Genetic Survey of Loggerhead Turtle Caretta Caretta Nesting Population

Marine Biodiversity Records, page 1 of 6. # Marine Biological Association of the United Kingdom, 2010 doi:10.1017/S175526721000014X; Vol. 3; e20; 2010 Published online Genetic survey of loggerhead turtle Caretta caretta nesting population in Tunisia olfa chaieb1,2, ali el ouaer2, fulvio maffucci3, mohamed nejmeddine bradai2, flegra bentivegna3, khaled said1 and noureddine chatti1 1Unite´ de Recherche: Ge´ne´tique, Biodiversite´ et Valorisation des Bioressources UR03ES09, Institut Supe´rieur de Biotechnologie de Monastir, Tunisie, 2Laboratoire de Biodiversite´ et Biotechnologie Marines, Institut National des Sciences et Technologies de la Mer, Tunisie, 3Stazione Zoologica ‘Anton Dohrn’, Villa Communale, Napoli, Italy

Genetic diversity of loggerhead turtles, Caretta caretta, nesting on the Kuriat Islands, the most important Tunisian nesting beach (central Mediterranean), was investigated using both nuclear and mitochondrial markers. Allozyme electrophoresis of 63 hatchlings from four different clutches showed a low genetic diversity. The genotypic composition of two clutches did not match Mendelian expectations suggesting the occurrence of multiple paternity. The analysis of 380 bp of the mitochondrial DNA control region revealed no genetic variability. Only one haplotype was described in our sample (N ¼ 16), which corresponds to the sequence of the most common haplotype found on the Mediterranean nesting beaches (CC-A2). The low genetic diversity detected by both mitochondrial and allozyme markers is discussed taking into account available data about past and present situations of loggerhead nesting activity in this site. Adequate conservation measures should be urgently taken to protect the nesting population in this area.

Keywords: Caretta caretta, allozymes, mitochondrial DNA, genetic diversity, multiple paternity, Kuriat Islands, central Mediterranean

Submitted 8 June 2009; accepted 1 December 2009

INTRODUCTION sea turtles (Ireland et al., 2003; Jensen et al., 2006; Theissinger et al., 2006) but there is some variability The loggerhead turtle, Caretta caretta, one of the seven extant between species or among different populations of the same sea turtle species, is circum-globally distributed in tropical species (Rieder et al., 1998; Crim et al., 2002). and warm-temperate regions and is currently listed as endan- In the Mediterranean Sea, loggerhead turtle nesting is gered by the IUCN (IUCN, 2007). Colonization of the almost confined to the eastern basin along the Greek, Mediterranean Sea by loggerhead turtles presumably occurred Turkish, Cyprus and Libyan coasts. A lower but regular after the last glacial era some 12,000 years ago (Bowen et al., nesting is also reported from several other countries in this 1993). Although this is a short period on an evolutionary region such as Egypt, Israel, Lebanon, Italy and Tunisia time scale, studies using both nuclear and mtDNA markers (Margaritoulis et al., 2003). The loggerhead is the only sea have shown that the Mediterranean population is reproduc- turtle species nesting along the Tunisian coasts and a mean tively and genetically independent from the Atlantic population of 19 nests are recorded annually (Jribi et al., 2006; Bradai (Bowen et al., 1993). A genetic structure was reported within & Jribi, 2007) in the main nesting site of the Kuriat Islands. the Mediterranean region by Schroth et al. (1996) and lately This site, consisting of two small islands located on the corroborated by Carreras et al. (2007) and Garofalo et al. eastern side of the country (Figure 1), is the unique one (2009) who distinguished genetically different nesting units, characterized by a regular nesting activity. In the past, it has most of them characterized by one exclusive mtDNA haplo- been reported that nesting was more intense and widespread type: Greece and the adjoining Ionian Islands, eastern Turkey, along Tunisian coasts mainly on the beaches of Nabeul, Israel, Cyprus and Italy. However, the overall variability of Hergla, Chebba, Kerkennah and Zarzis. However, nesting the mtDNA control region was low compared with that of has collapsed because of habitat degradation and intense the Atlantic populations (Carreras et al., 2007). fishing activities (Bradai et al., 2008). Genetic techniques have also permitted evaluation of the The Kuriat Islands have been the focus of many conserva- incidence of multiple paternity in the Mediterranean logger- tion activities. Since 1997, the nesting activity on the islands is head population. In Zakynthos (Greece), the biggest nesting monitored in the framework collaboration between the rookery in this basin, it has been estimated that 93% of the National Institute of Marine Sciences and Technologies nests have been fecundated by more than one male (INSTM), the Regional Activity Centre for Specially (Zbinden et al., 2007). This phenomenon is common among Protected Areas (RAC/SPA) and the Coastal Protection and Planning Agency (APAL). Major information about reproductive biology and ecology of the local nesting population has been gleaned from field surveys (Jribi et al., 2002). Corresponding author: O. Chaieb However, no information on genetic make-up of this small Email: [email protected] rookery is available.

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Table 1. Enzymes and electrophoretic conditions used. Names, enzyme commission numbers (EC), and abbreviations follow Murphy & Crabtree (1985).

Enzymatic systems EC number Locus Tissue

Esterase-3 3.1.1.2 Es-3 Heart Glutamate-oxaloactate transaminase 2.6.1.1 Got-1 Heart Glutamate-oxaloactate transaminase 2.6.1.1 Got-2 Heart Glucose-phosphate isomerase 5.3.1.9 Gpi-1 Liver Glucose-6-phosphate dehydrogenase 1.1.1.49 G6pd-1 Liver Isocitrate dehydrogenase 1.1.1.42 Idh-1 Liver Isocitrate dehydrogenase 1.1.1.42 Idh-2 Liver Lactate dehydrogenase 1.1.1.27 Ldh-1 Heart Lactate dehydrogenase 1.1.1.27 Ldh-2 Heart Malate dehydrogenase 1.1.1.37 Mdh-1 Liver Malate dehydrogenase 1.1.1.37 Mdh-2 Liver Malic enzyme 1.1.1.40 Mod-1 Liver Phosphogluconate dehydrogenase 1.1.1.44 Pgd-1 Liver Phosphogluconate dehydrogenase 1.1.1.44 Pgd-2 Liver Phosphoglucomutase 5.4.2.2 Pgm-1 Liver Sorbitol dehydrogenase 1.1.1.14 Sdh-1 Liver Fig. 1. Location of the analysed site: Kuriat Islands. Superoxyde dismutase 1.1.5.11 Sod-1 Liver

In this study, two genetic markers were used to characterize Mitochondrial DNA analysis the loggerhead turtles nesting on the Kuriat Islands. Allozymes were used in order to evaluate the incidence of Considering the small Tunisian nesting population size in multiple paternity in this population. We also analysed the comparison with the other Mediterranean rookeries, 54 rapidly evolving mtDNA control region, a marker of choice hatchlings were analysed over seven years of sampling cam- for marine turtle nesting population surveys (Fitzsimmons paigns (from 2002 to 2008), of which one individual comes et al., 1999), in order to assess genetic diversity. from the adjoining nesting site of Hergla (Figure 1). However, in order to avoid pseudoreplication, only 16 individuals belonging to different nesting females were considered for subsequent analysis (see Results below). These MATERIALS AND METHODS 16 individuals were sampled from clutches laid within a 15-day window, corresponding to the inter-nesting interval (Dodd, 1988), and over two years (2006 and 2007) as logger- Protein analysis head females return to nest each two or three years (Dodd, Four nests were sampled during two consecutive nesting 1988). seasons (N ¼ 14 and 25 (2002) and N ¼ 12 and 12 (2003)). Muscle or skin samples were collected from freshly dead After emergence, freshly dead hatchlings were sampled. embryos and stored at 2208C. Whole genomic DNA was iso- Samples of liver, heart, kidney, lung and gonad were lated by standard phenol chloroform extraction (Hillis et al., removed from each specimen and homogenized in an equal 1996). A fragment of 510 base pairs (bp) of the mitochondrial volume of an aqueous homogenizing buffer pH 6.8. After cen- DNA control region was amplified by polymerase chain reac- trifugation of resultant homogenates at 14,000 rpm for tion (PCR) using the primers L71 and H599 (Laurent et al., 35 minutes at 48C, the supernatants were stored at 2808C. 1998). The PCR protocol was 948C for 5 minutes, followed Seventeen loci coding for twelve enzymatic systems were exam- by 35 cycles at 948C for 1 minute, 568C for 1 minute and ined for polymorphism using horizontal starch gel (13%). 728C for 1 minute with a final extension at 728C for Electrophoresis procedures follow Pasteur et al. (1987). For 10 minutes. The PCR amplifications included negative all loci scored we used one buffer system Tris Citrate pH 6.7. control reactions to guard against contamination. The PCR Multiple loci encoding a single protein were designated with products were visualized in a 1% agarose gel and purified a hyphenated number indicating the relative migration of using Qiagen Qiaquick Spin PCR purification kit according their products, the slowest (less anodal) being designated as to the manufacturer’s instructions. Purified products were 1. Alleles were identified by their electromorph mobility rela- sequenced on a Beckman CEQ 2000XL automatic sequencer tive to that of the most common electromorph mobility using the Dye-Terminator cycle sequencing kit. assigned as 100. For each protein resolved, tissue type and Sequences were aligned using the BioEdit program v. 5.0.9 enzyme commission number (EC) are listed in Table 1. (Hall, 1999) and compared with previously 380 bp described Overall genetic variability was estimated, including allele loggerhead haplotypes published in the Archie Carr Centre frequencies, percentage of polymorphism at the 95% criterion of Sea Turtle Research DNA database (http://accstr.ufl.edu/). (P0.95) (a locus was polymorphic if the frequency of the most Genetic differentiation between our population and published common allele was lower than 95%), the observed and Mediterranean nesting populations of Greece, Turkey, Italy expected heterozygosities (Ho, H exp) and mean number of Cyprus, Lebanon and Israel (Table 2) (Carreras et al., 2007; alleles per locus (A) using the Genetix 4.03 software (Belkhir Garofalo et al., 2009) was verified with the exact test of et al., 2001). population differentiation (Raymond & Rousset, 1995) as genetic survey of tunisian loggerhead turtles 3

Table 2. Mitochondrial DNA haplotype distribution of loggerhead turtles occurring in different Mediterranean nesting sites, number of analysed individuals (N) and source.

Population N Mitochondrial DNA haplotypes Source

CC-A2 CC-A3 CC-A6 CC-A20 CC-A29 CC-A31 CC-A32

Greece Zakynthos 20 17 02 01 Carreras et al. (2007) Kyparissia 21 19 02 Encalada et al. (1998) Lakonikos 19 18 01 Carreras et al. (2007) Crete 19 19 Carreras et al. (2007) Turkey Eastern Turkey 32 19 13 Laurent et al. (1998) Western Turkey 16 15 01 Carreras et al. (2007) Italy Calabria 38 22 14 02 Garofalo et al. (2009) Cyprus 35 35 Encalada et al. (1998) Lebanon 09 09 Carreras et al. (2007) Israel 20 17 03 Carreras et al. (2007) Tunisia 16 16 Present study implemented in the software Arlequin v. 2.0.0 (Schneider Mitochondrial DNA analysis et al., 2000). The DNA investigation showed only one haplotype CC-A2 among 53 individuals, including the sample from the site of RESULTS Hergla. This haplotype is shared by both Mediterranean and Atlantic nesting populations although it occurs at much Protein analysis Table 3. Allele frequencies in the 17 loci and genetic parameters calculated Seven of the 17 scored loci were polymorphic (Got-1, Es-3, for the four clutches. G6pd-1, Idh-1, Mdh-1, Pgd-1 and Sdh-1) with three alleles in the Es-3 locus and two in each of the other six loci. Allelic fre- Locus Alleles Nests quencies, recorded for all the loci are provided in Table 3. A 1 2002 2 2002 1 2003 2 2003 low genetic difference was observed between the clutches N 5 14 N 5 25 N 5 12 N 5 12 with a presence of more or fewer alleles among polymorphic loci. Both clutches of 2002 and the first clutch of 2003 Es-3 100 0.0714 0.1200 0.0833 0.7500 showed five polymorphic loci, whereas the second clutch of 110 0.7857 0.6800 0.5000 0.2500 2003 showed only four polymorphic loci. 120 0.1429 0.2000 0.4167 0.0000 Got-1 90 0.4643 0.2400 0.3333 0.7500 Estimates of the genetic variability were provided by the fol- 100 0.5357 0.7600 0.6667 0.2500 lowing parameters: A, P and H reported in Table 3. The mean Got-2 220 1.0000 1.0000 1.0000 1.0000 number of alleles per locus (A) and the percentage of poly- G6pd-1 90 0.1429 0.3200 0.0000 0.0000 morphic loci (P0.95) were quite similar for all the four clutches 100 0.8571 0.6800 1.0000 1.0000 surveyed and were respectively on average of 1.32 and 0.27. The Idh-1 100 0.7143 0.4600 0.8333 1.0000 observed heterozygosity values were low and varied from 0.019 110 0.2857 0.5400 0.1667 0.0000 in the second clutch of 2002 to 0.088 in the first clutch of 2003. Mdh-1 100 1.0000 1.0000 0.5000 0.5000 The comparison between observed and expected heterozygosity 120 0.0000 0.0000 0.5000 0.5000 among the four clutches showed heterozygote deficiency. A Pgd-1 90 0.6429 0.7400 1.0000 1.0000 relatively high genetic heterozygosity (0.019 , Ho , 0.088) 100 0.3571 0.2600 0.0000 0.0000 Sdh-1 100 1.0000 1.0000 0.5000 0.5000 was observed in comparison with previous studies on Caretta 120 0.0000 0.0000 0.5000 0.5000 caretta in Queensland, Australia (Ho ¼ 0.016; Gyuris & Gpi-1 100 1.0000 1.0000 1.0000 1.0000 Limpus, 1988) and on green turtles Chelonia mydas (0.00 , Idh-2 100 1.0000 1.0000 1.0000 1.0000 Ho , 0.04; Bonhomme et al., 1987). Ldh-1 100 1.0000 1.0000 1.0000 1.0000 Moreover, the examination of the hatchling genotypes Ldh-2 100 1.0000 1.0000 1.0000 1.0000 revealed that in half of the clutches, the Es-3 locus exhibited Mdh-2 100 1.0000 1.0000 1.0000 1.0000 three alleles, and the genotypic composition of these two clutches Mod-1 100 1.0000 1.0000 1.0000 1.0000 did not correspond to Mendelian inheritance expected from the Pgd-2 100 1.0000 1.0000 1.0000 1.0000 null hypothesis of single paternity. Indeed, in the first clutch of Pgm-1 100 1.0000 1.0000 1.0000 1.0000 the season 2002, two homozygote genotypes have been Sod-1 100 1.0000 1.0000 1.0000 1.0000 100/100 110/110 Ho 0.038 0.019 0.088 0.069 observed: Es-3 and Es-3 and one heterozygote genotype 110/120 H exp 0.115 0.127 0.135 0.103 Es-3 . In the first clutch of 2003, two different homozygote 110/110 120/120 P 0.95 0.290 0.290 0.290 0.230 genotypes were present: Es-3 and Es-3 as well as two 100/110 110/120 A 1.350 1.350 1.350 1.230 different heterozygote genotypes Es-3 and Es-3 . Overall, these data support the idea that at least two males A, mean number of alleles per locus; H exp, expected heterozygosity; Ho, intervened in the fertilization of each female in both cases. observed heterozygosity; P0.95, level of polymorphism at 95%. 4 olfa chaieb et al.

higher frequencies in the Mediterranean region (Encalada years (Bradai, 1996; Jribi et al., 2002). In the past, significant et al., 1998; Laurent et al., 1998; Carreras et al., 2007). One nesting activity likely existed along southern Tunisian individual showed a heteroplasmy at Site 157 of 380 bp. beaches but then disappeared due to the intense fishing Heteroplasmy in sea turtles’ control region has been reported activity (Bradai, 1995). The Kuriat nesting site may have sur- previously (in loggerhead turtles by Laurent et al., 1998 and in vived because the islands are desert and are located 18 km green turtles by Formia, 2002) and the sequence was excluded from the mainland (Figure 1). However, in recent years, from the analysis. Only 16 individuals, representing the mean human activities consisting mainly of tourists’ tours to the number of different females nesting on the Kuriat Islands, islands during the summer and intense fishing activity (Jribi were considered for analyses. A comparison of haplotype fre- et al., 2002) raised our concern about the survival of the log- quencies indicated that the present population is not geneti- gerhead population at this site. cally different from the Mediterranean rookeries of Greece, The low genetic diversity observed in this study suggests Lebanon, Israel and Cyprus but significantly different from that strong conservation efforts should be taken. This area, those of eastern Turkey and of Calabria, Italy (P , 0.005). which constitutes one of the very few regular nesting sites for this species in the central Mediterranean, should be designated as a special protected area of high importance for the DISCUSSION marine biodiversity (Bradai, 1996). Recently, some encoura- ging results have been obtained owing to monitoring cam- As reported in previous studies carried out on different sea paigns. In fact the number of nests recorded has increased turtle species (Bonhomme et al., 1987; Gyuris & Limpus, since 1997 from 11 to 29 in 2007 (Jribi et al., 2006; Bradai 1988), a low genetic diversity has been detected by protein & Jribi, 2007). electrophoresis. This technique allowed detecting the presence This study has permitted us to obtain the first data on of multiple paternity in half of the clutches analysed, which genetic diversity of the Tunisian loggerhead turtle nesting indicated that multiple paternity is also common in this population. Analysing other Tunisian nesting sites, even small population. The lower frequency of multiple paternity those with irregular nesting activities, would be interesting reported here compared with that found by Zbinden et al. to assess a possible local genetic structuring which could (2007) in Greece and estimated at 93% using microsatellite potentially result in modification of management strategies. markers, can be explained by the low variability of the In this sense, recent genetic surveys of nesting populations genetic marker we used compared to microsatellites and by have uncovered genetic differences and restricted gene flow the small number of offspring analysed. However, it cannot between neighbouring Mediterranean rookeries suggesting be excluded that there is a different incidence of multiple the existence of several management units (Encalada et al., paternity between loggerhead populations in the 1998; Carreras et al., 2007; Garofalo et al., 2009). Moreover, Mediterranean region. Previous analyses of different popu- as mtDNA ignores male-mediated gene flow, analysing lations of the same sea turtle species have shown variable highly polymorphic and biparentally inherited nuclear levels of multiple paternity (Chelonia mydas: Fitzsimmons, markers (e.g. microsatellites), combined with the available 1998; Lee & Hays, 2004; Lepidochelys species: Hoekert et al., data on other Mediterranean nesting sites could provide 2002; Jensen et al., 2006). It has been documented that the additional insights into the phylogeography of this species rate of multiple paternity is likely to be affected by behaviour in the Mediterranean region. in the breeding area, mating opportunities along migration paths, sex-ratio of turtles at breeding sites (Zbinden et al., 2007) and breeding population size (Jensen et al., 2006). ACKNOWLEDGEMENTS Further analyses are required to understand the effective incidence of multiple paternity in the Mediterranean Sea. Such This study would have not been possible without the collabor- reproductive behaviour could be of great importance in ation of the staff of the following institutions: INSTM (Institut improving the genetic diversity of our population. In fact, it National des Sciences et Technologies de la Mer), the RAC/ has been admitted that multiple paternity has several benefits SPA (Regional Activity Centre for Specially Protected Areas) on genetic diversity of offspring by avoiding inbreeding and a and APAL (Agence de Protection et Ameńagement du possible genetic incompatibility and permitting the fertiliza- Littoral). We would like to thank I. Jribi, S. Karaa and tion of the ovocytes by the most competitive sperm S. Ben Hassine for their help and collaboration to provide (Stockley et al., 1993; Vala et al., 2000). samples from the Kuriat Islands. We are also grateful to The mitochondrial DNA analysis revealed the presence of the team of the Molecular Evolution Laboratory from the only one haplotype CC-A2 which is the haplotype most com- Stazione Zoologica of Naples, and especially to Professor monly found in the Mediterranean Sea. It was therefore, not G. Bernardi, for their assistance on molecular analysis. The possible to genetically differentiate the present population authors wish to acknowledge use of the Maptool program from the other Mediterranean nesting populations except (www.seaturtle.org) for the drawing of Figure 1. from the eastern Turkey and Calabria rookeries which are characterized by the high frequency of the haplotypes CC-A3 and CC-A20 respectively (Laurent et al., 1998; REFERENCES Garofalo et al., 2009). The low genetic diversity detected by both mitochondrial Belkhir K., Borsa P., Chikhi L., Raufaste N. and Bonhomme F. 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