Population Structure of the Thornback Ray (Raja Clavata L.) in British Waters ⁎ Malia Chevolot A, , Jim R

Journal of Sea Research 56 (2006) 305–316 www.elsevier.com/locate/seares

Population structure of the thornback ray (Raja clavata L.) in British waters ⁎ Malia Chevolot a, , Jim R. Ellis b, Galice Hoarau a, Adriaan D. Rijnsdorp c, Wytze T. Stam a, Jeanine L. Olsen a a Department of Marine Benthic Ecology and Evolution, Center for Ecological and Evolutionary Studies, University of Groningen, P.O. Box 14, 9750 AA Haren, The Netherlands b Centre for Environment Fisheries and Aquaculture Sciences (CEFAS), Lowestoft Laboratory, Pakefield Road, Lowestoft, Suffolk NR33 0HT, UK c Wageningen Institute for Marine Resources and Ecological Studies (IMARES), Animal Sciences Group, Wageningen UR, P.O. Box 68, 1970 AB IJmuiden, The Netherlands Received 27 July 2005; accepted 24 May 2006 Available online 17 June 2006

Abstract

Prior to the 1950s, thornback ray (Raja clavata L.) was common and widely distributed in the seas of Northwest Europe. Since then, it has decreased in abundance and geographic range due to over-fishing. The sustainability of ray populations is of concern to fisheries management because their slow growth rate, late maturity and low fecundity make them susceptible to exploitation as victims of by-catch. We investigated the population genetic structure of thornback rays from 14 locations in the southern North Sea, English Channel and Irish Sea. Adults comprised <4% of the total sampling despite heavy sampling effort over 47 hauls; thus our results apply mainly to sexually immature individuals. Using five microsatellite loci, weak but significant population differentiation was detected with a global FST = 0.013 (P < 0.001). Pairwise Fst was significant for 75 out of 171 comparisons. Although earlier tagging studies suggest restricted foraging distances from home areas, the absence of genetic differentiation between some distant populations suggests that a substantial fraction of individuals migrate over wide areas. Autumn/winter locations appear to have a lower level of differentiation than spring/summer, which could be due to seasonal migration. Management and conservation of thornback ray populations will be challenging as population structure appears to be dynamic in space and time. © 2006 Elsevier B.V. All rights reserved.

Keywords: Elasmobranchs; Genetic structure; Microsatellite; Thornback ray; Rajidae

1. Introduction Africa (Stehmann and Bürkel, 1994), though their taxonomic status in these waters is unclear. Around Thornback ray, Raja clavata, is a widely distributed the British Isles, thornback rays are most abundant in skate (Rajiformes: Rajidae) in the eastern Atlantic, coastal waters and large bays, including The Wash, ranging from Norway and Iceland to Northwest Africa, Outer Thames Estuary, Solent, Carmarthen Bay, Cardi- including the Mediterranean and Black Seas. They may gan Bay, Liverpool Bay and Solway Firth (Ellis et al., also occur in the Atlantic and Indian Oceans of southern 2005). Prior to the 1950s, thornback rays were common and widespread in the North Sea, but have declined ⁎ Corresponding author. thereafter in distribution and abundance (Walker and E-mail address: [email protected] (M. Chevolot). Heessen, 1996; Walker and Hislop, 1998).

Thornback rays are targeted in inshore longline and be rare, as well as between the English and French coasts gillnet fisheries in coastal waters of the British Isles, and across the English Channel. It was hypothesised that this are also an important component of the mixed demersal separation was probably due to the deeper waters and trawl fisheries. Despite R. clavata being one of the most strong currents in the Dover Strait (Walker et al., 1997). commercially valuable rays, the economic value of their Taken together, tagging studies suggest that R. clavata total catch is small in comparison to other demersal may have restricted ranges and potentially strong species (e.g. sole and plaice) and as such they have not localised population differentiation-particularly among been the focus of fisheries management. Between 1956 younger individuals. and 1995, R. clavata disappeared from the waters along To date, only a few population genetic studies based on the continental coasts of the North Sea and showed an nuclear markers have been conducted on elasmobranchs, overall decline of nearly 80% in this area (Walker and and all of these have been on sharks. Heist and Gold Heessen, 1996). Similarly, they are thought to have (1999) found no differentiation (three microsatellite loci) declined by approximately 45% between 1988 and 1997 in population structure of sandbar sharks (Carcharinus in the Irish Sea (Dulvy et al., 2000). This rapid decrease plumbeus) between the mid-Atlantic Bight and the has led to concern about the sustainability of ray western Gulf of Mexico. Schrey and Heist (2003) populations (Walker et al., 1997; Dulvy et al., 2000), observed evidence of population differentiation (four especially given their low fecundity, slow growth, and microsatellite loci) in shortfin mako shark (Isurus late maturity, which makes them potentially vulnerable oxyrinchus) between the North Atlantic and North Pacific. to exploitation (Brander, 1981; Heist, 1999). Feldheim et al. (2001) found weak but significant The fecundity of R. clavata is thought to be in the population structure (four microsatellite loci) in the range of 48–150 eggs per year (Holden, 1975; Ryland lemon shark (Negaprion brevirostris) in the western and Ajayi, 1984; Ellis and Shackley, 1995). Following Atlantic Ocean. Keeney et al. (2005) also found a weak mating in early spring, eggs are deposited regularly, with but significant population structure (8 microsatellite loci the spawning season occurring between February and and control region of the mtDNA) in the blacktip shark September (Holden, 1975), though the egg-laying (Carcharinus limbatus) between nine nursery areas periods of individuals may be more restricted (Holden (Northwestern Atlantic, Eastern, Western Gulf of Mexico, et al., 1971; Ellis and Shackley, 1995). Fertilised eggs North Yucatan and Belize). In studies where nuclear and are deposited as egg cases on the seabed and, after 4– mitochondrial markers were used, evidence for male- 5 months incubation, fully formed rays (ca. 8 cm disc biased dispersal and female philopatry has been suggested width) hatch (Ellis and Shackley, 1995). Tagging studies for white shark Carcharodon carcharias (Pardini et al., have shown that juveniles tend to remain in shallow 2001), shortfin mako (Schrey and Heist, 2003), lemon waters (10–30 m deep) for several years, with shark (Feldheim et al., 2001) and blacktip shark (Keeney recaptured individual close to the point of release et al., 2005). Only one population structure study has been (Walker et al., 1997). conducted on a Rajidae by Blake (1976) on thornback R. clavata matures at between 9 and 12 y of age rays in the Irish Sea using a eye-lens protein. No (Nottage and Perkins, 1983). At this stage, adults show differentiation was found, probably due to the lack of seasonal movements, from deeper waters (10 to 30 m) in polymorphism of the allozyme. winter, to shallower waters (<10 m) in spring, where they Here, we investigate the population genetic structure are presumed to mate and spawn (Walker et al., 1997). of R. clavata in the southern North Sea, English Despite these seasonal movements, past tagging experi- Channel, and Irish Sea using five microsatellite loci. ments indicated that 100% of the individuals remained The objectives were to determine the spatial scale of R. within a 60 nautical miles (nmi) area and 80% remained clavata population differentiation, assess the home within 20 nmi. This ‘home range’ has since been extended range based on assignment tests, and to compare to a maximum travelling distance of 70 nmi from studies migratory movements based on genetic data with using Data Storage Tags (DSTs) (Hunter et al., 2005b). those based on earlier tagging data. Their study also confirmed the seasonal movement pattern of thornback for mature and immature individuals 2. Material and methods (30–60 cm total length), and showed a philopatric behaviour as most individual rays were found in the 2.1. Sampling and DNA extraction same area during spring/summer time every year (Hunter et al., 2005a). Wider-ranging migrations between the A total of 483 immature rays were sampled from 14 southern North Sea and the English Channel appeared to locations during standard, annual bottom trawl surveys M. Chevolot et al. / Journal of Sea Research 56 (2006) 305–316 307 conducted by various fisheries institutes from 2002 to was determined using internal lane standard (GENES- 2004 (Table 1). Five out of the 14 locations were CAN™ 350 ROX™) and GENESCAN™ software. sampled at different time period (Fig. 1, Table 1). Adults were only rarely represented in the sampling (N = 20) 2.3. Data analysis and, therefore, not included in the population structure analysis. Individuals were classified as adult or The software package MICRO-CHECKER 2.2.1 (Van immature based on reproductive criteria, i.e., presence Oosterhout et al., 2004) was used to check for of fully differentiated shell glands for females and fully genotyping errors due to null alleles, stuttering, and developed testes and claspers for males (Stehmann, large allele drop-out for all loci and all locations. 1995). If reproductive data were not available, total Allelic diversity was calculated and corrected for length (<55 cm for immature) was substituted. sample size using GENCLONE β version (Arnaud-Haond Sampling was not easy as any single haul (fishing and Belkhir, available on request). In order to compare for 30 min at 4 nmi/h) provided only a small number of diversity among populations of different sample sizes, individuals (mean=4.7 individuals/haul, SD=5.8). the program performs a jacknife re-sampling of Single hauls producing >20 individuals occurred only individuals at each location. For each re-sampling, the at locations Ow-02, Tel-03, Te1-04, Ste-02. For all number of alleles is counted for a sample size from one others, pooling of hauls was necessary. Pooling rules individual to Nmax, Nmax being the maximum number of were guided by tagging studies which suggest that individuals in the location. Then, the mean allelic individuals generally remain within a 20-nmi area. diversity over all re-sampling procedures is calculated Using a conservative approach, we pooled hauls that for 1 to Nmax individuals. We estimated the allelic were close together, i.e., <7nmi apart. Exceptions were diversity with 1000 re-sampling procedures and cor- Lb-03, where hauls were <15 nmi and LyB-03 where rected for N = 16 individuals (smallest sample size, hauls came from same bay (<25 nmi) (Table 1). Table 1). Muscle tissue was collected from each individual and GENETIX 4.05 (Belkhir et al., 2004) was used for the preserved in 70% ethanol. Total genomic DNA was following calculations: Linkage disequilibrium for all extracted using either a modified CTAB (Hoarau et al., loci and for all locations using the LinkDis procedure 2002) or a silica-based extraction protocol (Elphinstone (Black and Krafsur, 1985); Observed (Hobs) and non- et al., 2003). biased expected (Hexp) heterozygosities (Nei, 1978); allelic richness for each locus individually and as a 2.2. Genotyping multilocus estimate per location; and single and multilocus FIS estimates using Weir and Cockerham f All individuals were genotyped for five microsatel- estimator (1984). Significance was tested against 3000 lite loci as described in Chevolot et al. (2005). PCR permutations. amplifications were performed in a 10-μL total volume Population differentiation was assessed using containing 1–3 μL (<1 ng) of extracted DNA, 1× Wright's FST (Wright, 1969) rather than Slatkin's RST reaction Buffer (Promega), 0.2 mM of each dNTP, 0.25 (Slatkin, 1995) because the latter estimate tends to have U Taq DNA polymerase (Promega), 1.5–3 mM MgCl2 a higher variance with fewer than 20 loci (Gaggiotti et and 0.4–0.66 μM primers. For each primer set, the al., 1999; Neigel, 2002). Global and pairwise FST was forward primer was fluorescently end-labelled with estimated using the Weir and Cockerham (1984) θ FAM or HEX. PCR amplifications were performed estimator with GENETIX 4.05 (Belkhir et al., 2004) and with either a PTC-100™ thermocycler (MJ Research, significance was tested against 3000 permutations. Inc.) or Mastercycler gradient cycler (Eppendorf). PCR Global θ among autumn/winter locations was estimated conditions were: initial denaturation for 1 min at 94 °C; as well as between spring/summer locations to test for followed by four cycles of: denaturation for 1 min at possible temporal differentiation between spring/sum- 94 °C, annealing at 52–60 °C for 1 min, and extension mer locations compared with autumn/winter locations. at 72 °C for 30 s, then 30 to 35 cycles of denaturation at Isolation by distance was tested using a Mantel test 94 °C for 20 s, annealing at 52–60 °C for 15 s and (Mantel, 1967) as implemented in GENETIX 4.05 with θ/ extension at 72 °C for 12 s, followed by a final (1-θ) as genetic distances and the log of geographical extension step at 72 °C for 10 min. distance (Rousset, 1997). Significance was tested PCR products were separated on a 6% polyacryla- against 10000 permutations. mide gel and visualised with an ABI Prism-377 A first-generation-migrants detection analysis was automatic sequencer (Applied Biosystems). Allele size performed using GENECLASS 2.0 (Cornuet et al., 1999; 308 M. Chevolot et al. / Journal of Sea Research 56 (2006) 305–316

Table 1 Sampling locations for Raja Clavata Locations (see Fig. 1) Code Sampling date Sources Lat Long Maximum Nr of hauls N b (° dec) (° dec) depth a (m) pooled Outer Wash Ow-02 Feb 2002 IBTS c 53.50 0.67 92 0 23 North Thames Estuary Nte2-02 April 2002 RIVO d 52.06 2.14 40 4 54 52.09 2.01 52.02 2.00 52.08 2.01 North Thames Estuary Nte1-03 July 2003 CEFAS e 52.15 1.70 20 5 25 52.26 1.69 52.29 1.77 52.36 1.75 52.48 1.85 Thames Estuary1 Te1-03 Feb 2003 IBTS 51.77 1.77 27 0 35 Thames Estuary1 Te1-04 Feb 2004 CEFAS 51.77 1.78 31 0 25 Thames Estuary2 Te2-03 July2003 CEFAS 52.13 1.73 24 2 21 51.69 1.37 Thames Estuary2 Te2-04 Aug 2004 CEFAS 51.68 1.44 24 2 21 51.72 1.28 South Thames Estuary Ste-02 Jan 2002 IBTS 51.72 1.75 26 0 33 East English Channel Eec-03 Nov 2003 IFREMER f 49.99 1.13 21 2 43 49.99 1.18 East English Channel Eec 04 July 2004 CEFAS 50.04 1.24 27 2 21 49.97 1.00 English Channel 1 Ec1-03 July 2003 CEFAS 50.70 −0.05 24 2 18 50.63 0.10 English Channel 2 Ec2-02 April 2002 RIVO 50.75 0.67 30 2 19 50.77 0.73 Lyme Bay Lyb-03 Sept 2003 CEFAS 50.24 −2.70 32 7 18 50.60 −3.11 50.55 −3.18 50.44 −3.31 50.24 −3.20 50.51 −3.44 50.52 −3.48 Carmarthen Bay Cb-03 Sept 2003 CEFAS 51.69 −4.48 24 3 16 51.60 −4.39 51.66 −4.54 Carmarthen Bay Cb-04 Sept 2004 CEFAS 51.57 −4.47 24 4 33 51.58 −4.34 51.66 −4.44 51.69 −4.63 51.67 −4.54 Tremadog Bay Tb-03 Sept 2003 CEFAS 52.33 −4.35 35 3 18 52.63 −4.29 52.72 −4.51 Caernarfon Bay Caeb-03 Sept 2003 CEFAS 53.15 −4.65 42 2 18 53.15 −4.60 Caernarfon Bay Caeb-04 Nov 2004 CEFAS 53.14 −4.65 46 0 16 Liverpool Bay Lb-03 Sept 2003 CEFAS 53.58 −3.36 37 3 25 53.62 −3.62 53.64 −3.30 Locations sampled during autumn/winter time are underlined; and locations sampled during spring/summer are not. a Maximum depth among pooled hauls. b Number of individuals per locations. c International Bottom Trawl Survey. d Netherlands Institute for Fisheries Research. e Center for Environment Fisheries and Aquaculture Sciences. f French Institute for Sea Research. M. Chevolot et al. / Journal of Sea Research 56 (2006) 305–316 309

Fig. 1. Sampling locations for Raja clavata. Locations sampled during autumn/winter time are underlined; and locations sampled during spring/ summer are not. The numbers represent the sampling year.

Piry et al., 2004) to estimate the ‘real-time’ migration The mean number of alleles per locus ranged from rate. For each individual the likelihood of being a 5.29 (Locus Rc-B3) to 21.3 (Locus Rc-B6) (Table 2). resident (i.e. born in the sampling location) or a The mean allelic richness per location was corrected for migrant from another reference population was sample size (N = 16) and varied from 7.9 to 10.4 with a estimated using the bayesian method of Rannala and mean of 9.1 (Table 2). The mean expected hetero- Mountain (1997). A Monte Carlo re-sampling with zygosity was relatively uniform among samples, 10000 simulated individuals following Paetkau et al. ranging from 0.678 to 0.763 (Table 2). Only locations (2004) simulation algorithm. The principle is to Nte2-02 and Lb-03 showed a significant departure from approximate the distribution of multilocus genotype Hardy-Weinberg equilibrium after sequential Bonfer- likelihoods in each reference population and then roni corrections (Table 2) (multilocus f = 0.089 and 0.12 compare the likelihood of the to-be-assigned indivi- respectively; corrected P= 0.0003 and 0.02). dual to that distribution. If the individual likelihood is inside the 95% likelihood distribution, then the null 3.2. Genetic differentiation hypothesis is accepted, i.e., that the individual is a resident. The global genetic differentiation among the locations was low (θ = 0.0126) but highly significant (P< 3. Results 0.001). Each of the five loci independently showed significant genetic differentiation (from θ=0.004 for B4 3.1. Data quality and genetic diversity to θ = 0.04 for B3). Subsequent pairwise multilocus θ estimates, without Bonferroni corrections, were signifi- None of the five microsatellite loci showed evidence cant in 75 out of 171 pairwise comparisons (Table 3) but of genotyping errors due to null alleles, stuttering or no clear pattern with geographic distance/nearest large allele drop-out. Pairwise comparisons between loci neighbour was observed to explain this differentiation. revealed no linkage disequilibrium after sequential With Bonferroni corrections, none of the pairwise com- Bonferroni corrections (Rice, 1989). parisons remained significant. Given the high number of 310

Table 2 Summary statistics of the genetic variability for five microsatellite loci at 19 sampling locations for Raja clavata

Locus Locations Ow-02 Nte2-02 Nte1-03 Te1-03 Te1-04 Te2-03 Te2-04 Ste-02 Eec-03 Eec-04 Ec2-02 Ec1-03 LyB-03 Cb-03 Cb-04 Tb-03 CaeB-03 CaeB-04 Lb-03 Mean NA/locus

Rc-B3 NA 4 5 4 6 5 3 2 5 6 4 4 5 6 5 5 5 4 3 6 5.29 305 (2006) 56 Research Sea of Journal / al. et Chevolot M. Hexp 0.602 0.496 0.552 0.544 0.617 0.537 0.514 0.725 0.617 0.498 0.627 0.593 0.693 0.622 0.584 0.595 0.635 0.632 0.558 Hobs 0.636 0.429 0.458 0.542 0.5 0.5 0.333 0.636 0.561 0.429 0.555 0.613 0.632 0.429 0.438 0.53 0.556 0.6 0.4 f −0.06 0.138 0.173 0.003 0.193 0.07 0.358 0.123 0.09 0.142 0.116 0.189 0.09 0.318 0.253 0.113 0.128 0.05 0.287 Rc-B4 NA 14 30 19 19 22 21 17 17 24 20 19 17 16 14 22 17 20 15 18 18.8 Hexp 0.886 0.940 0.94 0.938 0.958 0.957 0.92 0.912 0.938 0.952 0.95 0.94 0.935 0.938 0.9531 0.951 0.962 0.934 0.942 Hobs 0.739 0.87 0.875 0.914 0.91 1.000 0.81 0.939 0.954 0.857 1 0.944 0.78 0.875 0.933 0.833 0.944 0.938 0.826 f 0.169 0.08 0.07 0.026 0.053 −0.046 0.122 −0.031 −0.017 0.102 −0.054 −0.005 0.159 0.069 0.02 0.127 0.019 −0.004 0.126 Rc-B6 NA 19 24 18 25 18 21 17 23 27 16 22 18 20 21 21 18 21 16 24 21.3 Hexp 0.935 0.913 0.921 0.928 0.907 0.936 0.912 0.904 0.911 0.914 0.955 0.941 0.942 0.938 0.899 0.95 0.925 0.899 0.935 Hobs 0.957 0.865 0.917 0.886 0.84 0.952 0.85 0.97 0.977 0.8 0.947 1 0.947 0.938 0.9394 1 1 0.938 0.875 f −0.023 0.053 0.005 0.046 0.07 −0.018 0.06 −0.075 −0.073 0.127 0.008 −0.064 −0.006 0.000 −0.04 −0.055 −0.083 −0.04 0.066 Rc-E9 NA 6 8 6 6 4 5 4 7 5 5 5 6 7 3 5 6 3 3 3 5.43 Hexp 0.534 0.463 0.385 0.35 0.472 0.533 0.35 0.381 0.363 0.41 0.543 0.7 0.523 0.502 0.637 0.544 0.418 0.394 0.367 Hobs 0.609 0.389 0.32 0.314 0.458 0.429 0.3 0.375 0.325 0.47 0.632 0.667 0.473 0.438 0.54 0.625 0.389 0.462 0.333 f −0.143 0.161 0.171 0.103 0.03 0.2 0.146 0.016 0.105 −0.16 −0.168 0.049 0.09 0.132 −0.02 −0.154 0.07 −0.18 0.093 Rc-G2 NA 5 9 5 7 6 7 5 6 7 5 5 7 7 4 6 6 5 5 7 6.21 Hexp 0.691 0.677 0.598 0.694 0.74 0.483 0.691 0.638 0.641 0.653 0.666 0.606 0.724 0.635 0.617 0.638 0.674 0.587 0.66 Hobs 0.696 0.63 0.76 0.629 0.68 0.429 0.857 0.576 0.651 0.667 0.79 0.611 0.632 0.625 0.606 0.833 0.647 0.625 0.64 f −0.007 0.07 −0.279 0.095 0.08 0.115 −0.2 0.099 −0.016 −0.02 −0.192 −0.008 0.13 0.016 0.02 −0.318 0.041 −0.06 0.034 Mean NA 9.6 15.2 10.4 12.6 11 11.4 9 11.6 13.8 10 11 10.6 11.4 8.8 11.8 10.4 10.4 8.4 10.6 10.9 Mean Nc 8.3 9.2 8.6 9 8.9 9.8 7.9 8.7 8.8 8.9 10.1 10 10.4 8.8 8.8 9.8 9.9 8.4 9.2 9.1

Multilocus H 0.730 0.698 0.679 0.690 0.739 0.689 0.678 0.712 0.694 0.685 0.748 0.760 0.763 0.726 0.738 0.735 0.723 0.689 0.693 – exp 316 Multilocus f 0.003 0.089 0.019 0.049 0.080 0.040 0.070 0.018 0.000 0.060 −0.050 0.020 0.090 0.090 0.030 −0.040 0.020 −0.030 0.120 Underlined locations were sampled during autumn/winter. NA = Number of alleles, Nc = Number of allele corrected for sample size (N = 16 individuals), Hexp = Non-biased expected heterozygosity (Nei, 1978), Hobs = Observed heterozygosity, f = Inbreeding coefficient (Weir and Cockerham, 1984). Significant P-values are shown in bold. Only Nte2-02 and Lb-03 showed a significant heterozygote deficiency (corrected P = 0.0003 and P = 0.02 respectively) after sequential Bonferroni corrections. Table 3 Population differentiation among the sampling locations .Ceoo ta./Junlo e eerh5 20)305 (2006) 56 Research Sea of Journal / al. et Chevolot M. Pop Ow-02 Nte2-02 Nte1-03 Te1-03 Te1-04 Te2-03 Te2-04 Ste1-02 Eec-03 Eec-04 Ec2-02 Ec1-03 LyB-03 Cb-03 Cb-04 Tb-03 Cae-03 Cae-04 Lb-03 Ow-02 – −0.002 0.019 0.003 0.009 0.018 0.008 0.018 0.006 0.008 −0.001 0.020 0.006 −0.006 0.010 −0.005 0.004 0.009 0.011 Nte2-02 0.63 – 0.027 −0.001 0.015 0.017 0.013 0.028 0.006 −0.001 0.005 0.032 0.019 −0.005 0.009 −0.003 0.009 0.006 0.009 Nte1-03 0.02 0.001 – 0.034 0.028 −0.001 0.005 0.017 0.015 0.043 0.003 0.014 0.013 0.023 0.032 0.007 0.022 0.004 0.034 Te1-03 0.23 0.54 <0.001 – 0.016 0.027 0.015 0.019 0.004 −0.005 0.014 0.046 0.016 −0.002 0.013 0.010 0.005 0.010 0.013 Te1-04 0.11 0.01 0.003 0.016 – 0.027 0.003 0.025 0.003 0.013 0.011 0.027 0.003 0.001 0.012 0.021 0.018 0.018 −0.000 Te2-03 0.02 0.013 0.5 0.003 0.006 – 0.009 0.024 0.014 0.027 0.003 0.004 0.015 0.007 0.013 0.006 0.015 −0.002 0.028 Te2-04 0.15 0.03 0.22 0.02 0.28 0.15 – 0.01 0.003 0.024 0.010 0.022 0.001 0.004 0.026 0.014 0.014 0.016 0.011 Ste1-02 0.007 <0.001 0.013 0.002 0.003 0.004 0.05 – 0.016 0.025 0.019 0.022 0.001 0.014 0.028 0.021 0.026 0.008 0.027 Eec-03 0.14 0.06 0.012 0.15 0.24 0.027 0.26 0.005 – 0.006 0.009 0.027 0.001 0.001 0.016 0.013 0.02 0.007 0.000 Eec-04 0.13 0.57 0.001 0.81 0.06 0.007 0.012 0.001 0.15 – 0.015 0.042 0.020 −0.004 0.004 0.014 0.008 0.005 0.004 Ec2-02 0.49 0.15 0.29 0.023 0.09 0.3 0.11 0.007 0.08 0.037 – 0.014 0.011 −0.002 0.008 −0.001 0.004 0.004 0.017 Ec1-03 0.02 0.001 0.058 <0.001 0.017 0.25 0.036 0.01 0.003 <0.001 0.07 – 0.007 0.006 0.015 0.014 0.032 0.011 0.036 LyB-03 0.19 0.008 0.05 0.022 0.32 0.005 0.40 0.38 0.38 0.014 0.09 0.2 – 0.003 0.018 0.010 0.022 0.011 0.002 Cb-03 0.7 0.75 0.025 0.54 0.37 0.21 0.29 0.058 0.39 0.63 0.52 0.26 0.33 – −0.008 −0.005 −0.001 −0.003 0.002 Cb-04 0.06 0.02 0.001 0.016 0.055 0.004 0.006 < 0.001 0.002 0.21 0.12 0.037 0.023 0.71 – 0.010 0.008 0.001 0.021 Tb-03 0.75 0.65 0.15 0.06 0.017 0.17 0.07 0.008 0.04 0.038 0.94 0.05 0.11 0.83 0.07 – 0.004 0.002 0.015 Cae-03 0.23 0.07 0.015 0.18 0.023 0.02 0.057 0.003 0.004 0.15 0.27 0.003 0.01 0.49 0.15 0.25 – 0.009 0.030 –

Cae-04 0.14 0.17 0.25 0.07 0.047 0.5 0.067 0.15 0.14 0.25 0.28 0.14 0.11 0.55 0.40 0.34 0.14 0.018 – Lb-03 0.077 0.06 <0.001 0.022 0.43 0.006 0.10 0.002 0.44 0.26 0.03 0.003 0.34 0.35 0.009 0.046 0.003 0.048 – 316 The upper right matrix shows the pairwise multi-locus θ estimates (Weir and Cockerham, 1984). The lower left matrix shows P-values (after 3000 permutations). Significant P-values are shown in bold. Underlined locations were sampled during the winter. 311 312 M. Chevolot et al. / Journal of Sea Research 56 (2006) 305–316 comparisons (N = 171), a Bonferroni correction requires seemed to be more differentiated between them than an α = 0.00029, which in combination with small sample autumn/winter locations. size, increases type II errors and is, therefore, overly Results from the first-generation-migrants detection conservative. For these reasons, the application of analysis (Table 4) showed that about 80% (67–95%) of Bonferroni corrections to natural population studies is the individuals were residents (i.e. born in the sampling highly controversial (Bland and Altman, 1995; Perneger, locations). Individuals detected as migrants were not 1998; Moran, 2002; Garcia, 2003). The Mantel test for necessarily from neighbouring locations. A Kolmo- isolation by distance showed no significant correlation gorov-Smirnoff non-parametric test was performed to between genetic and geographic distances (P > 0.1). test for significant differences in the number of residents Five of the locations were sampled a second time to between spring/summer locations and autumn/winter test for temporal stability between years or between locations. No significance was detected (P >0.1)between seasons. Only location Te1 sampled during winter time sampling seasons. In order to check whether migrants in 2003 and 2004 showed a significant difference might have included individuals reaching maturity earlier between the two sampling years. The other locations (i.e., young adults), we made an a posteriori comparison Te2, Eec, Cb and Cae did not show significant of length class distribution between migrants and differences between the two years or the two seasonal residents using a Mann and Whitney ranking test. sampling periods (Table 3). Again, there was no significant difference (results not The global θ value among autumn/winter locations shown). The 20 adult samples (representing <4% of the (i.e. sampled between November and February) was 0.01 total catch) also did not assign to the areas where they and highly significant (P < 0.0001). If Ste-02 was were actually caught (results not shown). removed (which is an outlier as it is highly different from almost all locations), the global θ dropped to 0.006, but 4. Discussion remained significant at P = 0.01. The global θ value among spring/summer locations (i.e. sampled between Population structure is typically weak in most April and September) was higher (θ = 0.014) and highly commercial marine fishes (Waples, 1998). For baseline significant (P < 0.0001). Spring/summer locations characterisation, it is generally recommended that at ca.

Table 4 Results of first generation migrants detection using the Rannala and Mountain (1997) Bayesian approach Locations Ow- Nte2- Nte1- Te1- Te1- Te2- Te2- Ste- Eec- Eec- Ec2- Ec1- LyB- Cb- Cb- Tb- Cae- Cae- Lb- %of 02 02 03 03 04 03 04 02 03 04 02 03 03 03 04 03 03 04 03 migrants Ow-02 18 1 1 1 2 1 1 21.7 Nte2-02 0 47 1 1 1 2 1 1 12.9 Nte1-03 22 11112 Te1-03 1 30 1 1 1 1 14.3 Te1-04 22 11 1 12 Te2-03 15 1 2 1 1 1 28.6 Te2-04 1 2 16 1 1 23.8 Ste-02 1 1 29 1 1 12.1 Eec-03 2 1 38 1 1 1 11.6 Eec-04 18 1 1 1 14.3 Ec2-02 1 1 14 1 1 1 26.3 Ec1-03 1 13 1 1 2 27.8 LyB-03 1 1 1 1 15 21.1 Cb-03 1 14 1 12.5 Cb-04 1 1 1 30 9.1 Tb-03 17 1 5.6 Cae-03 1 1 1 12 2 1 33.3 Cae-04 1 1 2 12 25 Lb-03 1 1 1 1 21 16 Percentage of 78.3 87.1 78 85.7 78 71.4 76.2 87.9 88.4 85.7 73.7 72.2 78.9 87.5 90.9 94.4 66.7 75 84 residents Migrants denote proportion of individual within a sampling site for which the probability to be a resident was rejected at α = 0.05 after Paetkau et al. (2004) Monte-Carlo resampling method with 10000 simulated individuals. In bold are the number of individuals detected as residents. Underlined locations were sampled in winter time. M. Chevolot et al. / Journal of Sea Research 56 (2006) 305–316 313

50 adults/haul be sampled on the spawning grounds to population structure between rays and other demersal avoid large sampling variance (Waples, 1998). This is fishes, and the apparent spatial and temporal dynamics simply not possible for Raja clavata where adults of its population structure is probably due to behaviour. accounted for only 4% of the catch over 47 hauls. Even A wide variety of reproductive behaviours occur in with conservative pooling of hauls (or no pooling), the elasmobranchs, though detailed information about the number of individuals/location averaged around 25 social and mating behaviour of R. clavata is unknown. (range 16–54). A power analysis based on 124 total The few anecdotal observations that exist suggest that alleles from the five available loci indicated that sample some females remain in shallow waters most of the year sizes of around 30 were adequate to capture the overall while others migrate between in- and offshore areas allelic diversity (data not shown)—a condition just (Walker et al., 1997; Hunter et al., 2005a,b). The degree barely met in our study. The finding of significant to which such behaviour is related to feeding prefer- population structure in R. clavata is, therefore, likely to ences, niche partitioning or social/reproductive factors, be slightly underestimated at the pairwise level remains unknown. especially as two loci show very high levels of Thornback rays have been described as philopatric polymorphism, thus decreasing the power to detect species, mostly remaining in a small area (Holden, differentiation among samples due to size homoplasy 1975; Walker et al., 1997; Dulvy and Reynolds, 2002; (Hedrick, 1999; O'Reilly et al., 2004). This means that Whittamore and McCarthy, 2005), with a few tagged our ability to detect population structure is relatively individuals being found several hundred kilometres weak and appropriate caution should be exercised in from their release point (Walker et al., 1997; Hunter et interpreting the results. With these caveats in mind, al., 2005b). Our results clearly show that immature rays however, the most important general findings in the are ranging further than previously reported. The present study are that: (1) weak but significant global mismatch between tagging and genetic data is most population structure of thornback rays can be detected likely related to differences in the timeframe between even though subsequent pairwise θ estimates (with or the two types of data. Tagging data involve between 100 without Bonferroni corrections) show no obvious and 1000 individuals tracked for about 4 y, only one correlations with oceanographic current systems, bot- third to one half of a generation time. In contrast, genetic tom topography, isolation by distance or physical barrier data integrate several generations over a period of 100 to such as the Dover Strait; (2) population structure is 1000 y as population structure is the result of past and dynamic in space and time as annual and seasonal present processes (Hewitt, 1996). Given that the differences were detected in some cases. immature period lasts a decade (Nottage and Perkins, Most commercially important telosts have Type III 1983), longer-term monitoring and recovery of tags will life histories, characterised by high fecundity, external be necessary to accurately determine home ranges. fertilisation and a passive pelagic larval stage. This Thus, the use of genetic and tagging data provide leads to the prediction of large-scale panmixis and complementary sources of information on movement population structure that is shaped by oceanic currents, and migration pattern, which are necessary to integrate bathymetry and isolation by distance (for example, see short- and long-term movements for conservation and, Rocha-Olivares and Vetter (1999) on the rose thorn in the special case of thornback rays, to better under- rock fish, Pogson et al. (2001) and Ruzzante et al. stand the spatial and temporal dynamics of population (1998) for the Atlantic cod). In contrast, elasmobranchs structure in relationship to behaviour. are characterised by low fecundity, low juvenile The difference in the degree of population structure mortality, internal fertilisation and no passive pelagic between seasons suggests that winter/ autumn locations larval phase. Eggs are benthic and attached to the may be admixed: juveniles from different populations substrate (Holden, 1975). Young-of-the-year can gathered in a foraging area, whereas spring/summer actively swim, so that stock structure might be shaped locations are more isolated due to partial philopatry. more by individual or group behaviour leading to the This seasonal migration pattern has already been prediction of more localised population units. Although described with DST (Hunter et al., 2005a,b). Theoreti- population differentiation was not found at a local cally, admixed populations should show a Wahlund scale, as predicted, differentiation is detectable at a effect, i.e. a heterozygote deficiency due to pooling regional scale (British waters, hundreds of kilometres); individuals from different populations. None of the a smaller scale than for teleosts (usually thousands of locations showed a significant deficit in heterozygote kilometres) (Borsa et al., 1997; Hoarau et al., 2002; except for Nte2-02 and Lb-03 which is more likely due Nielsen et al., 2004). The difference in the scale of to the pooling of hauls. Again, we caution that a failure 314 M. Chevolot et al. / Journal of Sea Research 56 (2006) 305–316 to detect a Wahlund effect could be related to small individuals are often crucial to the maintenance of sample size (Chakraborty and Zhong, 1994). stocks due to both greater fecundity and higher-quality Although the results of the migrant detection analysis larvae (Heino and Godo, 2002; Heino et al., 2002; Grift should be treated very carefully (Rannala and Mountain, et al., 2003; Olsen et al., 2005). 1997), they support the general premise that most In conclusion, studying population structure of immature rays remain in their natal areas, but that some thornback rays will remain challenging-primarily in immature rays migrate to other locations, which relation to sampling requirements, which are difficult to explains the low level of genetic differentiation. There- overcome even with extra sampling effort and more loci. fore, given the apparent complex population differentia- Genetically based estimates of effective population size tion pattern associated with spatial and temporal and additional details of the breeding system with dynamism, defining the spatial scale of thornback ray respect to, e.g., multiple paternity, will provide addi- populations remains difficult. tional information about the long-term prospects of R. Genetic diversity (Hexp) was relatively uniform clavata. Coupling genetic data with more comprehen- among samples, as was the more sensitive measurement sive and long-term tagging studies, initiated from to recent demographic reductions-allelic richness (Spen- diverse locations around UK coastline, will collectively cer et al., 2000)(Table 2). There is, therefore, no help to build a more detailed picture of the behavioral apparent evidence of reduced genetic variation in dynamics and population structure of R. clavata. In any absolute terms or evidence for one location having case, the extreme vulnerability of rays to by-catch suffered more than another from a loss of genetic means that future management measures for rays will variation. Without a temporal comparison, however, it is have to be coupled with the establishment of ‘no take’ impossible to know whether or not genetic diversity has zones or other spatial management scenarios. decreased under fishing pressure. There are few biomass estimates for skates and rays and nothing is known Acknowledgements about effective population size (Ne)ofR. clavata, i.e., the number of individuals in the total population that We thank Jim Coyer for his comments on the contribute to the next generation. In commercial fish manuscript as well as J-P. Delpech (IFREMER such as New Zealand snapper (Hauser et al., 2002), Boulogne sur mer, France), H. Heessen (RIVO, North Sea cod (Hutchinson et al., 2003) and European IJmuiden, The Netherlands) for sampling and Sophie plaice (Hoarau et al., 2005), Ne has been shown to be Arnaud-Haond for her help in the analysis. We also three to five orders of magnitude smaller than the census thank the anonymous reviewers for their helpful sizes. Should a similar result be found in R. clavata, the comments on a previous version of this manuscript. impact of fisheries over the past 20 y may have been This research was supported by NWO-PRIORITEIT considerable. One way to estimate loss of genetic programme SUSUSE, Project Nr. 885-10-311. diversity and Ne would be to compare pre- and post- exploitation levels of genetic diversity using archived References vertebrae, an approach analogous to historical genotyping of otoliths in bony fish. 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Given the wide range Blake, K., 1976. Polymorphic forms of eye lens protein in the ray Raja of locations, seasonal timing and independent fishing clavata (Linnaeus). Comp. Biochem. Physiol., part B 54B, surveys, it is unlikely that we missed adults due to 441–442. — sampling artifacts. It is more likely that large rays have Bland, J.M., Altman, D.G., 1995. Multiple significance tests the Bonferroni method. Br. Med. J. 310, 170. been the victims of by-catch. From a conservation Borsa, P., Blanquer, A., Berrebi, P., 1997. Genetic structure of the perspective (Birkeland and Dayton, 2005), it is well flounders Platichthys flesus and P. stellatus at different geographic known that for many long-lived species, the older scales. Mar. Biol. 129, 233–246. M. Chevolot et al. / Journal of Sea Research 56 (2006) 305–316 315

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