Fish Sci (2016) 82:279–288 DOI 10.1007/s12562-015-0962-8

ORIGINAL ARTICLE Biology

Evaluation of the 5′ end of the 16S rRNA gene as a DNA barcode marker for the Cephalopoda

Gustavo Sanchez1 · Satoshi Tomano1 · Tetsuya Umino1 · Toshie Wakabayashi2 · Mitsuo Sakai3

Received: 7 October 2015 / Accepted: 12 December 2015 / Published online: 12 January 2016 © Japanese Society of Fisheries Science 2016

Abstract The present study seeks to incorporate a highly interspecific level may indicate high genetic variation of variable DNA barcode marker, additional to the stand- the region being proposed as compared to the standard of ard regions of the cytochrome oxidase I gene and the 3′ 16S rRNA. In addition, two well-supported clades and high end of the 16S large ribosomal subunit (16S rRNA), for a levels of divergence within Sthenoteuthis oualaniensis, more effective species-level identification among cephalo- Loliolus japonica and Sepia pharaonis suggest the occur- pods. Thus, we evaluated whether the 5′ end region of the rence of cryptic species. This study confirms the efficiency 16S rRNA gene can be suitable as a DNA barcode marker of the 5′ end region of the 16S rRNA gene as a DNA bar- among these taxa. Using a novel primer set, 28 different code marker which can be used along with the standard species were evaluated based on the pairwise intra- and DNA markers in future studies. interspecific distance and neighbor-joining (NJ) analysis. Except for Enteroctopus dofleini, we were able to obtain Keywords 16S rRNA · Cytochrome oxidase I · the sequences for the remaining species which formed Cephalopoda · Cryptic species · DNA barcoding highly supported clusters in the NJ tree. Divergence at the

Introduction Electronic supplementary material The online version of this article (doi:10.1007/s12562-015-0962-8) contains supplementary material which is available to authorized users. Inhabiting environments from tropical to polar regions, the Cephalopoda class contains an astonishing diversity * Tetsuya Umino of about 800 different species [1]. Members of this class, umino@hiroshima‑u.ac.jp especially squids, and octopus, are highly com- Gustavo Sanchez mercial and represent an important human diet resource [email protected] due to its high-quality protein [2–4]. According to the Food Satoshi Tomano and Agriculture Organization of the United Nations (FAO) satoshi‑tomano@hiroshima‑u.ac.jp statistics (FAO-Fishstat: http://www.fao.org/fishery/statis- Toshie Wakabayashi tics/software/fishstat/en, accessed January 15, 2015), world twakaba@fish‑u.ac.jp catches of this taxa reached 4 million tons during 2012. Mitsuo Sakai are found in all oceans, and world catches [email protected] could increase in the future when more areas farther from land begin to be exploited [5]. 1 Graduate School of Biosphere Science, Hiroshima University, Higashi‑Hiroshima, Hiroshima 739‑8528, Japan The commercial demand of this taxa includes various food products sold as fresh, frozen, canned or dried. Some 2 Department of Fisheries Science and Technology, National Fisheries University, Shimonoseki, Yamaguchi 759‑6595, concerns have arisen with the processed food industry, such Japan as the mislabeling and species substitution [6] because the 3 Tohoku National Fisheries Research Institute, Hachinohe, indistinguishable characteristics for a proper identifica- Aomori 031‑0841, Japan tion. Another concern is the difficulty of morphological

1 3 280 Fish Sci (2016) 82:279–288 identification, partly due to the lack of hard body parts in family. In addition, the 16S rRNA gene exhibits a unique coleoids species, exhibiting poor preservation of taxonomic copy in the mitochondrial genome of cephalopods and due characters after capture [7]. In addition, difficulty in iden- to its rRNA subunit nature, it is pre-amplified in many taxa, tifying early life stages of many cephalopods is still being which may facilitate its use in small or degraded samples addressed by researchers, because these stages are also [26]. Consequently, the 16S rRNA gene represents a poten- morphologically difficult to distinguish [8, 9]. tial mitochondrial region to explore in hopes of finding an Species identification concerns can be addressed by additional DNA barcode marker for cephalopods. molecular methods such as the DNA barcoding approach, The goal of this study was to evaluate the efficiency of which enables a DNA-based identification by the amplifi- the 5′ end region of the 16S rRNA gene as a DNA barcode cation of a short fragment of a gene. In fact, within the eco- marker among species. Tissues from 28 differ- nomically important group of cephalopods, molecular work ent species representing the extant Nautiloidea and Cole- has played an essential role by suggesting the presence of oidea taxa were analyzed with novel degenerate primer set. cryptic speciation in Loliolus beka [10], Sepia pharaonis For additional test of the DNA marker being proposed, we [11], Sepioteuthis lessoniana [12] and Sthenoteuthis ouala- analyzed one sample of processed industrial food (grilled niensis [13] and is, thus, a necessary tool in conservation squid) and four unknown immature individuals of Sepio- planning for species with high fishing pressure. teuthis spp. sampled in Okinawa Prefecture, Japan to dis- The key role of DNA-based identification is the use of criminate between the three species complex (sp. 1, sp. 2, gene fragments that enables discrimination between closely and sp. 3) of S. lessoniana. related species, or that presents high levels of interspecific divergence with low divergences at the intraspecific level. A 658-base pair region of the cytochrome oxidase I (COI) Materials and methods gene has been widely used to address many identification concerns. COI was initially reported in 2003 as the tar- Sample collection get gene for a global identification system of spe- cies using DNA barcoding [14, 15]. Since then, this gene In total, 114 samples representing 28 different species has become an important and very useful DNA marker with one to a maximum of eight individuals per taxa were for identification of cephalopod species, either by direct used in this study. Following previously reported keys, the sequencing [10, 12, 16–18] or polymerase chain reaction majority of the specimens were morphologically identified restriction fragment length polymorphism (PCR–RFLP [9, from fresh individuals, except for Watasenia scintillans and 19, 20]. However, Strugnell and Lindgren [21] highlighted Doryteuthis opalescens, which were both obtained as boiled some COI features that may be problematic if it is used individuals [3, 27–30]. Individuals belonging to S. lessoni- alone as a DNA barcode in cephalopods, such as the pos- ana species complex (sp. 1 and sp. 2) were morphologically sibility of low rates of evolution and gene duplication in and genetically identified in Tomano et al. [31]. Specimens some taxa. Thereby, it would be helpful to have an addi- that could not be identified to the species level were referred tional DNA marker that could provide information com- to as “sp.”. Immediately after morphological identification plementary to that based on the standard fragment of the of fresh individuals, approximately 2 cm of muscular tis- COI gene and with similar DNA barcoding characteristic sues from the tentacle or arm were stored in 99.5 % ethanol. to solve the above-mentioned morphological identification Tissues of individuals that correspond to Sepioteuthis sp. 3, problems. Opisthoteuthis sp., and Enteroctopus dofleini were obtained Cheng et al. [22] conducted nucleotide diversity com- from a private collection. Tissues of Architeuthis dux and parison of six mitochondrial genomes in Octopodiformes Nautilida species were provided from Miyajima Aquarium and reported that there are other genes with higher vari- (Hiroshima Prefecture) and Toba Aquarium (Mie Prefec- ability than the standard COI that may be more suitable ture) in Japan, respectively. Species used in this study and markers for DNA-based identification. From the analyzed sampling locations are shown in (Online Resource 1). genes by the authors, the 16S rRNA gene showed the For additional tests, one package of processed food (grilled highest nucleotide diversity peak. In cephalopods, species squid), directly acquired from a Japanese market and labeled identification based on this gene was limited to the ′3 end as Surumeika, which is referred to as Todarodes pacificus in region [23], which is commonly amplified with the univer- Japan, was evaluated to determine the performance of the sal primer set described by Palumbi et al. [24] or degen- proposing DNA marker in industrial food product. Also, four erate versions thereof. However Marin et al. [25] reported specimens identified as immature Sepioteuthis spp. were sam- that the 5′ end region of this gene is more variable than the pled in Okinawa Prefecture with the aim of classifying them standard 3′ end in the family Pectinidae (class Bivalvia), as members of any of the three species complexes reported thus being more suitable for DNA barcoding within this for S. lessoniana that co-occur in this location [32].

1 3 Fish Sci (2016) 82:279–288 281

Primer design in MAFFT v.7 with the default parameters. To check the reli- ability of the morphological identification, we used, when Full-length 16S rRNA gene was retrieved from the complete possible, the basic local alignment search tool (BLAST) to mitochondrial genome of 28 cephalopod species, includ- compare the sequences of this study with those available ing members of the subclasses Nautiloidea and Coleoidea, in GenBank based on the percentage of maximum identity. available in the GenBank database (Online Resource 2). Sequences were collapsed into haplotypes using the program Sequences were multi-aligned (5′–3′) with the free online DnaSP v.5.10. In addition, MEGA v.6.0 [36] was used to server MAFFT v.7 [33] with the default parameters. Because calculate intra- and interspecific sequence divergence using this region is characterized by its variable length, the final the Kimura two-parameter (K2P) distance model [37]. matrix was created by discarding the gaps presented in the To provide a graphical representation of the divergence borders of the multi-alignment. To explore the variability between species, a neighbor-joining (NJ) tree [38] of K2P along this matrix, nucleotide diversity was evaluated with distances was created in MEGA v.6.0 by performing 1000 sliding window analysis in DnaSP v.5.10. [34] excluding bootstrap replications with all of the gaps deleted because gaps and window/step sizes of 200/20 bp. Degenerate primer of difficulties in ascertaining conservative domains in sets flanking the more polymorphic region of the 16S rRNA highly variable regions. The sequence of Katharina tuni- gene were designed as follows: (1) a degenerate oligonucleo- cata (NC_001636.1) was used as the outgroup for this tide was only included when two or more nucleotide changes analysis. This sequence was aligned based on the alignment were present more than once at a specific site, otherwise the of our reporting haplotypes. more regular nucleotide was considered; and (2) a small tail was added to improve primer performance. Results DNA extraction, PCR, and sequencing Primer design and performance Genomic DNA was extracted from the tissues using TNES- urea buffer [35] followed by a standard phenol–chloro- Sliding window analysis clearly showed higher variability in form protocol. When necessary, DNA isolation was also the 5′ end compared with the 3′ end region of the 16S rRNA performed using a DNeasy blood and tissue kit (Qiagen, gene (Fig. 1). Thus, the forward CephaF (5′-GGTTACC Hilden, Germany). TTTTGYATAATGG-3′) and reverse CephaR (5′-TTCT Using the primer set designed in this study, PCR ampli- CACYGAGCAGGCYCRACTC-3′) were developed along fication was carried out in 10.05-µl reaction mixtures con- the most conservative sites of the 5′ end region (Fig. 2). taining 1 µl of template DNA (approximately 50 ng), 1 µl of After morphological identification, all sequences were 10 Ex Taq buffer, 0.8 µl of 2.5-mM dNTP mixture, 0.2 µl deposited separately in GenBank (accession numbers × of 10-µM forward/reverse primers, 0.05 µl of TaKaRa Ex LC063229–LC063341). Taq™ DNA (Takara Bio Inc., Shiga, Japan), and 6.8 µl of PCR products were recovered for almost all species but sterile distilled water. PCRs were conducted using a Mas- failed for Opisthoteuthis sp. and E. dofleini, even after a tercycler Gradient 96-well system (Eppendorf, Hamburg, DNA isolation performed with a DNeasy blood and tissue Germany) with the following conditions: initial dena- kit. Nevertheless, we were able to recover the sequences for turation at 95 °C (5 min); followed by 30 cycles of 95 °C Opisthoteuthis sp. after re-precipitation with NaCl (0.2 M (15 s), 50–58 °C (30 s) depending on template, and 72 °C final concentration) and 100 % ethanol of the DNA iso- (30 s); and a final extension at 72 °C (7 min). To confirm lated with the mentioned kit. Consequently, only Octopus amplification, PCR products were electrophoresed on 2 % dofleini was omitted from further analysis as it failed to agarose gels. In addition, PCR products were treated with amplify. These results indicated that DNA degradation or ExoSAP-IT (Affymetrix/USB Corporation, Cleveland, OH, the possible presence of other components in the DNA of E. USA) and then sequenced using a BigDye v3.1 Termina- dofleini may have prevented successful PCR amplification. tor Sequencing Kit (Applied Biosystems, CA, USA) with the forward primer on a genetic analyzer (ABI 3130 1, Species identification × Applied Biosystems, CA, USA). Amplicons of the region being proposed ranged from 476 Data analysis to 523 bp and corresponded to 57 unique haplotypes among the species analyzed (Online Resource 1). Our alignment Electropherograms were visually checked and manually matrix consisted of 616 base pairs, and the overall nucleo- edited with Chromas Lite v.2.1.1 (Technelysium Pty. Ltd, tide composition was A 36.5 %, T 42.2 %, C 5.3 %, = = = Helensvale, Australia). Sequence alignment was performed and G 16 %. = 1 3 282 Fish Sci (2016) 82:279–288

Fig. 1 Sliding window analysis showing nucleotide diversity in the alignment of the full- length 16S rRNA gene from 28 cephalopod species. The relative positions of the novel degener- ate primer set (CephaF and CephaR) and universal primer set of the 3′ end (16Sar and 16Sbr) are shown

Fig. 2 Primer developed to amplify the fragment of the 5′ end of the 16S rRNA gene. Degenerated nucleotides and the added tail are indicated in grey

Neighbor-joining tree topology depicted highly sup- of 5.0 %, with little (0.2 %) or no variation within clades. ported monophyletic clusters that are consistent with the Furthermore, S. pharaonis was represented by 4 haplotypes morphological classification (Fig. 3). S. oualaniensis was sorted into 2 clades with a supported value of 98 % (Fig. 3) represented by 7 haplotypes sorted into 2 clades with a sup- and divergence of 7.0 %, with little (0.1 %) or no variation ported value of 99 % (Fig. 3) and divergence of 9.0 %, with within clades. little (0.01 %) or no variation within clades. Loliolus japon- Other than the possible cryptic speciation in the mentioned ica was represented by 3 haplotypes separated into 2 clades taxa, we did not observe any overlapping between the intra- with bootstrap support of 100 % (Fig. 3) and divergence and interspecific distance at any other genus level (Table 1).

1 3 Fish Sci (2016) 82:279–288 283

Fig. 3 Neighbor-joining tree of 27 species. Haplotypes codes are listed in Online Resource 1. Numbers at nodes indicate topo- logical support in the form of bootstrap percentage values

1 3 284 Fish Sci (2016) 82:279–288

Table 1 Kimura two-parameter Level of comparison Taxa Intraspecific Interspecific distance of haplotypes at the genus level Min. (%) Max. (%) Mean (%) Min. (%) Max. (%) Mean (%)

Genus Nautilus 2 – – – – – 10.6 Sepioteuthis 3 – – 0.1 7.8 15.4 12.2 Uroteuthis 2 – – – – – 19.3 Sthenoteuthis* 2 – – 0.9 – – 9.0 Lolioulus* 3 0.2 0.2 0.2 5.0 11.7 9.3 Sepia* 6 0.0 0.6 0.26 7.0 33.5 23.4

Taxa with only one species were excluded * Intra- and interspecific divergence values adjusted for the possible cryptic species in S. oualaniensis, L. japonica and S. pharaonis

Fig. 4 Neighbor-joining tree for the identification of four individuals of Sepioteuthis spp. and one industrial food product (grilled squid)

The immature individuals classified as Sepioteuthis proposed showed higher variability compared with the 3′ spp. (individuals 1–4 and accession numbers LC063342– end of the 16S rRNA (Fig. 1). Dai et al. [10] reported mean LC063345) formed one cluster with Sepioteuthis sp. 2 intra- and interspecific values of 0.2 and 16.2 % based on with high bootstrap support of 100 % and no variation at the standard COI as well as 0.1–7.3 % based on the stand- the intraspecific level. The grilled squid (accession num- ard of the 16S rRNA, respectively, for the family Sepiidae, ber LC063346) was grouped within the T. pacificus cluster whereas the region being proposed only at the genus level with high bootstrap support of 100 % and little variation (Sepia) presented, on average, somewhat similar intraspe- (0.2 %) within the cluster (Fig. 4). cific values, but about 1.4- and 3.2-fold higher values than those reported with the standard DNA barcode markers, respectively. The higher variability of the 5′ end compared Discussion with the 3′ end region of the 16S rRNA was already con- firmed for scallops (family Pectinidae) [25]. These results This study confirms the efficiency of the′ 5 end of the provide some intuition that the genetic information from 16S rRNA gene as a DNA barcode marker for cephalo- the region being proposed in this study can be, in some pods. Based on sliding window analysis of the cephalopod cases, similar and, in other cases, higher than that based on sequences retrieved from the GenBank, the region being the standard COI, but most probably higher than that based

1 3 Fish Sci (2016) 82:279–288 285 on the 3′ end of the 16S rRNA. This intuition would be At the intraspecific level, sequences of the four individu- worth verifying with the incorporation of the region being als classified as Sepioteuthis sp. 1 showed tandem adenine proposed into further DNA barcoding studies. and thymine (AT) variation. This species is the only mem- The main disadvantage during the evaluation of the ber of the S. lessoniana species complex from Japan with region being proposed was the variable annealing tempera- such polymorphism and, except for one nucleotide indel ture used for PCR, that ranged from 50 to 58 °C even at the in the sequence of one individual, tandem repetitions is intraspecific level, probably due to the degeneracy of the the cause of their variation in amplicon size, which ranged primer set proposed [39]. Nevertheless, future analysis with from 476 to 487 base pairs. In future analysis, increasing a taxon-specific and non-degenerative primer sets could the number of individuals and sampling in different loca- probably improve the PCR performance. In addition, it is tions would be useful for understanding the relevance of unlikely that PCR failed to amplify E. dofleini as a result of this polymorphism in this species. On the other hand, at primer mismatching, because the amplification succeeded the interspecific level, the region being proposed was able across other widely divergent species. to detect possible cryptic speciation in three species: S. The main concern about the use of rRNA genes for bar- oualaniensis, L. japonica, and S. pharaonis, due to the high coding or phylogenetic analysis is the difficulty of aligning supported clades within each species and the high level of highly variable sequence regions from different taxa that divergences between the representative haplotypes. leads to ambiguously aligned nucleotide sites as a result of The purpleback flying squid S. oualaniensis is an oegop- the insertions or deletions (indels) [40–42]. In such analy- sid species of the family Ommastrephidae that has multiple sis, sequences of closely related species are easily aligned, intraspecific forms [48] and inhabits areas from approxi- whereas the opposite occurs when sequences of more distant mately 40°N to 40°S latitude in the tropical and subtropical species are evaluated [43]. Therefore, a correct alignment is waters of the Indo–Pacific region [49, 50]. Staaf et al. [13], critical to compare homologous sites. In this study, a multi- based on sequences of mtDNA ND2 and samples of this alignment with MAFFT v.7 of our total sequences led to the species collected in the central and eastern Pacific Ocean, appropriate number of haplotypes collapsed in DnaSP v.5.10 reported the presence of four well-supported genetic clades whereas the multi-alignment with other bioinformatics pack- named as: Equatorial, Eastern Typical, Central Typical and ages such as ClustalW [44] and Muscle [45] provided inac- Pacific Typical. Between the mentioned clades, the Equato- curate results (data not shown). Furthermore, our alignment rial clade represented the most divergent clade (14 %) com- with MAFFT v.7 resulted in high bootstrap values in accord- pared with the others. According to our BLAST queries, ance with the morphologically classified individuals, thereby haplotypes from S. oualaniensis clade 1 showed 98–99 % indicating good alignment performance among highly vari- identity with a sequence of the Pacific Typical clade (acces- able sites. However, it is worth mentioning that the difficulty sion number EU660576.1), whereas the haplotype from of multi-alignment in this study is only due to the analysis clade 2 showed 98 % identity with a sequence of the Equa- of widely divergent species. Thus, analysis of less divergent torial clade (accession number EU660577.1). These results species may have no multi-alignment issues with any of the clearly indicate that the region being proposed in this study mentioned alignment programs. This may represent a disad- successfully recovered a probable cryptic speciation in S. vantage of the region being proposed when the sequences of oualaniensis but in other geographical latitudes than the unknown specimens or only small piece of tissue (without above-mentioned study based on the mtDNA ND2. additional information) are going to be aligned. Within the genus Loliolus (family loliginidae), four dif- Slight length variation in the sequences from the region ferent species has been identified: L. japonica, L. uyii, L. being proposed was found among the analyzed species, sumatrensis, and L. beka [51]. These species inhabit the probably due to its non-coding characteristic. Lydeard et al. Indo–Pacific Ocean [52]. The lack of genetic information [46] reported a low guanine and cytosine (GC) content in GenBank databases did not allowed clear classification (25.5 %) of the 16S rRNA gene in Loligo bleekeri compared of the two clades presented in L. japonica. However, Loli- to other molluscs, which is close to the value obtained in this olus sp. 1 showed a divergence of 10.8 % from L. japon- study (21.3 % average GC content in cephalopods). Further- ica clade 1 and 11.11 % from clade 2. Surprisingly; the more, among the cephalopod taxa analyzed, the nautiloid divergence between both clades in L. japonica was 5.0 %, taxa clearly presented a higher average GC content (27.3 %) around half of the divergence value with Loliolus sp. 1, but compared with coleoids (20.7 %). This result may indicate a higher than the intraspecific values reported for the other possible relationship of GC content with divergence between species analyzed in this study. Although determining a these two subclasses. Future studies that include more spe- clear morphological description for the specimens belong- cies are needed to investigate whether this relationship is ing to these two clades is beyond the scope of this study, also observed in closely related species, as has been reported it is worth mentioning that Vecchione et al. [51] recom- among marine molluscs based on COI sequences [47]. mended examining the variability of certain morphological

1 3 286 Fish Sci (2016) 82:279–288 characteristics in L. japonica to distinguish new species, In this study, we demonstrated that the 5′ end region of whereas Dai et al. [10] reported similar findings for L. beka the 16S rRNA gene can be an appropriate DNA barcode (two clades) based on the standard COI and the 3′ region marker for cephalopods to complement the mentioned of the 16S rRNA DNA markers in samples collected from standard mitochondrial DNA markers. In addition, it is Chinese waters, suggesting the occurrence of cryptic spe- worth mentioning that the standard DNA barcode markers cies. Further studies with additional DNA barcoding mark- mentioned here already have large sequence databases and ers and more taxonomic scrutiny are necessary to confirm the region being proposed in this study seeks only to pro- these results. vide complementary molecular information for improving The pharaoh cuttlefish, S. pharaonis, is a neritic demer- the robustness of species-level identifications. sal squid widely distributed from East Africa to southern Japan [11]. Based on the standard mitochondrial (COI Acknowledgments This research was partly supported by “Grants- and the 3′ end of the 16S rRNA) and nuclear (rhodop- in-Aid for Scientific Research” (no. 26292106 for T.U.) and a “Grad- uate Student Scholarship for Foreign Student”, both from the Japa- sin) partial gene sequences, Anderson et al. [11] sug- nese Government through the Ministry of Education, Culture, Sport, gested the presence of up to five clades in this species: Science and Technology in Japan (Monbukagakusho: MEXT). We are Central Indian Ocean, Iranian, Northeastern Australia, also grateful to the following professors who kindly provided samples Western Indian Ocean, and Western Pacific. The sam- for this study: Dr. Taeko Miyazaki of Mie University with Sepioteu- this sp. 3, Dr. Ian G. Gleadall of Tohoku University with Opisthoteu- pling area of individuals belonging to the Western Pacific this sp. and Dr. John Bower of Hokkaido University with E. dofleini. clade was limited to the Gulf of Thailand and Taiwan. Dai We wish to thank Miyajima Aquarium (Hiroshima Prefecture) for et al. [10], based on the standard 3′ end region of the 16S providing samples of A. dux and Toba Aquarium (Mie Prefecture) rRNA, reported that samples collected in the South China for the samples of Nautilus macromphalus and Nautilus pompilius. We also wish to thank to Li Qiang and Dr. Zhizhi Liu from Shang- Sea belonged to the mentioned Western Pacific clade. hai Ocean University for providing samples of Uroteuthis chinensis, Our BLAST queries successfully classified clade 1 with Atsushi Tsuyuki from Hiroshima University for his support during 99 % identity to a sequence reported from Japan (acces- the sample collection in Japan, Dr. Lawrence Liao from Hiroshima sion number AP013076.1) [53]; however, specimens from University and his student Dan Anthony Uy Bataan from the Philip- pines for the great help with the collection of samples in their coun- the Philippines shared only 92 % identity with the same try. Finally, the authors gratefully acknowledge the criticism of two individual. Besides, both Japan and the Philippines sam- anonymous reviewers who greatly improved the manuscript. ples analyzed here shared 89 % identity with a sequence reported from the East China Sea (Zhoushan; accession number KC632521.1) [54] which shared 96 % identity References with the sequence reported by Dai et al. [10] (accession 1. Allcock AL, Lindgren A, Strugnell JM (2014) The contribution number JN315880.1). Thereby, our findings may indicate of molecular data to our understanding of cephalopod evolution that samples from Japan and the Philippines are not part and systematics: a review. J Nat Hist 49:1–49 of the Western Pacific clade but two new clades, prob- 2. Caddy JF (1983) Advances in assessment of world cephalopod ably because of the neritic characteristics of S. pharaonis resources. FAO Fisheries Technical Paper, vol 231. FAO, Rome 3. Roper CFE, Sweeney MJ, Nauen CE (1984) Cephalopods of the that may prevent this species to cross open oceans and the world: an annotated and illustrated catalogue of species of inter- Kuroshio current acting as a barrier between individuals est to fisheries. FAO Species Catalogue for Fishery Purpose, vol of this study and the ones from the previously mentioned 4. FAO, Rome Western pacific clade. A future study evaluating together 4. Zlatanos S, Laskaridis K, Feist C, Sagredos A (2006) Proxi- mate composition, fatty acid analysis and protein digestibility- all of the previously analyzed genes would help elucidate corrected amino acid score of three Mediterranean cephalopods. the findings in this species. Mol Nutr Food Res 50:967–970 In addition, we successfully identified specimens of 5. Iglesias J, Fuentes L, Villanueva R (2014) Cephalopod culture. Sepioteuthis spp. (individuals 1–4) collected in Okinawa Springer, Dordrecht 6. Galimberti A, De Mattia F, Losa A, Bruni LL, Federici S, Casir- Island as Sepioteuthis sp. 2, which may support the fact aghi M, Martellos S, Labra L (2013) DNA barcoding as a new that the region being proposed is not problematic when tool for food traceability. Food Res Int 50:55–63 different geographical locations are considered for species 7. Boyle P, Rodhouse P (2006) Cephalopods: ecology and fisheries. identification. Moreover, the correct classification of the Blackwell, Oxford 8. Boyle PR (1990) Cephalopod biology in the fisheries context. processed industrial food (grilled squid) within one highly Fish Res 8:303–321 supported cluster of T. pacificus indicates the potential util- 9. McKeown NJ, Robin J-P, Shaw PW (2015) Species-specific ity of the region being proposed for food traceability, where PCR–RFLP for identification of early life history stages of shorter amplicons have the best performance because of squid and other applications to fisheries research. Fish Res 167:207–209 DNA degradation in processed industrial samples. How- 10. Dai L, Zheng X, Kong L, Li Q (2012) DNA barcoding analy- ever, we encourage testing this region in a wider variety of sis of Coleoidea (: Cephalopoda) from Chinese waters. industrial food products to validate our current results. Mol Ecol Resour 12:437–447

1 3 Fish Sci (2016) 82:279–288 287

11. Anderson FE, Engelke R, Jarrett K, Valinassab T, Mohamed KS, 28. Yoshida M, Tsuneki K, Furuya H (2006) Phylogeny of selected Asokan PK, Zacharia PU, Nootmorn P, Chotiyaputta C, Dunning Sepiidae (Mollusca, Cephalopoda) based on 12S, 16S, and COI M (2010) Phylogeny of the Sepia Pharaonis species complex sequences, with comments on the taxonomic reliability of sev- (cephalopoda: Sepiida) based on analyses of mitochondrial and eral morphological characters. Zool Sci 23:341–351 nuclear DNA sequence data. J Molluscan Stud 77:65–75 29. Jereb P, Roper C (2010) Cephalopods of the world. An annotated 12. Cheng SH, Anderson FE, Bergman A, Mahardika GN, Much- and illustrated catalogue of cephalopod species known to date, lisin ZA, Dang TB, Calumpong HP, Mohamed KS, Sasikumar vol 2. Myopsid and Oegopsid Squids. FAO Species Catalogue G, Venkatesan V, Barber PH (2013) Molecular evidence for co- for Fishery Purpose. No. 4. FAO, Rome occurring cryptic lineages within the Sepioteuthis cf. lessoniana 30. Jereb P, Roper CFE, Norman MD, Julian KF (2014) Cepha- species complex in the Indian and Indo-West Pacific Oceans. lopods of the world. An annotated and illustrated catalogue of Hydrobiologia 725:165–188 cephalopod species known to date, vol 3. Octopods and Vam- 13. Staaf DJ, Ruiz-Cooley RI, Elliger C, Lebaric Z, Campos B, pire Squids. FAO Species Catalogue for Fishery Purpose. No. 4. Markaida U, Gilly WF (2010) Ommastrephid squids Sthenoteu- FAO, Rome this oualaniensis and Dosidicus gigas in the eastern Pacific show 31. Tomano S, Ueta Y, Kasaoka N, Umino T (2015) Stock identi- convergent biogeographic breaks but contrasting population fication and spawning depth of oval squid Sepioteuthis spp. in structures. Mar Ecol Prog Ser 418:165–178 Tanega-shima Island inferred by DNA markers. Acuacult Sci 14. Hebert PDN, Ratnasingham S, de Waard JR (2003) Barcod- 63:39–47 (In Japanese with English abstract) ing animal life: cytochrome c oxidase subunit 1 divergences 32. Izuka T, Numachi K, Segawa S, Okutani T (1994) Evidence among closely related species. Proc R Soc Lond B Biol Sci on the existence of three species in the oval squid, Sepioteu- 270:S96–S99 this lessoniana complex in Ishigaki Island, Okinawa, south- 15. Hebert PDN, Cywinska A, Ball SL, deWaard JR (2003) Bio- western Japan, by isozyme analyses. Jpn J Malacol Venus logical identifications through DNA barcodes. Proc Biol Sci 53:217–228 270:313–321 33. Katoh K, Standley DM (2013) MAFFT multiple sequence align- 16. Allcock AL, Barratt I, Eleaume M, Linse K, Norman MD, Smith ment software version 7: improvements in performance and usa- PJ, Steinke D, Stevens DW, Strugnell JM (2011) Cryptic spe- bility. Mol Biol Evol 30:772–780 ciation and the circumpolarity debate: a case study on endemic 34. Librado P, Rozas J (2009) DnaSP v5: a software for compre- Southern Ocean octopuses using the COI barcode of life. Deep hensive analysis of DNA polymorphism data. Bioinformatics Sea Res Part II Top Stud Oceanogr 58:242–249 25:1451–1452 17. Kaneko N, Kubodera T, Iguchis A (2011) Taxonomic study of 35. Asahida T, Kobayashi T, Saitoh K, Nakayama I (1996) Tissue shallow-water octopuses (Cephalopoda: Octopodidae) in Japan preservation and total DNA extraction form fish stored at ambi- and adjacent waters using mitochondrial genes with perspectives ent temperature using buffers containing high concentration of on octopus DNA barcoding. Malacologia 54:97–108 urea. Fish Sci 62:727–730 18. Williams RC, Newman SJ, Sinclair W (2012) DNA barcoding 36. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) in Nautilus pompilius (Mollusca: Cephalopoda): evolutionary MEGA6: molecular evolutionary genetics analysis version 6.0. divergence of an ancient species in modern times. Invertebr Syst Mol Biol Evol 30:2725–2729 26:548–560 37. Kimura M (1980) A simple method for estimating evolutionary 19. Wakabayashi T, Suzuki N, Sakai M, Ichii T, Chow S (2006) Iden- rates of base substitutions through comparative studies of nucle- tification of ommastrephid squid paralarvae collected in northern otide sequences. J Mol Evol 16:111–120 Hawaiian waters and phylogenetic implications for the family 38. Saitou N, Nei M (1987) The neighbor-joining method: a new Ommastrephidae using mtDNA analysis. Fish Sci 72:494–502 method for reconstructing phylogenetic trees. Mol Biol Evol 20. Taylor AL, McKeown NJ, Shaw PW (2012) Molecular identi- 4:406–425 fication of three co-occurring and easily misidentified octopus 39. Iserte JA, Stephan BI, Goñi SE, Borio CS, Ghiringhelli PD, species using PCR–RFLP techniques. Conserv Genet Resour Lozano ME (2013) Family-specific degenerate primer design: a 4:885–887 tool to design consensus degenerated oligonucleotides. Biotech- 21. Strugnell JM, Lindgren AR (2007) A barcode of life database for nol Res Int 2013:383646. doi:10.1155/2013/383646 the Cephalopoda? considerations and concerns. Rev Fish Biol 40. Lutzoni F, Wagner P, Reeb V, Zoller S (2000) Integrating ambig- Fish 17:337–344 uously aligned regions of DNA sequences in phylogenetic analy- 22. Cheng R, Zheng X, Ma Y, Li Q (2013) The complete mitochon- ses without violating positional homology. Syst Biol 49:628–651 drial genomes of two octopods Cistopus chinensis and Cistopus 41. Noé L, Kucherov G (2004) Improved hit criteria for DNA local taiwanicus: revealing the phylogenetic position of the genus Cis- alignment. BMC Bioinform 5:149 topus within the order Octopoda. PLoS ONE 8:e84216 42. Chu KH, Li CP, Qi J (2006) Ribosomal RNA as molecular bar- 23. Chapela MJ, Sotelo CG, Calo-Mata P, Perez-Martin RI, Rehbein codes: a simple correlation analysis without sequence alignment. H, Hold GL, Quinteiro J, Rey-Mendez M, Rosa C, Santos AT Bioinformatics 22:1690–1701 (2002) Identification of cephalopod species (Ommastrephidae 43. Lemey P, Salemi M, Vandamme A-M (2009) The Phyloge- and Loliginidae) in seafood products by Forensically Informative netic Handbook: a practical approach to phylogenetic analysis Nucleotide Sequencing (FINS). J Food Sci 67:1672–1676 and hypothesis testing, 2nd edn. Cambridge University Press, 24. Palumbi S, Martin A, Romano S, Mcmillan WO, Stice L, Cambridge Grabowski G (1991) The simple fool’s guide to PCR, version 20. 44. Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: University of Hawaii Press, Honolulu improving the sensitivity of progressive multiple sequence align- 25. Marín A, Fujimoto T, Arai K (2014) The variable 5′ end of the ment through sequence weighting, position-specific gap penal- 16S rRNA gene as a novel barcoding tool for scallops (Bivalvia, ties and weight matrix choice. Nucleic Acids Res 22:4673–4680 Pectinidae). Fish Sci 81:73–81 45. Edgar RC (2004) MUSCLE: multiple sequence alignment 26. Tautz D, Arctander P, Minelli A, Thomas RH, Vogler AP (2003) with high accuracy and high throughput. Nucleic Acids Res A plea for DNA . Trends Ecol Evol 18:70–74 32:1792–1797 27. Okutani T (2015) Cuttlefish and squid of the world. Seizando- 46. Lydeard C, Holznagel WE, Schnare MN, Gutell RR (2000) Shoten Publishing, Tokyo Phylogenetic analysis of molluscan mitochondrial LSU rDNA

1 3 288 Fish Sci (2016) 82:279–288

sequences and secondary structures. Mol Phylogenet Evol Jorgensen E, Sukramongkol N (2005) Systematics of Indo-West 15:83–102 Pacific Loliginids. Phuket Mar Biol Cent Res Bull 66:23–26 47. Layton KKS, Martel AL, Hebert PD (2014) Patterns of DNA 52. Vecchione M, Brakoniecki TF, Natsukari Y, Hanlon RT (1998) barcode variation in Canadian marine molluscs. PLoS ONE A provisional generic classification of the family loliginidae. 9:e95003 Smithson Contrib Zool 586:215–222 48. Nesis KN (1993) Population structure of oceanic ommastre- 53. Kawashima Y, Nishihara H, Akasaki T, Nikaido M, Tsuchiya phids, with particular reference to Sthenoteuthis oualaniensis: K, Segawa S, Okada N (2013) The complete mitochondrial a review. In: Okutani T, O’Dor RK, Kubodera T (eds) Recent genomes of deep-sea squid (Bathyteuthis abyssicola), bob-tail advances in cephalopod fishery biology. Tokai University Press, squid ( patagonica) and four giant cuttlefish (Sepia Tokyo, pp 375–383 apama, S. latimanus, S. lycidas and S. pharaonis), and their 49. Xinjun C, Bilin L, Siquan T, Weiguo Q, Xiaohu Z (2007) Fishery application to the phylogenetic analysis of Decapodiformes. Mol biology of purpleback squid, Sthenoteuthis oualaniensis, in the Phylogenet Evol 69:980–993 northwest Indian Ocean. Fish Res 83:98–104 54. Wang W, Guo B, Li J, Qi P, Wu C (2014) Complete mitochon- 50. Fang Z, Xu L, Chen X, Liu B, Li J, Chen Y (2015) Beak growth drial genome of the common cuttlefishSepia pharaonis (Sepi- pattern of purpleback flying squid Sthenoteuthis oualanien- oidea, Sepiidae). Mitochondrial DNA 25:198–199 sis in the eastern tropical Pacific equatorial waters. Fish Sci 81:443–452 51. Vecchione M, Shea E, Bussarawit S, Anderson F, Alexeyev D, Lu C-C, Okutani T, Roeleveld M, Chotiyaputta C, Roper C,

1 3