A SURVEY OF THE GENETIC DIVERSITY OF THE LOGGERHEAD AND GREEN SEA TURTLES OF THE SULTANATE OF OMAN Joshua S. Reece1, A. Alansari2, H. M. Kalkvik3, A. Alkindi2, I. Mahmoud2, A. Y. Al Kiyumi4, A. E. Elshafie2, Michael P. Jensen5, and C. L. Parkinson3 1Department of Biology, California State University, 2555 E. San Ramon Ave, Fresno CA 93740 USA 2Sultan Qaboos University, Department of Biology, Al-Khod 36, Postal Code 123, Muscat, Sultanate of Oman 3University of Central Florida Department of Biology, 4000 Central Florida Blvd., Orlando, FL 32816-2368 USA 4Ministry of Nature Conservation, P.O. Box 323, Postal Code 113, Muscat, Sultanate of Oman 5Southwest Fisheries Science Center, National Marine Fisheries Service, NOAA, 8901 La Jolla Shores Drive, La Jolla, CA 92037 USA Reece, J. S., A. Alansari, H. M. Kalkvik, A. Alkindi, I. Mahmoud, A. Y. Al Kiyumi, A. E. Elshafie, Michael P. Jensen, and C. L. Parkinson. 2016. A survey of the genetic diversity of the Loggerhead and Green Sea Turtles of the Sultanate of Oman. Bulletin of the Florida Museum of Natural History 54(6):104−117. ABSTRACT The Sultanate of Oman hosts large annual nesting aggregations of up to 23,000 Loggerhead Sea Turtles (Caretta caretta) and 10,000 Green Sea Turtles (Chelonia mydas). Both populations have until now been poorly sampled (eight samples for C. caretta and fifteen for C. mydas). Genetic diversity information is needed for both species because these populations are increasingly threatened as the coastlines of Oman become more developed. It is currently unclear how diverse these rookeries are relative to other rookeries in the Atlantic, Mediterranean, Indian, or Pacific oceans. We explored mitochondrial DNA (mtDNA) diversity in 100 C. caretta from Masirah Island and 42 C. mydas from Ras al Hadd, Oman. We also analyzed C. caretta with four nuclear DNA (nDNA) microsatellites to investigate nuclear diversity. Loggerhead populations in Oman showed extremely low mtDNA diversity, but high nDNA diversity, suggesting isolation of genetically impoverished matrilineal lines, but relatively high male-mediated gene flow. In contrast, C. mydas populations in Oman consist of two clusters of distinct haplotypes that yield extremely high estimates of haplotype and nucleotide diversity relative to other global rookeries. These results describe the diversity of C. caretta and C. mydas rookeries in Oman with implications for global conservation. Key words: Caretta caretta, Chelonia mydas, genetic diversity, Indian Ocean, nesting aggregation, phy- logeography, rookery, sea turtles. Published On-line: December 7, 2016 Open Access Download at https://www.flmnh.ufl.edu/bulletin/publications/ ISSN 2373-9991 Copyright © 2016 by the Florida Museum of Natural History, University of Florida. All rights reserved. Text, images and other media are for nonprofit, educational, or personal use of students, scholars, and the public. Any commercial use or republication by printed or electronic media is strictly prohibited without written permission of the museum. REECE ET AL.: Genetic diversity of sea turtles from the Sultanate of Oman 105 INTRODUCTION Loggerhead Sea Turtles (Caretta caretta) are distributed globally and nest in temperate and subtropical regions of the Mediterranean, Atlantic, Pacific, and Indian oceans (Bolten and Witherington, 2003). The two largest known rookeries consist of over 10,000 females nesting per year, and are located in south Florida in the United States and on Masirah Island in Oman (Indian Ocean) (Ross, 1978; Baldwin et al., 2003; Ehrhart et al., 2003). Loggerhead Sea Turtles are threatened by global climate change, loss of nesting habitat, hunting and bycatch (Witherington, 2003; Witt et al., 2010; Reece et al., 2013). Caretta caretta is currently listed by IUCN (www.redlist.org) as Vulnerable, and as Threatened under the U.S. Endangered Species Act (NMFS and USFWS, 1991a). The rookery at Masirah Island, Oman, contains up to 23,000 nesting females annually on a single 10-kilometer beach (Ross, 1978; Ross and Banwari, 1982; Baldwin et al., 2003); a number comparable to the largest rookeries in the Caribbean (Ehrhart et al., 2003). Assessment of genetic diversity and genetic structure has been widely used in management of listed species (Frankham et al., 2002; Frankham, 2010). Although the south Florida C. caretta rookery has been extensively characterized for genetic diversity (Bowen et al., 2005; Shamblin et al., 2012, 2014), the Oman rookery has not been thoroughly surveyed for genetic diversity at mitochondrial DNA (mtDNA) or nuclear DNA (nDNA) levels (but see Bowen et al., 1994). Green Sea Turtles (Chelonia mydas) are also globally distributed in tropical and subtropical oceans and share many of the same threats as the Loggerhead Sea Turtle (NMFS and USFWS, 1991a, 1991b, 1998). This species is listed as Endangered by the IUCN and was recently downgraded to Threatened under the US Endangered Species Act (NMFS and USFWS, 1991a). Chelonia mydas nest in significant numbers in Florida, throughout the Caribbean, and Hawaii; the largest known aggregations are at Ascension Island (mid-Atlantic) and Raine Island, Australia. They also nest in significant numbers in Oman, where up to 44,000 nests (representing over 10,000 females) were recorded in 2005. Genetic data on C. mydas aggregations in Oman is also sparse, but include a study of four microsatellite loci to examine patterns of male-mediated gene flow (Roberts et al., 2004), an RFLP survey (Karl et al., 1992), and a mtDNA control region sequence analysis of 15 individuals (Bowen et al., 1992), all of which were used to examine global phylogeography. Chelonia mydas in Oman share close ties to Austral-Asian rookeries. Male-mediated gene flow occurs over much larger distances than female-mediated gene flow. However, information on comparative levels of genetic diversity for both species is lacking. The genetic diversity of C. caretta and C. mydas in Oman is of interest both for understanding biogeography and for long-term management (NMFS and USFWS, 1991a, 1991b, 1998). We inventory and describe the population genetic diversity of the C. caretta rookery at Masirah Island, Oman, based on a sample of 100 nesting females, and the C. mydas rookery at Ras al Hadd, Oman, based on 42 nesting females. These samples more than double the most extensive sampling for both species from Oman for genetic data to date (Bowen et al., 1992; Bowen et al., 1994). We assessed genetic diversity for C. caretta using markers that would allow for comparison with the most extensive characterization of Atlantic rookery population genetic diversity available at the time of the study, which included a short (391 bp) fragment of the mtDNA control region and four nuclear microsatellite loci (Bowen et al., 2005). We describe mtDNA and nDNA diversities at Masirah Island relative to other rookeries globally. We assessed C. mydas genetic diversity based solely on mtDNA control region haplotypes comparable to those published for the Atlantic, Indian and Pacific Ocean rookeries. These data represent an important improvement over the current state of knowledge for sea turtles in Oman. MATERIAL AND METHODS Sample ColleCtion During summer 2004, a single researcher (JSR) collected tissues from 100 C. caretta adult nesting females over 10 km of northeastern Masirah Island, Oman (58.8905° N, 20.4551° E) and from 106 BULLETIN FLORIDA MUSEUM NATURAL HISTORY VOL. 54(6) 42 adult C. mydas over two km of Ras al Hadd, Oman (22.5080° N, 59.7643° E). We obtained tissue samples from the dorsal medial portion of the front right flipper using a six mm biopsy punch. We collected all C. caretta samples over the course of three nights and inspected each individual for sign of previous biopsy to avoid double sampling. In most cases, we biopsied females after they completed the nesting process, and when possible, flipper tags were applied by local Omani researchers. We sampled C. mydas over the course of a single evening, using the same methods to avoid double sampling of any individual. Tissue samples were stored in lysis buffer for later DNA extraction using DNeasy Tissue Kits (Qiagen). mitoChondrial dna iSolation and amplifiCation Because C. caretta rookeries were globally described by Encalada et al. (1998) based on a 391 base pair (bp) fragment of the mtDNA D-loop, we amplified this fragment using primers TCR- 5 and TCR-6 (Norman et al., 1994). Subsequent to sequences being generated for this study, a longer fragment (817 bp) has been developed (Abreu-Grobois et al., 2006) that provides greater resolution (Shamblin et al., 2012). We did not use this fragment because it was not available at the time these data were generated and because the majority of studies available for comparison today used the shorter 391 bp fragment (Bowen et al., 1995; Encalada et al., 1998; Hatase, et al. 2002; Boyle et al., 2009; Garofalo et al., 2009; Chaieb et al., 2010; Shamblin et al., 2011). We evaluated C. mydas nesting populations at the same locus, but using a 487 bp fragment amplified by primers CR-1 and CR-2 (Norman et al., 1994) that have been used extensively to characterize C. mydas populations in the Atlantic Ocean (Encalada et al., 1996; Bjorndal et al., 2005, 2006), Indian (Bourjea et al., 2007) and Pacific oceans (FitzSimmons et al., 1996; Dethmers et al., 2006; Formia et al., 2006). We subjected purified DNA to polymerase chain reaction (PCR) in 25 µL reactions by denaturing at 93° C for three min, followed by 39 cycles of (1) DNA denaturing at 93° C for 30 s, (2) primer annealing at 52° C for 30 s, and (3) primer extension at 72° C for 30 s, with a final primer extension cycle at 72° C for 10 min with both positive and negative PCR controls. We visualized PCR products on an agarose gel and purified the 391 bp (C. caretta) and 487 bp (C. mydas) fragments using MinElute Gel Extraction Kits (Qiagen). We sequenced the purified products on an ABI 3100 automated sequencer in both directions following manufacturer’s protocols. All mtDNA analyses were conducted in 2004 at Sultan Qaboos University in Muscat, Oman. miCroSatellite amplifiCation for loggerhead Sea turtleS Microsatellite loci were chosen to be comparable to a previous inventory of Atlantic rookeries by Bowen et al. (2005). Additional loci have become available subsequent to the generation of the data in this study (Shamblin et al., 2009), but comparative data are not yet available for other rookeries. Carreras et al. (2007) surveyed the Mediterranean for three of the four microsatellite loci used in this study, but due to lack of the fourth microsatellite locus, these data were not included in our analyses. We used the primers CC141 (FitzSimmons et al., 1996), CC176 (Moore and Ball, 2002), CC7 (FitzSimmons, 1998), and DC107 (Bowen et al., 2005). Amplification conditions for the multiplexed microsatellites consisted of 15 μL reactions containing 0.4 units of Sigma Taq, 1.5 μL 10x reaction buffer, 1.5 mM MgCl2, 0.2 μM dNTPs, 200 nM primers and 6 ng DNA. The thermocycler programs for the PCR reactions were 93° C for 3 min, 8 cycles of 92° C for 30 s, annealing temperature for 55 s (54° C, CC141; 55° C, DC107; 56° C, CC7; 60° C, CC176), 72° C for 1 min 25 s, followed by 30 cycles of 89° C for 30 s, annealing for 55 s (52° C, CC141; 53° C, DC107; 54° C, CC7; 58° C, CC176), 72° C for 1 min 25 s, with a final extension of 72° C for 10 min. Forward primers were labeled fluorescently, and samples were genotyped on a Beckman CEQ 8000 using 20 μL dilution of PCR reactions in SLS buffer. The microsatellite fragment length was scored using CEQ 8000 software. We included the same loci as Bowen et al. (2005), and to ensure identical binning of microsatellite loci and comparability of datasets from their study and our own sampling, we acquired the original tissue samples from REECE ET AL.: Genetic diversity of sea turtles from the Sultanate of Oman 107 eight C. caretta used in their study to standardize allele scoring of the microsatellite loci. The eight individuals were genotyped, and the fragment lengths acquired from our analysis were compared to those reported in Bowen et al. (2005). The eight individuals represented 31–55% of the identified alleles of the four loci from their original study. We believe this provided a sufficient sampling of alleles to ensure consistent scoring between the two microsatellite datasets and comparability of datasets. We attempted to acquire additional samples scored in the previous study, but were unsuccessful. This limitation would have no effect on estimates of diversity. mitoChondrial dna analySeS We manually edited sequence data in SEQUENCHER v.4.2 (www.genecodes.com) and aligned the sequence data in GENEDOC (Nicholas and Nicholas, 1997). Sequences from several previous studies were incorporated with our own sampling. To compare our findings with globally published estimates of C. caretta genetic diversity, we incorporated eight previously published global or regional surveys of major C. caretta nesting aggregations and eight surveys of major C. mydas rookeries (see results). For both C. caretta and C. mydas, we inventoried mitochondrial haplotype diversity (h [Nei, 1987]), nucleotide diversity (π [Nei, 1987]), and average number of nucleotide differences (k [Tajima, 1983]) per population for the mitochondrial control region in DnaSP v.5.0 (Rozas et al., 2003). For each of the above metrics (except k), we computed 1000 coalescent simulations using the coalescent calculator in DNAsp and recorded 95% confidence intervals for each metric for the Oman rookery. We then compared these distributions to the values calculated in the same way for rookeries previously surveyed. We used Tajima’s D (Tajima, 1989) to test the hypothesis that control region variation does not differ from neutral expectations or show evidence of large demographic shifts. For C. caretta, we described nDNA diver- sity based on microsatellites using the program GENALEX v.6 (Peakall and Smouse, 2006) to summarize the number of alleles (A), number of effective alleles (AE), mean expected heterozygosity per locus (HE), mean observed heterozygosity per locus (HO), and overall fit to Hardy-Weinberg equilibrium expectations. Genetic diversity at these loci was compared to previously surveyed rook- eries in the western Atlantic (Bowen et al., 2005). Table 1. Haplotypes and numbers of individuals for C. caretta and C. mydas sampled in Oman. Haplotype names referenced here correspond with nomenclature used by the University of the Florida Archie Carr Center for Sea Turtle Research database. Haplotype Number of Individuals Caretta caretta (n = 100) CC-Oman1 (new) 1 CC-Oman2 (new) 1 CCA11 98 Chelonia mydas (n = 42) CM-Oman1 (new) 1 CM-Oman2 (new) 1 CMP49 1 CMP62 15 CMP71 8 CMP73 16 108 BULLETIN FLORIDA MUSEUM NATURAL HISTORY VOL. 54(6) RESULTS genomiC diverSity in the maSirah iSland Caretta Caretta rookery We identified low mtDNA diversity in the Masirah Island C. caretta rookery with three mitochondrial haplotypes in 100 individuals (Table 1). The most common haplotype (CC-A11) occurred in 98 individuals. The two remaining haplotypes were novel, each found in a single individual, and were distinct from the common haplotype CC- A11 by a single transition mutation at sites 119 and 314, respectively. Haplotype diversity (h) for the Masirah Island C. caretta rookery was 0.04 (95% CI = 0.0–0.4 from coalescent simulations), which is among the lowest documented haplotype diversity for a major C. caretta rookery surveyed in this dataset. Nucleotide diversity (π) was 0.0001 (95% CI = 0.0–0.1) and the average number of nucleotide differences (k) was 0.04. Tajima’s D for the Masirah Island (D = -1.38) rookeries was not significant at α = 0.05. Oman has among the lowest C. caretta mitochondrial DNA diversity globally (Table 2) in terms of both haplotype diversity (Fig. 1) and nucleotide diversity (Fig. 2). No microsatellite loci deviated from H-W equilibrium at an α = 0.05 after a Bonferroni correction. In contrast to mtDNA diversity, the Masirah Island rookery showed similar levels of nuclear marker diversity not only to the similarly sized south Florida rookery (Table 3), but also relative to the entire northwestern Atlantic aggregation combined. We compared observed and expected heterozygosity among all four microsatellite loci between Masirah and the south Florida rookery, both similarly sized, and between all northwestern Atlantic rookeries combined. Neither metric yielded significantly different comparisons (t-test, p ≥0.1). mitoChondrial genetiC diverSity for Chelonia mydas at raS al hadd rookery We observed six haplotypes in 42 sampled individuals (Table 1). These haplotypes form two clusters, one that includes three haplotypes unique to Oman (CMP71, CMP73, and one novel haplotype- CM-Oman1) and separated by 14 mutational steps from a second cluster that contains three haplotypes, two unique to Oman (CMP62 and a novel haplotype CM-Oman2), and one shared between Oman and other rookeries throughout Austral-Asia (CMP49). Haplotype diversity was 0.71 (95% CI = 0.59–0.93 from 1000 coalescent simulations). Nucleotide diversity was 0.02 (95% CI = 0.01 to 0.03) and the average number of nucleotide differences was 8.6 (Table 4). Tajima’s D was significant and positive (D = 2.5) indicating recent population contraction. Overall, the C. Figure 1. Caretta caretta haplotype diversity for this study (Oman) and previously published surveys of rookeries listed in Table 2. Whiskers represent standard errors where available from published studies. The abbreviations CC and CM correspond to C. caretta and C. mydas, respectively. REECE ET AL.: Genetic diversity of sea turtles from the Sultanate of Oman 109 Table 2. Caretta caretta diversity metrics for this study and eight others. Metrics reported here include ocean basin, haplotype diversity (h) and standard error (SE), nucleotide diversity (π) and standard error (SE), the number of individuals sampled (n), and relevant citation. Rookery Ocean Basin h SE π SE n Citation Oman Indian 0.04 0.027 0.0001 0.00007 100 This study Tunisia Mediterranean 0 0 0 16 Chaieb et al. (2010) Australia Pacific 0 0 26 Bowen et al. (1995) Japan Pacific 0.212 0.097 0.00277 0.00127 26 Eastern Australia Pacific 0.095 0.028 101 Boyle et al. (2009) New Caledonia Pacific 0.133 0.081 27 NWFL Atlantic 0.44 0.09 0.02 0.0035 42 Encalada et al. (1998) S. Florida Atlantic 0.59 0.033 0.0294 0.00096 50 NEFL/NC Atlantic 0.04 0.0019 105 Mexico Atlantic 0.65 0.093 0.0028 0.00054 20 Greece Mediterranean 0.18 0 21 All Atlantic Atlantic 0.67 0.027 0.023 0.001 249 Calabria Mediterranean 0.533 0.04 47 Garofalo et al. (2009) Minabe Pacific 0.06 0.0008 102 Hatase et al. (2002) Miyazaki Pacific 0.41 0.006 46 Fukiagehama Pacific 0.09 0.0013 22 Yakushima Pacific 0.24 0.0034 89 Brazil Atlantic 0.138 0.0327 0.000372 0.000606 190 Reis et al. (2010) Volusia Co FL Atlantic 0.181 0.104 0.0087 0.0052 21 Shamblin et al. (2011) Miami-Dade Co FL Atlantic 0.416 0.09 0.02 0.0109 22 St. Johns Co FL Atlantic 0.333 0.111 0.0134 0.0075 27 Flagler Co FL Atlantic 0.272 0.089 0.0132 0.0073 32 N Volusia Co FL Atlantic 0.252 0.092 0.0083 0.0049 37 Collier Co FL Atlantic 0.53 0.035 0.0242 0.0126 40 S Georgia Atlantic 0.047 0.044 0.0022 0.0018 43 Broward Co FL Atlantic 0.479 0.058 0.0206 0.0108 48 Palm Beach Co FL Atlantic 0.615 0.069 0.0169 0.009 49 Sarasota Co FL Atlantic 0.571 0.061 0.0232 0.0121 57 Canaveral FL Atlantic 0.492 0.054 0.022 0.0115 58 Dry Tortugas Atlantic 0.254 0.074 0.0068 0.0041 58 Melbourne Beach, FL Atlantic 0.451 0.044 0.0201 0.0105 106 110 BULLETIN FLORIDA MUSEUM NATURAL HISTORY VOL. 54(6) Figure 2. Caretta caretta nucleotide diversity for this study (Oman) and previously published surveys of rookeries listed in Table 2. Data represent means and standard errors where available from published studies. Table 3. Microsatellite population genetic statistics for Caretta caretta, including the sample size (n), number of alleles (A), the effective number of alleles (AE), observed heterozygosity (HO), and expected heterozygosity (HE). None of the comparisons observed and expected heterozygosities were significantly different at α = 0.05. Population Locus n A AE HO HE North Carolina DC107 26 7 4.418 0.769 0.774 CC176 26 14 4.952 0.923 0.798 CC141 27 11 6.284 0.741 0.841 CC7 26 10 4.135 0.769 0.758 South Carolina DC107 24 6 4.299 0.750 0.767 CC176 24 12 4.235 0.750 0.764 CC141 24 11 6.261 0.833 0.840 CC7 24 11 4.702 0.875 0.787 Georgia DC107 39 7 4.261 0.821 0.765 CC176 39 15 5.828 0.872 0.828 CC141 39 11 6.364 0.821 0.843 CC7 39 12 5.895 0.872 0.830 Florida DC107 288 10 4.652 0.795 0.785 CC176 289 29 5.289 0.775 0.811 CC141 290 15 7.650 0.828 0.869 CC7 290 18 5.128 0.766 0.805 Brazil DC107 81 8 3.647 0.741 0.726 CC176 81 14 4.528 0.790 0.779 CC141 81 11 3.266 0.704 0.694 CC7 81 8 2.368 0.580 0.578 Oman DC107 115 15 2.587 0.583 0.613 CC176 108 12 3.283 0.713 0.695 CC141 112 11 6.018 0.839 0.834 CC7 115 14 4.606 0.774 0.783 REECE ET AL.: Genetic diversity of sea turtles from the Sultanate of Oman 111 Table 4. Chelonia mydas diversity metrics for this study and eight others. Metrics reported here include ocean basin, haplotype diversity (h) and standard error (SE), nucleotide diversity (π) and standard error (SE), number of individuals sampled (n), and relevant citation. Rookery Ocean Basin h SE π SE n Citation Oman Indian 0.706 0.017 0.0215 0.00133 42 This study Trinidade Atlantic 0.5046 0.0522 0.0012 0.0011 99 Bjorndal et al. (2006)Atol das Rocas Atlantic 0.5196 0.0763 0.0012 0.0011 53 Fernando de Noronha Atlantic 0.2333 0.1256 0 0 16 Tortugaro Atlantic 0.16 0.02 0.0034 0.0022 433 Bjorndall et al. (2005) Total NMC Indian 0.3964 0.01962 191 Bourjea et al. (2007) Total SMC Indian 0.3425 0.0221 53 Europa Indian 0.1174 0.0076 33 Juan de Nova Indian 0.5632 0.036 20 Nosy Iranja Indian 0 0 13 Mayotte Indian 0.4524 0.0231 41 Mohéli Indian 0.3708 0.0133 34 Glorieuses Indian 0.3441 0.0168 39 Cosmoledo Indian 0.3871 0.021 31 Aldabra Indian 0.4646 0.0249 26 Farquhar Indian 0.7143 0.0342 7 Tromelin Indian 0.241 0.0132 44 Total SW Indian Ocean Indian 0.5063 0.0289 288 Pacific Pacific 0.71 0.02 0.034 0.017 272 Indian Indian 0.7 0.03 0.019 0.01 107 Dethmers et al. (2006) Australasia Indo-Pacific 0.88 0.01 0.041 0.02 714 Southeast Asia Indo-Pacific 0.8 0.01 0.006 0.004 335 Cyprus Mediterranean 0.22 0.16 0.00042 10 Florida Atlantic 0.56 0.047 0.0013 24 Mexico Atlantic 0.82 0.058 0.0057 20 Costa Rica Atlantic 0.13 0.11 0.00028 15 Aves Atlantic 0.25 0.18 0.0053 8 Surinam Atlantic 0.26 0.14 0.0056 15 Encalada et al. (1996) Brazil Atlantic 0.68 0.085 0.0017 16 Ascension Atlantic 0.35 0.12 0.00077 20 Guinea Bissau Atlantic 0 0 0 0 19 Atlantic overall Atlantic 0.83 0.005 147 Poilão Atlantic 0 0 0 0 51 Ascension Atlantic 0.26 0.081 0.0006 0.0007 50 Bioko Atlantic 0.184 0.068 0.0004 0.0006 50 Principe Atlantic 0.533 0.172 0.0011 0.0012 6 Formia et al. (2006) São Tome Atlantic 0.584 0.127 0.003 0.0021 20 Comoros Indian 0.733 0.12 0.0261 0.0154 10 Atlantic overall Atlantic 0.22 0.041 0.0007 0.0008 178 Aves Atlantic 0.186 0.088 0.0039 0.0025 Lahanas et al. (1998) Costa Rica Atlantic 0.1627 0.0231 0.0033 0.0022 433 Seminoff (2002) 112 BULLETIN FLORIDA MUSEUM NATURAL HISTORY VOL. 54(6) mydas rookery in Oman appears to have relatively high haplotype (Fig. 3) and nucleotide diversity (Fig. 4) compared to other rookeries globally. DISCUSSION This study represents the first detailed survey of genetic diversity in Oman’s C. caretta and C. mydas rookeries. We find that mitochondrial DNA diversity in the C. caretta sample is low and among the lowest ever surveyed compared to similarly sized rookeries in the Atlantic (Tables 1–2; Figs. 1–2). This is despite the tremendous size of the Masirah Island rookery, which may be equal in size to the largest Atlantic C. caretta rookeries (Ross, 1978; Ross and Banwari, 1982; Baldwin et al., 2003; Ehrhart et al., 2003). However, nuclear diversity at four microsatellite loci is high (Table 3), suggesting an even greater discrepancy between nuclear and mitochondrial diversity than has been observed in Atlantic rookeries (Bowen et al., 2005). This disparity likely results from the effective population size differences between mtDNA and nDNA, but also from the geographic isolation of maternally-inherited mitochondrial DNA through natal homing, whereas nuclear material is exchanged through male-mediated gene flow with other rookeries. The three haplotypes we observed in Oman are closely related to one very common allele in the Atlantic (CCA1), but are at least 16 mutational steps from most remaining Atlantic haplotypes. We surveyed C. mydas for mitochondrial DNA only and found levels of genetic diversity that are high relative to other rookeries in the Indian Ocean, Atlantic Ocean, or entire Austral- Asian region (Figs. 3–4). Haplotype diversity in Figure 3. Chelonia mydas haplotype diversity for this study (Oman) and previously published surveys of rookeries listed in Table 4. Data represent means and standard errors where available from published studies. Figure 4. Chelonia mydas nucleotide diversity for this study (Oman) and previously published surveys of rookeries listed in Table 4. Data represent means and standard errors where available from published studies. REECE ET AL.: Genetic diversity of sea turtles from the Sultanate of Oman 113 Oman (0.71) is similar to that reported for Mexico (0.82), and Brazil (0.68), and higher than rookeries in Florida (0.56) and Costa Rica (0.13), Ascension Island (0.35), and the Mediterranean (Encalada et al., 1996). Overall haplotype diversity for the Atlantic is 0.83 with nucleotide diversity lower than what we observed in Oman (see also Encalada et al., 1996; Bjorndal et al., 2005). Haplotype and nucleotide diversity also rank high relative to other rookeries sampled in the southwest Indian Ocean, which range from 0 to 0.71 for haplotype diversity and 0 to 0.03 for nucleotide diversity (the rookery at Farquhar, North of Madagascar, being the most diverse; Bourjea et al., 2007). African rookeries typically have lower levels of genetic diversity, with the exception of the Comoros (h =0.733, π = 0.026) (Formia et al., 2006). Our focus in this work is the comparative diversity of C. mydas rookeries in Oman, but preliminary analyses (not shown here) reveal close associations of mtDNA haplotypes and some shared haplotypes with rookeries in the southwestern Indian Ocean (Bourjea et al., 2007) and Austral-Asia (Dethmers et al., 2006). Use of microsatellite loci would make it possible to more accurately identify and estimate levels of connectivity among Oman and other rookeries throughout the Indian and Pacific Oceans. However, it is clear that Oman hosts a C. mydas rookery that is genetically diverse with a mixture of unique and shared mitochondrial haplotypes (Table 1). Additional sampling is warranted to provide a clearer assessment of genetic diversity in this region. We demonstrated that Oman’s C. caretta show low mitochondrial DNA diversity. It is unclear whether the shared haplotypes between Oman and the western Atlantic are a result of gene flow from the Indian to the Atlantic Ocean or vice versa. However, it is clear that of the two clades of C. caretta present in the Atlantic (Encalada et al., 1998; Reece et al., 2005; Shamblin et al. 2014), one is less diverse and shares recent ancestry with the Omani rookery. Available evidence indicates that the CCA11 lineage present in Oman may represent a recent re-invasion of the Indo-Pacific by an Atlantic lineage (see Shamblin et al., 2014). Chelonia mydas in Oman appear to not have suffered as severe of a population contraction and/ or loss of genetic diversity as C. caretta. These changes are potentially mitigated by lower nest site fidelity in C. mydas and female-mediated gene flow with other rookeries in the Indo-Pacific and the southern Atlantic (Bourjea et al., 2007). The C. caretta rookery at Masirah Island has low levels of mitochondrial DNA and high levels of nuclear DNA diversity, suggesting a recent colonization or demographic shift and/or high nest site fidelity. However, male-mediated gene flow may be substantial, allowing for an overall diverse genetic makeup of the rookery. It is unclear how unique the genetic variants that make up Oman’s C. caretta rookery are in the Indian Ocean. Although most of the C. caretta nesting in Oman are concentrated at Masirah Island, other rookeries do exist elsewhere in Oman and neighboring Yemen. None has been characterized for genetic diversity. Threats faced by Oman’s rookeries include rapid coastal development and loss of dune structure due to introduced goats, which defoliate the dunes and rob them of the root structures on which the dunes depend (JSR, pers. obs.). Loss of dunes leads to urban lights being visible from the berm, which causes disorientation of adults and hatchlings (JSR, pers. obs.). Chelonia mydas face additional threats, including some harvesting for food, although not on a commercial level (Oman Ministry of Environment and Climatic Affairs, pers. comm.). Oman’s C. mydas are genetically diverse. Their genetic makeup is similar to other rookeries in the Pacific and Indian Oceans, such that females may recruit to different nesting beaches over the course of their reproductive lives or these shared haplotypes may be an artifact of common ancestry. Determining the degree of connectivity among rookeries is critical for their future management. For example, the impacts of local threats to rookeries may be ameliorated or exacerbated by the degree of connectivity with surrounding rookeries. That information is necessary for conservation practitioners to develop effective management strategies. Future studies in Oman will benefit from the increased 114 BULLETIN FLORIDA MUSEUM NATURAL HISTORY VOL. 54(6) phylogeographic resolution of longer mtDNA fragments (Abreu-Grobois et al., 2006; Shamblin et al., 2012), additional microsatellite data (Shamblin et al., 2009), and genomic advances (Tikochinski et al., 2012). Satellite tagging and physical tags will yield additional information on the movements of C. mydas throughout the Indo-Pacific and south Atlantic. Lastly, although this study characterized a single geographically localized rookery in Oman for each species, additional sampling and increased nuclear microsatellite sampling may yield greater insight into the management of distinct nesting beaches on a regional scale, which may or may not be genetically similar genetically to those sampled here. ACKNOWLEDGMENTS Funding for this project was provided by grants to JSR from the University of Central Florida Biology Department and the Caribbean Conservation Corporation Mel Stark Award, and funds awarded to CLP from UCF startup package, NSF grant DEB–0416000, and a Florida Fish and Wildlife Conservation Commission/Caribbean Conservation Corporation License Plate Grant. Samples of Oman Loggerhead Sea Turtles were collected under permit number 09/2003 issued by the Director General of Nature Conservation, Sultanate of Oman, and CITES permit 04US076985/9 issued by the U.S. Department of Interior. Transportation, housing, collecting assistance, laboratory space and logistics of field work were provided by Sultan Qaboos University. We acknowledge Shoaib Al-Zidjali and the Hematology Department at Sultan Qaboos University Hospital for sequencing assistance. We thank the turtle rangers at Ras al Hadd and Masirah Island who provided invaluable assistance through the Department of Nature Conservation. We also thank Perran Ross for his valuable advice in preparation for this project. We thank Earl Possardt and Blair Witherington and the U. S. Fish and Wildlife Service and Nicolas Pilcher for their assistance in the field and coordination of travel. 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