Mitochondrial haplotype diversity in the tortoise species Testudo graeca from North Africa and the Middle East

Background: To help conservation programs of the endangered spur-thighed tortoise and to gain better insight into its systematics, genetic variation and evolution in the tortoise species Testudo graeca (Testudines: Testudinidae) was investigated by sequence analysis of a 394-nucleotide fragment of the mitochondrial 12S rRNA gene for 158 tortoise specimens belonging to the subspecies Testudo graeca graeca, Testudo graeca ibera, Testudo graeca terrestris, and a newly recognized subspecies Testudo graeca whitei. A 411-nucleotide fragment of the mitochondrial D-loop was additionally sequenced for a subset of 22 T. graeca, chosen because of their 12S gene haplotype and/or geographical origin. Results: Haplotype networks generated by maximum-likelihood and neighbor-joining analyses of both the separate and the combined sequence data sets suggested the existence of two main clades of Testudo graeca, comprising Testudo graeca from northern Africa and Testudo graeca from the Turkey and the Middle East, respectively. Conclusion: Mitochondrial DNA haplotyping suggests that the tortoise subspecies of T. g. graeca and T. g. ibera are genetically distinct, with a calculated divergence time in the early or middle Pleistocene. Other proposed subspecies could not clearly be recognized based upon their mt haplotypes and phylogenetic position, and were either part of the T. g. graeca or of the T. g. ibera clade, suggesting that genetic evidence for the existence of most of the 15 proposed subspecies of T. graeca is weak