First observation of multiple paternity in loggerhead sea turtles, Caretta caretta, nesting on Dalyan Beach, Turkey

First observation of multiple paternity in loggerhead sea turtles, Caretta caretta, nesting on Dalyan Beach, Turkey

Journal of Experimental Marine Biology and Ecology 488 (2017) 60–71 Contents lists available at ScienceDirect Journal of Experimental Marine Biology...

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Journal of Experimental Marine Biology and Ecology 488 (2017) 60–71

Contents lists available at ScienceDirect

Journal of Experimental Marine Biology and Ecology journal homepage: www.elsevier.com/locate/jembe

First observation of multiple paternity in loggerhead sea turtles, Caretta caretta, nesting on Dalyan Beach, Turkey Fikret Sari a,⁎, Aylin Koseler b, Yakup Kaska a a b

Pamukkale University, Faculty of Science and Arts, Department of Biology, Denizli, Turkey Pamukkale University, Faculty of Medicine, Department of Biophysics, Denizli, Turkey

a r t i c l e

i n f o

Article history: Received 31 August 2016 Received in revised form 28 November 2016 Accepted 29 November 2016 Available online xxxx Keywords: Caretta caretta Dalyan Beach Inter-nesting mating Microsatellite Multiple paternity Sperm storage

a b s t r a c t Sea turtles are promiscuous breeders with both males and females mating multiply. Due to this mating system, multiple paternity (MP) occurs in sea turtle clutches, and the frequency of MP varies greatly within and among species. In this study, the paternity of a population of loggerhead sea turtles (Caretta caretta) nesting on Dalyan Beach in Turkey was investigated using two highly polymorphic microsatellite markers (CcP2F11 and CcP7C06). Tissue samples collected from randomly selected hatchlings (a total of 522 hatchlings) from two to three successive clutches (a total of 25 clutches) of 10 nesting females were used for paternity analysis with an average sampling effort of 28.2% of offspring per clutch. Evidence of MP in seven out of 10 females (70%) was found, and it was detected that four out of these seven females mated with at least two males, whereas the remaining three females with at least three males. By analysing the successive clutches of females, it was detected that both the number and genotype of contributing sires was same in all clutches of a given female, possibly due to the lack of successful inter-nesting mating in this population. The high frequency of MP implies the possible high genetic diversity within this population. This study indicates that the density of individuals may be the reason of the high frequency of MP in this relatively small population because mating takes place mainly in a narrow area in Dalyan. The possible mating behaviours of the sea turtles and their population structures were discussed in light of the high frequency of MP within this population reported for the first time. © 2016 Elsevier B.V. All rights reserved.

1. Introduction If a species lives in marine habitats, it is very difficult to observe individuals while mating, and it is usually impossible to determine which mating individuals are actually successful (Karl, 2008). Molecular techniques provide an important tool for determining which individuals are contributing to species persistence in the lack of directly observing reproductive success, and molecular studies reveal aspects of mating system of an organism that otherwise would be impossible to detect (Karl, 2008). Specification of mating system of a species is an important part of understanding its natural history (Bjorndal et al., 1983; Wright, 1931). Mating systems are especially important within small populations because they can affect the genetic effective population size and the evolution of that species (Arden and Kapuscinski, 2002; Charlesworth, 2009; Frankham, 1995; Vucetich et al., 1997). Small population size and a skewed ratio of males to females that are available to mate at any nesting season, also known as operational sex ratio (OSR), may decrease genetic variation and the ability of that population to adapt to new environmental pressure (Kvarnemo and Ahnesjö, 1996; ⁎ Corresponding author. E-mail addresses: fi[email protected], [email protected] (F. Sari).

http://dx.doi.org/10.1016/j.jembe.2016.11.018 0022-0981/© 2016 Elsevier B.V. All rights reserved.

Montgomery et al., 2000). It is very important to estimate population size, population structure, and reproductive behaviour accurately in order to improve current conservation projects and make effective management decisions on endangered species. In populations whose mating system is polyandrous, multiple paternity (MP) affects the effective population size (Sugg and Chesser, 1994) and the genetic variability within a population (Baer and Schmid-Hempel, 1999). For decades, MP studies have been carried out in sea turtle species (i.e., Figgener et al., 2016; FitzSimmons, 1998; Harry and Briscoe, 1988; Jensen et al., 2006; Theissinger et al., 2009) as well as other species, and they provide valuable information regarding mating patterns and enable to understand population structure (Jensen et al., 2006). The frequency of multiple mating is critical for understanding of the evolution of the mating systems and for the conservation of endangered populations (Kichler et al., 1999; Moore and Ball, 2002). Sea turtles are promiscuous breeders, with both males and females mating with multiple mates (FitzSimmons, 1998; Hamann et al., 2003; Miller, 1997), and males engage in intense and aggressive courtship behaviour to gain access to females (Lee and Hays, 2004). It is well known that MP is evident in all sea turtle species: loggerhead (Lasala et al., 2013; Zbinden et al., 2007), green (Alfaro-Núñez et al., 2015; FitzSimmons, 1998), leatherback (Figgener et al., 2016; Stewart and

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Dutton, 2011), olive ridley (Duran et al., 2015; Jensen et al., 2006), Kemp's ridley (Kichler et al., 1999), flatback (Theissinger et al., 2009), and hawksbill (Joseph and Shaw, 2011; Phillips et al., 2013). Frequency of the MP shows inter- and intra-specific variability (Jensen et al., 2006), and the percentage of clutches with MP can vary from 0 to N 90% across these species. The factors causing these differences in MP among sea turtle species and between solitary and arribada rookeries include sperm storage (FitzSimmons, 1998; Pearse and Avise, 2001), sex ratio (Bollmer et al., 1999), inter-nesting interval (Kalb and Owens, 1994), abundance and density of individuals (Ireland et al., 2003; Jirotkul, 1999). Not only short term sperm storage but also sperm competition have been proven to be important aspects of sea turtle mating system (FitzSimmons, 1998; Phillips et al., 2013). Increased offspring viability and genetic diversity, fertilisation assurance and procurement of compatible gametes have been suggested to be some of the benefits of this behaviour (FitzSimmons, 1998; Jennions and Petrie, 2000; Uller and Olsson, 2008). The loggerhead sea turtles (Caretta caretta) are long lived, and generally reach sexual maturity between 20 and 35 years of age (Conant et al., 2009; Snover, 2002); however, they typically reach sexual maturity earlier in the Mediterranean due to slower growth presumably as an adaptation to particular conditions (Casale et al., 2011). As a result, adult loggerheads in the Mediterranean have significantly smaller body size than the other populations around the world (Dodd, 1988; Margaritoulis et al., 2003). After sexual maturation, males are able to mate annually, whereas females mate every 2–3 years (Conant et al., 2009; Hays et al., 2010; Wibbels et al., 1990). Both females and males migrate asynchronously from foraging areas to breeding areas several weeks to months prior to the nesting season (Limpus, 1985). Males arrive a few weeks in advance of the females, but some males appear to be nonmigratory and may reside in breeding areas throughout the year (Henwood, 1987). Courtship and mating of loggerheads start in late March, and take place nearshore of adjacent nesting beaches. Females can store sperm and mate with more than one male in a breeding season (Moore and Ball, 2002; Pearse and Avise, 2001; Sakaoka et al., 2013), and may lay more than one clutch in a season with intervals of approximately two weeks (Dodd, 1988). The loggerhead turtles have a circum-global distribution in temperate and subtropical regions including the whole of the Mediterranean (Bolten, 2003; Dodd, 1988). On the 2015 IUCN Red List, the loggerhead turtle is globally categorized as vulnerable (Casale and Tucker, 2015), while the loggerhead turtle in the Mediterranean is categorized as least concern (Casale, 2015). This categorization of the Mediterranean loggerheads demonstrates that long-term monitoring and conservation projects have recently started to give the results. The loggerhead is the most abundant sea turtle in the Mediterranean, having evolved local populations (Margaritoulis et al., 2003). Loggerhead turtles nesting in the Mediterranean have been shown to have diverged genetically from those in the Atlantic (Bowen et al., 1993; Laurent et al., 1993, 1998), although high numbers of loggerheads from the Atlantic enter the Mediterranean (Carreras et al., 2006; Laurent et al., 1998). The Mediterranean loggerheads are confined almost exclusively to the eastern basin, and the main nesting concentrations are found in Greece, Turkey, and Cyprus (Margaritoulis et al., 2003). Genetic analyses from several nesting areas in Turkey show differentiation among rookeries (Schroth et al., 1996). Loggerhead populations in the Mediterranean are fairly small in comparison with the world population; however, a significant part of the Mediterranean nesting population uses Turkey's beaches, and this demonstrates the importance of protecting sea turtles in this country. Most of the behavioural knowledge of loggerhead mating systems comes from nesting beach-based studies. Although adult female sea turtles are more accessible nearshore or on the nesting beaches to direct observation (Bowen et al., 2004; Dodd, 1988), adult males are less accessible to direct observation as they generally remain in open waters. In this respect, MP studies have great importance, and they provide

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insights into the mating system and the population size and/or density of loggerheads. While several MP studies were conducted previously in different nesting loggerhead turtle populations (Bollmer et al., 1999; Harry and Briscoe, 1988; Lasala et al., 2013; Moore and Ball, 2002; Tedeschi et al., 2015; Zbinden et al., 2007), there was no MP study carried out in the Mediterranean loggerhead population nesting on not only Dalyan Beach but also any other Turkish beach. Dalyan Beach is one of the most important reproductive sites of the loggerhead turtles in terms of annual nest number (Canbolat, 2004), nesting density (Canbolat, 2004), hatching success and high proportion of male hatchlings produced (Sarı and Kaska, 2015), protection status (Türkozan and Yılmaz, 2008), and regular sea turtle surveys (Sarı and Kaska, 2015) not only for Turkey but for the Mediterranean region. This beach is therefore presumably the most appropriate place for conducting the studies of MP and population size on loggerheads. In this study, the paternity of the loggerhead sea turtle population nesting on Dalyan Beach (Turkey)—for the first time at a sea turtle nesting beach in Turkey—was investigated using microsatellite analysis. It was aimed to quantify the frequency of MP, to use this frequency of MP for estimating population size of the nesting females of Dalyan Beach, and to demonstrate the skew in male contributions within clutch and among the successive clutches of a female. In light of the findings, the questions of whether the mating takes place prior to nesting season, or internesting mating that contributes to the hatchling genotypes occur, and whether correlations exist between the number of sires per clutch and female size, nesting date, and hatching success were also discussed. 2. Materials and methods 2.1. Study site The Mediterranean coasts of Turkey are important nesting grounds for both loggerhead and green sea turtles (Chelonia mydas) (Türkozan and Kaska, 2010). Based on nest numbers, Turkey holds the second most important loggerhead sea turtle stocks in the Mediterranean (Margaritoulis et al., 2003). Female loggerhead sea turtles nest on the beaches of Turkey from late April until mid-August, and these nests hatch from late June until early October. Dalyan Beach has been a part of Köyceğiz-Dalyan Specially Protected Area since 1988, and is one of the best protected nesting beaches in Turkey. The beach is used by both loggerhead sea turtles and the Nile softshelled turtles (Trionyx triunguis), and the reproduction of these two endangered species on the same beach increases the importance of the beach (Türkozan and Yılmaz, 2008). Public access is not allowed on Dalyan Beach from 20:00 to 08:00 h during the nesting and hatching season. Dalyan Beach has a length of 4.5 km, and is located in the south-west of Turkey (Fig. 1). The beach is a crescent-shaped fine-sand dune. Behind the western two thirds of the beach is an extensive wetland with a labyrinth of reedy channels, and the wetland complex at the western edge of the beach opens to the sea through a channel. There is a small lake, named Alagöl, in the wetland, and mating of the loggerheads take place in this lake as well as the nearshore of the sea and labyrinth of reedy channels. Majority of the mating activities occurs in Alagöl and the labyrinth of reedy channels (Kaska, personal communication). Behind the eastern third is a small lake, named Iztuzu Lake, which is separated from the Dalyan estuary by a mountain ridge. There are several sand dunes at the lake side of the beach. 2.2. Sample collection This study was approved by the Pamukkale University Animal Ethics Committee (No: PAUHDEK-2014/016), and samples from nesting female turtles and their offspring were obtained during the 2014 nesting season from mid-May to mid-September on the beach.

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F. Sari et al. / Journal of Experimental Marine Biology and Ecology 488 (2017) 60–71

Fig. 1. Map of the study site showing general view of Dalyan Beach (Turkey), and the location of Sea Turtle Research Centre (DEKAMER).

Adult nesting and non-nesting females were identified and tagged using metal flipper tags during nightly patrols with the Monitoring and Conservation Project of Sea Turtles on Dalyan Beach. Using sterile procedures, tissue samples were taken from nesting females of known identity for each individual female by removing surface skin (b0.5 cm2) from the trailing edge of the fore-flipper with a scalpel after females finished laying the observed clutch. After female turtle size (curved carapace length [CCL]) was recorded, nest locations were marked, and nests were screened against mammal predation with wire cages (1 × 1 m) with a mesh size of 9 cm. These nests were checked during their incubation periods. Approximately 45 days after nesting, screens were placed around nests to contain hatchlings of each nest, and these nests were monitored between 19:00 and 07:00 h for emergent activity. Screens were lifted at dawn so that hatchlings were able to escape in case of unattended emergence during the day. On hatching, tissue biopsies (b3 × 5 mm) were taken from a random sample of hatchlings in each clutch from the trailing edge of the carapace using a different sterile scalpel for each individual hatchling. Nests were allowed to hatch naturally, and were excavated when no further hatchlings had emerged for 48 h, or after 5 days since the first hatchling emerged, whichever was sooner. Live and dead hatchlings found inside the nest, as well as dead embryos from unhatched eggs, were also randomly sampled. After the excavation, empty eggshells, unfertilized eggs, and dead-in-egg embryos in the nests were counted, and the hatching success for each nest was calculated by dividing the number of empty eggshells to the number of total number of eggs. On Dalyan Beach, 10 nesting female loggerheads with multiple clutches and a total of their 522 hatchlings from 25 out of 433 nests laid in the entire nesting season (5.8%) were sampled in this study to examine paternity. Each tissue sample was kept in 96% ethanol at −20 °C until DNA extraction.

2.3. DNA extraction and microsatellite genotyping DNA was prepared from approximately 50 mg of a skin tissue sample following Sari et al. (2015). After DNA isolation, 5 μl of the DNA extracts were run on 1% agarose gels with ethidium bromide to check for quality and yield. Some samples were diluted 1:10 for further analysis. All DNA extracts were stored at −20 °C. Two tetranucleotide microsatellite loci (CcP2F11 and CcP7C06) designed for C. caretta (Shamblin et al., 2009) were used to genotype each female and its offspring by amplifying their DNA through a Polymerase Chain Reaction (PCR). One of each primer pair was fluorescently labelled with FAM and HEX. Each microsatellite locus was amplified in a 25 μl total reaction volume with a final concentration of 0.3 mM total dNTP (Thermo Scientific), 0.2 μM each of a corresponding labelled forward- and an unlabelled reverse-primer, 1.5 mM MgCl2 (Thermo Scientific), 1 × Taq buffer with (NH4)2SO4 (Thermo Scientific), 5 units Taq DNA polymerase (Thermo Scientific), and 5 μl of the template DNA solution. For both CcP2F11 and CcP7C06, thermal cycling was initiated with a denaturation step of 3 min at 94 °C, followed by 40 cycles of a 30 s denaturation at 94 °C, an 1 min annealing at 55 °C, and an 1 min extension at 72 °C, and followed by a final extension step of 7 min at 72 °C. Prior to fragment analysis, the PCR product for each sample was checked for amplification using 2% agarose gels with ethidium bromide. The PCR products were sent to an external laboratory (GATC Biotech, Germany), where fragment analysis was performed on an ABI 3730 DNA Analyzer (Applied Biosystems, Life Technologies Corporation, CA). Results from the DNA Analyzer were visualized using Peak Scanner Software (Applied Biosystems), and then allele sizes were assigned. To cross-check allele scoring, a number of samples were selected to re-amplify using the same template DNA and to rescore, and then allelic differences were looked for in the resulting genotypes. The hatchling genotype dataset was also examined to look for allelic stutter, large

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allele dropout, and null alleles using Micro-Checker 2.2.3 (van Oosterhout et al., 2004). 2.4. Data analyses For the 10 nesting females, allele frequencies were generated, and Hardy–Weinberg equilibrium and expected heterozygosity at two microsatellite loci were assessed using the software GENEPOP on the web (http://genepop.curtin.edu.au/; Rousset, 2008). The observed heterozygosity (H) at a specific locus was calculated following the formula in Hanotte et al. (1991): 2



H ¼ 1−∑ pi ; where p is the frequency of the ith allele for n alleles. To calculate the probability of being able to detect MP with individual loci (d) and across two loci together (D), formulae previously published by Westneat et al. (1987) were used. To estimate the probability that two unrelated individuals within the population share the same genotype at a locus (q), unbiased formula previously published by Waits et al. (2001) was used: qunbiased ¼



  n3 2a2 2 −a4 −2n2 ða3 þ 2a2 Þ þ nð9a2 þ 2Þ−6 =½ðn−1Þðn−2Þðn−3Þ;

where n is the sample size, ai equals Σpij, and pj is the frequency of the jth allele. To calculate the probability that two unrelated individuals share a common genotype across two loci (Q), the probabilities derived from each locus were multiplied (Hanotte et al., 1991; Waits et al., 2001). The probability of detecting MP with a given sample size was further assessed with regard to possible skewed sire contributions for two loci that were used to score females and their offspring using the PrDM software. PrDM integrates the information on the number of loci, allele frequencies within population, sample size of offspring, and the mother's genotype (Neff and Pitcher, 2002). Paternal alleles were manually inferred through simple autosomal Mendelian inheritance by comparing the known genotypes of the offspring to the genotype of their mothers and excluding the alleles of the mother. These remaining paternal alleles formed suspected paternal genotypes. The presence of three or more paternal alleles at two loci was assumed to have been derived from MP. After confirming the genotypes of the female nesters and accounting for the maternal alleles in the genotypes of the hatchlings, the genotypes of the contributing male turtles were reconstructed. In addition to evaluating paternity and reconstructing male genotypes manually, the program GERUD 2.0 (Jones, 2005) was used to reconstruct the genotypes of the alleged sires from the observed paternal alleles in the hatchling dataset and to confirm the manual assessment of paternity. The total number of observed sires across all analysed clutches of one female was considered the observed mating frequency. As a quantity, that takes into account the actual paternities of contributing males, the effective number of mates (me) was determined for each female using the formula published by Starr (1984):

me ¼ 1=∑p2i −1;

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season, the minimum number of sires per nest and the hatching success. Relations between the number of sires and each of these variables were tested by Pearson correlation. Differences in female size and hatching success among the groups formed based on the minimum number of sires in the nests were analysed by Kruskal-Wallis test. All these statistical analyses were performed using Minitab 16 (Minitab Inc., State College, PA). P values b 0.05 were considered statistically significant. 3. Results 3.1. Population data For 10 nesting female turtles, summary statistics are provided in Table 1. All 10 nesting females were genotyped at two loci with the probability of sharing a genotype across all loci being 1.5 × 10−3. Although the number of females analysed was only 10, both loci were highly polymorphic, with eight and 10 alleles found at microsatellites CcP2F11 and CcP7C06, respectively. Expected heterozygosities (He) were 0.85 for CcP2F11 and 0.89 for CcP7C06, while observed heterozygosities (Ho) were slightly lower for both CcP2F11 (0.81) and CcP7C06 (0.85). Allele frequencies at both loci were in agreement with Hardy– Weinberg equilibrium (P N 0.05). The probability of detecting multiple fathers (d) was 0.62 at CcP2F11 and 0.70 at CcP7C06, but when two loci were combined, the probability of detection (D) rose to 0.89. 3.2. Paternity analysis The two microsatellite loci were amplified consistently in all samples. Summary statistics for 10 nesting females and their hatchlings are listed in Table 2. Two to three clutches per female were assessed, and sample sizes ranged from eight to 30 hatchlings, with a mean of 20.9 hatchlings. The proportion of hatchlings per clutch analysed for paternity ranged from 8.3 to 74.1%, with a mean of 28.2%. Genotypes from six to 10 hatchling samples per female were replicated (average = 7.7 hatchlings per female, total = 77; 14.9%), and the genotyping error rate was found as 0 because no genotyping error was detected. No evidence of allelic stutter or large allele dropout was found in the hatchling dataset, and no null alleles were detected at either of the two loci. To assess the accuracy of detecting MP with a given sample size in the case of a skewed contribution by fathers with two loci used in this study, the probability to detect MP (PrDM) was calculated based on the allele frequency data for the whole population for different paternal skews. PrDM in clutches sired by two males assuming equal paternal contributions was high (PrDM = 0.96) when sampling 15 offspring, and PrDM in clutches sired by three males assuming equal paternal contributions was slightly higher (PrDM = 0.98) when sampling only 10. In clutches with two sires, a skewed paternal contribution of 1:2 would still give a high PrDM (N 0.94) when sampling 15 offspring, while a very skewed case of 1:9 would reduce the PrDM to 0.69. In clutches with three sires, a skewed paternal contribution of 1:1:2 would still give a high PrDM (N0.96) when sampling 10 offspring, while a very skewed case of 1:1:8 would reduce the PrDM to 0.79. Sampling size in total of two or three different clutches per nesting female in this study ranged from 40 to 74. It was therefore assumed that the probability of not detecting MP in nests where it did occur was small.

where pi is the proportion of offspring in a clutch sired by male i, with following correction for sampling error by Pamilo (1993):  ∑pi 2 ¼ N∑yi 2 −1 =ðN–1Þ; where yi are the observed contributions by each sire, and N is the number of sampled offspring. In this study, it was also tested if statistically significant correlations exist between the minimum number of sires and the female size (CCL), the minimum number of sires per nest and the nesting date in the

Table 1 The number of alleles per locus, the expected heterozygosity (He), observed heterozygosity (Ho), Hardy-Weinberg P-value (HW), the probability of two females having the same genotype at a locus (q) and the probability of being able to detect MP at each locus (d) for 10 nesting females on Dalyan Beach. Locus

Number of alleles

He

Ho

HW

q

d

CcP2F11 CcP7C06

8 10

0.85 0.89

0.81 0.85

0.09 0.95

5.6 × 10−2 2.7 × 10−2

0.62 0.70

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Table 2 Summary statistics for 10 nesting turtles and the nests laid by them on Dalyan Beach. Female Tag ID number of female

Female CCL (cm)

Nest ID

F1

TR-A0215

70

F2

TR-Y0036

81

F3

TR-Y0097

80

F4

TR-Y0458

76

F5

TR-Y0474

69

F6

TR-Y0475

79

F7

TR-Y0482

82

F8

TR-Y0485

88

F9

TR-Y0487

69

F10

TR-Y0563

75

F1-1 F1-2 F2-1 F2-2 F3-1 F3-2 F4-1 F4-2 F5-1 F5-2 F5-3 F6-1 F6-2 F6-3 F7-1 F7-2 F7-3 F8-1 F8-2 F8-3 F9-1 F9-2 F10-1 F10-2 F10-3

Nest date

09.06.2014 19.06.2014 13.06.2014 09.07.2014 18.05.2014 04.06.2014 25.05.2014 06.06.2014 19.05.2014 01.06.2014 25.06.2014 19.05.2014 02.06.2014 23.06.2014 28.05.2014 10.06.2014 26.06.2014 31.05.2014 06.07.2014 21.07.2014 04.06.2014 09.07.2014 28.06.2014 10.07.2014 23.07.2014

Clutch size (number of eggs)

Number of hatchlings hatched from eggs

Hatching success (%)

Number of genotyped hatchlings

Sampling effort per clutch (%)

Number of paternal alleles

78 80 104 84 106 83 118 84 96 75 68 50 79 73 92 82 27 122 103 58 85 62 66 71 118

69 76 102 81 30 46 100 78 73 63 54 49 78 71 86 79 23 120 90 51 76 55 64 68 98

88.5 95.0 98.1 96.4 28.3 55.4 84.7 92.9 76.0 84.0 79.4 98.0 98.7 97.3 93.5 96.3 85.2 98.4 87.4 87.9 89.4 88.7 97.0 95.8 83.1

20 20 20 20 20 20 20 21 8 18 28 19 24 29 19 20 20 22 22 30 20 20 20 21 21

25.6 25.0 19.2 23.8 18.9 24.1 16.9 25.0 8.3 24.0 41.2 38.0 30.4 39.7 20.7 24.4 74.1 18.0 21.4 51.7 23.5 32.3 30.3 29.6 17.8

3 3 4 4 2 2 4 4 2 2 2 4 5 5 4 4 4 6 5 5 2 2 4 5 4

All sampled hatchlings (n = 522) from 25 clutches of 10 different females were genotyped and analysed consistently. The observed maternal and hatchling and inferred paternal genotypes per locus are provided in Table 3. For three of the 10 nesting females analysed, there was no evidence of MP within their clutches (seven clutches) and paternal genotype inference was straightforward. Extra paternal alleles were detected in seven out of 10 females (70%), confirming that MP was observed. For all seven of these turtles, MP was evident in all clutches (18 clutches) across the season. Based on the manual paternal genotype assignments, two sires were detected in nine clutches of four females, while three sires in nine clutches of three females. The average number of sires was calculated as 2.1 per clutch. Results from GERUD2.0 confirmed the manual assessment of paternity; hatchling genotypes from three of the females were consistent with only one sire, while hatchlings from seven other females had multiple sires. Four out of seven females (57%) had two mates, but three females (43%) had three mates. During the inference of paternal genotypes, 20 individual males were identified, resulting in an operational sex ratio of 2:1 (male:female). No inferred paternal genotype was identical, which indicates that 20 males had mated with just one female each, and 20 different males contributed to the clutches of the 10 females analysed in this study. The probability of two males sharing an identical genotype was 5.7 × 10−4 for this group of males. 3.3. Paternal contribution In all clutches of females, where MP was detected among their offspring, the contribution of each sire was in general unequal within and between clutches (Fig. 2). Between the clutches laid by the same female, the frequency of contributing sires differed in general, but the composition did not differ. Both the number and genotype of contributing sires was the same in all clutches of a given female. This means that the same one or more sires contributed to the subsequent clutches of a given female. In all clutches of multiply mated females, a majority of offspring was sired by a single male (primary sire), while the remaining offspring was sired by one or two different males (secondary and tertiary sires). On average, primary sires contributed 62.7% to clutches laid by

CcP2F11 CcP7C06 4 4 3 3 2 2 3 3 1 2 2 6 5 5 4 4 4 5 5 5 2 2 5 6 5

Minimum number of fathers 2 2 2 2 1 1 2 2 1 1 1 3 3 3 2 2 2 3 3 3 1 1 3 3 3

a particular female; this value ranged from 45.2% to 78%. Secondary sires contributed 30.9% (13.5%–40.3%) to clutches of a particular female, while tertiary sires contributed 7.2% (12.2%–14.5%). Effective mating frequencies (me) ranged from 1.39 to 2.86 in each clutch in which MP was detected, and from 1.54 to 2.75 over the sequences of clutches laid by a given female. They were lower than the observed number of males contributing to clutches, with some exceptions (i.e., in the second clutch of female F1). 3.4. The relationships between the number of sires and different variables The CCL of 10 females sampled in this study ranged from 69 cm to 88 cm, with a mean of 76.9 ± 6.3 cm. Mean size of the females mated with only one male was 72.7 ± 6.4 cm (69 cm–80 cm), while mean sizes of the females mated with two and three males were respectively 77.3 ± 5.5 cm (70 cm–82 cm) and 80.7 ± 6.7 cm (75 cm–88 cm). No statistically significant female size differences were found among three groups which were formed based on the minimum number of males (Kruskal-Wallis test: P N 0.05). When the relationship between the number of sires and the female size was analysed, a non-significant increasing trend in the number of sires with the increased female size was found (Pearson correlation: P N 0.05) (Fig. 3a). In 2014 nesting season, 433 loggerhead sea turtle nests were laid on Dalyan Beach. The first nest was laid on 24th of April, while the last nest on 9th of August. The dates of the first and the last nests sampled in this study were 18th of May and 23rd of July, respectively. Based on statistical analysis, a non-significant increasing trend was observed in the number of sires in the nests within the nesting season from the beginning towards the end (Pearson correlation: P N 0.05) (Fig. 3b). For instance, the number of sires was one in the first sampled nest, whereas it was three in the last sampled nest (Table 2). The hatching success of the 25 nests sampled in this study ranged from 28.3% to 98.7%, with a mean of 86.9 ± 15.5%. Mean hatching success for seven nests with one sire was 71.6 ± 22.3%, with a range from 28.3% to 89.4%, while mean successes for nine nests with two and three sires were respectively 92.0 ± 4.8% (84.7%–98.1%) and 93.7 ± 5.9% (83.1%–98.7%). Statistically significant hatching success differences were found among three groups which were formed based on

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Table 3 The observed maternal and hatchling genotypes together with the inferred paternal genotypes per locus present in 25 clutches of 10 female loggerheads on Dalyan Beach. Female ID

Maternal genotypes

Hatchling genotypes (number of hatchlings)

Inferred paternal genotypes

CcP2F11

CcP7C06

CcP2F11

CcP2F11

CcP7C06

F1

264/276

258/274

256/264 256/276 260/264 260/276 264/268 268/276

(5) (4) (2) (4) (11) (14)

F2

268/268

286/298

252/268 260/268 268/268 268/288

(6) (9) (18) (7)

F3

248/256

290/290

F4

264/272

278/302

F5

252/280

286/290

F6

256/264

258/290

248/264 248/272 256/264 256/272 248/264 248/272 260/264 264/264 264/272 264/276 272/276 252/260 252/280 260/280 280/280 256/260 256/264 256/268 256/272 256/276 260/264 264/264 264/268 264/272 264/276

(11) (10) (10) (9) (10) (8) (2) (6) (8) (5) (2) (12) (17) (13) (12) (12) (11) (3) (9) (9) (5) (9) (2) (8) (4)

F7

264/276

282/286

F8

268/268

274/294

252/264 252/276 256/264 256/276 264/264 264/268 264/276 268/276 256/268 260/268 264/268 268/268 268/272 268/284

(5) (1) (5) (5) (3) (13) (5) (22) (5) (35) (7) (20) (4) (3)

F9

264/264

270/274

252/264 264/268

(21) (19)

F10

256/268

274/290

256/256 256/260 256/268 256/272 256/276 260/268 268/268

(11) (14) (22) (5) (3) (3) (4)

CcP7C06

Minimum number of fathers

258/258 258/274 258/282 258/290 258/298 274/282 274/290 274/298 258/286 258/298 270/286 270/298 286/298 298/298 286/290 290/294

(8) (8) (7) (3) (2) (3) (5) (4) (5) (6) (6) (5) (10) (8) (17) (23)

260/268 256/268

290/298 258/282

2

260/288 252/268

258/270 258/298

2

264/272

286/294

1

274/278 274/302 278/286 278/290 286/302 290/302

(11) (6) (7) (5) (10) (2)

248/264 260/276

274/286 274/290

2

282/290 286/290 290/290

(24) (22) (8)

260/280

282/290

1

258/270 258/274 258/278 258/282 258/294 258/298 270/290 274/290 278/290 282/290 290/294 290/298 258/282 258/286 274/282 274/286 282/286 282/290 286/286 286/290 270/274 270/294 274/274 274/278 274/282 274/290 274/294 278/294 282/294 290/294 258/270 258/274 270/286 274/286 258/274 258/290 266/290 274/274 274/286 274/290 274/294 274/298 286/290 290/294 290/298

(10) (8) (6) (3) (7) (2) (5) (11) (9) (1) (6) (4) (7) (3) (14) (12) (11) (4) (4) (4) (3) (2) (8) (5) (18) (4) (12) (2) (17) (3) (16) (12) (6) (6) (1) (3) (5) (5) (10) (1) (4) (14) (8) (3) (8)

260/272 264/276 264/268

270/274 278/294 282/298

3

252/268 256/264

274/286 258/290

2

260/268 264/284 256/272

274/282 278/290 270/290

3

252/268

258/286

1

256/272 260/268 268/276

274/298 286/294 258/266

3

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Fig. 2. The relative paternal contribution from observed sires for each clutch and across all clutches laid by each of the seven nesting females that exhibited multiple paternity (MP) on Dalyan Beach, Turkey. The different colours represent the proportion of offspring that each sire has contributed per clutch and per total. The first colour in the bottom of each column represents the primary sire or the sire that contributed to most offspring until the last colour in the top representing the sire with the smallest offspring contribution. The effective mating frequency (me) accounts for the actual paternities of contributing males based on the frequency of their contribution. There were 17 different males that contributed to the clutches with MP and none of them were identical.

the minimum number of sires (Kruskal-Wallis test: H = 9.75, df = 2, P b 0.01). When the relationship between the number of sires and hatching success was analysed, statistically significant positive correlation was detected; increased number of sires resulted in an increased hatching success (Pearson correlation: r25 = 0.56, P b 0.01) (Fig. 3c). 4. Discussion This is the first paternity study on Dalyan Beach that documents the presence of high frequency of MP in the loggerhead population nesting on this beach. It is concluded that at least 70% of female loggerheads nesting on Dalyan exhibited MP in their clutches, with a minimum of two contributing males for 57% of them, and a minimum of three contributing males for the remaining 43%. Although this study has some weaknesses such as sample size, study period, and number of loci used for genotyping, several aspects of the results provide compelling evidence that high levels of MP occurs in the loggerheads nesting on

Dalyan Beach. PrDM in clutches sired by multiple sires assuming even skewed paternal contributions was high enough when sampling at least 15 offspring. Because N15 offspring was sampled in general, it is concluded that the probability of not detecting MP in nests where it did occur is small. In addition, the minimum number of sires in the nests was detected with relatively small sample size (5.8% of the total nests). The frequency of MP for the population nesting on this beach is therefore more likely to be higher than detected in this study. Taken together, the high level of MP and the relatively high number of sires per clutch here (2.1 sires) suggests that female loggerheads nesting on Dalyan Beach may be under intense mating pressure, and MP is therefore common in the mating system of these loggerheads (Jensen et al., 2006). This high level of MP also suggests that effective population size and diversity may be high for this population (Zbinden et al., 2007). It can be concluded that this study contributes to what is known about loggerheads nesting on this beach by examining paternity within multiple clutches of females across the nesting season and by

F. Sari et al. / Journal of Experimental Marine Biology and Ecology 488 (2017) 60–71

Fig. 3. The number of sires in relation to (A) female size (CCL), (B) nesting date, and (C) hatching success.

assessing paternal genotypes. This study is of great importance for understanding of mating system and population structure of this population. For more accurate MP results, it is more appropriate if MP studies on loggerheads are carried out for at least three years because the female loggerheads breed and nest every 2–3 years (Hays et al., 2010). MP has previously been documented in loggerhead sea turtles across the globe. Harry and Briscoe (1988) found 38% MP in clutches of loggerheads sampled in Queensland, Australia. In Florida, two independent studies determined that just over 30% of nests showed multiple paternal contributions (Bollmer et al., 1999; Moore and Ball, 2002). In Greece, Zbinden et al. (2007) detected multiple paternal contributions in the nests of 14 out of 15 females (93%). Lasala et al. (2013) determined that 75% of clutches sampled on the small nesting beach on Wassaw Island, Georgia had multiple sires. Lastly, Tedeschi et al. (2015) investigated the levels of MP in loggerhead sea turtles from three rookeries in Western Australia and found highly variable rates of MP among the rookeries which they sampled (25% on Bungelup Beach, 36% on Dirk Hartog Island, and 86% on Gnaraloo Bay). The frequency of MP detected in this study was within the range demonstrated in these previous studies on loggerheads. The results of this study show that each analysed female mated with a different male. This study suggests that the mating system of this

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nesting population may be polyandrous and not polygynous. Although some male loggerheads are nonmigratory, and reside in Dalyan throughout the year even after mating instead of migrating (Kaska, personal communication), it was detected that no male sired the clutches of more than one female. This finding implies that the Dalyan population may be a male-biased population in any breeding season. It can be suggested that the chance of a female is likely to be higher to encounter different males, whereas the chance of a male is likely to be lower or almost unavailable to encounter different females. In addition to learning more about the mating system of loggerhead turtles, assessing the paternal genotypes enabled to examine the adult males; otherwise it would be very hard to study. To date, most population estimates are either based on the number of females observed on nesting beaches or derived from the number of nests laid on a beach divided by the average clutch frequency of females at that beach (Canbolat, 2004; Margaritoulis et al., 2003). By assessing the genotypes of males that successfully mated with females at Dalyan and produced hatchlings, the number of males contributing to this population can be counted and therefore a more realistic estimate of the adult population size can be obtained. Accounting for the male component of the populations has important consequences for determining population viability. Hatchling and juvenile sex ratios in sea turtles are relatively easy to determine, and have been found to be strongly female-biased (Godfrey et al., 1996; Hawkes et al., 2007; Mrosovsky et al., 2002; Sarı and Kaska, 2015), with some exceptions (Baptistotte et al., 1999; Steckenreuter et al., 2010). Apart from these sex ratios, it is critical to understand the OSRs of adult populations. If the sex ratios of hatchling and juvenile sea turtles are female-biased, it can be expected that this could lead to a skewed OSR among adults with a strong female bias. Although the majority of the hatchlings produced on Dalyan Beach are female (Sarı and Kaska, 2015), male-biased OSR finding in this study (2:1) demonstrates that feminisation of the sexually mature Dalyan population is not true. It can therefore be suggested that male hatchlings are more resistant to environmental threats such as predation and diseases than the females and that chance of them to reach the adulthood is higher than that of females. On the other hand, even if the adult sex ratio is female-biased in a population, male turtles may balance out female-biased sex ratio with shorter reproductive and migratory cycles of one year instead of every second or third year, returning to breed more frequently than females (Hays et al., 2010; Stewart and Dutton, 2011). It can therefore be stated that MP can indicate a balanced OSR. Recent MP studies have confirmed the general trend of higher levels of MP related with larger populations. Joseph and Shaw (2011) found 20% MP in a small population of hawksbill sea turtles from Sabah Turtle Islands, Malaysia, while Joseph (2006) determined MP levels of 71% in a larger population of green turtles from the same area. Although this is the case, some conflicting data have also appeared; the smaller population, the higher levels of MP (Duran et al., 2015; Lasala et al., 2013; Zbinden et al., 2007). In this case, the question is if there is another factor affecting the levels of MP in a population either apart from or together with the abundance of individuals. One explanation for detecting extremely high rates of MP in small populations suggested by Zbinden et al. (2007) is that the most influencing factor is the density of breeding individuals (females and males) within the mating area rather than the actual number of them. The same explanation in leatherbacks (Dermochelys coriacea) was also suggested by Figgener et al. (2016). For instance, Jensen et al. (2006) determined that olive ridley turtles nesting in arribada-congregations had a significantly higher level of MP (92%) in their clutches than those nesting solitarily (30%), leading them to conclude that the number of animals at the breeding site had a direct effect on the incidence of MP, while Lasala et al. (2013) found MP in 75% of the clutches in a small loggerhead nesting beach on Wassaw Island, Georgia despite a population size consisting of b200 females. In light of the results of this study and similar studies, it can be suggested that density and abundance of individuals are likely to be

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key determinants of the frequency of MP, but the density of individuals is the most influencing factor of this frequency for the population nesting on this beach. The high level of MP detected in this study can be used in order to estimate population size of females nesting on Dalyan Beach. When a regression graph produced by Jensen et al. (2006) is used to calculate the breeding population size corresponding to the frequency of MP found in the present study (70%), the result exceeds at least 20,000 individuals, but data from beach monitoring projects strongly demonstrates that the actual population size is much lower than this value. Female breeding population size can be estimated as the total number of clutches laid in the population, divided by the mean clutch frequency, adjusted by the estimated remigration interval (Ireland et al., 2003). From data of Sarı and Kaska (2015) and unpublished reports, approximately 2900 annual loggerhead nests appear to be laid based on the mean of annual nest numbers for each nesting beach throughout the Mediterranean coastline of Turkey. It is assumed that female loggerheads nest on average three times a season (Canbolat 2004), and nest in general every three years once they reach sexual maturity (Hays et al. 2010). It can therefore be estimated that approximately 2900 female loggerheads nest on Turkish beaches. Even if the calculation of the mean annual nest number is based on the maximum of annual nest numbers for each nesting beach, the estimated breeding population size would be approximately 5600 females, but this value is still much b20,000 calculated using the regression by Jensen et al. (2006). As for Dalyan Beach, breeding population size is estimated to be 500 females with a bounteous approach. Taken together, actual number of individuals cannot be the best explanation for increased frequency of MP in this case. Thus, the best explanation can be the density of animals in the breeding area as mentioned above, although the density of population nesting on this beach has not been studied extensively and calculated. Individuals of the Dalyan population mate mostly in Alagöl and the labyrinth of reedy channels (Kaska, personal communication) (Fig. 1), and hence it can be mentioned that this population has a high density resulting with high level of MP. It can be concluded that small population size could have a precise effect on the mating system of loggerhead sea turtles in nature and that frequency of MP is likely to be density dependent. Mating of loggerheads is assumed to occur several weeks to months prior to onset of the nesting season (Limpus, 1985). While females remain in the vicinity of the nesting beaches, males migrate long distances from breeding areas to foraging grounds after mating (Limpus and Reed, 1985) with some exceptions (Henwood, 1987). In this case, it is possible to encounter both males and females near the nesting beaches throughout the world early in the nesting season. At least in Dalyan, it is known that there are some males which reside in the area throughout the year, and use here for both breeding and feeding (Kaska, personal communication). The question is if successful inter-nesting mating events take place in Dalyan population, or if all successful multiple mating events of a female occur prior to the nesting season. In leatherbacks, Stewart and Dutton (2011) reported that no inter-nesting mating event that contributed to the hatchling genotypes did occur in their dataset; however, Figgener et al. (2016) determined that mating took place between the successive clutches of some females that they sampled. Once the clutches exhibiting MP were examined (Fig. 3), it was observed that both the primary and secondary sires (or the secondary and tertiary sires together with the primary sire in some clutches) contributed to all clutches for any given female. The different clutches of each female shared the same composition of males, meaning that same male/ males sired all the successive clutches of each female. This finding indicates that mating with males took place prior to the nesting season, and there was no mating event that contributed to the hatchling genotypes between two successive nesting events on Dalyan Beach. This study supports the hypothesis of sperm storage, agreeing with the result of Stewart and Dutton (2011). In turtles, sperm storage of up to four years has been reported (Ewing, 1943), and sperm storage across

seasons has been shown in genetic studies of freshwater turtles (Johnston et al., 2006; Pearse and Avise, 2001; Roques et al., 2006). In addition, sperm storage within seasons has been documented for some sea turtle species using genetic markers (Crim et al., 2002; FitzSimmons, 1998; Harry and Briscoe, 1988; Kichler et al. 1999; Sakaoka et al., 2013); however, sperm storage across seasons is possible in sea turtles as well. If the fertilisation from the mating of previous season occurs, there is a possible loss of sperm through time in storage tubules of females, or older sperm may be less viable (Ewing, 1943; FitzSimmons, 1998). If successful multiple mating occurs in the mating system of an organism, sperm from different males may compete to fertilise a single clutch of eggs. Sperm competition may therefore be an important factor in the evolution of reproduction of many organisms, though the certain mechanism determining sperm success is not fully understood (Jones and Clark, 2003). Since the timing of copulation (after or before ovulation and production of eggs) plays a crucial role in successful fertilisation (FitzSimmons, 1998), there might be mechanisms of sperm competition that enables the sperm of a single male to sire the biggest portion of offspring in a given clutch or among all clutches during a nesting season (FitzSimmons, 1998). In this study, unequal contribution of sires within one clutch and in following clutches per female was found. One primary male throughout all clutches of a multiply mated female was detected, and both this primary male and the secondary and tertiary males were the same individuals in all successive clutches female. In addition, effective mating frequencies (me) were in general close to the observed mating frequencies in the nests, except in the first nest of female F7 and all nests of female F8. This indicates that the contribution of sires was not strongly skewed, but strongly skewed paternal contribution was the case for these exception nests. When these findings are taken into account together, it can be suggested that this study points out sperm competition. Because sperm competition taking place is expected to cause each clutch to show one primary male in high frequency and secondary and tertiary males in low frequencies (FitzSimmons, 1998). It has been suggested that a low male contribution to a clutch could be the result of poor competitor sperm, or residual sperm stored from a previous nesting season (Stewart and Dutton, 2011). For female F8 in this study, there were both secondary and tertiary males, but their contributions (10 and nine hatchlings, respectively) were minimum compared to that of the primary male (55 hatchlings) across the season. Furthermore, effective mating frequencies of three nests of this female were lower, ranging from 1 to 1.5 compared to the observed frequencies. These results raise two possibilities: (1) the mating with three males occurred prior to nesting season in 2014, and these secondary and tertiary sires were poor competitors in terms of reproductive success, or (2) this female mated with these secondary and tertiary sires in the previous breeding season and stored the sperm of them, and mated with only primary sire in 2014. Although this turtle was tagged the first time in 2014, it can be assumed due to large body size (a CCL of 88 cm) that the female was remigrant, and there was sperm remaining from the previous breeding season. Nevertheless, it is necessary to test the second alternative above for more precise result on sperm storage. Since this study was conducted for only one year, it was impossible to test the second possibility. As mentioned above, female sea turtles nest every three years (Hays et al., 2010). It will be better if a similar study investigating the successive clutches of the females is carried out in 2017 on the same beach because the turtles that nested in 2014 will be returning to the beach in 2017. Hatchlings of the females in this study will therefore be able to be targeted to investigate sperm storage between consecutive nesting seasons. It is known that nesting females continually grow, suggesting that older females are generally larger (Casale et al., 2011). Pelvic opening structure of the species constrains egg size and hence offspring size; larger turtles can therefore produce more eggs, since they can accumulate more resources, and/or bigger eggs, because of their larger pelvic

F. Sari et al. / Journal of Experimental Marine Biology and Ecology 488 (2017) 60–71

opening (Wilbur and Morin, 1988). There is a positive correlation between egg size and hatchling body size, and larger hatchlings have a higher survival rate (Packard and Packard, 1988). Larger females also have a higher capacity to hold eggs (van Buskirk and Crowder, 1994). For these reasons, one can expect that bigger females should be more attractive to males. In addition, it is reasonable that larger females encounter more males than do smaller females at least for sea turtles, independent of male behaviour (Zbinden et al., 2007). Larger females are likely to swim faster than smaller females, and may thus arrive in the breeding area earlier, and therefore spend more time for mating (Zbinden et al., 2007). Taken together, it is necessary to test if there is a correlation between female size and minimum number of sires detected in the nests on Dalyan Beach, although some studies have tested this correlation for different species and for different populations. For instance, Zbinden et al. (2007) and Lasala et al. (2013) independently determined for loggerheads that the number of sires was positively correlated to female body size, but Lee and Hays (2004) found females of multiply sired clutches of green sea turtles to be larger than those laying clutches with only one sire detected, although the result was not statistically significant. In this study, it was detected that there was not a statistically significant correlation between the number of sires and female size, but an increasing trend was observed in the number of sires with the increased female size. This finding suggests possible positive correlation between them. The reason for not detecting statistically significant relationship may be the number of females sampled in this study as only 10 out of approximately 150 females that nested on Dalyan Beach in 2014 were sampled. It is concluded that more accurate results about this relationship would be obtained, if almost all these females were sampled. In this study, no significant correlation was detected between the number of sires and nesting date, but it was observed that the number of sires was in a relatively increasing trend from the beginning towards the end of the nesting season. This is consistent with the hypothesis of sperm mixing at the beginning of the season (Pearse et al., 2002; Uller and Olsson, 2008) rather than stratification; otherwise, it would be possible to see a skew in the number of sires per clutch by per female over the course of the season. Recent studies on successive clutches of individual female leatherback and hawksbill sea turtles (Phillips et al., 2013; Stewart and Dutton, 2011) support this allegation of sperm mixing rather than stratification. The increasing trend of the number of sires detected in this study may be caused by sperm competition (FitzSimmons, 1998). According to this finding, it can be suggested that the number of sires is likely to be lower at the beginning of nesting seasons, and to be higher towards the end, although “new” sires (sires present in a clutch but not in the previous clutch) were not observed in any of the successive clutches of any females. It has been suggested that MP may be favoured as long as it increases the variability and viability of the offspring in the populations (Lee and Hays, 2004; Uller and Olsson, 2008; Wright et al., 2013). These outcomes are not necessarily related. In this study, it was assumed that one of the simplest ways to investigate viability of the offspring was to examine hatching success, which was defined here as the frequency of hatchlings that successfully emerged from the eggs. It was found that there was a statistically significant positive correlation between the number of sires and hatching success despite the relatively small sample size. This means that an increased number of sires resulted in an increased hatching success. Similar results exist for several species of reptiles (Roques et al., 2006; Zbinden et al., 2007) and amphibians (Osikowski and Rafinski, 2001). On the other hand, there are some studies in which no relationship between the number of sires and hatching success has been reported in painted turtles (Chrysemys picta) (Pearse and Avise, 2001; Pearse et al., 2002) and in loggerheads (Lasala et al., 2013). This finding points out that more sires contribute to both the variability and the viability of hatchlings in terms of alleles related to viability. It should be taken into account that the number of sires is not the solely factor influencing hatching success. Hatching success in sea turtle

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nests are affected by several ecological factors such as nest temperature (Yntema and Mrosovsky, 1982), particle size of the sand (Mortimer, 1990), limited gas exchange in the nest (Ackerman, 1980), level and frequency of tidal inundation (Foley et al., 2006), and distance to the vegetation (Ditmer and Stapleton, 2012). For instance, if a nest falls into inundation, or is predated, hatching success in this nest is likely to be lower, or it is possible that no hatchling is able to emerge, despite the number of sires.

5. Conclusion MP studies are of great importance as they provide valuable information about reproductive behaviour of sea turtles which is difficult to observe directly. MP levels of sea turtle populations should be taken into account for management and conservation strategies, since they influence effective population size and diversity. Genetic diversity plays a key role in the ability of the species to adapt themselves to environmental changes such as global warming and their survival in the future. New insights into the mating system of loggerheads provided by the MP studies could contribute to the management and assessment of the genetic health of global populations. This study is the first MP study carried out on loggerhead sea turtles nesting in Turkey, and thus can be considered as an important and preliminary study concerning loggerhead mating system in this region. High frequency of MP is evident in the population nesting on Dalyan Beach, and it could be an indicator of genetic diversity in this population. It is inferred that MP levels depend on density of individuals rather than actual number of them for this beach; however, further and comprehensive studies are needed to confirm the results of the present study.

Acknowledgements This study was supported by Pamukkale University Scientific Research Projects Coordination Unit as a Ph.D. thesis project (grant no: 2014FBE027). We would like to thank Turkish Ministry of Environment and Urbanization General Directorate of the Protection of Natural Assets for the permission to work on the beach. We would also like to thank Dr. Kelly R. Stewart and Christine Figgener for their helps for some calculations. References Ackerman, R.A., 1980. Physiological and ecological aspects of gas exchange by sea turtle eggs. Am. Zool. 20 (3), 575–583. Alfaro-Núñez, A., Jensen, M.P., Abreu-Grobois, F.A., 2015. Does polyandry really pay off?: the effects of multiple mating and number of fathers on morphological traits and survival in clutches of nesting green turtles at Tortuguero. PeerJ PrePrints. 3, p. e1112. Arden, W.R., Kapuscinski, A.R., 2002. Demographic and genetic estimates of effective population size (Ne) reveals genetic compensation in steelhead trout. Mol. Ecol. 12, 35–49. Baer, B., Schmid-Hempel, P., 1999. Experimental variation in polyandry affects parasite loads and fitness in a bumble-bee. Nature 397, 151–154. Baptistotte, C., Scalfoni, J.T., Mrosovsky, N., 1999. Male-producing thermal ecology of a southern loggerhead nesting beach in Brazil: implications for conservation. Anim. Conserv. 2, 9–13. Bjorndal, K.A., Meylan, A.B., Turner, B.J., 1983. Sea turtles nesting at Melbourne Beach, Florida, I. Size, growth and reproductive biology. Biol. Conserv. 26, 65–77. Bollmer, J.L., Irwin, M.E., Rieder, J.P., Parker, P.G., 1999. Multiple paternity in loggerhead turtle clutches. Copeia 1999 (2), 475–478. Bolten, A.B., 2003. Introduction: the loggerhead sea turtle—a most excellent fish. In: Bolten, A.B., Witherington, B.E. (Eds.), Loggerhead Sea Turtles. Smithsonian Books, Washington, DC, pp. 1–3. Bowen, B.W., Avise, J.C., Richardson, J.I., Meylan, A.B., Margaritoulis, D., Hopkins-Murphy, S.R., 1993. Population structure of loggerhead turtles (Caretta caretta) in the Northwestern Atlantic Ocean and Mediterranean Sea. Conserv. Biol. 7 (4), 834–844. Bowen, B.W., Bass, A.L., Chow, S.M., Bostrom, M., Bjorndal, K.A., Bolten, A.B., Okuyama, T., Bolker, B.M., Epperly, S., Lacasella, E., Shaver, D., Dodd, M., Hopkins-Murphy, S.R., Musick, J.A., Swingle, M., Rankin-Baransky, K., Teas, W., Witzell, W.N., Dutton, P.H., 2004. Natal homing in juvenile loggerhead turtles (Caretta caretta). Mol. Ecol. 13 (12), 3797–3808. Canbolat, A.F., 2004. A review of sea turtle nesting activity along the Mediterranean coast of Turkey. Biol. Conserv. 116, 81–91.

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