GENETIC AND GROWTH PROFILES OF THREE SPECIMENS OF SWORDFISH (XIPHIAS GLADIUS) TAGGED AND RECAPTURED IN THE NORTH ATLANTIC

SCRS/2006/119 Col. Vol. Sci. Pap. ICCAT, 60(6): 1964-1973 (2007) GENETIC AND GROWTH PROFILES OF THREE SPECIMENS OF SWORDFISH (XIPHIAS GLADIUS) TAGGED AND RECAPTURED IN THE NORTH ATLANTIC P. Kasapidis 1, X. Valeiras 2, A. Antoniou 1, G. Kotoulas 1, B. García-Cortés 3 and J. Mejuto 3 SUMMARY This paper presents an analysis of the genetic and growth patterns of three swordfish specimens (two males and one female) tagged with traditional tags and later recaptured over a period of time ranging from 2.7 to 5.4 years. Two of these specimens were tagged and recaptured in the NW Atlantic, while the third individual was tagged and recaptured in the NE Atlantic. These three specimens were genotyped for four micro-satellite loci and were analysed together with additional genotyped specimens from Atlantic and Mediterranean areas using a Bayesian cluster analyses. The results suggest that the three recaptured swordfish have a genetic profile that is characteristic of the Atlantic. The growth patterns were seen to differ in two of the specimens analysed (male and female): the male was assumed as a single ring pattern, while the female was assumed as a mostly double ring pattern. The age estimation of these two specimens according to these premises could be accommodated to previous growth studies. The third specimen which is known to be a male at least 6 years of age, tagged and recaptured in the NW Atlantic, was not possible to accommodate to previous growth studies. The examination of genetic data in combination with tagging-recapture information may facilitate the study of aspects related to individual and stock specific growth and elucidate the patterns of ring formation on the hard parts (spines). It would be advisable to enhance the tagging-recapture protocols of swordfish and to include the routine sampling of tissues and hard parts in all the recaptures made by the different fleets. RÉSUMÉ Le présent document fournit une analyse des schémas génétiques et de croissance de trois spécimens d espadon (deux mâles et une femelle) marqués avec des marques traditionnelles et ensuite récupérés au cours d une période allant de 2,7 à 5,4 ans. Deux de ces spécimens ont été marqués et récupérés dans l Atlantique Nord-Ouest, tandis que le troisième a été marqué et récupéré dans l Atlantique Nord-Est. On a déterminé le génotype de ces trois spécimens pour quatre locus microsatellites et on les a analysés avec d autres spécimens dont le génotype avait été déterminé et qui provenaient de zones de l Atlantique et de la Méditerranée, à l aide d une analyse par grappes bayésienne. Les résultats suggèrent que les trois espadons récupérés ont un profil génétique caractéristique de l Atlantique. On a observé que les schémas de croissance différaient chez deux des spécimens analysés (mâle et femelle) : on a postulé que le mâle avait un schéma à anneau unique, tandis que la femelle présentait principalement un schéma à doubles anneaux. Selon ces prémisses, l estimation de l âge de ces deux spécimens était conforme aux études de croissance antérieures. Le troisième spécimen, dont on sait qu il s agit d un mâle d au moins 6 ans, marqué et récupéré dans l Atlantique Nord-Ouest, ne concordait pas avec les études de croissance antérieures. L examen des données génétiques conjointement avec les informations de marquage-récupération pourrait faciliter l étude des aspects relatifs à la croissance individuelle et spécifique du stock et expliquer les schémas de formation des anneaux sur les pièces dures (épines). Il serait recommandable de renforcer les protocoles de marquage-récupération de l espadon et d inclure l échantillonnage systématique des tissus et pièces dures dans toutes les récupérations effectuées par les différentes flottilles. 1 Hellenic Centre for Marine Research, Institute of Marine Biology and Genetics, Thalassokosmos, 71500 Gournes, Crete, Greece. 2 Instituto Español de Oceanografía, P.O. Box 240, 39080 Santander, España. 3 Instituto Español de Oceanografía, P.O. Box 130, 15080 A Coruña, España. 1964

RESUMEN Se analizan patrones genéticos y de crecimiento de tres peces espada (dos machos y una hembra) marcados con marcas tradicionales los cuales fueron recapturados entre 2.7 y 5.4 años más tarde. Dos de los individuos fueron marcados-recapturados en el Atlántico NW mientras que el tercero fue marcado-recapturado en el Atlántico NE. Estos tres individuos fueron genotipados mediante cuatro microsatélite loci y analizados conjuntamente con especímenes genotipados del Atlántico y Mediterráneo usando análisis Bayesianos tipo de cluster. Los resultados sugieren que el perfil genético de estos tres peces espada es Atlántico. Los patrones de crecimiento fueron identificados como distintos en dos de los peces analizados (macho y hembra): el macho fue asumido con un patrón de anillos simples, mientras que la hembra fue asumida con un patrón mayoritariamente de anillos dobles. La estimación de la edad de ambos bajo estas premisas podría ser acomodada a estudios de crecimiento realizados previamente. El tercer individuo, del que se conoce que es un macho de al menos unos 6 años de edad, marcado y recapturado en el Atlántico NW, no fue posible acomodarlo a estimaciones previas de crecimiento. El examen de datos genéticos en combinación con información de marcado-recaptura puede facilitar el estudio de aspectos relacionados el crecimiento tanto individual como del stock y elucidar los patrones de formación de anillos en las partes duras (espinas). Se sugiere la conveniencia de potenciar los protocolos de marcado-recaptura del pez espada e incorporar rutinariamente la toma de muestras de tejido y de partes duras en todas las recapturas realizadas por las diferentes flotas. KEYWORDS Swordfish, tagging, recapture, genetic, growth 1. Introduction Tagging-recapture (traditional tagging) techniques have been used as a tool in the identification of the migratory patterns of tuna and tuna- like species, and on the basis of these movements and exchanges, to promote possible stock units for assessment purposes. However, these tagging-recapture techniques are somewhat limited given the fact that they are dependent upon fishery activity. Some of the limitations that must be considered when interpreting the results include the following: the different tagging intensity and the different spatial-temporal patterns applied to the experiments, as well as the different intensity of fishing operations among the zones and fleets and their respective reporting rates. Despite the complexity of the techniques used to tag swordfish, owing to the extreme fragility of this fish, these tagging activities have continued to a greater or lesser extent in the North Atlantic for decades with traditional tags. Tagging has been carried out since the mid-1950s particularly in the NW Atlantic (Anonymous, 2003). Tagging in the NE Atlantic is a relatively recent activity. However, the results are promising, providing a more complex view of the migratory pattern of the swordfish in the North Atlantic (García-Cortés et al. 2003). In the South Atlantic, tagging has been scarce and sporadic, although in recent years some of the fleets have been given incentives, which has produced some interesting recaptures (Anonymous in press). While tagging-recapture data are useful, this technique does not provide information on the place of origin of the tagged individual, although in some cases the origin may be deduced on the basis of the life or migratory history of the individual. Moreover, owing to the difficulty involved in the reporting of recaptured animals, swordfish that have been tagged and recaptured are seldom analysed in terms of their hard parts (otoliths, spines, etc.), parasite content or other indicators that would be of additional interest. In this sense, the availability of samples of the hard parts and muscle tissue of these tagged and recaptured individuals would contribute to the knowledge of their respective profiles in terms of genetics and growth, making it possible to compare them with previously identified population profiles. The analyses carried out on the hard parts of tagged and recaptured fishes are useful not only in the validation of growth studies, but they also may contribute to formulate hypotheses on which to base the ring formation patterns that are assumed to be an indication of age, particularly during the period in which the tagged fish is at liberty between the time it was tagged and recaptured-. 1965

Molecular genetic markers may potentially be used as tags, in the same way that physical, chemical, microbial and parasite tags are used in the study of dispersal patterns (Thorrold et al. 2002). Genetic markers, like microsatellite DNA loci, are now used to identify individuals and to assign them to populations of origin. Nevertheless, for the successful identification of the population of origin of an individual by means of genetic markers, a good knowledge of both the number and structure of populations is required (Hansen et al. 2001). This paper analyses three specimens of swordfish that were tagged and recaptured and from which samples of their spines and muscle tissue were taken for growth and genetic analyses. The purpose of this study is to discuss the individual results obtained on these fish specimens and relate them to previously described population patterns, in addition to recommending protocols for action to all the fleets that will be applicable to future recaptures of tagged-recaptured swordfish. 2. Material and methods 2.1 Fish sampling Three tagged swordfish were recaptured by commercial surface longline vessels in the North Atlantic. The swordfish tagging program of the IEO, in collaboration with the Spanish longline fleet, made it possible to obtain information on recapture location and other data as well as biological samples (first anal fin and muscle tissue) from each recaptured fish. Information on the tagging and release of specimens was provided by the respective tagging programs. Measurements of lower jaw fork length (LJFL to the nearest cm) and round weight (kg) were recorded for each fish. The size/weight data during releasing activities could be estimated, so inconsistencies between both data are even possible in some cases. The tree recaptured animals were sexed and the first anal fin and muscular tissue (heart) samples were taken for storage and analysis. 2.2 Genetic analysis 2.2.1 Genotyping Total genomic DNA was extracted from the three recaptured specimens (sample tissue codes: Sw42, Sw801, CR1366 corresponding to tags BF40729, ES-10205 and HM57896, respectively) using standard procedures (Sambrook et al. 1989). The individuals under study were genotyped for four microsatellite loci (XgA, Xg47, Xg51, Xg195), which had been previously developed for the species (Kasapidis et al. submitted). Polymerase chain reaction (PCR) conditions and primer sequences are reported elsewhere (Kasapidis et al. submitted). The forward primer was fluorescently labeled with Texas-Red and the PCR products were electrophoresed on a Vistra 725 automated sequencer. Allele size was determined manually using as a reference individuals, whose allele size had been determined by comparison with standard fragments. The genetic dataset comprised the three recaptured individuals together with 1432 specimens from the Atlantic and western Mediterranean which had been previously genotyped for the same loci (Kasapidis et al. submitted, Kotoulas et al. submitted). Information on the samples used is given in Table 1 and Figure 1. 2.2.2 Statistical analysis Individuals were grouped into potential population units according to their geographic origin as shown in table 2 and Figure 1. A Bayesian clustering method, implemented in Structure v 2.1 software (Pritchard et al. 2000), was used to infer population structure and assign individuals to populations probabilistically. This model-based method estimates the most appropriate number (K) of populations needed for interpreting the observed genotypes. The program assigns individuals on the basis of their genotypes, and provides the proportion of an individual genome that originated from a certain population (cluster). In this analysis the number of K was estimated, using the admixture model (thus without using prior population information), by fixing prior values of K = 1-9 and comparing the Ln likelihood of the data. 2.3 Spine preparation Methodology based on Berkeley and Houde (1983), Ehrhardt et al. (1996), Esteves et al. (1995) and Tserpes and Tsimenides (1995) was used in this study. Anal fins of the three recaptured specimens were thawed in the laboratory and the second spine was removed. The second spine was immersed briefly in boiling water for one minute to clean the spine radius of tissue. These tissues were carefully removed with a scalpel and tweezers avoiding damage to the surface of the spine. The spine was washed with water and left to dry completely. A 1966

cross section of about 0.5 mm thick was cut using an ISOMED 5000 Cutter. Two cut points were used: one cut located at a distance equivalent to the maximum width of the condyle base, and a second point located midway between the above distance. Both distances were measured above the line of maximum condyle width. In each cut location of the spine, two consecutive cuts were made in order to choose the best one when taking the reading. The sections were washed in a 70% ethanol solution. They were later mounted onto labelled holders and embedded in Eukitt highly transparent mounting resin. The clearest of the two sections was examined and read twice using a profile projector (model: Nikon 4C) with transmitted light at 20x magnification. The distance from the focus to the distal edge of each growth band (annulus) was measured and recorded. 3. Results 3.1 Tagging-recapture data of each swordfish The three recaptured swordfish (Table 1) present different life events. The swordfish with codes HM and BF (female and male, respectively) were tagged and recaptured in regions of the NW Atlantic, while the male swordfish specimen with code ES was tagged and recaptured in the NE Atlantic. Although this does not verify their respective origins or the time they spent in these regions over the course of their lives at liberty after being tagged, it may however suggests different life events that occurred randomly or owing to the selection of preferred areas. If all of these specimens were from the North Atlantic, then they all would probably originate from the spawning grounds of the NW Atlantic the only zones reported to date where reproduction processes of the swordfish have been described or from the Mediterranean regions, whose genetic profile has been identified as highly characteristic. 3.2 Genetic assignment of each recaptured swordfish The Bayesian Clustering Analysis performed by Structure v 2.1 indicated the presence of two distinct clusters, as had been previously demonstrated (Kotoulas et al. submitted; REEB and BLOCK submitted). One cluster consists of individuals originating mainly from the Mediterranean while the second cluster of individuals originated mainly from the Atlantic. In total, 71.1 % of the individuals under study were assigned to one of the two clusters with a membership coefficient higher than 0.80, while the remaining 28.9% were considered as unassigned. From the assigned individuals, 1.01 % of the Atlantic cluster had been sampled in the Mediterranean (mainly in the Alboran Sea), while 11.8 % of the Mediterranean cluster had been sampled in the Atlantic. In our case, the three recaptured specimens analysed, BF40729, ES-10205 and HM57896, were assigned to the Atlantic cluster with membership coefficients of 0.86, 0.83 and 0.96, respectively. The proportion of membership of each pre-defined population in each of the 2 clusters (Atlantic and Mediterranean) for individuals with a membership coefficient above 0.80, as well as the proportion of unassigned individuals, is shown in Table 2 and Figure 1. 3.3 Ageing of each recaptured swordfish Data on ageing of tree swordfish are given in Table 3 and Figure 2. The tagging-recaptured information provides help for the age interpretation. Fish HM057896 was considered as a 4 years old female. The age interpretation assumed for this fish could be accommodated in the range of growth estimations provided by other papers on swordfish ageing from spine readings (Berkeley and Houde 1983, Riehl 1984, Ehrhardt et al. 1996, Esteves et al. 1995 and Tserpes and Timenides 1995). It also agrees with the expected ageing from growth estimations using tagging data (Restrepo 1990). This fish was 2.7 years old at liberty and had increased 36 cm length during that time, most likely in area BIL94A. Fish ES10205 was considered as a 4 year old male. Annuli were counted taken into consideration a pattern of multiple growth rings (approximately 2 rings per year) in the cross section of the anal spine. This fish was 4 years at liberty and had increased 85 cm length, most likely in area BIL94B. This age interpretation agrees with the age range by LJFL reported in growth studies done by the authors mentioned above. Fish BF40729 was considered as being a 6 year old male. The age interpretation can not be accommodated within the range of other authors regarding length and back-calculated age. According to the sizes of tagging and recapture and considering most of the previous growth papers, this male fish should be about 3 or 4 year old when recaptured and probably around one year old when tagged. However, 6 single growth annuli have been identified in cross sections of the fin spine. Furthermore, this swordfish was 5.4 years at liberty (1958 days) and 1967

had increased 45 cm in length. The age interpretation does not agree with the length of the fish, although the size and diameter of the spine is in accordance with the LJFL reported. However, the size vs. weight data during releasing is not agreeable. The geographical position data indicates that this fish was probably released in the area BIL 93, very close to the border of area BIL 92, and recaptured in area BIL94A. 4. Discussion A number of genetic studies have revealed the presence of three stocks of swordfish in the Atlantic and the Mediterranean: a North Atlantic, South Atlantic and Mediterranean stock (Kotoulas et al. 1995, Alvarado- Bremer et al. 1999, Rosel and Block 1996, Chow and Takeyama 2000, Greig et al. 2000, Alvarado-Bremer et al. 2005), with genetic differences between north and south Atlantic stocks being small but statistically significant. The Bayesian clustering approach used in this study detected only two clusters, as it was not possible to distinguish between the two Atlantic stocks mentioned above. This could be due to the limited number of microsatellite loci used in this study. However, the same data when analysed by classical population genetics approaches (Fisher s exact tests, Fsts) revealed small but statistically significant differences between north and south Atlantic samples (Kasapidis et al. submitted). The three recaptured swordfish used in this paper were clearly assigned to the Atlantic cluster. The future use of a larger number of microsatellite loci is expected to increase the resolution power of the genetic analysis by allowing a finer within Atlantic assignment. Moreover the results of the readings of the hard parts of three studied specimens highlight the difficulties entailed in finding an explanation for the growth patterns of the swordfish and in putting forth a hypothesis with a common base regarding the formation and periodicity of the rings that appear on these hard parts. These growth rings on spine sections are interpreted as being age-related, despite the fact that the three specimens originate from the Atlantic, according to their respective genetic profiles. Specimens HM 057896 and ES-10205 (a female and a male, respectively) seem to exhibit different ring formation patterns (single vs. double). The period each of these specimens spent at liberty is known, which force or help to interpret the rings pattern in the male specimen ES as being double. This male might possibly have spent its period at liberty in the NE Atlantic. The growth pattern of the BF male specimen is difficult to interpret on the basis of the published growth studies of the Atlantic swordfish. The time spent at liberty (5.4 years) would suggest that it must be at least 6 years old, which agrees with a reading of the single annuli pattern in the spine. Nonetheless, its recapture size is considerably smaller that would be expected for this age-sex, according to previous growth studies. Additionally, a complete consensus was not achieved in some of the annuli-spines interpretation among authors. The complex migratory pattern of the swordfish and the differences found between sex and areas makes it even harder to define a common pattern in the formation of growth rings. The selection of samples from one zone of origin as opposed to another could affect the ring patterns and growth results obtained. Genetic analyses that use an appropriate number of markers could help provide a more accurate definition of the origin (population) of the samples analysed for growth, or at the very least, they may be able to help rule out samples from undesirable origins that might cause additional problems when attempting to define the patterns of ring formation on the hard parts. In this study we have excluded the Mediterranean as a plausible origin of the tree recaptured swordfish. The Mediterranean swordfish have been described as different growth rates from the Atlantic (Tserpes and Tsimenides 1995). However, the difficulty to interpret the different individual growth patters in the hard parts of the Atlantic swordfish will require further studies. Based on studies of the individual patterns of each fish specimen with known origin it will be possible to formulate more consistent hypotheses regarding ring formation on the hard parts (spines) of swordfish. In general, by gathering information obtained from genetics and tagging-recapture experiments, we will be able to shed more light on different aspects of swordfish biology that remain largely unknown (i.e. reproductive biology, behavioural biology, migration etc.). In order to undertake this task, the tagging-recapture programs being carried out by the different fleets of the Atlantic (North and South) and the Mediterranean will need to be reinforced and the protocols will have to be fitted, intensified and expanded. In addition to the collection of the recommended traditional data during the recapture (size, geographical position, sex, etc.), in our opinion it would be advisable to take biological samples at least of the recaptures that are obtained of this species, samples that would include at least the muscle tissue and the hard parts used for growth (first anal fin). In order to do this, it will be necessary to provide guidelines to all the people involved in this process and to be able to rely on the collaboration of all the scientists and fleets catching swordfish. 1968

Acknowledgments The authors would like to thank Marta Ruiz for their collaboration on spines preparation and to the skippers and crew members of the Spanish surface longline fleet for their great help and collaboration in voluntarily tagging and reporting information as well as for their collaboration on this and other scientific tasks. Special thanks go out to all the staff involved in the IEO projects (SWOATL, SHKLL and SHKLL03) through which this research and other works were made possible. References ANONYMOUS. 2003. Report of the 2002 Atlantic Swordfish Stock Assessment Session (Madrid, September 9 to 13. 2002). Col. Vol. Sci. Pap. ICCAT, 55(4):1289-1415. ANONYMOUS. 2007. Report of the 2006 ICCAT Swordfish Stock Structure Workshop (Crete, Greece, 13-15 March, 2006). Col. Vol. Sci. Pap. ICCAT. SCRS/2006/015. ALVARADO BREMER, J.R., J. Mejuto, J. Gómez-Márquez, F. Boán, P. Carpintero, J. M. Rodriguez, J. Viñas, T. W. Greig and B. Ely. 2005. Hierarchical analysis of genetic variation of samples from breeding and feedding grounds confirm the genetic partitioning of the northwest Atlantic and South Atlantic populations of swordfish (Xiphias gladius L.). Journal of Experimental Marine Biology and Ecology 327:167-182. ALVARADO BREMER, J.R., J. Mejuto, J. Gómez-Márquez, J. Vinas and F. Boán. 1999. Hierarchical analysis of nucleotide diversity reveals extremely low levels of mitochondrial DNA gene flow between northeast Atlantic and Mediterranean swordfish populations. Col. Vol. Sci. Pap. ICCAT, 49(1): 467-475. BERKELEY, S.A. and E.D. Houde. 1983. Age determination of broadbill swordfish, Xiphias gladius, from the Straits of Florida, using anal fin spine sections. NOAA Technical Report NMFS, 8: 137-143. CHOW, S. and H. Takeyama. 2000. Nuclear and mitochondrial DNA analyses reveal four genetically separated breeding units of the swordfish. Journal of Fish Biology 56: 1087-1098. EHRHARDT, N.M., R.J. Robbins and F. Arocha. 1996. Age validation and growth of swordfish, Xiphias gladius, in the northwest Atlantic. Col. Vol. Sci. Pap. ICCAT, 45(2): 358-367. ESTEVES, E., P. Simoes, H.M. Da Silva and J.P. Andrade. 1995. Ageing of swordfish, Xiphias gladius Linnaeus, 1758, from the Azores, using sagittae, anal-fin spine and vertebrae. Arquipélago, Ciencias Biológicas e Marinhas, 13A: 39-51. GARCÍA-CORTÉS, B., J. Mejuto and M. Quintans. 2003. Summary of swordfish (Xiphias gladius) recaptures carried out by the Spanish surface longline fleet in the Atlantic Ocean. Col. Vol. Sci. Pap. ICCAT, 55: 1476-1484. GREIG, T.W., J.R. Alvarado Bremer and B. Ely. 2000. Nuclear markers provide additional evidence for population subdivision among Atlantic swordfish. Col. Vol. Sci. Pap. ICCAT, 51(5): 1637-1640. HANSEN, M.M., E. Kenchington and E.E. Nielsen. 2001. Assigning individual fish to population using microsatellite DNA markers. Fish and Fisheries 2: 93-112. KASAPIDIS, P., J. Mejuto, G. Tserpes, A. Antoniou, B. Garcia-Cortes, P. Peristeraki, K. Oikonomaki, G. Kotoulas and A. Magoulas. 2007. Genetic structure of the swordfish (Xiphias gladius) stocks in the Atlantic using microsatellite DNA analysis. ICCAT Swordfish Stock Structure Workshop (Crete, Greece, 13-15 March, 2006). Col. Vol. Sci. Pap. ICCAT. SCRS/2006/035. KOTOULAS, G., A. Magoulas, N. Tsimenides and E. Zouros. 1995. Marked mitochondrial DNA differences between Mediterranean and Atlantic populations of the swordfish, Xiphias gladius. Molecular Ecology 4: 473-481. 1969

KOTOULAS, G., J. Mejuto, A. Antoniou, P. Kasapidis, G. Tserpes, C. Piccineti, P. Peristeraki, B. Garcia- Cortes, K. Oikonomaki, J. M. De la Serna and A. Magoulas. 2007. Global genetic structure of the swordfish Xiphias gladius, as revealed by microsatellite markers. ICCAT Swordfish Stock Structure Workshop (Crete, Greece, 13-15 March, 2006). Col. Vol. Sci. Pap. ICCAT. SCRS/2006/034. PRITCHARD J.K., M. Stephens and P. Donnelly P. 2000. Inference of Population Structure Using Multilocus Genotype Data. Genetics, 155: 945 959 REEB, C.A., and B. Block. 2007. Ten microsatellite loci show that Mediterranean swordfish are genetically unique from other populations worldwide. ICCAT swordfish stock structure workshop (Crete, Greece, 13-15 March, 2006). Col. Vol. Sci. Pap. ICCAT. SCRS/2006/028. RESTREPO, V. 1990. An update of swordfish tagging data for use in growth analyses. Col. Vol. Sci. Pap. ICCAT, 32(2): 360-370. RIEHL, M.W. 1984. Age and growth estimation of northwest Atlantic broadbill swordfish, Xiphias gladius, using fin-spines. Thesis for Bachelor of Science with Honours in Biology, mount Allison University. 33pp. ROSEL, P. and B.A. Block. 1996. Mitochondrial control region variability and global population structure in the swordfish, Xiphias gladius. Marine Biology, 125: 11-22. SAMBROOK, J., E. Fritsch and T. Maniatis. 1989. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, New York. THORROLD, S.R., G.P. Jones, M.E. Hellberg, R. Burton, S.E. Swearer, J.E. Niegel, S.G. Morgan and R.R. Warner. 2002. Quantifying larval retention and connectivity in marine populations with artificial and natural markers. Bulletin of Marine Science 70: 291 308. TSERPES, G. and N. Tsimenides. 1995. Determination of age and growth of swordfish, Xiphias gladius L., 1758, in the eastern Mediterranean using anal-fin spines. Fishery Bulletin, 93(3): 594-602. 1970

Table 1. General data on tagged and recaptured swordfish used in this paper. Date: day/month/year. Sex: 1= male, 2= female. TAG Code HM057896 ES-10205 BF40729 Tagging location 47º68'N / 42º58'W 33º31'N / 19º55'W 18º73' N / 66º93' W Tagged date 08/09/2003 06/02/1996 23/12/1993 Tagged LJFL (cm) 114 65 96 Tagged round weight (kg) 15.8 3.0 20.0 Recapture location 41º43'N / 39º29'W 39º23'N / 19º20'W 41º47'N / 40º26'W Recapture date 04/05/2006 19/02/2000 05/05/1999 Recapture LJFL (cm) 150 150 141 Recapture round weight (kg) 38 38 37 Sex 2 1 1 Years at liberty 2.7 4.0 5.4 LJFL increment (cm) 36 85 45 Table 2. Sample name, geographical coordinates of samples, sample size (N), and proportion of membership of each pre-defined sample in each of the two clusters (Atlantic and Mediterranean) revealed by a Bayesian clustering approach, implemented in Structure v2.1. In each cluster only assigned individuals with a membership coefficient above 0.80 were considered, while those with lower coefficients were treated as unassigned. Sample name Coordinates N Atlantic cluster Mediterranean cluster Unassigned Northwest Atlantic (NWA) 30 to 48 º N / 31 to 50ºW 448 0.621 0.031 0.348 Northeast Atlantic (NEA) 30 to 45ºN / 05 to 31ºW 363 0.576 0.077 0.347 Mid-Atlantic (MIDA) 05ºS to 05ºN / 05 to 35ºW 167 0.596 0.055 0.349 Spanish Mediterranean (MED) 35 to 40ºN / 03ºW to 07ºE 393 0.003 0.847 0.150 Alboran Sea (ALB) 35 to 36ºN / 02 to 05ºW 64 0.078 0.734 0.188 Table 3. Estimated age for the present study from spine readings and estimated ages for LJFL from other studies in the Atlantic and Mediterranean Sea. Sex: 1= male, 2= female. TAG Code LJFL cm Sex Berkeley and Riehl Houde (1984) (1983) Ehrhardt (1990, 1992) Esteves et al (1995) Tserpes and Tsimenides (1995) Restrepo (1990)* HM057896 150 2 4 4-5 3-4 4 4 4 4 ES-10205 150 1 4 4 4 4 4-5 3-4 4 BF40729 141 1 3-4 3-4 3 3-4 3-4 3 6 * Tagging-recapture information. Present study 1971

45 o ME 30 o NW NE ALB 15 o 0 o -15 o MID -60 o -45 o -30 o -15 o 0 o Figure 1. Geographic locations of the samples used for the genetic analysis. The size of the grey circles is proportional to the number of individuals collected in 5 x 5 degrees squares. Pies represent the proportion of membership of each pre-defined population in each of the two clusters (Atlantic= white, Mediterranean= black, unassigned=grey), revealed by Structure v2.1.arrows yellow, red and green indicate each tagged-recaptured fish. 1972

SWO HM057896 SWO ES10205 SWO BF40729 Figure 2. Sections of three second anal fin of the three tagged and recaptured swordfish. Annuli interpretation used to estimate the age of this fish are shown. 1973