Genetic markers have been widely used in marine turtles to assess population structuring and origin of individuals in common feeding grounds, which are key elements for understanding their ecology and for developing conservation strategies. However, these analyses are very sensitive to missing information, especially from abundant nesting sites. Kyparissia Bay (western Greece) hosts the second largest Mediterranean nesting aggregation of the loggerhead turtle (

El uso de marcadores genéticos ha sido muy extendido en tortugas marinas con el fin de determinar la estructura poblacional y el origen de individuos en zonas comunes de alimentación; elementos clave para entender su ecología y desarrollar estrategias efectivas de conservación. Sin embargo, este tipo de análisis es muy sensible a la falta de información de ciertas zonas de nidificación, especialmente de aquellas muy abundantes. El perfil genético de las tortugas nidificantes de la bahía de Kyparissia (Grecia occidental) todavía no ha sido descrito usando la versión extendida del marcador mitocondrial (mtDNA) usado históricamente en esta especie, a pesar de ser la segunda zona de nidificación más abundante de todo el Mediterráneo. Con el fin de cubrir este vacío de información, se secuenciaron 36 individuos nidificantes de la bahía de Kyparissia y se compararon las frecuencias de haplotipos obtenidas con datos publicados de otras zonas de nidificación del Mediterráneo. Los resultados confirmaron la conexión entre Kyparissia y otras zonas de nidificación de Grecia así como el aislamiento de este grupo de Grecia occidental con el resto de zonas de nidificación del Mediterráneo. Como consecuencia de este aislamiento, todo parece indicar que este abundante grupo de zonas de nidificación (casi el 30% de la producción del Mediterráneo) no podría contribuir de forma significativa a la recuperación del número de hembras nidificantes en otras poblaciones en declive del Mediterráneo.

The loggerhead marine turtle (

Furthermore, this genetic structuring has been used in the determination of the origin of turtles in feeding grounds. A loggerhead sea turtle may undertake vast migrations across the oceans during its life (

The Mediterranean Sea hosts an independent regional management unit (

Despite these recent efforts, one notable omission from profiling using longer sequences is the breeding aggregation of Kyparissia Bay, which represents the second largest loggerhead nesting aggregation in the region (

In this study, we analysed the mtDNA diversity of the Kyparissia Bay nesting population using the 800-bp fragment with a primer set that includes the traditional 380-bp mtDNA fragment (

Samples from the Kyparissia Bay nesting area (KYP,

We amplified a long (~800 bp) fragment of the mitochondrial DNA (mtDNA) control region using the primers LCM15382 (5’-GCTTAACCCTAAAGCATTGG-3’) and H950 (5’-GTCTCGGATTTAGGGGTTT-3’) (

We reviewed all published long (~800 bp) and short (~380 bp) mtDNA haplotype frequencies in Mediterranean nesting areas (_{st}). Significance of differentiation was also tested by computing the exact test, based on haplotype frequencies (

Finally, we used the program POWSIM 4.1 (_{0}, thus accepting _{1} (no differentiation among populations), and is an essential complement to assess the reliability of the non-significant pairwise comparisons. The program simulates the divergence of different populations and estimates the probability of false negatives by resampling them. Thus, statistical power is expressed as the proportion of non-significant outcomes (1000 replicates) of the diverged populations. We simulated different levels of divergence (_{st}

A total of 36 sequences were obtained from females at the Kyparissia Bay nesting aggregation, and three haplotypes were found, when either the short or the long fragment was considered. These haplotypes were the widespread CC-A2/CC-A2.1 (Genebank EU179445, (

CC-A2 | CC-A3 | CC-A6 | CC-A10 | Cc-A13 | CC-A20 | CC-A26 | CC-A29 | CC-A31 | CC-A32 | CC-A43 | CC-A50 | CC-A52 | CC-A53 | CC-A65 | CC-A68 | n | Ref. | ||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|

Italy | Calabria -CAL | 22 | 14 | 2 | 38 | A | |||||||||||||

Lampedusa -LAM# | 2 | 2 | B | ||||||||||||||||

Tunisia | Tunisia-TUN | 16 | 16 | C | |||||||||||||||

Libya | Misurata-MIS | 13 | 1 | 14 | D | ||||||||||||||

Sirte -SIR | 28 | 2 | 4 | 1 | 35 | D | |||||||||||||

Sirte-SIR # | 21 | 3 | 1 | 2 | 27 | E | |||||||||||||

Sirte-SIR # | 7 | 7 | B | ||||||||||||||||

Greece | Zakynthos-ZAK | 16 | 2 | 1 | 19 | E-F | |||||||||||||

Kyparissia-KYP | 33 | 2 | 1 | 36 | G | ||||||||||||||

Kyparissia-KYP# | 19 | 2 | 21 | H | |||||||||||||||

Lakonikos-LAK | 18 | 1 | 19 | E-F | |||||||||||||||

Greece general # | 10 | 1 | 11 | B | |||||||||||||||

Rethymno, Crete-CRE | 20 | 20 | E-F | ||||||||||||||||

Cyprus | Cyprus-CYP | 44 | 1 | 45 | E | ||||||||||||||

Cyprus-CYP# | 35 | 35 | B | ||||||||||||||||

Turkey | Dalyan-DLY | 25 | 15 | 40 | I | ||||||||||||||

Dalaman-DLM | 5 | 15 | 20 | I | |||||||||||||||

Western Turkey-WTU | 60 | 9 | 1 | 1 | 1 | 72 | I | ||||||||||||

Mid Turkey-MTU | 46 | 1 | 47 | I | |||||||||||||||

Eastern Turkey-ETU | 60 | 16 | 76 | I | |||||||||||||||

Turkey General# | 19 | 13 | 32 | B | |||||||||||||||

Lebanon | Lebanon-LEB | 17 | 2 | 19 | E | ||||||||||||||

Israel | Israel -ISR | 17 | 2 | 19 | E | ||||||||||||||

Israel -ISR ^{#} |
6 | 6 | B | ||||||||||||||||

Total | 559 | 76 | 7 | 1 | 1 | 14 | 5 | 2 | 3 | 1 | 1 | 1 | 1 | 1 | 2 | 1 | 676 |

A, ^{#} Sample set discarded for analysis after filtering process. WTU, MTU and ETU are groups of nesting beaches as defined in

CC-A2.1 | CC-A2.8 | CC-A2.9 | CC-A3.1 | CC-A3.2 | CC-A6.1 | CC-A13.1 | CC-A20.1 | CC-A26.1 | CC-A29.1 | CC-A31.1 | CC-A32.1 | CC-A43.1 | CC-A50.1 | CC-A52.1 | CC-A53.1 | CC-A65.1 | CC-A68.1 | n | Ref. | ||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|

Italy | Calabria-CAL | 22 | 14 | 2 | 38 | A | |||||||||||||||

Libya | Misurata-MIS | 12 | 1 | 1 | 14 | B | |||||||||||||||

Sirte-SIR | 16 | 12 | 2 | 4 | 1 | 35 | B | ||||||||||||||

Sirte-SIR # | 11 | 10 | 3 | 1 | 2 | 27 | C | ||||||||||||||

Greece | Zakynthos-ZAK | 16 | 2 | 1 | 19 | C | |||||||||||||||

Kyparissia-KYP | 33 | 2 | 1 | 36 | D | ||||||||||||||||

Lakonikos-LAK | 18 | 1 | 19 | C | |||||||||||||||||

Rethymno, Crete-CRE | 16 | 4 | 20 | C | |||||||||||||||||

Cyprus | Cyprus-CYP | 44 | 1 | 45 | C | ||||||||||||||||

Turkey | Dalyan-DLY | 25 | 15 | 40 | E | ||||||||||||||||

Dalaman-DLM | 5 | 15 | 20 | E | |||||||||||||||||

Western Turkey-WTU | 60 | 16 | 76 | E | |||||||||||||||||

Mid Turkey-MTU | 46 | 1 | 47 | E | |||||||||||||||||

Eastern Turkey-ETU | 60 | 8 | 1 | 1 | 1 | 1 | 72 | E | |||||||||||||

Lebanon | Lebanon-LEB | 17 | 2 | 19 | C | ||||||||||||||||

Israel | Israel -ISR | 15 | 2 | 2 | 19 | C | |||||||||||||||

Total | 366 | 4 | 25 | 62 | 1 | 5 | 14 | 5 | 2 | 3 | 3 | 1 | 1 | 1 | 1 | 1 | 2 | 1 | 546 |

A,

Nesting area | ~380bp | ~800bp | Population size | ||||
---|---|---|---|---|---|---|---|

Φ_{st} |
h | π | Φst | h | π | nests/year (range) | |

KYP | - | 0.160(0.080) | 0.0004(0.0007) | - | 0.160 (0.080) | 0.0002 (0.0003) | 621 (286-927) |

CAL | 0.255 A,B | 0.541(0.049) | 0.0015(0.0014) | 0.255 ^{A,B} |
0.540 (0.049) | 0.0007 (0.0007) | 17 (15-20) |

TUN | -0.012 | 0.000(0.000) | 0.0000(0.0000) | - | - | - | 18 |

MIS | 0.007 | 0.143(0.119) | 0.0004(0.0006) | 0.022 | 0.275 (0.148) | 0.0004(0.0004) | 592 |

SIR | 0.048 ^{A,b} |
0.353(0.097) | 0.0010(0.0011) | 0.317 ^{A,B} |
0.676 (0.050) | 0.0011(0.0009) | 343 |

ZAK | 0.018 | 0.292(0.127) | 0.0010(0.0011) | 0.018 | 0.292 (0.127) | 0.0005 (0.0005) | 1,244 (833-2,018) |

LAK | -0.036 | 0.105(0.092) | 0.0003(0.0005) | -0.036 | 0.105 (0.092) | 0.0001 (0.0003) | 197 (107-288) |

CRE | -0.003 | 0.000(0.000) | 0.0000(0.0000) | 0.135 ^{b} |
0.337 ( 0.110) | 0.0004 (0.0005) | 324 (166-516) |

CYP | 0.019 | 0.044(0.042) | 0.0001(0.0003) | 0.019 | 0.044 (0.042) | 0.0001 (0.0002) | 515 |

DLY | 0.291 ^{A,B} |
0.481(0.042) | 0.0013(0.0012) | 0.291 ^{A,B} |
0.481 (0.042) | 0.0006 (0.0006) | (57-330) |

DLM | 0.695 ^{A,B} |
0.395(0.101) | 0.0010(0.0011) | 0.695 ^{A,B} |
0.395 ( 0.101) | 0.0005 (0.0006) | (69-112) |

WTU | 0.131 ^{A,B} |
0.337(0.055) | 0.0009(0.0010) | 0.131 ^{A,B} |
0.337 ( 0.055) | 0.0004 (0.0005) | (169-523) |

MTU | 0.021 | 0.043(0.040) | 0.0001(0.0003) | 0.021 | 0.043 ( 0.040) | 0.0001 (0.0002) | (136-1165) |

ETU | 0.062 ^{a,b} |
0.293(0.065) | 0.0009(0.0010) | 0.058 ^{a,b} |
0.297 ( 0.067) | 0.0004 (0.0005) | (212-936) |

LEB | 0.043 | 0.199(0.112) | 0.0005(0.0008) | 0.043 | 0.199 ( 0.112) | 0.0002 (0.0004) | 60 (40-122) |

ISR | 0.043 | 0.199(0.112) | 0.0005(0.0008) | 0.063 ^{a,b} |
0.374 (0.130) | 0.0005 (0.0005) | 57 |

Abbreviations as _{st}, genetic distance compared with Kyparissia; h, haplotype diversity; π, nucleotide diversity. Bold values indicate a genetic distance significantly different (p<0.05) from 0 using either ^{a}, chi-square test or ^{b}, exact test. Uppercase of these tests (^{A} or ^{B}) denotes p-values remaining significant after FDR correction. No long (~800 bp) information exists from TUN (Tunisia). Population sizes (nests/year) obtained from (

With the addition of our results, we compiled a total of 676 individuals with genetic information for the short fragment and 546 individuals with genetic information for the long fragment (

Once WGR had been grouped (

CAL | TUN | MIS | SIR | WGR | CRE | CYP | DLY | DLM | WTU | MTU | ETU | LEB | ISR | |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|

CAL | - | - | 0.198 ^{A,B} |
0.338 ^{A,B} |
0.293 ^{A,B} |
0.243 ^{A,B} |
0.313 ^{A,B} |
0.334^{ A,B} |
0.568^{ A,B} |
0.295^{ A,B} |
0.319^{ A,B} |
0.258^{ A,B} |
0.224^{ A,B} |
0.212^{ A,B} |

TUN | 0.225 ^{A,B} |
- | - | - | - | - | - | - | - | - | - | - | - | - |

MIS | 0.206 ^{A,B} |
0.010 | - | 0.171 ^{A} |
0.027 | 0.089 | 0.057 | 0.150^{ b} |
0.559^{ A,B} |
0.031 | 0.061^{ a} |
-0.012 | -0.037 | -0.013 |

SIR | 0.228 ^{A,B} |
0.020 | -0.005 | - | 0.371 ^{A,B} |
0.278 ^{A,B} |
0.370 ^{A,B} |
0.331^{ A,B} |
0.496^{ A,B} |
0.340^{ A,B} |
0.377^{ A,B} |
0.318^{ A,B} |
0.257^{ A,B} |
0.166^{ A,B} |

WGR | 0.293 ^{A,B} |
0.003 | 0.009 | 0.062 ^{A,B} |
- | 0.146 ^{A,B} |
0.032 ^{a} |
0.304^{ A,B} |
0.689^{ A,B} |
0.134^{ A,B} |
0.033^{ a} |
0.065^{ A,B} |
0.040^{ a} |
0.080^{ A,b} |

CRE | 0.244 ^{A,B} |
0.000 | 0.026 | 0.031 | 0.011 | - | 0.212 ^{A,B} |
0.275^{ A,B} |
0.615^{ A,B} |
0.179^{ A,B} |
0.218^{ A,B} |
0.123^{ A,B} |
0.121 | 0.106^{ A,b} |

CYP | 0.313 ^{A,B} |
-0.028 | 0.036 | 0.064 ^{A,B} |
0.032 | -0.021 | - | 0.352^{ A,B} |
0.788^{ A,B} |
0.151^{ A,B} |
0.000 | 0.065^{ a,b} |
0.084 | 0.105^{ A,B} |

DLY | 0.334 ^{A,B} |
0.265 ^{A,B} |
0.156 ^{b} |
0.193 ^{A,B} |
0.304 ^{A,B} |
0.284 ^{A,B} |
0.352^{ A,B} |
- | 0.215^{ A,b} |
0.049^{ a} |
0.357^{ A,B} |
0.109^{ A,B} |
0.128^{ b} |
0.242^{ A,B} |

DLM | 0.568 ^{A,B} |
0.714 ^{A,B} |
0.602 ^{A,B} |
0.556 ^{A,B} |
0.689 ^{A,B} |
0.737 ^{A,B} |
0.788^{ A,B} |
0.215^{ A,b} |
- | 0.446^{ A,B} |
0.793^{ A,B} |
0.500^{ A,B} |
0.572^{ A,B} |
0.588^{ A,B} |

WTU | 0.295 ^{A,B} |
0.107 | 0.020 | 0.079 ^{A,B} |
0.134 ^{A,B} |
0.118 ^{a,b} |
0.151^{ A,B} |
0.049 | 0.446^{ A,B} |
- | 0.154^{ A,B} |
0.004 | 0.003 | 0.136^{ A,B} |

MTU | 0.319 ^{A,B} |
-0.029 | 0.038 | 0.066 ^{A,B} |
0.033 | -0.021 | 0.000 | 0.357^{ A,B} |
0.793^{ A,B} |
0.154^{ A,B} |
- | 0.066^{ A,b} |
0.088 | 0.109^{ A,B} |

ETU | 0.266 ^{A,B} |
0.034 | -0.026 | 0.039 ^{a,b} |
0.068 ^{A,B} |
0.043 | 0.070^{ A,b} |
0.114^{ A,B} |
0.516^{ A,B} |
0.004 | 0.072^{ a,b} |
- | -0.028 | 0.077^{ A,b} |

LEB | 0.224 ^{A,B} |
0.043 | -0.059 | 0.011 | 0.040 | 0.059 | 0.084 | 0.128^{ b} |
0.572^{ A,B} |
0.003 | 0.088^{ A} |
-0.028 | - | 0.056 |

ISR | 0.224 ^{A,B} |
0.043 | 0.029 | 0.047^{ a} |
0.052 | 0.059 | 0.084 | 0.258^{ A,B} |
0.651^{ A,B} |
0.127^{ A,B} |
0.088 | 0.065^{ a} |
0.056 | - |

Abbreviations as^{a}, chi-square test or ^{b}, exact test. Uppercase of these tests (^{A} or ^{B}) denotes p-values remaining significant after FDR correction. No long (~800 bp) information exists from TUN (Tunisia). WTU, MTU and ETU are groups of nesting beaches as defined in (

The analysis of statistical power showed that the long mtDNA marker had low power in detecting low differentiation (e.g. F_{st}<0.01) but high power in detecting high differentiation (e.g. F_{st}<0.04) (

The present study confirmed the findings of previous work (_{st} statistics or the PCA. As a consequence, we strongly recommend the use of this new set of primers in future loggerhead turtle genetic studies, especially for foraging areas where information is thus far only available for the short fragment.

The nesting area of Kyparissia Bay represents almost 9% of total nesting in the Mediterranean, considering that recent estimates suggested a reproductive output of approximately 7200 nests per year for the region (

As a consequence of this grouping, the overall structuring of Mediterranean nesting areas resulting from our study did not change significantly in relation to previous studies using the long fragment (

Many genetic studies have been conducted in the Mediterranean nesting beaches over the last two decades (

The existence of orphan haplotypes (haplotypes not detected in nesting areas but in feeding grounds) is one of the shortcomings of the mixed stock analysis and leads to mis-assignations. For instance, several individuals exhibiting the CC-A10 haplotype have been found in feeding grounds within the Mediterranean Sea, such as northeastern Spain (

These results have implications for the conservation of the species as there are large differences in abundance and conservation status among Mediterranean nesting aggregations. While the Greek nesting beaches host the largest aggregations, populations such as those found on the Levantine Mediterranean shores have extremely low numbers of nesting females (

Knowledge of the genetic structuring of the Mediterranean nesting beaches has been improving since the first studies (

We wish to thank Brian Ground and the ARCHELON volunteers in Greece who collected the samples. The authors wish to acknowledge use of the Maptool program in Figure 1 of this paper. Maptool is a product of SEATURTLE.ORG (information is available at