1 Heredity 1999 Vol: 83(3):280-289. DOI: 10.1038/sj.hdy.6885520

Allozyme differentiation in the cuttlefish Sepia officinalis (Mollusca: Cephalopoda) from the NE Atlantic and Mediterranean

The Atlantic–Mediterranean area has recently been proposed as a new phylogeographical area on the basis of concordance of genetic differentiation patterns observed in several marine species. However, additional taxa need to be studied to establish the phylogeographical relationship between the Atlantic and Mediterranean. Eleven samples of the cuttlefish Sepia officinalis around the Iberian Peninsula, one from the Canary Islands, and another from Fiumicino (Italy) were screened for 33 allozyme loci. Genetic variability was low in all samples (He between 0.022 and 0.076). Intersample genetic differentiation was high (FST=0.220), mainly because of genetic variation in the non-Iberian samples. One locus (PEPD*), diagnostic between the Italian sample and all others, suggests the possible existence of hitherto unrecognized species or subspecies of Sepia in the Mediterranean Sea. The 11 Iberian samples exhibited moderate genetic differentiation (FST=0.100), which could be explained on the basis of genetic differentiation between Atlantic and Mediterranean samples. Significant clines in allele frequencies were observed for five out of six polymorphic loci. These results support a model of secondary intergradation (i.e. secondary contact of populations that were previously differentiated in isolation) similar to that previously proposed for other marine species from the Atlantic–Mediterranean area.

Mentions
Figures
Figure 1: Samples of Sepia officinalis collected in fishing ports along the Atlantic and Mediterranean coasts. Sample codes are indicated in Table 1. Figure 2: UPGMA dendrograms showing relationships among 13 samples of Sepia officinalis based on Nei's (1978) and Cavalli-Sforza & Edwards's (1967) arc genetic distances. The cophenetic correlations were ra=0.982 and rb=0.970. Sample codes are indicated in Table 1. N, the most northern Iberian samples; W, north-western and western Iberian samples; S and E, southern and eastern Iberian samples, respectively. Figure 3: Correlation between Cavalli-Sforza & Edwards's (1967) arc genetic distance and geographical distance (km) for 13 samples of Sepia officinalis. o: Fium with every one of the other 12 samples. r=0.721, P < 0.001 (Mantel test). Figure 4: Allele frequency variation at polymorphic loci ESTD*, IDDH*, IDHP*, MEP*, OPDH-1* and OPDH-2* for 11 Iberian samples of Sepia officinalis against geographical distance. RoBo is at 0 km and Rosa is at 31 km ( 100). All loci, except IDHP*, displayed significant geographical allozyme variation around the Iberian Peninsula.
Altmetric
References
  1. Avise, J. C.Molecular Markers, Natural History and Evolution. Chapman & Hall, New York , (1994) .
    • . . . Such variation in allele frequencies may be the result of secondary contact of cuttlefish populations that were isolated in the past and became differentiated in allopatry (i.e. secondary intergradation; Endler, 1977; Avise, 1994) . . .
    • . . . The results presented for S. officinalis in this paper support the importance of the Atlantic–Mediterranean area in phylogeographical structuring within marine species (Sanjuan et al., 1996a), as has been similarly suggested for the Gulf of Mexico/Atlantic coasts of North America and the Indian Ocean/Malayan Provinces (Avise, 1994; Palumbi, 1994). . . .
  2. Bianco, P. G.. Potential role of the palaeohistory of the Mediterranean and Paratethys basins on the early dispersal of Euro-Mediterranean freshwater fishes. Ichthyol Expl Freshw, 1: 167-184 , (1990) .
    • . . . This is compatible with what is known of the historical hydrography of this area: intermittent physical barriers, precluding colonization and migration, existed as recently as one million years ago (Maldonado, 1985 and references therein); recent lowering of sea level by 100–200 m during Quaternary glaciations resulted in the partial or total closure of the Straits (Bianco, 1990) . . .
  3. Brierley, A. S., Allcock, A. L., Thorpe, J. P. and Clarke, M. R.. Biochemical genetic evidence supporting the taxonomic separation of Loligo edulis and Loligo chinensis (Cephalopoda: Teuthoidea) from the genus Loligo. Mar Biol, 127: 97-104 , (1996) .
    • . . . The study of 33 enzyme loci in 13 samples of S. officinalis showed that this species has low levels of allozyme variability; the mean He (0.057 0.022) falls below the average for invertebrates and molluscs (0.122 and 0.145, respectively; Ward et al., 1992), and also for marine molluscs (0.147; Fujio et al., 1983), but within the range for cephalopods (see Brierley et al., 1996; Sanjuan et al., 1996b; and references therein). . . .
  4. Carvalho, G. R. and Hauser, L.. Molecular genetics and the stock concept in fisheries. Rev Fish Biol Fish, 4: 300-326 , (1994) .
    • . . . Allozyme polymorphisms have proved to be effective for estimating population divergence and identifying discrete fish and cephalopod stocks (Carvalho & Hauser, 1994 and references therein) . . .
  5. Cavalli-Sforza, L. L. and Edwards, A. W. F.. Phylogenetic analysis: models and estimation procedures. Evolution, 32: 550-570 , (1967) .
    • . . . Nei's (1978) (DN) and Cavalli-Sforza & Edwards's (1967) arc (Darc) genetic distances among samples were computed . . .
    • . . . The correlation coefficient between the matrix of Cavalli-Sforza & Edwards's (1967) arc genetic distances and geographical distances was calculated and its probability was estimated by means of a Mantel test based on 1000 permutations . . .
    • . . . Genetic distances of Nei (1978) (DN) and Cavalli-Sforza & Edwards (1967) (Darc) are presented in Table 4 . . .
    • . . . UPGMA dendrograms showing relationships among 13 samples of Sepia officinalis based on Nei's (1978) and Cavalli-Sforza & Edwards's (1967) arc genetic distances . . .
    • . . . Correlation between Cavalli-Sforza & Edwards's (1967) arc genetic distance and geographical distance (km) for 13 samples of Sepia officinalis. o: Fium with every one of the other 12 samples. r=0.721, P < 0.001 (Mantel test). . . .
  6. Endler, J. A.Geographic Variation, Speciation, and Clines. Princeton University Press, Princeton, NJ , (1977) .
    • . . . Such variation in allele frequencies may be the result of secondary contact of cuttlefish populations that were isolated in the past and became differentiated in allopatry (i.e. secondary intergradation; Endler, 1977; Avise, 1994) . . .
  7. FAO (FOOD AND AGRICULTURE ORGANIZATION)Yearbook Fishery Statistics1992 no. 74. FAO, Rome , (1994) .
    • . . . Sepia officinalis (Linnaeus 1758; Cephalopoda: Sepiidae) is an important fishery resource for European and North African countries (annual captures are around 70 000 metric tonnes; FAO, 1994) . . .
  8. Fujio, Y., Yamanaka, R. and Smith, P. J.. Genetic variation in marine molluscs. Bull Jap Soc Sci Fish, 49: 1809-1817 , (1983) .
    • . . . The study of 33 enzyme loci in 13 samples of S. officinalis showed that this species has low levels of allozyme variability; the mean He (0.057 0.022) falls below the average for invertebrates and molluscs (0.122 and 0.145, respectively; Ward et al., 1992), and also for marine molluscs (0.147; Fujio et al., 1983), but within the range for cephalopods (see Brierley et al., 1996; Sanjuan et al., 1996b; and references therein). . . .
  9. Guerra, A.. Mollusca, Cephalopoda. In: Ramos, M. A. et al(eds) Fauna Ibérica vol. 1. Museo Nacional de Ciencias Naturales, CSIC, Madrid , (1992) .
    • . . . It is distributed along the NE Atlantic continental margin, from the Baltic Sea to Senegal, and throughout the Mediterranean Sea (Guerra, 1992) . . .
  10. Maldonado, A.. Evolution of the Mediterranean basins and a reconstruction of the Cenozoic palaeoceanography. In: Margalef, R. (ed.) Western Mediterranean, pp. 18-61. Pergamon Press, London , (1985) .
    • . . . This is compatible with what is known of the historical hydrography of this area: intermittent physical barriers, precluding colonization and migration, existed as recently as one million years ago (Maldonado, 1985 and references therein); recent lowering of sea level by 100–200 m during Quaternary glaciations resulted in the partial or total closure of the Straits (Bianco, 1990) . . .
  11. Murphy, R. W., Sites, C. W.Jr, Buth, D. G. and Haufler, C. H.. Proteins: isozyme electrophoresis. In: Hillis, D. M. and Moritz, C. (eds) Molecular Systematics. pp. 51-120. Sinauer Associates, Sunderland, MA , (1996) .
    • . . . Standard horizontal starch gel electrophoresis was carried out (Murphy et al., 1996) . . .
    • . . . For ALPDH, G6PDH and GAPDH the electrode buffer was Tris-Citrate pH 8.0 (gel buffer dilution 1:9), and histochemical staining recipes were as in Murphy et al. (1996). . . .
  12. Nei, M.. Estimation of average heterozygosity and genetic distance from a small number of individuals. Genetics, 89: 583-590 , (1978) .
  13. Nei, M.Molecular Evolutionary Genetics. Columbia University Press, New York , (1987) .
    • . . . The genetic structure of samples was analysed by means of F-statistics (Nei, 1987) . . .
    • . . . Mediterranean samples exhibited the highest values for the variability indices, although He was not significantly different between Mediterranean and Atlantic samples [Student's t11=0.710, 0.40 < P < 0.50; Nei (1987)]. . . .
  14. Nesis, K. N.Cephalopods of the World. T. H. F. Publications, Neptune City, NJ , (1987) .
    • . . . These results suggest that the individuals from Fium are genetically distinct (no gene flow between these and the other samples), and may even comprise a separate and hitherto unrecognized subspecies or species of Sepia. Both Nesis (1987) and Khromov (pers. comm.) have suggested that, based on morphological data, the notion of a subspecies of S. officinalis in the Mediterranean Sea (S. officinalis mediterranea Nini 1884) is valid . . .
  15. Palumbi, S. R.. Genetic divergence, reproductive isolation, and marine speciation. Ann Rev Ecol Syst, 25: 547-572 , (1994) .
    • . . . The results presented for S. officinalis in this paper support the importance of the Atlantic–Mediterranean area in phylogeographical structuring within marine species (Sanjuan et al., 1996a), as has been similarly suggested for the Gulf of Mexico/Atlantic coasts of North America and the Indian Ocean/Malayan Provinces (Avise, 1994; Palumbi, 1994). . . .
  16. Pérez-Losada, M., Guerra, A. and Sanjuan, A.. Allozyme electrophoretic technique and phylogenetic relationships in three species of Sepia (Cephalopoda: Sepiidae). Comp Biochem Physiol B, 114: 11-18 , (1996) .
    • . . . Samples of mantle muscle were prepared for electrophoresis using methods previously described for Sepia species (Pérez-Losada et al., 1996) . . .
    • . . . Detailed electrophoretic conditions and histochemical staining recipes for most of the enzymes are described in Pérez-Losada et al. (1996) . . .
  17. Rice, W. R.. Analyzing tables of statistical tests. Evolution, 43: 223-225 , (1989) .
    • . . . The sequential Bonferroni technique (Rice, 1989) was used to adjust significance levels for multiple simultaneous comparisons. . . .
  18. Rodríguez, J.Oceanografía Del Mar Mediterráneo. Pirámide, Madrid , (1982) .
    • . . . This asymmetrical gene flow could be maintained by the unidirectional marine surface circulation southwards along the coast of the Iberian Peninsula and into the Mediterranean Sea across the Straits of Gibraltar (Rodríguez, 1982). . . .
  19. Rohlf, F. J.Ntsys-Pc. Numerical Taxonomy and Multivariate Analysis System Version 1.80. Exeter Software, New York, NY , (1994) .
    • . . . The Mantel test was carried out using the NTSYS-PC computer program (Rohlf, 1994). . . .
  20. Roldán, M. I., García-Marín, J. L., Utter, F. M. and Pla, C.. Population genetic structure of European hake, Merluccius merluccius. Heredity, 81: 327-334 , (1998) .
    • . . . Studies on several different marine fishes and molluscs (see Sanjuan et al., 1996a, 1997 and references therein; Roldán et al., 1998) have revealed geographically concordant intersample genetic differentiation between Atlantic and Mediterranean samples . . .
  21. Saitou, N. and Nei, M.. The neighbour-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol, 4: 406-425 , (1987) .
    • . . . These values were then used to construct UPGMA (unweighted pair-group method using arithmetic averages; Sneath & Sokal, 1973) and neighbour-joining (Saitou & Nei, 1987) trees. . . .
  22. Sanjuan, A., Comesaña, S. and De Carlos, A. (1996a). Macrogeographic differentiation by mtDNA restriction site analysis in the SW European Mytilus galloprovincialis Lmk. J Exp Mar Biol Ecol, 198: 89-100 , .
    • . . . Studies on several different marine fishes and molluscs (see Sanjuan et al., 1996a, 1997 and references therein; Roldán et al., 1998) have revealed geographically concordant intersample genetic differentiation between Atlantic and Mediterranean samples . . .
    • . . . The results presented for S. officinalis in this paper support the importance of the Atlantic–Mediterranean area in phylogeographical structuring within marine species (Sanjuan et al., 1996a), as has been similarly suggested for the Gulf of Mexico/Atlantic coasts of North America and the Indian Ocean/Malayan Provinces (Avise, 1994; Palumbi, 1994). . . .
  23. Sanjuan, A., Pérez-Losada, M. and Guerra, A. (1996b). Genetic differentiation in three Sepia species (Mollusca: Cephalopoda) from Galician waters (Northwest Iberian Peninsula). Mar Biol, 126: 253-259 , .
    • . . . The one previous allozyme-based study on Sepia officinalis (Sanjuan et al., 1996b) found no significant genetic differences between samples on either side of a subsurface oceanographic front off the NW Iberian Peninsula . . .
    • . . . The study of 33 enzyme loci in 13 samples of S. officinalis showed that this species has low levels of allozyme variability; the mean He (0.057 0.022) falls below the average for invertebrates and molluscs (0.122 and 0.145, respectively; Ward et al., 1992), and also for marine molluscs (0.147; Fujio et al., 1983), but within the range for cephalopods (see Brierley et al., 1996; Sanjuan et al., 1996b; and references therein). . . .
  24. Sanjuan, A., Zapata, C. and Álvarez, G.. Genetic differentiation in Mytilus galloprovincialis Lmk. throughout the world. Ophelia, 47: 13-31 , (1997) .
    • . . . Studies on several different marine fishes and molluscs (see Sanjuan et al., 1996a, 1997 and references therein; Roldán et al., 1998) have revealed geographically concordant intersample genetic differentiation between Atlantic and Mediterranean samples . . .
  25. Sneath, P. H. and Sokal, R. R.Numerical Taxonomy. W. H. Freeman, San Francisco , (1973) .
    • . . . These values were then used to construct UPGMA (unweighted pair-group method using arithmetic averages; Sneath & Sokal, 1973) and neighbour-joining (Saitou & Nei, 1987) trees. . . .
  26. Swofford, D. L. and Selander, R. B.. Biosys-1: a Fortran program for the comprehensive analysis of electrophoretic data in population genetics and systematics. J Hered, 72: 281-283 , (1981) .
    • . . . BIOSYS-1 (Swofford & Selander, 1981) and Zaykin & Pudovkin (1993) computer programs were used to perform most of the genetic analyses . . .
  27. Thorpe, J. P.. Enzyme variation, genetic distance and evolutionary divergence in relation to levels of taxonomic speciation. In: Oxford, G. S. and Rollinson, D. (eds) Protein Polymorphism. Adaptive and Taxonomic Significance. pp. 131-152. Academic Press, London , (1983) .
    • . . . Nevertheless, the unbiased genetic identity (I; Nei, 1978) between Fium and the other 12 samples (0.954–0.946) is of the order generally considered to be indicative of conspecific populations (Thorpe, 1983) . . .
  28. Ward, R. D., Skibinski, D. O. F. and Woodwark, M.. Protein heterozygosity, protein structure and taxonomic differentiation. Evol Biol, 26: 73-159 , (1992) .
    • . . . The study of 33 enzyme loci in 13 samples of S. officinalis showed that this species has low levels of allozyme variability; the mean He (0.057 0.022) falls below the average for invertebrates and molluscs (0.122 and 0.145, respectively; Ward et al., 1992), and also for marine molluscs (0.147; Fujio et al., 1983), but within the range for cephalopods (see Brierley et al., 1996; Sanjuan et al., 1996b; and references therein). . . .
  29. Zaykin, D. V. and Pudovkin, A. I.. Two programs to estimate significance of 2values using pseudo-probability tests. J Hered, 84: 152-152 , (1993) .
    • . . . BIOSYS-1 (Swofford & Selander, 1981) and Zaykin & Pudovkin (1993) computer programs were used to perform most of the genetic analyses . . .
Expand