Journal of Experimental Marine Biology and Ecology 267 (2002) 139 – 145 www.elsevier.com/locate/jembe

Molecular evidence of male-biased dispersal in loggerhead turtle juveniles Paolo Casale a,*, Luc Laurent b, Guido Gerosa c, Roberto Argano d a via Antonio Calderara 29, I-00125 Rome, Italy BioInsight, Biologie de la Conservation, B.P. 2132, F-69603 Villeurbanne, France c Chelon, Marine Turtle Conservation and Research Program, Via Val Padana 134/B, I-00141 Rome, Italy d Dipartimento di Biologia Animale e dell’Uomo, Universita` ‘‘La Sapienza’’, I-00185 Rome, Italy b

Received 13 June 2001; received in revised form 27 August 2001; accepted 17 September 2001

Abstract Serum testosterone levels and mtDNA haplotypes were obtained from 65 juvenile loggerhead turtles (Caretta caretta, L.) incidentally caught in the central Mediterranean. The group of specimens carrying a haplotype specific for the northwest Atlantic had higher testosterone levels, and so included more males, than the other one. Since primary sex ratios of northwest Atlantic colonies are strongly skewed towards females, results indicate a male bias among Atlantic turtles entering the Mediterranean. This demonstrates for the first time a sex-biased dispersal of specimens in the pelagic phase, an important factor to be considered in conservation programs. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Caretta caretta; Dispersal; Mediterranean Sea; Sex ratio

1. Introduction Sea turtles are endangered species whose biology is poorly known, especially that of males and the juvenile phase of both sexes. While adult females, eggs, and hatchlings are relatively well studied when on reproductive beaches, less is known on life at sea. Mixed stock analyses are revealing that juveniles from different and distant nesting colonies mix together in feeding areas (Laurent et al., 1993, 1998; Bowen et al., 1996), but sex-related aspects of their dispersal are still unknown.

*

Corresponding author. Tel./fax: +39-06-5245-6820. E-mail address: [email protected] (P. Casale).

0022-0981/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 2 - 0 9 8 1 ( 0 1 ) 0 0 3 6 5 - 3

140

P. Casale et al. / J. Exp. Mar. Biol. Ecol. 267 (2002) 139–145

Sea turtles are able to cover great distances. In most species, small juveniles mainly frequent pelagic oceanic habitats, then, at a larger size, they actively move to demersal neritic habitats, possibly making seasonal migrations between summer and winter areas (Musick and Limpus, 1997). As adults, they migrate between the same or different seasonal foraging areas of juveniles, and also between foraging and reproductive areas (Musick and Limpus, 1997). So far, the latter was the only kind of movement thought to be influenced by sex; females are known to reproduce at a lower frequency than males (Miller, 1997), with a consequent difference in migration frequency. There also seem to be differences in the seasonal timing of these migrations (Plotkin et al., 1996; Miller, 1997). In the last decade, mtDNA markers have demonstrated the origin of turtles in foraging areas, because different nesting sites are identified by different frequencies of mtDNA haplotypes, a consequence of homing behaviour of adult females (Meylan et al., 1990; Bowen et al., 1994). Recent studies showed that a large number of western Atlantic juvenile loggerhead turtles (Caretta caretta) enter the Mediterranean (Laurent et al., 1993, 1998), probably following the North Atlantic gyre. Specimens originating from western Atlantic nesting assemblages occur in the eastern part of the gyre (Azores and Madeira) in proportions close to the number of nests produced at their natal nesting sites (Bolten et al., 1998). It was hypothesized that this Atlantic juvenile pelagic movement is male-biased, as a partial explanation of the low matriarchal gene flow observed between the Mediterranean and Atlantic nesting populations (Laurent et al., 1993). In the Mediterranean, Atlantic and local specimens share pelagic habitats (Laurent et al., 1998) and this gives the opportunity of investigating possible sex-biased dispersal, if Mediterranean (local) and Atlantic (distant) specimens are compared through their serum testosterone levels, known to be higher in juvenile males than females (Wibbels et al., 1987, 1991).

2. Materials and methods Blood samples were obtained from the cervical sinus (Owens and Ruiz, 1980) of 65 turtles incidentally caught by fishing vessels from Lampedusa Island, Italy (35°31V N; 12°35V E) between 30th June and 20th September in 1991, 1992, and 1993, and centrifuged to separate serum and cells. Size range of turtles was 29– 59 cm Standard Curved Carapace Length, lower than the minimum recorded for a nesting female in the Mediterranean (Broderick and Godley, 1996). Testosterone and mtDNA data were separately analyzed as part of two studies dealing with different topics. Serum samples were analyzed by a radioimmunoassay method to obtain testosterone levels (for details see Casale et al., 1998). An aliquot for each sample was extracted with diethyl ether, dried under nitrogen, then reconstituted with tris/gel buffer. Each sample was then divided into two aliquots. Tritiated testosterone and testosterone antiserum were diluted with tris/gel buffer and added to tubes with samples and to those with known concentrations of testosterone. Unbound tritiated testosterone was separated by adding dextran-coated charcoal, then the tubes were vortexed, incubated, and centrifuged. The supernatant and scintillation cocktail were poured together and counted in a scintillation spectrometer.

P. Casale et al. / J. Exp. Mar. Biol. Ecol. 267 (2002) 139–145

141

Table 1 Haplotypes found in the sample (n = 65) and their frequencies at nesting assemblages Sample haplotype

n

Nesting assemblage

Frequency (n)

A1

39

Mediterranean North-West Florida South Florida NEFL-NC Mexico Mediterranean North-West Florida South Florida Mexico Mexico – North-West Florida South Florida – North-West Florida South Florida NEFL-NC –

84.8% (92) 9.5% (42) 48% (50) 1% (105) 55% (20) 14.1% (92) 4.8% (42) 4% (50) 10% (20) 5% (20) – 4.8% (42) 2% (50) – 81% (42) 44% (50) 99% (105) –

A3

3

A4 A5 A7

1 2 1

A8 C1/C2

1 17

C3

1

Corresponding haplotype code B B B B C C C I – G G – A A A –

Mediterranean and Atlantic frequencies are calculated from Laurent et al. (1998) and Encalada et al. (1998), respectively. Haplotype codes used by the latter study are shown for correspondence. NEFL-NC: North – East Florida/Georgia/South Carolina/North Carolina.

From blood cells, the total DNA was extracted and PCR amplification of the middle third part (452 –458 bp in length) of the control region was obtained with two primers designed for this purpose. Each sample was sequenced in both directions and the observed polymorphic sites defined different haplotypes (for details see Laurent et al., 1998). Haplotypes C1 and C2 (Table 1) correspond to the same haplotype known from nesting sites (A; Table 1), due to a shorter mtDNA region studied by these works (Encalada et al., 1998). Therefore, we treated these two haplotypes as one.

3. Results Eight haplotypes were observed (Table 1), none endemic to the Mediterranean. Haplotype C1/C2 is known to occur at high proportions in northwestern Atlantic nesting sites but is not found in the Mediterranean (Encalada et al., 1998; Laurent et al., 1998). Therefore, we assumed the specimens with this haplotype (n = 17; 26.2%) to be of Atlantic origin (Group I). Haplotypes A1 and A3 are found at high proportions in both the Atlantic and the Mediterranean, and we considered specimens with these haplotypes (n = 42; 64.6%) as a group of mixed origin (Group II). The other five haplotypes were represented by 1 – 2 specimens and either not found in nesting sites (A5, A8, C3) or found at very low frequencies in Atlantic ones (A4, A7). We conservatively excluded all these specimens

142

P. Casale et al. / J. Exp. Mar. Biol. Ecol. 267 (2002) 139–145

Fig. 1. Frequency distribution of testosterone levels (logarithmic scale) of specimens of Groups I and II.

(n = 6; 9.2%) from the analysis, the former because their origin is unknown, the latter because we cannot exclude the possibility that they also occur at low frequencies in Mediterranean nesting sites. Group I (Atlantic) had higher levels of testosterone than Group II (Atlantic and Mediterranean) (Fig. 1). In spite of the mixed origin of Group II, which weakened the comparison, a strong difference was observed (Median test; Fisher’s exact test, p < 0.01). We found no difference by month of capture between Group I and II specimens (v2 = 0.59; df = 2; p = 0.75), excluding possible seasonal affects. Testosterone is known to be positively correlated with age (Owens, 1997), but we found no difference in size between Group I and II specimens (Median test, Fisher’s exact test; p = 0.20). Present knowledge does not show interpopulation differences in male and female ranges of testosterone levels (Owens, 1997), therefore, the only alternative explanation of these results is that Group I had a higher proportion of males than Group II.

4. Discussion Sex determination of sea turtles depends on incubation temperature, so different sex ratios are produced at different nesting sites (Mrosovsky, 1994). If the two groups had sex ratios representative of their populations, according to present results, the Atlantic population should be skewed towards males or at least much more skewed than the Mediterranean one, but this is not the case. In fact, haplotype C1/C2 (Group I) is found in three nesting assemblages only (South Florida; North-East Florida/Georgia/South Carolina/North Carolina; North-West Florida; frequencies of haplotype C1/C2 are shown in Table 1), whose relative contribution in nests is 90.5%, 8.8%, and 0.6%, respectively (Turtle Expert Working Group, 1998). A primary sex ratio of 1– 13% of males is estimated to be produced in SFL (Mrosovsky and Provancha, 1992), 44 – 55% in NEFL-NC (Mrosovsky et al., 1984; Webster and Gouveia, 1988), while it is unknown for NWFL. So, even assuming a 100% male production in NWFL, it seems very likely that a minority

P. Casale et al. / J. Exp. Mar. Biol. Ecol. 267 (2002) 139–145

143

(10 – 21%), not a majority, of C1/C2 (Group I) males are produced, and therefore, it is not possible to explain present results with a lower proportion of males produced in the Mediterranean. Hence, present findings (i) demonstrate that the juvenile class of a sea turtle is not necessarily representative of the whole population as far as sex ratio is concerned, (ii) show that this difference is bigger for specimens far from their natal areas (north-western Atlantic) and consists in a male bias, and (iii) strongly suggest that juvenile males disperse farther than females, and so move the beginning of sexual differences in behaviour from the adult to the small juvenile class. This also suggests that the gene flow from the Atlantic to the Mediterranean would be mainly mediated by males. It should be taken into account that the above conclusions are based on one population only (Atlantic) and on the assumption of no bias in catchability. Certainly, investigations on other populations are highly desirable. Sex-biased dispersal has generally been explained by three major hypotheses: avoidance of resource competition; avoidance of intrasexual competition for mates; inbreeding avoidance (Johnson and Gaines, 1990). A recent game-theory kin-selection model predicted male-biased dispersal in species where competition with relatives of the same sex is greater among males than among females (Perrin and Mazalov, 2000). This is the case for polygynous/promiscuous species, if the female reproductive output is limited by intrinsic factors more than by the availability of breeding opportunities (Perrin and Mazalov, 2000). In such a situation, male competition for females is greater than female competition for resources and this selects for male-biased dispersal. This tendency is also reinforced by the selective pressure for inbreeding avoidance, which acts on both sexes making an unbiased dispersal unstable (Perrin and Mazalov, 2000). Life history of sea turtles, as well as that of many mammal species with male-biased dispersal, seems to fit well within the model above. In fact, sea turtles are promiscuous and males compete for females (Miller, 1997). Moreover, the physiological processing of food seems to limit female reproduction more than availability of resources (Miller, 1997). Naturally, sexbiased dispersal patterns cannot be evident between close populations sharing the same adult areas, because actual dispersors could be those transferring to different areas, depending on proximate mechanisms of dispersal. For instance, three populations of Chelonia mydas in North and East Australia share an extensive common adult foraging area (Fitzsimmons et al., 1997), and the observed interpopulation gene flow can be explained by relaxing the mating area fidelity of males rather than a definitive shift between mating areas which are all included in the normal adult feeding range (Fitzsimmons et al., 1997). On the other hand, a male-mediated gene flow is suspected also between distant C. mydas populations (Karl et al., 1992), which are less likely to share adult areas. Present findings have important consequences for current research and conservation practices. Sex ratio studies usually prefer juveniles to adults because they are thought not to have sex-biased dispersal (Wibbels et al., 1987), but in the light of present results, sex ratio data should be interpreted in a more complex context. In turn, sex ratio information could improve future conservation programmes because it is plausible that males and females have different values for population viability, and the human impact on populations could be different according to the sex ratio occurring in the exploited area.

144

P. Casale et al. / J. Exp. Mar. Biol. Ecol. 267 (2002) 139–145

Furthermore, present results suggest that in areas far from nesting sites, sex ratios would be skewed towards males, while near nesting sites, sex ratios would be determined by the relative contributions of local and distant populations. As a consequence, if females are assumed to have more value for the population viability, human impacts on juvenile habitats (e.g., fishing activity) would be more harmful for populations with close nesting sites than for those with distant ones. For instance, despite the fact that some Mediterranean fisheries are estimated to interact with about the same number of Atlantic and Mediterranean specimens in their pelagic phase (Laurent et al., 1998), the worst consequences probably affect the Mediterranean population. Certainly, more data on sex ratios of different stocks sharing the same area are desirable in order to optimize conservation strategies.

References Bolten, A.B., Bjorndal, K.A., Martins, H.R., Dellinger, T., Biscoito, M.J., Encalada, S.E., Bowen, B.W., 1998. Transatlantic developmental migrations of loggerhead sea turtles demonstrated by mtDNA sequence analysis. Ecol. Appl. 8 (1), 1 – 7. Bowen, B.W., Kamezaki, N., Limpus, C.J., Hughes, G.H., Meylan, A.B., Avise, J.C., 1994. Global phylogeography of the loggerhead turtle (Caretta caretta) as indicated by mitochondrial DNA haplotypes. Evolution 48, 1820 – 1828. Bowen, B.W., Bass, A.L., Garcia-Rodriguez, A., Diez, C.E., Van Dam, R., Bolten, A., Bjorndal, K.A., Miyamoto, M.M., Ferl, R.J., 1996. Origin of hawksbill turtles in a Caribbean feeding area as indicated by genetic markers. Ecol. Appl. 6 (2), 566 – 572. Broderick, A.C., Godley, B.J., 1996. Population and nesting ecology of the green turtle, Chelonia mydas, and the loggerhead turtle, Caretta caretta, in northern Cyprus. Zool. Middle East 13, 27 – 46. Casale, P., Gerosa, G., Argano, R., Barbaro, S., Fontana, G., 1998. Testosterone titers of immature loggerhead sea turtles (Caretta caretta) incidentally caught in the central Mediterranean: a preliminary sex ratio study. Chel. Conserv. Biol. 3 (1), 90 – 93. Encalada, S.E., Bjorndal, K.A., Bolten, A.B., Zurita, J.C., Schroeder, B., Possardt, E., Sears, C.J., Bowen, B.W., 1998. Population structure of loggerhead turtle (Caretta caretta) nesting colonies in the Atlantic and Mediterranean as inferred from mitochondrial DNA control region sequences. Mar. Biol. 130, 567 – 575. Fitzsimmons, N.N., Limpus, C.J., Norman, J.A., Goldizen, A.R., Miller, J.D., Moritz, C., 1997. Philopatry of male marine turtles inferred from mitochondrial DNA markers. Proc. Natl. Acad. Sci. U. S. A. 94 (16), 8912 – 8917. Johnson, M.L., Gaines, M.S., 1990. Evolution of dispersal: theoretical models and empirical tests using birds and mammals. Annu. Rev. Ecol. Syst. 21, 449 – 480. Karl, S.A., Bowen, B.W., Avise, J.C., 1992. Global population genetic structure and male-mediated gene flow in the green turtle (Chelonia mydas)—RFLP analyses of anonymous nuclear loci. Genetics 131 (1), 163 – 173. Laurent, L., Lescure, J., Excoffier, L., Bowen, B., Domingo, M., Hadjichristophorou, M., Kornaraki, L., Trabuchet, G., 1993. Etude ge´ne´tique des relations entre les populations me´diterrane´enne et atlantique d’une tortue marine (Caretta caretta) a` l’aide d’un marqueur mitochondrial. C. R. Acad. Sci. Paris 316, 1233 – 1239. Laurent, L., Casale, P., Bradai, M.N., Godley, B.J., Gerosa, G., Broderick, A.C., Schroth, W., Schierwater, B., Levy, A.M., Freggi, D., Abd El-Mawla, E.M., Hadoud, D.A., Gomati, H.E., Domingo, M., Hadjichristophorou, M., Kornaraky, L., Demirayak, F., Gautier, C., 1998. Molecular resolution of marine turtle stock composition in fishery bycatch: a case study in the Mediterranean. Mol. Ecol. 7, 1529 – 1542. Meylan, A.B., Bowen, B.W., Avise, J.C., 1990. A genetic test of the natal homing versus social facilitation models for green turtle migration. Science 248, 724 – 727. Miller, J.D., 1997. Reproduction in sea turtles. In: Lutz, P.L., Musick, J.A. (Eds.), The Biology of Sea Turtles. CRC Press, Boca Raton, FL, pp. 51 – 81. Mrosovsky, N., 1994. Sex ratios of sea turtles. J. Exp. Zool. 270 (1), 16 – 27.

P. Casale et al. / J. Exp. Mar. Biol. Ecol. 267 (2002) 139–145

145

Mrosovsky, N., Provancha, J., 1992. Sex ratio of hatchling loggerhead sea turtles: data and estimates from a 5year study. Can. J. Zool. 70 (3), 530 – 538. Mrosovsky, N., Hopkins-Murphy, S.R., Richardson, J.I., 1984. Sex ratio of sea turtles: seasonal changes. Science 225, 739 – 741. Musick, J.A., Limpus, C.J., 1997. Habitat utilization and migration in juvenile sea turtles. In: Lutz, P.L., Musick, J.A. (Eds.), The Biology of Sea Turtles. CRC Press, Boca Raton, FL, pp. 137 – 163. Owens, D.W., 1997. Hormones in the life history of sea turtles. In: Lutz, P.L., Musick, J.A. (Eds.), The Biology of Sea Turtles. CRC Press, Boca Raton, FL, pp. 315 – 341. Owens, D.W., Ruiz, G.J., 1980. New methods of obtaining blood and cerebrospinal fluid from marine turtles. Herpetologica 36 (1), 17 – 20. Perrin, N., Mazalov, V., 2000. Local competition, inbreeding, and the evolution of sex-biased dispersal. Am. Nat. 155 (1), 116 – 127. Plotkin, P.T., Owens, D.W., Byles, R.A., Patterson, R., 1996. Departures of male olive ridley turtles (Lepidochelys olivacea) from a nearshore breeding ground. Herpetologica 52 (1), 1 – 7. Turtle Expert Working Group, 1998. An assessment of the Kemp’s ridley (Lepidochelys kempii) and loggerhead (Caretta caretta) sea turtle populations in the Western North Atlantic. NOAA Tech. Mem. NMFS-SEFSC409, 96 pp. Webster, W.D., Gouveia, J.F., 1988. Predicting hatchling sex ratios in loggerhead sea turtles (Caretta caretta) by incubation duration. Schroeder, B.A. (Comp.) Proceedings of the Eighth Annual Workshop on Sea Turtle Conservation and Biology, Forth Fisher, NC, 24 – 26 February 1988. NOAA Tech. Mem. NMFS-SEFC, Miami, FL, pp. 127 – 128. Wibbels, T., Owens, D.W., Morris, Y.A., Amoss, M.S., 1987. Sexing techniques and sex ratios for immature loggerhead sea turtles captured along the atlantic coast of the United States. In: Witzell, W.N. (Ed.), Ecology of East Florida Sea Turtles, Cape Canaveral, FL, Feb. 26 – 27, 1985. NOAA Tech. Rep. NMFS-53, NOAA NMFS-SEFC, Miami, FL, pp. 65 – 74. Wibbels, T., Martin, R.E., Owens, D.W., Amoss Jr., M.S. 1991. Female-biased sex ratio of immature loggerhead sea turtles inhabiting the Atlantic coastal waters of Florida. Can. J. Zool. 69 (12), 2973 – 2977.