004; ML analysis in Lindgren, 2010), to Oegopsida (e.g. Allcock et al., 2011), and, in the most recent ones, as sister to all remaining Decapodiformes (maximum parsimony analysis in Lindgren, 2010; Lindgren et al., 2012; Strugnell & Nishiguchi,

2007; Sutton et al., 2016), although in all cases with low or no statistical support.

Members of the family Idiosepiidae are the smallest among Decapodiformes, have strong sexual dimorphism, and present specific morphological traits, the most obvious being the adhesive gland (von Byern, Söller & Steiner, 2012; Nishiguchi et al., 2014).

These peculiarities likely hampered the identification of morphological synapomorphies with other Decapodiformes.

The next branch diverging in the reconstructed decapodiform tree corresponds to the genus (Fig. 3; the same relative position was recovered in Kawashima et al. 2013; Groth et al., 2015; Zhang et al. 2016), which represents the family , many of whose members are well known for their light-producing bacterial symbionts

(Nishiguchi, Lopez & Boletzky, 2004). In the past, this family was placed with families

Sepiadariidae and Sepiidae (and sometimes also Spirulidae) in the order Sepioidea due to strong morphological similarities (Young et al., 1998). However, the hypothesized close phylogenetic relationship has been challenged (e.g. Lindgren et al., 2012, and references therein) in agreement with our reconstructed tree, which rejects the monophyly of Sepioidea, as well as a close phylogenetic relationship between Sepiidae and Sepiolidae. Instead, our results support the previously proposed order Sepiida

(Sepiidae) whereas testing the monophyly of the order Sepiolida would await incorporation of members of the family Sepiadariidae into molecular phylogenetic analyses. The remaining Decapodiformes formed two main clades, one with the eight taxa of the family Loliginidae (Myopsida) and the other with the representatives of

Bathyteuthidae (Bathyteuthoidea), Spirulidae (Spirulida), and of families belonging to

Oegopsida (Architeuthidae, Enoploteuthidae, and Ommastrephidae) (Fig. 3). For many years, the relative position of Myopsida was the subject of an important debate, with different authors advocating for their closer relationship either to Oegopsida or to

Sepiida and Sepiolida (Young et al., 1998; Lindgren et al., 2012). Our results support the first hypothesis (Myopsida+Oegopsida. i.e. Teuthida; Naef, 1921-23) and agree with most recent molecular phylogenies (Allcock et al., 2011; Lindgren et al., 2012; Zhang et al., 2016) as well as a phylogeny that incorporated fossil data (Sutton, et al., 2016).

Within Loliginidae, Sepioteuthis was recovered as sister group to a clade that includes

Loligo and Doryteuthis as sister to a non-monophyletic Uroteuthis due to the inclusion of Loliolus (Fig. 3). The generic classification of Loliginidae is still under debate

(Vecchione et al., 1998; Anderson, 2000) and our results indicate the need of having a robust phylogeny as framework to make taxonomic decisions in this complex group.

The reconstructed phylogeny strongly supports a close relationship of pelagic

Bathyteuthoidea, Spirulida and Oegopsida (Fig. 3). The close affinity of

Bathyteuthoidea and Oegopsida has been recovered in several previous studies (e.g.

Lindgren, 2010; Allcock et al., 2011; Lindgren et al., 2012; Kawashima et al., 2013;

Groth et al., 2015; Zhang et al., 2016). The families Bathyteuthidae and

Chtenopterygidae include small deep-sea squids with some typical features of

Oegopsida such as paired oviducts and suckers without circularis muscles (Allcock et al., 2011). However, the most interesting result in this part of the tree is the relative phylogenetic position of Spirulida (Fig. 3). This group contains a single mesopelagic species, the ram's horn squid, Spirula spirula, which is best characterized by having an internal chambered and coiled shell that functions as buoyancy organ (Warnke &

Keupp, 2005). Spirulida has been variously placed as sister group of all other

Decapodiformes (Allcock et al., 2011) or as sister of Bathyteuthoidea and Oegopsida

(Lindgren et al., 2012). To our knowledge, the phylogenetic position here recovered for

Spirulida has never been proposed before, although is weakly supported (BP = 71%, PP

= 0.85) and thus should be considered tentative.

Oegopsida was recovered as a monophyletic group, although with poor support

(BP = 66%, PP = 0.8). This lineage shows great morphological diversity and contains about 25 families, more than all other coleoids together (Young et al., 1998). Here, only three of these families could be included in the phylogenetic analyses, limiting out ability to draw conclusions. Other studies (e.g. Lindgren, 2010) with a larger taxon sampling reported higher statistical support of Oegopsida monophyly. The , family Architeuthidae, was recovered as the sister group of Enoploteuthidae and

Ommastrephidae (Fig. 3). Within the latter, two main lineages were recovered, one including Illex and Todarodes, and the other including Ommastrephes as sister group of

Dosidicus and Sthenotheutis (Fig. 3). The long branch exhibited by Watasenia

(Enoploteuthidae), indicating higher substitution rates, introduces biases in the phylogenetic reconstruction, and this taxon has been variously recovered as sister group to all other Oegopsida (Allcock et al., 2011) or more strikingly within Ommastrephidae

(Zhang et al., 2016) based on complete mt genomes. A recent BI phylogeny based on a multilocus data set including mt and nuclear data recovered Architeuthidae sister to

Enoploteuthidae to the exclusion of Ommastrephidae (Lindgren, 2010). However, this relationship was not supported in maximum parsimony and ML analyses (Lindgren,

2010; see also Lindgren et al., 2012). Overall, the reconstructed trees showed high resolution (all but three nodes had statistical support above 75% BP and 1 PP). Therefore, our results strongly support the usefulness of complete mt genomes for within-class phylogenetic reconstruction under probabilistic methods with appropriate best-fit evolutionary models provided that: (1) lineage diversity is maximized through a dense and strategic taxon sampling; (2) evolutionary (substitution) rates are relatively homogeneous among lineages; and (3) amino acid and nucleotide sequence data are used to resolve deep and shallow phylogenetic relationships, respectively. Under these criteria, potential negative effects of including missing data from those representatives for which not complete mt genomes are available seems to be ameliorated, as long as the number of such representatives is kept to an essential minimum. Here, we had to introduce missing data

(partial gene sequences added about 20% of the complete mt genomes) for genera

Opistoteuthis, Argonauta, Japetella, Thaumeledone, and Spirula. All of these genera but Spirula belong to the Octopodiformes, and phylogenetic relationships within this group received strong statistical support except for the node grouping Japetella and

Thaumeledone, which had the worst support (BP=48%, PP=0.89) of all nodes in the two reconstructed trees, likely because of both tips having shorter sequences. In the case of

Spirula, the closest two nodes in that part of the tree received low support (BP=68-71%,

PP=0.80-0.85). Here, the local lack of resolution might be directly related to the lack of sequence data for Spirula. The nodes corresponding to the early divergences within

Decapodiformes are rather short and thus require gathering the largest data sets as possible to ensure having phylogenetically informative positions. Hence, for this particular node, the sequencing of the complete mt genome of Spirula spirula would be crucial to determine its relative phylogenetic position with respect to Bathyteuthoidea and Oegopsida.

Cephalopod mitochondrial genome rearrangements

Molluscs are widely known for having frequent gene rearrangements in their mt genomes (Stöger & Schrödl, 2013; Osca et al., 2014) and some cephalopods are particularly prone to depart from the ancestral gene order of molluscan mt genomes

(Yokobori et al., 2004; Akasaki et al., 2006; Kawashima, et al., 2013; Hall et al., 2016).

Here, we double-checked genome annotations available in GenBank using the program

MITOS (Bernt et al., 2013) and mapped mt genome organizations of main lineages onto the reconstructed phylogeny to further understand the evolution of mt gene rearrangements within Cephalopoda (Fig. 4).

The general pattern that emerges from the comparative analysis is that all mt genomes belonging to Octopodiformes (Yokobori et al., 2004; Yokobori et al., 2007) retain the ancestral order of molluscs (Stöger & Schrödl, 2013; Osca et al., 2014), whereas Nautiloidea and Decapodiformes have undergone considerable gene rearrangements (Kawashima et al., 2013) that generally follow the Tandem Duplication and Random Loss model (TDRL model; Moritz, Dowling & Brown, 1987; San Mauro et al., 2006). First, a large block of genes or even the whole mt genome is duplicated; then, redundant genes are lost randomly; and finally additional sporadic translocations of tRNA genes occur. In some cases, the purging of copied genes is not complete, and partial or completely duplicated genes are retained as relicts of the duplication event

(Yokobori et al., 2004; Kawashima et al., 2013). An overview of the different cephalopod mt gene orders show that the integrity of certain blocks is normally maintained during rearrangements: (1) cox1, cox2, trnD, atp9, atp6; (2) trnF, nad5, trnH, nad4, nad4L, trnT; (3) trnS (tga), cob, nad6, trnP, nad1; and (4) rrnL, trnV, rrnS

(Fig. 4). The reconstructed phylogeny sheds light on the evolution of gene rearrangements within cephalopods. The two representatives of Nautiloidea (Boore, 2006; Groth et al.,

2015) share the same genome arrangement, which evolved from the molluscan ancestral gene order (Kawashima et al., 2013; Stöger & Schrödl, 2013; Osca et al., 2014) through

TDRL of a large fragment spanning at least from atp6 gene to the cluster MCYWQG of tRNA genes (Boore, 2006; Fig. 4). In addition, a translocation of the trnT gene needs to be postulated. The mt genomes of species belonging to the family Sepiidae share all the same organization (Akasaki et al., 2006; Yokobori et al., 2007; Kawashima et al.,

2013), which likely derived from the molluscan ancestral gene order through TDRL of a block spanning at least from trnF to nad3 genes, plus the subsequent translocation of the trnD gene (Fig. 4). The mt genome of the representative of the family Idosepiidae

(Hall et al., 2016) shows the output of a TDRL event involving a block of genes spanning at least from atp6 to trnR genes, and posterior translocations of nad5-trnH, trnW, trnM, trnY, rrnL and rrnS genes (Fig. 4). The mt genome of Semirossia

(Sepioliidae) has a very similar organization, which only differs in the translocation of the trnS (tga) gene and of a block including trnR, nad5, trnH, trnM, trnY, trnK, and atp6 genes (Fig. 4). This similarity suggests that the above-mentioned TDRL event may have occurred in the common ancestor of all non-sepiidae Decapodiformes. The mt genomes of members of the family Loliginidae (Myopsina) show a very distinct gene arrangement (Fig. 4). All share the same genome organization but Sepioteuthis, in which the block with trnI, rrnL, trnV, rrnS, and trnW genes has been translocated. The consensus Loliginidae mt genome order has many rearrangements compared with the molluscan ancestral gene order (Fig. 4), and thus it is not possible to infer whether more than one TDRL event may have occurred, how many genes were affected, and the number of subsequent translocation events. While the blocks (trnF. nad5, nad4, nad4L, trnT), and (trnS (tga), cob, nad6, trnP, nad1) are maintained, the block (cox1, cox2, trnD, atp8, atp6) is disintegrated (Fig. 4).

The ancestor of (Bathyteuthoidea) and Oegopsina likely underwent whole mt genome duplication and posterior random gene loss (Fig. 4). A block of genes including cox1, cox2, trnD, atp8, atp6, and cox3 was not purged by selection and is maintained as a duplicated copy (Fig. 4). The duplicated genes are very similar or even identical in sequence except in the case of cox3 in the Bathyteuthis mt genome, in which the second copy has several indels and it is likely non-functional (Kawashima et al.,

2013). The relative position of the duplicated blocks is the same in Bathyteuthidae and

Architeuthidae but exchanged in Enoploteuthidae and Ommastrephidae (Kawashima, et al., 2013).

Timing of the major cladogenetic events in Cephalopoda

The reconstructed time tree dates the split between Nautiloidea and Coleoidea about

375 Mya (Fig. 5), although with a relatively large credible interval (95% highest posterior density; 450-310). The separation of Octopodiformes and Decapodiformes was estimated to have occurred at 194 (210-179) Mya. Within Octopoda, the split between Cirrata and Incirrata was dated about 146 (178-162) Mya. Within

Decapodiformes, the successive separations of the different main lineages were dated between 117-85 (128-78) Mya (Fig. 5). Increase in lineage diversity within Incirrata,

Sepiida, Myopsina and Oegopsina started at the end of the and continued during the Paleogene (Fig. 5).

Several previous studies have dated cephalopod diversification using reconstructed trees based on different sequence data sets and inference methods

(Strugnell et al., 2006; Kröger et al., 2011; Warnke et al., 2011). The divergence between Nautiloidea and Coleoidea has been estimated between 453-415 Mya (Kröger et al., 2011; Warnke et al., 2011). Our estimate is about 50 Myr younger but the credible interval extends back to 450 Mya. The differences in estimates may stem from the very different calibration points and taxon samplings used in previous studies

(emphasizing outgroup taxa) and the present one (emphasizing ingroup taxa). In any case, our results agree with the hypothesis that Early Paleozoic cephalopod fossils should be interpreted as members of the cephalopod stem group prior to the divergence of Nautiloidea and Coleoidea (Kröger et al., 2011). A similar pattern is found for the estimations for the divergence of Octopodiformes and Decapodiformes, which varied between 275-245 Mya in previous studies (Kröger et al., 2011; Warnke et al., 2011) and here are considerably younger. However, our estimate for the divergence between

Cirrata and Incirrata is only slightly younger than that of Strugnell et al. (2006).

Comparisons of divergence times within Decapodiformes are more difficult to elucidate because internal phylogenetic relationships here reconstructed greatly differ from those in the only previous molecular clock study focused in this group (Strugnell, et al.,

2006). In any case, the divergences of main decapodiform lineages in that study were estimated to have occurred roughly between 362-155 Mya using an autocorrelated molecular clock whereas our estimates were much younger, 117-85 Mya, and more congruent with time trees including fewer decapodiform representatives (150-140 Mya;

Kröger et al., 2011; Warnke et al., 2011). Overall, our estimates are also more in agreement with earlier studies based exclusively on the fossil record (Naef, 1921-23;

Young et al., 1998).

AUTHORS’ CONTRIBUTIONS

J.U.E. performed phylogenetic analyses; R.Z. designed the research and wrote the paper.

ACKNOWLEDGEMENTS

We thank David Reid, Janet R. Voight, Annie R. Lindgren, and an anonymous reviewer for their insightful comments on a previous version of the manuscript. We are grateful to Jesús Marco and Luis Cabellos who provided access to the supercomputer

Altamira at the Institute of Physics of Cantabria (IFCA-CSIC), member of the Spanish

Supercomputing Network, for performing phylogenetic analyses. This work was supported by the Spanish Ministry of Science and Innovation CGL2013-45211-C2-2-P to RZ and BES-2011-051469 to JEU. REFERENCES

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LEGENDS TO FIGURES

Figure 1. Consensus tree depicting higher phylogenetic relationships of Cephalopoda.

Internal phylogenetic relationhips of Decapodiformes are depicted as a main

polytomy. Lineages whose relative phylogenetic position is particularly

controversial are shown in grey.

Figure 2. Phylogenetic relationships of Cephalopoda based on complete mt genomes

(protein coding genes analyzed at the amino acid level plus rRNA genes).

The reconstructed ML phylogram using Nautiloidea as outgroup is shown.

Numbers at nodes are statistical support values for ML (BP)/ BI (PP). The

asterisk indicates species for which no complete mt genomes are available.

Scale bar indicates substitutions/site.

Figure 3. Phylogenetic relationships of Decapodiformes based on complete mt

genomes (protein coding genes analyzed at the nucleotide level plus rRNA

genes). The reconstructed ML phylogram using Sepiida as outgroup is

shown. Numbers at nodes are statistical support values for ML (BP)/ BI (PP).

The asterisk indicates the species for which no complete mt genomes is

available. Scale bar indicates substitutions/site.

Figure 4. Mitochondrial gene orders of the main lineages of Cephalopoda. The genes

encoded in the major and minor strands are shown in the top and bottom

lines, respectively. Colors denote blocks of genes whose integrity is relatively

conserved despite rearrangements. The gene organization of the mt genome

of Octopodiformes corresponds to that inferred for the ancestral Mollusca.

Figure 5. Chronogram of Cephalopoda based on complete mt genomes (protein coding

genes analysed at the amino acid level plus rRNA genes) and using a

supertree that combines trees shown in Figs. 1 and 2. A Bayesian uncorrelated relaxed lognormal clock with four fossil-based calibration priors

(A-D) was used in BEAST. Horizontal bars represent 95% credible intervals

for time estimates; dates are in millions of years.

Teuthida Oegopsida Myopsida Spirulida Decapodiformes Sepiolida Sepiida Idiosepiida Bathyteuthoidea Vampyromorpha Bolitaenidae Vitreledonellidae Incirrata Amphitretidae Octopodidae Octopodiformes Argonautoidea Octopoda Cirrata Nautiloidea Oegopsida Spirulida 97/1 Bathyteuthoidea Myopsida Sepiolida Idiosepioidea

99/1 Sepia pharaonis 97/1 Sepia aculeata DECAPODIFORMES 100/1 Sepia lycidas 100/1 95/1 Sepia esculenta Sepiella japonica Sepiida 100/1 Sepiidae Sepiella inermis 100/1 100/1 Sepia officinalis

Sepia apama

100/1 Sepia la

100/1 Octopus bimaculatus 100/1 Octopus bimaculoides

100/1 80/1 Octopus vulgaris

Amphioctopus aegina 93/1 99/1 Amphioctopus fangsiao

87/1 Cistopus taiwanicus 100/1 Cistopus chinensis

76/1 Octopus minor 48/0.89 OCTOPODIFORMES Japetella diaphana* Incirrata Thaumeledone gunteri* 100/1 79/1 Octopus conispadiceus Octopoda 95/1 * Cirrata Opisthoteuthis massyae* 76/1 Vampyromorpha Vampyroteuthis infernalis

Naulus macromphalus NAUTILOIDEA Allonaulus scrobiculatus 0.3 0.3 Sthenoteuthis oualaniensis 87/1

100/1 Dosidicus gigas

100/1 Ommastrephes bartramii Ommastrephidae

Illex argennus 76/1 75/1 Todarodes pacificus OEGOPSIDA 66/0.80 Watasenia scinllans Enoploteuthidae

71/0.85 Architeuthis dux Architeuthidae

96/1 Spirula spirula* SPIRULIDA

Bathyteuthis abyssicola BATHYTEUTHOIDEA Bathyteuthidae

Uroteuthis edulis 88/1

Uroteuthis chinensis 88/1 84/1 Loliolus beka 100/1 100/1 Loliolus uyii Loliginidae

100/1 Uroteuthis duvaucelii 77/1 Doryteuthis opalescens 100/1

MYOPSIDA 100/1 Loligo bleekeri

100/1 Sepioteuthis lessoniana

Semirossia patagonica SEPIOLIDA Sepiolidae

Idiosepius sp. IDIOSEPIIDA Idiosepiidae

Sepia apama SEPIIDA Sepiidae Sepia lamanus 3.0 3.0 Ommastrephidae cox1 cox2 D atp8 atp6 T cox3 A N I nad3 cox1 cox2 D atp8 atp6 cox3 K R S1 nad2 nad5 H nad4 nad4L S2 cob nad6 P nad1 L2 L1 rrnL M Y W G E F V rrnS C Q Enoploteuthidae cox1 cox2 D atp8 atp6 T cox3 A N I nad3 cox1 cox2 D atp8 atp6 cox3 K R S1 nad2 nad5 H nad4 nad4L S2 cob nad6 P nad1 L2 L1 rrnL Y W G E F V rrnS M C Q Architeuthidae

cox1 cox2 D atp8 atp6 T cox3 K R I S1 nad2 cox1 cox2 D atp8 atp6 cox3 A N nad3 nad5 H nad4 nad4L S2 cob nad6 P nad1 L2 L1 rrnL Y W G E F V rrnS M C Q Bathyteuthidae

cox1 cox2 D atp8 atp6 T cox3 K R S1 nad2 cox1 cox2 D atp8 atp6 cox3 A N I nad3 nad5 H nad4 nad4L S2 cob nad6 P nad1 L2 L1 rrnL Y W G E F V rrnS M C Q

Other Loliginidae cox1 N cox2 R T A D atp8 atp6 cox3 nad3 I K S1 nad2 C Y E M F nad5 nad4 nad4L L2 G H L1 S2 cob nad6 P nad1 Q rrnL V rrnS W

Sepioteuthis cox1 N cox2 R T I A D atp8 atp6 cox3 nad3 K S1 nad2 C Y E M F nad5 nad4 nad4L L2 G rrnL V rrnS W H L1 S2 cob nad6 P nad1 Q

Semirossia cox1 cox2 D atp8 T R K atp6 cox3 A N I nad3 S1 nad2 cob nad6 P nad1 L2 C Q S2 F nad4 nad4L W L1 V G E rrnL rrnS nad5 H M Y

Idiosepiidae cox1 cox2 D atp8 T R K atp6 cox3 A N I nad3 S1 nad2 cob nad6 P nad1 L2 C Q nad5 H M Y F nad4 nad4L S2 W L1 V G E rrnL rrnS

Sepiidae cox1 cox2 atp8 atp6 N I nad3 D T cox3 K A R S1 nad2 F nad1 L2 L1 rrnL V rrnS C Y Q G nad5 H nad4 nad4L S2 cob nad6 P M W E

Octopodiformes cox1 cox2 D atp8 atp6 T cox3 K A R N I nad3 S1 nad2 (Ancestral Mollusca) F nad5 H nad4 nad4L S2 cob nad6 P nad1 L2 L1 rrnL V rrnS M C Y W Q G E

Nauloidea cox1 cox2 D atp8 T atp6 cox3 K A R N I nad3 S1 nad2 F L2 L1 rrnL V rrnS M C Y W Q G nad5 H nad4 nad4L S2 cob nad6 P nad1 E Sthenoteuthis oualaniensis D Dosidicus gigas Ommastrephes bartramii Todarodes pacificus Illex argennus Watasenia scinllans Architeuthis dux Spirula spirula Bathyteuthis abyssicola Uroteuthis chinensis Uroteuthis edulis Loliolus beka Loliolus uyii B Uroteuthis duvaucelii Heterololigo bleekeri Doryteuthis opalescens Sepioteuthis lessoniana Semirossia patagonica Idiosepius sp. Sepia aculeata Sepia pharaonis Sepia lycidas C Sepia esculenta Sepiella japonica Sepiella inermis Sepia officinalis Sepia lamanus Sepia apama Octopus bimaculoides Octopus bimaculatus Octopus vulgaris Amphioctopus fangsiao Amphioctopus aegina Cistopus chinensis Cistopus taiwanicus Octopus minor Japetella diaphana Thaumeledone gunteri A Octopus conispadiceus Argonauta nodosa Opisthoteuthis massyae Vampyroteuthis infernalis 300 275 250 225 200 175 150 125 100 75 50 25 Allonaulus scrobiculatus Naulus macromphalus

27450 27400 27350 27300 27250 27200 27150 27100 27050 27000 26950 26900 26850 26800 26750 26700 26650 26600 26550 26500 26450 26400 26350 26300 26250 26200 26150 26100 26050 26000 25950 25900 25850 25800 25750 25700 25650 25600 25550 25500 25450 25400 25350 25300 25250 25200 25150 25100 25050 25000 24950 24900 24850 24800 24750 24700 24650 24600 24550 24500 24450 24400 24350 24300 24250 24200 24150 24100 24050 24000 23950 23900 23850 23800 23750 23700 23650 23600 23550 23500 23450 23400 23350 23300 23250 23200 23150 23100 23050 23000 22950 22900 22850 22800 22750 22700 22650 22600 22550 22500 22450 22400 22350 22300 22250 22200 22150 22100 22050 22000 21950 21900 21850 21800 21750 21700 21650 21600 21550 21500 21450 21400 21350 21300 21250 21200 21150 21100 21050 21000 20950 20900 20850 20800 20750 20700 20650 20600 20550 20500 20450 20400 20350 20300 20250 20200 20150 20100 20050 20000 19950 19900 19850 19800 19750 19700 19650 19600 19550 19500 19450 19400 19350 19300 19250 19200 19150 19100 19050 19000 18950 18900 18850 18800 18750 18700 18650 18600 18550 18500 18450 18400 18350 18300 18250 18200 18150 18100 18050 18000 17950 17900 17850 17800 17750 17700 17650 17600 17550 17500 17450 17400 17350 17300 17250 17200 17150 17100 17050 17000 16950 16900 16850 16800 16750 16700 16650 16600 16550 16500 16450 16400 16350 16300 16250 16200 16150 16100 16050 16000 15950 15900 15850 15800 15750 15700 15650 15600 15550 15500 15450 15400 15350 15300 15250 15200 15150 15100 15050 15000 14950 14900 14850 14800 14750 14700 14650 14600 14550 14500 14450 14400 14350 14300 14250 14200 14150 14100 14050 14000 13950 13900 13850 13800 13750 13700 13650 13600 13550 13500 13450 13400 13350 13300 13250 13200 13150 13100 13050 13000 12950 12900 12850 12800 12750 12700 12650 12600 12550 12500 12450 12400 12350 12300 12250 12200 12150 12100 12050 12000 11950 11900 11850 11800 11750 11700 11650 11600 11550 11500 11450 11400 11350 11300 11250 11200 11150 11100 11050 11000 10950 10900 10850 10800 10750 10700 10650 10600 10550 10500 10450 10400 10350 10300 10250 10200 10150 10100 10050 10000 9950 9900 9850 9800 9750 9700 9650 9600 9550 9500 9450 9400 9350 9300 9250 9200 9150 9100 9050 9000 8950 8900 8850 8800 8750 8700 8650 8600 8550 8500 8450 8400 8350 8300 8250 8200 8150 8100 8050 8000 7950 7900 7850 7800 7750 7700 7650 7600 7550 7500 7450 7400 7350 7300 7250 7200 7150 7100 7050 7000 6950 6900 6850 6800 6750 6700 6650 6600 6550 6500 6450 6400 6350 6300 6250 6200 6150 6100 6050 6000 5950 5900 5850 5800 5750 5700 5650 5600 5550 5500 5450 5400 5350 5300 5250 5200 5150 5100 5050 5000 4950 4900 4850 4800 4750 4700 4650 4600 4550 4500 4450 4400 4350 4300 4250 4200 4150 4100 4050 4000 3950 3900 3850 3800 3750 3700 3650 3600 3550 3500 3450 3400 3350 3300 3250 3200 3150 3100 3050 3000 2950 2900 2850 2800 2750 2700 2650 2600 2550 2500 2450 2400 2350 2300 2250 2200 2150 2100 2050 2000 1950 1900 1850 1800 1750 1700 1650 1600 1550 1500 1450 1400 1350 1300 1250 1200 1150 1100 1050 1000 950 900 850 800 750 700 650 600 550 500 450 400 350 300 250 200 150 100 50 0 Cretaceous Palaeogene Neo Table 1. Mitochondrial (mt) DNA data analyzed in this study. Length in bp, Genbank accesion number and reference author.

Complete mt genomes Species Orden Family bp GenBank Acc. No.Reference Nautilus macromphalus Nautilidae 16258 NC_007980 Boore, 2006 Allonautilus scrobiculatus Nautilida Nautilidae 16132 NC_026997 Groth et al., 2015 Vampyroteuthis infernalis Vampyromorpha Vampyroteuthidae 15617 NC_009689 Yokobori et al., 2007 Octopus bimaculatus Octopoda Octopodidae 16084 NC_028547 Domínguez-Contreras et al., 2015 Octopus vulgaris Octopoda Octopodidae 15744 NC_006353 Yokobori et al., 2004 Octopus bimaculoides Octopoda Octopodidae 15733 KU295559 Farfan et al., 2016 (unpublished) Octopus conispadiceus Octopoda Octopodidae 16027 KJ789854 Ma et al., 2014 Cistopus taiwanicus Octopoda Octopodidae 15793 NC_023257 Cheng et al., 2013 Cistopus chinensis Octopoda Octopodidae 15706 KF017606 Cheng et al., 2013 Callistoctopus minor Octopoda Octopodidae 15974 NC_015896 Cheng et al., 2012 Amphioctopus fangsiao Octopoda Octopodidae 15979 AB240156 Akasaki et al., 2006 Amphioctopus aegina Octopoda Octopodidae 15545 KT428877 Zhang et al., 2015 Todarodes pacificus Teuthida Ommastrephidae 20254 NC_006354 Yokobori et al., 2004 Sthenoteuthis oualaniensis Teuthida Ommastrephidae 20306 NC_010636 Staaf et al., 2010 Ommastrephes bartramii Teuthida Ommastrephidae 20308 NC_020348 Wakabayashi and Yanagimoto, 2013 (unpublished) Illex argentinus Teuthida Ommastrephidae 20278 NC_026908 Jiang et al., 2015 Dosidicus gigas Teuthida Ommastrephidae 20324 NC_009734 Staaf et al., 2010 Watasenia scintillans Teuthida Enoploteuthidae 20093 NC_007893 Akasaki et al., 2006 Bathyteuthis abyssicola Teuthida Bathyteuthidae 20075 NC_016423 Kawashima et al., 2013 Architeuthis dux Teuthida Architeuthidae 20331 NC_011581 Elliger et al., 2010 (unpublished) Uroteuthis chinensis Teuthida Loliginidae 17353 NC_028189 Jiang et al., 2015 (unpublished) Uroteuthis duvaucelii Teuthida Loliginidae 17413 NC_027729 Jiang et al., 2015 (unpublished) Uroteuthis edulis Teuthida Loliginidae 17360 NC_017746 Takemoto and Yamashita, 2012 (unpublished) Sepioteuthis lessoniana Teuthida Loliginidae 16631 NC_007894 Akasaki et al., 2006 Loliolus beka Teuthida Loliginidae 17483 NC_028034 Jiang et al., 2015 (unpublished) Loliolus uyii Teuthida Loliginidae 17134 NC_026724 Jiang et al., 2015 (unpublished) Heterololigo bleekeri Teuthida Loliginidae 17211 NC_002507 Sasuga et al., 1999 Doryteuthis opalescens Teuthida Loliginidae 17387 NC_012840 Elliger et al., 2012 (unpublished) Semirossia patagonica Sepiida Sepiolidae 17086 NC_016425 Kawashima et al., 2013 Idiosepius sp. Sepiida Idiosepiidae 16183 KF647895 Hall et al., 2016 Sepiella inermis Sepiida Sepiidae 16191 NC_022693 Wang et al., 2015 Sepiella japonica Sepiida Sepiidae 16172 NC_017749 Takemoto and Yamashita, 2012 (unpublished) Sepia aculeata Sepiida Sepiidae 16219 NC_022959 Guo et al., 2016 Sepia lycidas Sepiida Sepiidae 16244 NC_022468 Kawashima et al., 2013 Sepia latimanus Sepiida Sepiidae 16225 NC_022467 Kawashima et al., 2013 Sepia apama Sepiida Sepiidae 16184 NC_022466 Kawashima et al., 2013 Sepia pharaonis Sepiida Sepiidae 16208 NC_021146 Wang et al., 2014 Sepia esculenta Sepiida Sepiidae 16199 NC_009690 Yokobori et al., 2007 Sepia officinalis Sepiida Sepiidae 16163 NC_007895 Akasaki et al., 2006

Partial mt genes Species Order Family cox1* cox3 cob rrnS rrnL Opisthoteuthis massyae Octopoda Opisthoteuthidae AY545187 EU071451 –– AY545079 AY545103 Argonauta nodosa Octopoda Argonautidae AY557517 AJ628206 AJ628170 GU288526 AY545104 Japetella diaphana Octopoda Bolitaenidae AY557518 EU071453 –– AY545093 AJ252766 Thaumeledone gunteri Octopoda Octopodidae EU148466 EU148470 EU148465 EU071443 AF299266 Spirula spirula Spirulida Spirulidae AF000066 AJ966787 –– AY545097 AY293659

*mitochondrial gene abbreviations follow Boore, 1999 0,64Uroteuthis_edulis 0,99Uroteuthis_duvaucelii

Supplementary material: 1 Uroteuthis_chinensis

1 Loliolus_uyii PhyloBayes aminoacids matrix 0,99 Loliolus_beka Maxdiff: 0.10554 0,990,62Loligo_bleekeri Doryteuthis_opalescens 0,9 Meandiff: 0.00899 Sepioteuthis_lessoniana

0,54 Semirossia_patagonica Ideosepius_sp.

0,92Sthenoteuthis_oualanie 1 Ommastrephes_bartramii 0,6 0,99 Dosidicus_gigas

Todarodes_pacificus 0,620,65 Illex_argentinus 0,59 Watasenia_scintillans

Architeuthis_dux 0,53 Spirula_spirula 0,99 Bathyteuthis_abyssicol

0,99Sepia_pharaonis 0,94 Sepia_aculeata

1 Sepia_lycidas

0,65 Sepia_esculenta

1 Sepia_latimanus 1 Sepia_apama

1 Sepiella_japonica 1 Sepiella_inermis

Sepia_officinalis

0,99Octopus_bimaculoides 1 0,98 Octopus_bimaculatus

Octopus_vulgaris 0,57 0,58 Amphioctopus_fangsiao 0,54 Amphioctopus_aegina 0,7 Argonauta_nodosa

0,720,96 Cistopus_taiwanicus Cistopus_chinensis

Octopus_minor 1 0,6 Octopus_conispadiceus

0,93 Japetella_diaphana Thaumeledone_gunteri 0,86 Opisthoteuthis_massyae

Vampyroteuthis_inferna

1 Allonautilus_scrobicul Nautilus_macromphalus

2.0