INTRODUCTIONTop

Neon flying squid, Ommastrephes bartramii, is widely distributed around the world’s oceans extending from the subtropics to temperate waters in the northern and southern hemispheres except in equatorial waters (Rodhouse 2005Rodhouse P.G. 2005. World squid resources. Review of the state of world marine fishery resources, FAO Tech. Rep. C2:175., Lefkaditou et al. 2011Lefkaditou E., Peristeraki P., Chartosia N., et al. 2011. Recent findings of Ommastrephes bartramii (Cephalopoda: Ommastrephidae) in the eastern Mediterranean and the implication on its range expansion. Mediterr. Mar. Sci. 12(2): 413-428.). Despite its huge potential abundance, it only supports a commercial fishery in the northwest Pacific Ocean. The exploitation of this epipelagic species was started in 1974 (Chen et al. 2009Chen X.J., Liu B.L., Wang Y.G. 2009. Cephalopod of the world. Marine Press, Beijing, China, 1064 pp.), with roughly 200000 to 300000 t landed during the 1980s by Japan, Korea and Taiwan (Burke et al. 1993Burke W.T., Freeberg M., Miles E.L. 1993. United Nations Resolutions on driftnet fishing: An unsustainable precedent for high seas and coastal fisheries management. Ocean Dev. Int. Law. 25: 127-186.). Mainland China started fishing this squid in 1993, and mainly fished in the western waters 160° east of the North Pacific. Recently, commercial squid jigging vessels of mainland China have mainly fished in the areas 150°E to 170°W and 40° to 46°N from May to October with annual catches of between 50000 and 100000 t.

The population structure of O. bartramii has been studied by many researchers for putative seasonal cohorts (Murata 1990Murata M. 1990. Oceanic resources of squids. Mar. Behav. Phys. 18: 19-71., Murata and Hayase 1993Murata M., Hayase S. 1993. Life history and biological information on flying squid (Ommastrephes bartramii) in the North Pacific Ocean. Bull. Int. Nat. North Pacific Comm. 53: l47-182.), rates of infection by helminth parasites (Bower and Margolis 1991Bower S.M., Margolis L. 1991. Potential use of helminth parasites in stock identification of flying squid, Ommastrephes bartramii, in North Pacific waters. Can. J. Zool. 69(4): 1124-1126., Nagasawa et al. 1998Nagasawa K., Mori J., Okamura H. 1998. Parasites as biological tags of stocks of neon flying squid (Ommastrephes bartramii) in the North Pacific Ocean, In: Okutani, T. (1998), Contributed papers to International Symposium on Large Pelagic Squids, July 18-19, 1996, for JAMARC’s 25th anniversary of its foundation, pp. 49-64. ) and mantle length distribution (Murata 1990Murata M. 1990. Oceanic resources of squids. Mar. Behav. Phys. 18: 19-71., Yatsu et al. 1998aYatsu A., Tanaka H., Mori J. 1998a. Population structure of the neon flying squid, Ommastrephes bartramii, in the North Pacific. In: Okutani T. (ed.), Contributed Papers to International Symposium on Large Pelagic Squids. Japan Marine Fishery Resources Research Center, Tokyo, pp. 31-48. ). Murakami et al. (1981)Murakami K., Watanabe Y., Nakata J. 1981. Growth, distribution and migration of flying squid (Ommastrephes bartrami) in the North Pacific. In: Mishima, S. (ed.), Pelagic animals and environments around the Subarctic Boundary in North Pacific. Hokkaido University, Research Institute of North Pacific Fisheries, Hakodate, pp. 161-179 (In Japanese with English abstract) identified the four stocks based on the squid body sizes extra-large (LL), large (L), small (S) and extra-small (SS). Chen et al. (2002)Chen X.J., Tian S.Q., Ye X.C. 2002. Study on population structure of flying squid in Northwestern Pacific based on gray system theory. J. Shanghai Fish. Univ. 11(4): 335-341. (In Chinese with English Abstract) found that two populations existed in the waters of 165°E westward through nine body variables based on the Grey System Theory. A geographic pattern of population genetic variability was observed in O. bartramii, with major genetic differentiation attributable to inconsistency in allele frequency distribution and in levels of genetic variation between the squid from the western and eastern parts of the species, which covers a wide area in the North Pacific Ocean (Katugin 2002Katugin O.N. 2002. Patterns of genetic variability and population structure in the North Pacific squids Ommastrephes bartramii, Todarodes pacificus and Berryteuthis magister. Bull. Mar. Sci. 71(1): 383-420.). Generally, in the North Pacific, the population of O. bartramii mainly comprises two cohorts: a) the autumn cohort hatching from September to February and b) the winter-spring cohort hatching from January to May (Katugin 2002Katugin O.N. 2002. Patterns of genetic variability and population structure in the North Pacific squids Ommastrephes bartramii, Todarodes pacificus and Berryteuthis magister. Bull. Mar. Sci. 71(1): 383-420., Chen and Chiu 2003Chen C.S., Chiu T.S. 2003. Variations of life history parameters in two geographical groups of the neon flying squid, Ommastrephes bartramii, from the North Pacific. Fish. Res. 63: 349-366., Ichii et al. 2004Ichii T., Mahapatra K., Sakai M., et al. 2004. Differing body size between the autumn and the winter–spring cohorts of neon flying squid (Ommastrephes bartramii) related to the oceanographic regime in the North Pacific: a hypothesis. Fish. Oceanogr. 13: 295-309, Bower and Ichii 2005Bower J.R., Ichii T. 2005. The red flying squid (Ommastrephes bartramii): A review of recent research and the fishery in Japan. Fish. Res. 76(1): 39-55., Chen et al. 2011Chen F., Chen X.J., Lu H.J., et al. 2011. Comparison of biological characteristics of Ommastrephes bartarmii between two different areas in the east central waters of North Pacific Ocean. J. Shanghai Fish. Univ. 20(5): 759-764. (In Chinese with English Abstract)). The autumn cohort consists of the central stock and east stock, which separate near 160°W. The winter-spring cohort also comprises the west stock and central-east stock, which separate near 170°E (Ichii et al. 2004Ichii T., Mahapatra K., Sakai M., et al. 2004. Differing body size between the autumn and the winter–spring cohorts of neon flying squid (Ommastrephes bartramii) related to the oceanographic regime in the North Pacific: a hypothesis. Fish. Oceanogr. 13: 295-309, Bower and Ichii 2005Bower J.R., Ichii T. 2005. The red flying squid (Ommastrephes bartramii): A review of recent research and the fishery in Japan. Fish. Res. 76(1): 39-55.). Although these two cohorts overlap geographically, they have been caught in different areas at separate times (Bower and Ichii 2005Bower J.R., Ichii T. 2005. The red flying squid (Ommastrephes bartramii): A review of recent research and the fishery in Japan. Fish. Res. 76(1): 39-55.). The autumn cohort (NE stock) distributed in the waters east of 170°E from May to June and the winter-spring cohort (NW stock) distributed in the waters west of 165°E from July to November have been the main fishing target for Chinese squid jigging fleets since 1998 (Chen and Chiu 2003Chen C.S., Chiu T.S. 2003. Variations of life history parameters in two geographical groups of the neon flying squid, Ommastrephes bartramii, from the North Pacific. Fish. Res. 63: 349-366., Wang and Chen 2005Wang Y.G., Chen X.J. 2005. The Resource and Biology of Economic Oceanic Squid in the World. Ocean Press, Beijing, pp. 79-295. ).

Stock identification is critical for an effective fishery management to avoid overfishing and promote the sustainable development of fisheries (Cadrin and Silva 2005Cadrin S.X., Silva V.M. 2005. Morphometric variation of yellowtail flounder. ICES J. Mar. Sci. 62: 683-694.). Stock structures are often identified and verified on the basis of their different life history strategies and genetic structures. However, morphometric traits are still often used in this field (Sajina et al. 2011Sajina A.M., Chakraborty S.K., Jaiswar A.K., et al. 2011. Stock structure analysis of Megalaspis cordyla (Linnaeus, 1758) along the Indian coast based on truss network analysis. Fish. Res. 108: 100-105). Traditional measurements are based on the conventional orthogonal method, which uses length and width to describe the variables for species with rigid body forms. Unlike fish and many crustaceans, squid have a flexible soft body without a hard surface structure. The cylindrical mantle cavity varies during locomotion and respiration, and the stretched arms and tentacles are also broken easily, often by hooks during the jigging capture process (Cabanellas-Reboredo et al. 2011Cabanellas-Reboredo M., Alós J., Palmer M., et al. 2011. Simulating the indirect handline jigging effects on the European squid (Loligo vulgaris) in captivity. Fish. Res. 110(3): 435-440., Kurosaka et al. 2012Kurosaka K., Yamashita H., Ogawa M., et al. 2012. Tentacle-breakage mechanism for the neon flying squid (Ommastrephes bartramii) during the jigging capture process. Fish. Res. 121: 9-16.). Thus, measurements based on the soft parts of squid are challenging and frequently contain errors, and an alternative structure should be used to separate stocks on the basis of body morphology. However, these measurements are fairly reliable if the approach is correct, as in the studies of Loliginidae by Pierce et al. (1994)Pierce G. J., Hastie L. C., Guerra A., et al. 1994. Morphometric variation in Loligo forbesi and Loligo vulgaris: regional, seasonal, sex, maturity and worker differences. Fish. Res. 21(1): 127-148. and of Ommastrephidae by Martínez et al. (2002)Martínez P., Sanjuan A., Guerra A. 2002. Identification of Illex coindetii, I. illecebrosus and I. argentines (Cephalopoda: Ommastrephidae) throughout the Atlantic Ocean by body and beak characters. Mar. Biol. 141: 131-143..

Hard structures, including the statolith, beak (mandibular) and gladius (pen), which contain a series of ecological information during its mysterious life history, have gradually been used for their stable and constant configuration (Bizikov and Arkhipkin 1997Bizikov V. A., Arkhipkin A. I. 1997. Morphology and microstructure of the gladius and statolith from the boreal Pacific giant squid Moroteuthis robusta (Oegopsida; Onychoteuthidae). J. Zool. 241(3): 475-492., Piatkowski et al. 2001Piatkowski U., Pütz K., Heinemann H. 2001. Cephalopod prey of king penguins (Aptenodytes patagonicus) breeding at Volunteer Beach, Falkland Islands, during austral winter 1996. Fish. Res. 52(1): 79-90., Ikeda et al.2003Ikeda Y., Arai N., Kidokoro H., et al. 2003. Strontium: calcium ratios in statoliths of Japanese common squid Todarodes pacificus (Cephalopoda: Ommastrephidae) as indicators of migratory behavior. Mar. Ecol. Prog. Ser. 251(1): 169-179., Jackson and Domeier 2003Jackson G.D., Domeier M.L. 2003. The effects of an extraordinary El Niño/La Niña event on the size and growth of the squid Loligo opalescens off Southern California. Mar. Biol. 142(5): 925-935., Guerra et al. 2010Guerra A., Rodríguez-Navarro A.B., González A.F., et al. 2010. Life-history traits of the giant squid Architeuthis dux revealed from stable isotope signatures recorded in beaks. ICES J. Mar. Sci. 67(7): 1425-1431., Ruiz-Cooley et al. 2013Ruiz-Cooley R.I., Ballance L.T., McCarthy M.D. 2013. Range Expansion of the Jumbo Squid in the NE Pacific: δ15N Decrypts Multiple Origins, Migration and Habitat Use. PloS One. 8(3): e59651.). As calcified structures embedded in cartilage, a pair of statoliths is an indispensable part of the acceleration receptor system that controls the movement and direction of the cephalopod (Hanlon and Messenger 1996Hanlon R.T., Messenger J.B. 1996. Cephalopod Behaviour. Cambridge University Press Cambridge, UK, 232 pp., Arkhipkin and Bizikov 2000Arkhipkin A.I., Bizikov V.A. 2000. Role of the statolith in functioning of the acceleration receptor system in squids and sepioids. J. Zool. 250(1): 31-55.). They are also used in studies of species identification (Clarke 1978Clarke M.R. 1978. The cephalopod statolith-introduction to its form. J. Mar. Biol. Ass. U.K. 58: 701-712., Dommergues et al. 2000Dommergues J.L., Neige P., Boletzky S.V. 2000. Exploration of morphospace using procrustes analysis in statoliths of cuttlefish and squid (Cephalopoda: Decabrachia)-evolutionary aspects of form disparity. Veliger-berkeley, 43(3): 265-276., Lombarte et al. 2006Lombarte A., Rufino M.M., Sánchez P. 2006. Statolith identification of Mediterranean Octopodidae, Sepiidae, Loliginidae, Ommastrephidae and Enoploteuthidae based on warp analyses. J. Mar. Biol. Ass. U. K. 86(04): 767-771.), age estimation (Villanueva 1992Villanueva R. 1992. Interannual growth differences in the oceanic squid Todarodes angolensis Adam in the northern Benguela upwelling system, based on statolith growth increment analysis. J. Exp. Mar. Biol. Ecol., 159(2): 157-177., Sánchez 1995Sánchez P. 1995. Age and growth of Illex coindetii. ICES J. Mar. Sci. 199: 441-444, Arkhipkin and Shcherbich 2012Arkhipkin A.I., Shcherbich Z.N. 2012. Thirty years’ progress in age determination of squid using statoliths. J. Mar. Biol. Ass. U. K. 92(06): 1389-1398.), growth pattern (Yatsu 2000Yatsu A. 2000. Age estimation of four oceanic squids, Ommastrephes bartramii, Dosidicus gigas, Sthenoteuthis oualaniensis, and Illex argentinus (Cephalopoda, Ommastrephidae) based on statolith Microstructure. Jpn. Agri. Res. Quart. 34: 75-80., Yatsu et al. 1997Yatsu A., Midorikawa S., Shimada T., et al. 1997. Age and growth of the neon flying squid, Ommastrephes bartrami, in the North Pacific Ocean. Fish. Res. 29: 257-270) and trace elements (such as strontium) (Durholtz et al. 1997Durholtz M.D., Lipinski M.R., Przybylowicz W.J., et al. 1997. Nuclear microprobe mapping of statoliths of chokka squid Loligo vulgaris reynaudii d’Orbigny, 1845. Biol. Bull. 193(2): 125-140., Ikeda et al. 1996Ikeda Y., Arai N., Sakamoto W., et al. 1996. PIXE analysis of trace elements in squid statoliths: comparison between Ommastrephidae and Loliginidae. International Journal of PIXE. 6: 537-542., 1997Ikeda Y., Arai N., Sakamoto W., et al. 1997. Comparison on trace elements in squid statoliths of different species’ origin as available key for taxonomic and phylogenetic study. International Journal of PIXE. 7: 141-146., Yatsu et al. 1998bYatsu A., Mochioka N., Morishita K., et al. 1998b. Strontium/calcium ratios in statoliths of the neon flying squid, Ommastrephes bartrami (Cephalopoda) in the North Pacific Ocean. Mar. Biol. 131: 275-282.) to determine life history and relevant environmental conditions. As the main feeding organ, the beak can be easily preserved, and is non-corroding, so it has been used in the studies of chemical structure (Miserez et al. 2010Miserez A., Rubin D., Waite J.H. 2010. Cross-linking Chemistry of Squid Beak. J. Biol. Chem. 285(49): 38115-38124.), aging and growth (Yatsu et al. 1997Yatsu A., Midorikawa S., Shimada T., et al. 1997. Age and growth of the neon flying squid, Ommastrephes bartrami, in the North Pacific Ocean. Fish. Res. 29: 257-270, Raya and Hernández-González 1998Raya C.P., Hernández-González C.L. 1998. Growth lines within the beak microstructure of the octopus Octopus vulgaris Cuvier, 1797. S. Afr. J. Mar. Sci. 20(1): 135-142., Yatsu and Mori 2000Yatsu A., Mori J. 2000. Early growth of the autumn cohort of neon flying squid, Ommastrephes bartramii, in the North Pacific Ocean. Fish. Res. 45: 189-194., Raya et al. 2010Raya C.P., Bartolomé A., García-Santamaría M.T., et al. 2010. Age estimation obtained from analysis of octopus (Octopus vulgaris Cuvier, 1797) beaks: Improvement and comparisons. Fish. Res. 106: 171-176, Castanhari and Tomás 2012Castanhari G., Tomás A.R.G. 2012. Beak increment counts as a tool for growth studies of the common octopus Octopus vulgaris in southern Brazil. Bol. Inst. Pesca, São Paulo 38(4): 323-331, Perales-Raya et al. 2014Perales-Raya C., Jurado-Ruzafa A., Bartolomé A., et al. 2014. Age of spent Octopus vulgaris and stress mark analysis using beaks of wild individuals. Hydrobiologia 725(1): 105-114.), species identification (Smale et al. 1993Smale M.J., Clarke M.R., Klages N.T.W., et al. 1993. Octopod beak identification—resolution at a regional level (Cephalopoda, Octopoda: Southern Africa). S. Afr. J. Mar. Sci. 13(1): 269-293.), biomass estimation (Lu and Ickeringill 2002Lu C.C., Ickeringill R. 2002. Cephalopod beak identification and biomass estimation techniques: tools for dietary studies of southern Australian finfishes. Mus. Victoria Sci. Rep. 6: 1-65.), trophic dynamics (Cherel and Hobson 2005Cherel Y., Hobson K.A. 2005. Stable isotopes, beaks and predators: a new tool to study the trophic ecology of cephalopods, including giant and colossal squids. Proc. R. Soc. Lond. B. 272: 1601-1607., Cherel et al. 2009Cherel Y., Ridoux V., Spitz J., et al. 2009. Stable isotopes document the trophic structure of a deep-sea cephalopod assemblage including giant octopod and giant squid. Biol. Lett. 5: 364-367.) and paralarval ontogeny (Uchikawa et al. 2009Uchikawa K., Sakai M., Wakabayashi T., et al. 2009. The relationship between paralarval feeding and morphological changes in the proboscis and beaks of the neon flying squid Ommastrephes bartramii. Fish. Sci. 75(2): 317-323.).

The measurement of hard structures has been the basis of these studies. Radial measurement is a simple way to analyse the relationship between statolith shape and growth pattern (Arkhipkin 2003Arkhipkin A.I. 2003. Towards identification of the ecological lifestyle in nektonic squid using statolith morphometry. J. Mollus. Stud. 69: 171-178., Ma et al. 2009Ma J., Chen X.J., Liu B.L., et al. 2009. Morphologic Features of Statolith for Ommastrephes bartramii in the Northwest Pacific Ocean. Periodical of Ocean University of China 39(2): 215-220. (In Chinese with English abstract), Chen et al. 2010Chen X.J., Ma J., Liu B.L., et al. 2010. Effects of sexual maturity and body size on statolith shape of Ommastrephes bartramii in the Northwest Pacific Ocean. J. Fish. China. 30(6): 928-934. (In Chinese with English abstract)). The beak also has specific characteristics and has been used in species identification (Clarke 1986Clarke M.R. 1986. A handbook for the identification of cephalopod beaks. Clarendon Press, Oxford, UK, 273 pp., Ogden et al. 1998Ogden R.S., Allcock A.L., Wats P.C., et al. 1998. The role of beak shape in octopodid taxonomy. S. Afr. J. Mar. Sci. 20(1): 29-36., Lu and Ickeringill 2002Lu C.C., Ickeringill R. 2002. Cephalopod beak identification and biomass estimation techniques: tools for dietary studies of southern Australian finfishes. Mus. Victoria Sci. Rep. 6: 1-65., Xavier and Cherel 2009Xavier J.C., Cherel Y. 2009. Cephalopod beak guide for the southern ocean. British Antarctic Survey Press, Cambridge, UK, 129 pp.). It has been proved that hard structures with a stable form can perform better than soft tissues (mantle, arm, tentacle, etc.) in squid population division (Martínez et al. 2002Martínez P., Sanjuan A., Guerra A. 2002. Identification of Illex coindetii, I. illecebrosus and I. argentines (Cephalopoda: Ommastrephidae) throughout the Atlantic Ocean by body and beak characters. Mar. Biol. 141: 131-143.). Some new methods (landmarks and outline study) have also revealed that hard structures give good results in identity analysis (Dommergues 2000Dommergues J.L., Neige P., Boletzky S.V. 2000. Exploration of morphospace using procrustes analysis in statoliths of cuttlefish and squid (Cephalopoda: Decabrachia)-evolutionary aspects of form disparity. Veliger-berkeley, 43(3): 265-276., Neige and Dommergues 2002Neige P., Dommergues, J.L. 2002. Disparity of beaks and statoliths of some coleoids a morphometric approach to depict shape differentiation. Gabh. der Geol. Bun. 57: 393-399, Lombarte et al. 2006Lombarte A., Rufino M.M., Sánchez P. 2006. Statolith identification of Mediterranean Octopodidae, Sepiidae, Loliginidae, Ommastrephidae and Enoploteuthidae based on warp analyses. J. Mar. Biol. Ass. U. K. 86(04): 767-771., Neige 2006Neige P. 2006. Morphometrics of hard structures in cuttlefish. Vie Milieu 56(2): 121-127., Crespi-Abril et al. 2010Crespi-Abril A.C., Morsan E.M., Barón P.J. 2010. Analysis of the ontogenetic variation in body and beak shape of the Illex argentinus inner shelf spawning groups by geometric morphometrics. J. Mar. Biol. Ass. U. K. 90(03): 547-553.). Some researchers may be cautious about using the beak as an identification material (Xavier et al. 2007Xavier J.C., Clarke M.R., Magalhães M.C., et al. 2007. Current status of using beaks to identify cephalopods: III International Workshop and training course on Cephalopod beaks, Faial Island, Azores. Arquipélago 24: 41-48.), but it is still important for stock identification (Xavier et al. 2011Xavier J.C., Phillips R.A., Cherel Y. 2011. Cephalopods in marine predator diet assessments: why identifying upper and lower beaks is important. ICES J. Mar. Sci. 68(9): 1857-1864.). Therefore, the phenotypic characteristics, such as statoliths and beaks, can play a supportive role for stock identification and investigation.

A morphological variation of cephalopods has occurred among the species due to different genetic structures and populations, induced mainly by oceanographic environmental factors (Adkison 1995Adkison M.D. 1995. Population differentiation in Pacific salmons: local adaptation genetic drift, or the environment? Can. J. Fish. Aquat. Sci. 52(12): 2762-2777.). Multivariate analyses of morphometric data have been used in some cephalopods at intraspecies (Martínez et al. 2002Martínez P., Sanjuan A., Guerra A. 2002. Identification of Illex coindetii, I. illecebrosus and I. argentines (Cephalopoda: Ommastrephidae) throughout the Atlantic Ocean by body and beak characters. Mar. Biol. 141: 131-143.) and interspecies (Wolff 1984Wolff G.A. 1984. Identification and estimation of size from the beaks of 18 species of cephalopods from the Pacific Ocean. NOAA Technical Report NMFS 17: 1-50) levels for the evaluation of taxonomic and geographic variations. Stocks inhabiting different environments can impact their growth patterns, including the shapes of statolith and beak. It is common that the separation of stocks is only based on one material and then compared with the results obtained from different materials (Martínez et al. 2002Martínez P., Sanjuan A., Guerra A. 2002. Identification of Illex coindetii, I. illecebrosus and I. argentines (Cephalopoda: Ommastrephidae) throughout the Atlantic Ocean by body and beak characters. Mar. Biol. 141: 131-143.), and the approach of integrating two materials in the stock separation is rarely used. In this study we integrated the morphological variables of statolith and beaks to compare their differences between stocks and between the sexes and to establish a discriminant function for two seasonal cohorts in the North Pacific Ocean. Our aims were to identify different cohorts of O. bartramii by hard structures (statolith and beaks). This integrated approach can also be used for other species when different body structures are available in the stock identifications.

MATERIALS AND METHODSTop

The survey was carried out in the waters of 150°E-177°W and 38°-44°N from May to October in 2010 to 2012 by the Chinese squid jigging vessel Jinhai 827. The samples were collected randomly from the daily catches and frozen on board immediately for future analysis. The sampling station is shown in Figure 1.

A total of 570 individuals were collected. The mantle length (ML) was measured to the nearest 0.1 mm after thaw in the laboratory, and the sex was identified by their entirely different gonad structure. The sexual maturity stages were determined according to Lipinski and Underhill (1995)Lipinski M. R., Underhill L. G. 1995. Sexual maturation in squid: quantum or continuum. S. Afr. J. Mar. Sci. 15: 207-223. Statoliths and beaks were dissected according to Raya et al. (2010)Raya C.P., Bartolomé A., García-Santamaría M.T., et al. 2010. Age estimation obtained from analysis of octopus (Octopus vulgaris Cuvier, 1797) beaks: Improvement and comparisons. Fish. Res. 106: 171-176 and Chen et al. (2013)Chen X.J., Li J.H., Liu B.L., et al. 2013. Age, growth and population structure of Jumbo flying squid, Dosidicus gigas, off the Costa Rica Dome. J. Mar. Biol. Ass. U. K. 93:567-573. As a result, a subsample of 406 pairs of statoliths and beaks were prepared for the analysis. A pair of beaks were precisely paired with a pair of statoliths which had been picked from the same squid sample (Table 1).

Photos were taken using a charge-coupled device (CCD, connecting device) for right statolith under a 50× optical microscope (Olympus) and nine parameters of statolith morphology, i.e. total statolith length (TSL), maximum width (MW), dorsal dome length (DDL), lateral dome length (LDL), dorsal lateral length (DLL), rostrum lateral length (RLL), rostrum length (RL), rostrum width (RW) and wing length (WL), were measured by using the image analysis software WT-Tiger3000 (Fig. 2A). The parameters of each statolith were measured to the nearest 0.01µm. Beaks were also measured by vernier caliper, which included upper hood length (UHL), upper crest length (UCL), upper rostrum length (URL), upper rostrum width (URW), upper lateral wall length (ULWL), upper wing length (UWL), lower hood length (LHL), lower crest length (LCL), lower rostrum length (LRL), lower rostrum width (LRW), lower lateral wall length (LLWL), and lower wing length (LWL) (Fig. 2B). All the data for beaks were measured to the nearest 0.01 mm. Measures of these variables were obtained independently by the two readers. The average was used if the range of the counts for the same sample was within 5% of the error; otherwise it was measured again and the average of the variables of each reader was used for the same sample (Francis and Mattlin 1986Francis R.I.C.C., Mattlin R.H. 1986. A possible pitfall in the morphometric application of discriminant analysis: measurement bias. Mar. Biol. 93(2): 311-313., Chen et al. 2013Chen X.J., Li J.H., Liu B.L., et al. 2013. Age, growth and population structure of Jumbo flying squid, Dosidicus gigas, off the Costa Rica Dome. J. Mar. Biol. Ass. U. K. 93:567-573).

A Student t-test was conducted to compare differences between the geographic stock and sexes. All of the variables were subjected to normal distribution (Kolmogorov-Smirnov test, P>0.05). Considering the impact of allometric growth (Moltschaniwskyj 1995Moltschaniwskyj N.A. 1995. Changes in shape associated with growth in the loliginid squid Photololigo sp.: a morphometric approach. Can. J. Zool. 73(7): 1335-1343., Lombarte et al. 1997Lombarte A., Sanchez P., Morales-Nin B. 1997. Intraspecific shape variability in statoliths of three cephalopod species. Vie Milieu 47: 165-169., O’Dor and Hoar 2000O’Dor R.K., Hoar J.A. 2000. Does geometry limit squid growth? ICES J. Mar. Sci. 57(1): 8-14), raw data standardization should be done before the analysis. The normalization method, as introduced by Lleonart (2000)Lleonart J., Salat J., Torres G.J. 2000. Removing allometric effects of body size in morphological analysis. J. Thero. Biol. 205: 85-93., was used to standardize morphological variables of statoliths and beaks. The accuracy of this method has been demonstrated in related investigations (Pineda et al. 2002Pineda S.E., Hernández D.R., Brunetti N.E., et al. 2002. Morphological identification of two Southwest Atlantic Loliginid squids: Loligo gahi and Loligo sanpaulensis. Rev. Invest. Desarr. Pesq. 15: 67-84., Vega et al. 2002Vega M.A., Rocha F.J., Guerra A., et al. 2002. Morphological difference between the Patagonian squid Loligo gahi populations from the Pacific and Atlantic Oceans. Bull. Mar. Sci. 71(2): 903-913, Lefkaditou and Bekas 2004Lefkaditou E., Bekas P. 2004. Analysis of beak morphometry for the horned octopus Eledone cirrhosa (Cephalopoda: Octopoda) from the Thracian Sea (NE Mediterranean). Mediterr. Mar. Sci. 5(1): 143-149., Chen et al. 2012Chen X.J., Lu H.J., Liu B.L., et al. 2012. Species identification of Ommastrephes bartramii, Dosidicus gigas, Sthenoteuthis oualaniensis and Illex argentinus (Ommastrephidae) using beak morphological variables. Sci. Mar. 76(3): 473-481.). The TSL in statolith and UHL in beak were chosen as the independent variables to standardize the other variables of beaks (Chen et al. 2012Chen X.J., Lu H.J., Liu B.L., et al. 2012. Species identification of Ommastrephes bartramii, Dosidicus gigas, Sthenoteuthis oualaniensis and Illex argentinus (Ommastrephidae) using beak morphological variables. Sci. Mar. 76(3): 473-481.). The standardized morphometric variables were represented by adding a lower case letter “s” after each variable, i.e. MWs, DDLs, LDLs, DLLs, RLLs, RLs, RWs, WLs; or UCLs, URLs, ULWLs, UWLs, LHLs, LCLs, LRLs, LLWLs and LWLs.

A stepwise discriminant analysis (SDA) was performed to select the significant standardized morphological variables based on the statolith, beak and the combination of the two structures (P<0.05; Rencher 2002Rencher A.C. 2002. Methods of Multivariate Analysis, 2nd edition. John Wiley & Sons, Inc, New York.), and the classification functions were developed for the three different materials (i.e., statolith, beak, and their combinations). Finally, a leave-one-out cross-validation (the Jackknife method) was used to determine rates of successful classification of squid from the two stocks for different uses of the hard structures.

RESULTSTop

Sexual dimorphism and variation of different cohorts in hard structure sizes

The morphometric variables of beaks and statoliths in both of the two cohorts are shown in Table 2 and Table 3. Significant differences were found in the beak variables between sexes for the NE cohort (P<0.05). The NW cohort also shows significant differences for all variables (P<0.05) except for UWL (P>0.05). Apparently, sexual dimorphism of statolith variables is similar to that of the beak for the NE cohort, but t-tests showed no significant differences between males and females in the NW cohort (P>0.05). The above results indicated that between-sex differences were greater for the NE stock than for the NW stock (Tables 2 and 3).

For a given sex, different stocks also revealed different patterns with different hard structures. Female individuals had significant differences in beak morphology between stocks (t-test, P<0.01), but had no significant differences in male squids (P>0.05), with the exception of UHL, UWL and LRW between stocks (P<0.05). The morphometric characteristics of statoliths showed significant difference between NE and NW cohorts in beak shape for female squids (P<0.05). DDL and WL showed no differences in male squids (P>0.05), but the other variables of statoliths were significantly different between stocks (P<0.05) (Table 3).

Discriminant analysis using standardized beak variables

We divided the squid samples into four groups based on geography and sex. The beak was chosen as the only analytical material first. Stepwise discriminant analyses showed that the six variables, URW_S, UWL_S, LCL_S, ULWL_S, LRW_S and URL_S, could explain the morphological features among the four groups. The Wilks λ was decreased from 0.551 to 0.419 (Table 4). Three canonical functions effectively separated these four groups, explaining 82.1%, 15.6% and 2.3% of the total variance. The distribution of the four groups on function 1 overlapped except for western females, which could be more easily identified (Fig. 3). The cross-validation rate was 36.0% for the western males (NW-M), 43.6% for the western females (NW-F), 68.7% for the eastern males (NE-M) and 74.6% for the eastern females (NE-F), showing slightly lower values than the original ones (Table 4). The classification functions with coefficients are presented in Table 5.

Discriminant analysis using standardized statolith variables

When referring to statoliths, RL_S, LDL_S, RLL_S, RW_S and WL_S were chosen as the most important variables from ten morphological parameters for the population discrimination (Table 6). The total Wilks λ was 2.626 for the five variables. Canonical functions 1 and 2, explaining 80.9% and 18.5%, respectively, together explained almost 100% of all the groups (99.4%). The distribution of the NE-M revealed little overlap with the other three groups on function 1 (Fig. 4). Therefore, the highest cross-validation rate was 86.6% for the NE-M, and the remainder was 36.0% for the NW-M, 37.6% for the NW-F and 50.8% for the NE-F. The classification result was similar to that of the beak (Table 6). The classification functions with coefficients are presented in Table 7.

Discriminant analysis with combination of standardized beak and statolith variables

When combined with the two hard structures, the SDA results showed that eight variables (URW_S, MW_S, RLL_S, UWL_S, LLWL_S, DLL_S, LCL_S and RL_S) were identified effectively among all the four groups within geography and sex. Total Wilks λ was 1.630, decreasing sharply from 0.551 to 0.127 (Table 8). Canonical function 1 showed the highest rate of 86.5%, followed by 12.3% and 1.2% for canonical functions 2 and 3, respectively (Table 8). The different geographical stocks were effectively separated by function 1, and there was little overlap between sexes in the NW stock (Fig. 5). Overall, the successful classification rate was 71.7% with 53.5% for the NW-M, with the remainder for the NW-F at 56.4%, 95.5% for the NE-M and 81.4% for the NE-F respectively, over 15% higher than in the classification with either hard structure alone. The classification functions with coefficients are presented in Table 9.

DISCUSSIONTop

Since the 1960s, the structures and morphological characteristics of the statolith and beak of cephalopods have attracted much interest (Clarke 1962Clarke M.R. 1962. The identification of cephalopod “beaks” and the relationship between beak size and total body weight. Bull. Br. Mus. nat. Hist. Zool. 8: 419-480, 1978Clarke M.R. 1978. The cephalopod statolith-introduction to its form. J. Mar. Biol. Ass. U.K. 58: 701-712., 2003Clarke M.R. 2003. Potential of statoliths for interpreting coleoid evolution: a brief review. Ber. Palä. Abh. 3: 37-47., Villanueva 1992Villanueva R. 1992. Interannual growth differences in the oceanic squid Todarodes angolensis Adam in the northern Benguela upwelling system, based on statolith growth increment analysis. J. Exp. Mar. Biol. Ecol., 159(2): 157-177., Arkhipkin 2003Arkhipkin A.I. 2003. Towards identification of the ecological lifestyle in nektonic squid using statolith morphometry. J. Mollus. Stud. 69: 171-178., Chen et al. 2012Chen X.J., Lu H.J., Liu B.L., et al. 2012. Species identification of Ommastrephes bartramii, Dosidicus gigas, Sthenoteuthis oualaniensis and Illex argentinus (Ommastrephidae) using beak morphological variables. Sci. Mar. 76(3): 473-481.). For this reason we chose the statolith and beak as materials in this study for their rigid characteristics and wide usage. Meanwhile, data standardization can effectively remove the influence of allometric growth. There are also other sorts of data standardization, such as log-transformation. Some studies have already proved that the method of data standardization used in this study is more effective than the original data, and there are other methods for identification with a low rate of misclassification (Chen et al. 2012Chen X.J., Lu H.J., Liu B.L., et al. 2012. Species identification of Ommastrephes bartramii, Dosidicus gigas, Sthenoteuthis oualaniensis and Illex argentinus (Ommastrephidae) using beak morphological variables. Sci. Mar. 76(3): 473-481., Fang et al. 2012Fang Z., Chen X.J., Lu H.J., et al. 2012. Morphological differences in statolith and beak between two spawning stocks for Illex argentinus. Acta. Ecol. Sin. 32(19): 5986-5997. (In Chinese with English Abstract))

Sexual dimorphism usually occurs in cephalopods (Mercer et al. 1980Mercer M.C., Misra R.K., Hurley G.V. 1980. Sex determination of the Ommastrephid squid Illex illecebrosus using beak morphometric. Can. J. Fish. Aquat. Sci. 37: 283-286, Pineda et al. 2002Pineda S.E., Hernández D.R., Brunetti N.E., et al. 2002. Morphological identification of two Southwest Atlantic Loliginid squids: Loligo gahi and Loligo sanpaulensis. Rev. Invest. Desarr. Pesq. 15: 67-84., Vega et al. 2002Vega M.A., Rocha F.J., Guerra A., et al. 2002. Morphological difference between the Patagonian squid Loligo gahi populations from the Pacific and Atlantic Oceans. Bull. Mar. Sci. 71(2): 903-913, Chen et al. 2012Chen X.J., Lu H.J., Liu B.L., et al. 2012. Species identification of Ommastrephes bartramii, Dosidicus gigas, Sthenoteuthis oualaniensis and Illex argentinus (Ommastrephidae) using beak morphological variables. Sci. Mar. 76(3): 473-481.). Our study also found this difference in the NE stock in both the statolith and beak (P<0.05), and this was mainly affected by the sex-segregated migration whereby males are separated from northward migrating females, which stay at the spawning/nursery ground to avoid cannibalism (Yatsu 1997Yatsu A., Midorikawa S., Shimada T., et al. 1997. Age and growth of the neon flying squid, Ommastrephes bartrami, in the North Pacific Ocean. Fish. Res. 29: 257-270, O’Dor and Dawe 1998O’Dor R.K., Dawe E.G. 1998. Illex illecebrosus. In: Rodhouse P.G., Dawe E.G., O’Dor R.K. (eds), Squid recruitment dynamics. FAO Fish Tech Pap 376, Rome, pp. 77-104., Ichii et al. 2009Ichii T., Mahapatra K., Sakai M., et al. 2009. Life history of the neon flying squid: effect of the oceanographic regime in the North Pacific Ocean. Mar. Ecol. Prog. Ser. 378: 1-11.). This meant that we could not find more male individuals in our study areas. With the disparity of latitude distribution, environmental conditions can eventually affect the morphology of hard structures. This was also reflected in the results from the discriminant analysis (Tables 2 and 3).

The NW stock revealed a different situation in morphometric characteristics. Most beak characteristics varied according to sex (P<0.05); however, none of the statolith variables showed differences between the sexes (P>0.05). Both sexes of the NW stock had lived in the same area, and shared a similar oceanographic environment with a single migration pattern. Therefore, the difference in the beaks might have been caused by asynchronous maturation, which is a type of reproductive strategy commonly found in other cephalopods (Rocha et al. 2001Rocha F., Guerra A., González A.F. 2001. A review of reproductive strategies in cephalopods. Biol. Rev. 76: 291-304.). However, this sexual dimorphism was small, as is shown by the overlapped dots in the discriminant analysis (Figs 3 and 4).

The population structure of O. bartramii in the Pacific Ocean has been discussed in previous studies (Murakami et al. 1981Murakami K., Watanabe Y., Nakata J. 1981. Growth, distribution and migration of flying squid (Ommastrephes bartrami) in the North Pacific. In: Mishima, S. (ed.), Pelagic animals and environments around the Subarctic Boundary in North Pacific. Hokkaido University, Research Institute of North Pacific Fisheries, Hakodate, pp. 161-179 (In Japanese with English abstract), Murata 1990Murata M. 1990. Oceanic resources of squids. Mar. Behav. Phys. 18: 19-71., Bower and Margolis 1991Bower S.M., Margolis L. 1991. Potential use of helminth parasites in stock identification of flying squid, Ommastrephes bartramii, in North Pacific waters. Can. J. Zool. 69(4): 1124-1126.), and the two main seasonal cohorts (the NE stock with large size located east of 170°E and the NW stock with small size located west of 170°E, Chen and Chiu 2003Chen C.S., Chiu T.S. 2003. Variations of life history parameters in two geographical groups of the neon flying squid, Ommastrephes bartramii, from the North Pacific. Fish. Res. 63: 349-366.) lived in different habitats. One of the reasons for the difference in statolith (both sexes) and beak (female) morphology is a separated migration trajectory. The NW stock occurs in the subtropical frontal zone (STFZ) with rich productivity, whereas NE stock occurs in the subtropical domain which is less productive because it is far from the transition zone chlorophyll front (TZCF). When the TZCF shifts northward in spring, the migration pattern of the NW stock moves with the change of the TZCF, but the NE stock remains southward to the north of the TZCF until summer or autumn (Ichii et al. 2009Ichii T., Mahapatra K., Sakai M., et al. 2009. Life history of the neon flying squid: effect of the oceanographic regime in the North Pacific Ocean. Mar. Ecol. Prog. Ser. 378: 1-11.). Therefore, the NW stock is of large size in the enhanced productivity water, feeding on myctophid (Symbolophorus californiensis, Ceratoscopelus warmingi) and squid (Onychoteuthis borealijaponica, Gonatus berryi). The small-sized NE stock feed on euphausiids, amphipods and fish (Maurolicus imperatorius) (Ichii et al. 2004Ichii T., Mahapatra K., Sakai M., et al. 2004. Differing body size between the autumn and the winter–spring cohorts of neon flying squid (Ommastrephes bartramii) related to the oceanographic regime in the North Pacific: a hypothesis. Fish. Oceanogr. 13: 295-309). This disparity of feeding may induce variation in the beak morphology (Uchikawa et al. 2009Uchikawa K., Sakai M., Wakabayashi T., et al. 2009. The relationship between paralarval feeding and morphological changes in the proboscis and beaks of the neon flying squid Ommastrephes bartramii. Fish. Sci. 75(2): 317-323.). The high rate of classification also demonstrated the difference between the two stocks (Fig. 5).

Many studies have focused on interspecies identification, with relatively high classification rates using beaks (Martínez et al. 2002Martínez P., Sanjuan A., Guerra A. 2002. Identification of Illex coindetii, I. illecebrosus and I. argentines (Cephalopoda: Ommastrephidae) throughout the Atlantic Ocean by body and beak characters. Mar. Biol. 141: 131-143., Pineda et al. 2002Martínez P., Sanjuan A., Guerra A. 2002. Identification of Illex coindetii, I. illecebrosus and I. argentines (Cephalopoda: Ommastrephidae) throughout the Atlantic Ocean by body and beak characters. Mar. Biol. 141: 131-143., Chen et al. 2012Chen X.J., Lu H.J., Liu B.L., et al. 2012. Species identification of Ommastrephes bartramii, Dosidicus gigas, Sthenoteuthis oualaniensis and Illex argentinus (Ommastrephidae) using beak morphological variables. Sci. Mar. 76(3): 473-481.). The total correct classification rate was nearly 50% for both statoliths and beaks, but increased to about 70% for the combination of both hard parts. Thus, adding more relevant variables can increase the success of classification, using SDA to improve the accuracy of classification by retaining some highly correlated variables although the results of correct classification are still low compared with other studies of cephalopods (Martínez et al. 2002Martínez P., Sanjuan A., Guerra A. 2002. Identification of Illex coindetii, I. illecebrosus and I. argentines (Cephalopoda: Ommastrephidae) throughout the Atlantic Ocean by body and beak characters. Mar. Biol. 141: 131-143., Chen et al. 2012Chen X.J., Lu H.J., Liu B.L., et al. 2012. Species identification of Ommastrephes bartramii, Dosidicus gigas, Sthenoteuthis oualaniensis and Illex argentinus (Ommastrephidae) using beak morphological variables. Sci. Mar. 76(3): 473-481.). The result was credible as the first three variables entered the combination group, which all entered the beak-based and statolith-based SDA (Table 8).

There are still two sub-stocks in the northwest Pacific (central stock and east stock) and northeast Pacific (west stock and central-east stock) (Bower and Ichii 2005Bower J.R., Ichii T. 2005. The red flying squid (Ommastrephes bartramii): A review of recent research and the fishery in Japan. Fish. Res. 76(1): 39-55.). The relatively high successful classification rates demonstrate that morphology of hard parts in sub-stocks showed less variation and it is hard to separate them using only morphology of hard structures.

In conclusion, hard structures, such as statolith and beak in cephalopod, are credible materials to identify at an intraspecies level. The NE stock of O. bartramii showed sexual dimorphism in hard parts, but the NW stock showed no significant difference by sex. The among-stock differences were greater in the female group than in the male group. The classification approach could separate the two stocks, especially the NE stock, with more than 80% accuracy. Meanwhile, data standardization and variable combination could help improve the accuracy of the classification. As the overlapped dots showed in the results, we must be cautious of over-interpretation of phenotype-based analysis, which is more reliable when combined with genetic methods.

ACKNOWLEDGEMENTSTop

This work was funded by the National Science Foundation of China (NSFC41306127 and NSFC41276156), the National Science Foundation of Shanghai (13ZR1419700), the Innovation Programme of Shanghai Municipal Education Commission (13YZ091), the State 863 projects (2012AA092303), the Funding Program for Outstanding Dissertations in Shanghai Ocean University, the Funding Scheme for Training Young Teachers in Shanghai Colleges and the Shanghai Leading Academic Discipline Project (Fisheries Discipline). Y Chen’s involvement was supported by SHOU International Center for Marine Studies and Shanghai 1000 Talent Program.

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