Molecular genetic investigation for differentiated populations has been characterised as a powerful tool for conservation purposes (Crandall et al. 2000; Moritz, 1994). However, length of the sequences, selected markers and sample size play an important role in differentiating populations (Monzón-Argüello et al. 2010; Clusa et al. 2013). Using extended mtDNA 815bp haplotype sequences, even independent management units (MUs) have been identified within the Mediterranean RMU (Shamblin et al. 2014), namely: (i) Calabria-Italy (CAL), (ii) western Greece (WGRC), (iii) Crete-Greece (CRT), (iv) Libya (LIBY), (v) Dalyan-Dalaman-Turkey (DLYDAL), (vi) western Turkey (TKW), (vii) eastern Mediterranean (EMED, including middle and eastern Turkey, Cyprus, Israel, and Lebanon).
According to our knowledge so far, the highest diversity with seven haplotypes was reported from nesting sites was Turkey (Yilmaz et al. 2011, Carreras et al. 2014, Clusa et al. 2014 and references therein). It is followed by Libya with five haplotypes (Said et al. 2012), Greece with four haplotypes (Yilmaz et al. 2011, Carreras et al. 2014), Cyprus (Clusa et al. 2013) and Lebanon (Yilmaz et al. 2011) with two haplotypes, and Tunisia with one haplotype (Chaieb et al. 2010). Although recent sporadic loggerhead turtle nest records were available from Albania (Piroli and Haxhi, 2020), genetic characterization was not available to date.
Libyan coasts and information about the nesting sites are identified as the major knowledge gap in the Mediterranean (Casale et al. 2018). In addition, Libya is a significant place as it is the area where the loggerhead turtle first colonised in the Mediterranean (Clusa et al. 2013). The results show that more effort needs to be put into sampling, especially along the largely unexplored Libyan coasts as two novel haplotypes have been found in this area. In total nine haplotypes were found. The second most frequent haplotype found in Misratah and Sirte, Libya was CC-A2.9 which occurs frequently in Israel and Libyan rookeries (Saied et al. 2012; Clusa et al. 2014). One specimen from a nesting site in Misratah, Libya was detected carrying the rare haplotype CC-A10.4. This haplotype was previously only recorded occasionally at Tyrrhenian nesting sites (Garofalo et al. 2016a; Mafucci et al. 2016). It derives from the CC-A10 haplotype (380 bp mtDNA sequence) which is previously observed only once in Greece (Laurent et al. 1998). For CC-A10.4 it was also predicted that this haplotype probably originated not only from Tyrrhenian rookeries but also from Mediterranean colonies (Tolve et al. 2018). The origin of the specimens found in the Adriatic (Yilmaz et al. 2012, Tolve et al. 2018, Bertuccio et al. 2019) could be in Libya. Both haplotypes, CC-A29.1 and CC-A10.4 were only found once in Turkey and Libya, respectively which is why the sampling size in these rookeries needs to be increased. Another very rare haplotype is CC-A68.1 which was found only once at the nesting location in Sirte, Libya in 2009. In this study two individuals carried CC-A68.1 from Sirte, Libya. Splendiani et al. (2017), who used samples of rescued Caretta caretta found along the Southern Adriatic coast of Italy, described the haplotype CC-A71.1 for the first time which is why they were not able to determine the origin of it. However, by creating a phylogenetic tree it was possible for them to see the phylogenetic relatedness to CC-A26.1 which is exclusive to Libya (Shamblin et al. 2014). For this reason, Splendiani et al. (2017) suggested that CC-A71.1 could be from a Libyan rookery. Our findings support this proposition since one specimen from Sirte, Libya carrying this haplotype was detected.
The 104 samples analysed from Greece, including the Management Unit of Western Greece and Crete, included all known haplotypes in these rookeries. In Western Greece 71 (95.9%) and in Crete 22 (73.3%) of the loggerheads carried the haplotype CC-A2.1. Four individuals from Chania, Crete carried CC-A2.8 while one individual from Mavrovouni, Lakonikos Bay carried this haplotype, which was known to be endemic to the Cretan rookery. One individual from Crete carried a haplotype CC-A3.1, another one CC-A32.1. Haplotype CC-A6.1 was carried by loggerheads from Kotychi and Crete. Both haplotypes, CC-A6.1 and CC-A32.1 were known to be endemic to Western Greece. One specimen from Crete and one from Koroni had haplotype CC-A31.1 originating from Greek and Calabrian rookeries but also found in sporadic Sicilian nesting sites (Garofalo et al. 2016b).
In this study in total five haplotypes were detected at main Turkish nesting sites of which two individuals belong to the same, new haplotype. The most frequent haplotype in Turkey, which is also the most common in the Mediterranean in general, was CC-A2.1 followed by CC-A3.1 also a common haplotype. The discovery of the CC-A29.1 haplotype at a nesting beach in Turkey, more precisely in western Turkey (Çıralı) shows, as has been proposed before (Tolve et al. 2018), that the origin of this haplotype must not only be in Israel, but also in other, poorly sampled or unknown nesting sites. This finding reinforces the results of MSA from the Adriatic Sea (Tolve et al. 2018): Western Turkish rookeries, which despite the fact that they are much more abundant and closer to the Adriatic Sea, showed a medium contribution probability to the Adriatic stock (Tolve et al. 2018). Two samples from Slovenia (northern Adriatic) carried the haplotype CC-A29.1. If CC-A29.1 would also be included as a haplotype of Western Turkish origin, the posterior probability would probably increase, and the contribution would not be similar or smaller than the ones of Israeli nesting areas. Haplotype CC-A50.1 is another very rare haplotype which was identified for the first time in Cyprus (Clusa et al. 2013). We are not aware that this haplotype was detected again afterwards. One specimen from a Turkish rookery (Kazanlı) carried this haplotype.
Turkey is not only a foraging site for loggerhead sea turtles originating from Cyprus but also a new nesting site. A review of general migratory routes of 63 adult loggerhead turtles released mainly from Greece and Cyprus showed that only a few of them oriented to Turkish coasts (Luschi and Casale, 2014). The tracking studies from Northern Cyprus showed that only early nesters visited other Turkish rookeries (Snape et al. 2016). MSA of an eastern Turkish foraging ground made by Türkozan et al. (2018) showed a local contribution (62%) of Cyprus to the western subdivision.
For Tunisian rookeries (Kuriat islands), only the short mtDNA control region fragment (500 bp) has been used so far, and only CC-A2.1 has been found (Chaieb et al. 2010). Analysis using the short fragment have shown no significant differences between Libyan and Tunisian rookeries, but due to several hundred kilometres of separation Shamblin et al. (2014) suggested that these might be demographically isolated nesting populations. A distinction, with the shorter D-loop sequence, between the haplotype CC-A2.9 and the widely distributed haplotype CC-A2.1 is not possible (Splendiani et al. 2017), and given the fact that CC-A2.9 is common in Libya, it has been proposed that reanalysing the Tunisian samples using the longer D-loop fragment is crucial (Shamblin et al. 2014). So far only for MSA the long fragment was used (Sami et al. 2011). In this study the long fragment was used showing that most loggerheads from the Tunisian rookery carry the common haplotype CC-A2.1 (39), however one specimen also carried haplotype CC-A2.9, and another one was CC-A3.1. Based on the detection of the CC-A2.9 haplotype at a Tunisian rookery, classification of Tunisia and Libya as one Management Unit (MU) is reasonable (see Shamblin et al. 2014).
In Albania, evidence of the occurrence of nesting was apparent with infrequent reports of hatchling sightings along the coastline, but it was not until 2018 that the first official loggerhead nest was confirmed (Piroli and Haxhi, 2020) which is why, until now, no molecular genetic analysis was done. In this study all eight samples analysed carried the cosmopolitan haplotype CC-A2.1. We have included both one hatchling and the other samples into the analyses to see if there is any different haplotype. We have also included 2 samples from Egypt in the same line to see the presence of additional haplotypes present in the Mediterranean as project partners provided samples.
Eight loggerheads were of Lebanese origin. The haplotypes they carried are consistent with previous findings, as haplotype CC-A2.1 and CC-A3.1 were found (Saied et al. 2012; Clusa et al. 2014).
Differences in polymorphism measures of different Mediterranean countries were observed. While these differences may reflect a discrepancy in sample size for most comparisons, higher values were measured in Libya than in Turkey, even though fewer loggerhead sea turtle samples were used there. Furthermore, two new haplotypes were discovered in Libya, thus our findings support Saied et al. (2012) claim that Libya has an important with wide range of haplotypes for loggerhead sea turtle which is why the protection of this assembly is essential in order to conserve the Mediterranean stock.
This study has provided new insights into the population structure of the loggerhead sea turtle in the Mediterranean Sea. Haplotypes previously thought to be endemic to certain nesting sites, but now found at other rookeries as well, and due to the assignment of an ''orphaned'' haplotype to a nesting area, it is suggested that MSA should be repeated, as these new findings could change the contribution of some rookeries to certain stocks (e.g. Adriatic Sea).