Acta Herpetologica 4(2): 119-123, 2009 Phylogeographic link between Sicilian and Corso-Sardinian Testudo h. hermanni confirmed Gabriele Giacalone1,2, Mario Lo Valvo1, Uwe Fritz3 1 Laboratorio di Zoologia applicata, Dipartimento di Biologia Animale, Università degli Studi di Pa- lermo, Via Archirafi 18, I-90123 Palermo, Italy. 2 Present address: Dipartimento di Botanica, Università degli Studi di Catania, Via Antonino Longo 19, I-95125 Catania, Italy. 3 Museum of Zoology (Museum für Tierkunde), Senckenberg Dresden, A.B. Meyer Building, Königs-brücker Landstr. 159, D-01109 Dresden, Germany. Corresponding author. E-mail: uwe.fritz@senckenberg.de. Submitted on: 2008, 12th October; revised on 2009, 20th August; accepted on 2009, 31st August. Abstract. Our study confirms that Sicilian and Corso-Sardinian Testudo h. herman- ni share a certain mtDNA haplotype, while tortoises from peninsular Italy harbour slightly different haplotypes. When the fossil record is considered, this striking pattern agrees well with the idea of local extinction in Corsica and Sardinia and later replace- ment by tortoises originating elsewhere, either by natural oversea dispersal or translo- cation by man. Keywords. Testudo hermanni, phylogeography, Corsica, Sardinia, Sicily. Traditionally, the fauna of the islands Corsica, Sardinia and Sicily is considered to constitute the Tyrrhenian faunistic province (La Greca, 1995). However, while Corsica and Sardinia are known to share many endemic or closely related taxa, Sicily represents a dis- tinct entity (Lanza and Vanni, 1987; Lanza, 1988; Cheylan, 1992). Most of Sicily’s zooge- ographic affinities point at the Italian peninsula (Cheylan, 1992), although some endemic taxa are known (Stöck et al., 2008), one of which (Bufo siculus) suggests a link to North Africa. Also other taxa support this relationship between Sicily and North Africa (La Gre- ca, 1990, 1995; Cheylan, 1992). Fritz et al. (2006) demonstrated that two Sicilian Testudo hermanni hermanni har- boured a mitochondrial haplotype also occurring on Corsica and Sardinia, an unexpect- ed finding when it is considered that tortoises from the Italian peninsula possess distinct haplotypes. In order to find out whether the two individuals studied by Fritz et al. (2006) might represent introductions from Corsica or Sardinia, we obtained blood samples of T. h. hermanni from five other Sicilian populations (Fig. 1) and sequenced most of the mito- chondrial cytochrome b gene (cyt b), the marker used by Fritz et al. (2006). In addition, we sequenced part of the faster evolving mitochondrial control region (CR; Table 1) from two Sicilian, three Corsican, and two Sardinian tortoises. 120 G. Giacalone, M. Lo Valvo and U. Fritz Agrigento Palermo Catania Siracusa Messina 1 2 3 4 5 6 7 Fig. 1. Collection sites of Sicilian Testudo hermanni hermanni samples used in this study and in Fritz et al. (2006). Site numbers refer to Table 1. Table 1. Geographic origin of Testudo hermanni samples used in this study, Sicilian samples from Fritz et al. (2006) and the studied mtDNA fragments. Sample numbers refer to the Tissue Collection of the Museum of Zoology Dresden (MTD T); site numbers, Fig. 1. Sample Site Cyt b CR Reference 2140 1 – Castellammare del Golfo (Palermo), Sicily + - Fritz et al. (2006) 2141 2 – Balestrate (Palermo), Sicily + - Fritz et al. (2006) 4078 3 – M. M. Belsito (Madonie), Sicily + - This study 4075 4 – Collesano-Campofelice (Madonie), Sicily + - This study 4077 4 – Collesano-Campofelice (Madonie), Sicily + - This study 4082 4 – Collesano-Campofelice (Madonie), Sicily + - This study 4074 5 – Caronia (Nebrodi), Sicily + - This study 4079 6 – S. Agata di Militello (Nebrodi), Sicily + + This study 4083 7 – Augusta (Siracusa), Sicily + - This study 4084 7 – Augusta (Siracusa), Sicily + + This study 4085 7 – Augusta (Siracusa), Sicily + - This study 4086 7 – Augusta (Siracusa), Sicily + - This study 1921 Porto Vecchio, Corsica - + This study 1927 Casabianda, Corsica - + This study 1936 Fontane du Salario (Ajaccio), Corsica - + This study 1114 Porto Palmas (Sassari), Sardinia + + Cyt b: Fritz et al. (2006), CR: this study 1115 Bay of Porto Ferro (Sassari), Sardinia + + Cyt b: Fritz et al. (2006), CR: this study 121Phylogeography of Testudo hermanni Laboratory procedures followed Fritz et al. (2006). For amplification and sequenc- ing of an approximately 1000 bp long fragment of cyt b the primers CytbG (Spinks et al., 2004), mt-f-na, mt-c-For2, and mt-E-Rev (Fritz et al., 2006) were used; for an approxi- mately 850 bp long CR fragment, the primers Thr-L15569 and Phe-H26 (Palkovacs et al., 2003). An ABI 3130 Genetic Analyzer was used for automatic sequencing in both direc- tions. Obtained sequences were checked by eye and aligned. Cyt b sequences were com- pared with published data (Fritz et al., 2006). Using tcs 1.21 (Clement et al., 2000), a parsi- mony network was constructed from a 1008 bp long alignment including all 31 previously published Western Mediterranean T. h. hermanni sequences (Fritz et al., 2006) plus our 10 new sequences from Sicily (Fig. 2). The alignment of the CR sequences comprised 855 bp. All of our 10 Sicilian T. h. hermanni harboured the same cyt b haplotype (H5) as the two Sicilian tortoises studied by Fritz et al. (2006). With respect to the CR, all Corsican and Sardinian tortoises shared the same haplotype; this haplotype was also found in one Sicilian tortoise. The second Sicilian individual (MTD T 4079) possessed a similar haplotype differ- ing in only one mutational step (position 815 of our alignment: T instead of A). The two CR haplotypes are deposited under accession numbers FN298446-FN298447 in GenBank. Compared to the eastern subspecies T. h. boettgeri, the phylogeographic structure of Western Mediterranean T. h. hermanni is strikingly shallow (Fritz et al., 2006). This is unex- pected because the fossil record provides evidence for its long presence and wide distribu- tion in the Western Mediterranean (Delfino, 2002; Morales Pérez and Sanchis Serra, 2009). Amongst others, fossils are known from the Middle Pleistocene of Corsica (Hervet, 2000, Fig. 2. Parsimony network of mtDNA haplotypes (cyt b fragment, 1008 bp) of Western Mediterranean Testudo hermanni hermanni, using the dataset of Fritz et al. (2006) and 10 new Sicilian sequences. Hap- lotype nomenclature follows Fritz et al. (2006). Symbol size corresponds to haplotype frequency; missing haplotypes, dots. Each line between symbols represents one mutation step. The arrow indicates the con- nection to Eastern Mediterranean T. h. boettgeri haplotypes. Black symbols, peninsular Italy and southern France (Var); dark grey, Sicily; light grey, Corsica and Sardinia; white, Spain (i: introduced populations or populations known to comprise also allochthonous individuals, n: native population). H1: n = 12, H2: n = 1, H3: 7, H4: n = 1, H5: n = 18, H6: n = 1, H7: n = 1. When only Western Mediterranean haplotypes are considered, H1 is under coalescent theory ancestral to H2-H7 (outgroup probability of H1: 0.3175). 122 G. Giacalone, M. Lo Valvo and U. Fritz 2001; Hervet and Salotti, 2000) and from the Plio-Pleistocene boundary of Sardinia (Abbaz- zi et al., 2004). On the Italian peninsula, the oldest findings date back to the Pliocene; the oldest fossils from Sicily are from the Middle Pleistocene (Delfino, 2002), suggesting that Sicily was colonized later than Corsica and Sardinia. Consequently, some extent of genetic differentiation of the island tortoises and a closer relationship of the Sicilian and the penin- sular Italian populations should be expected, but not between Sicily and Corso-Sardinia. The weak genetic differentiation of Western Mediterranean tortoises might be related to a major climatically caused extinction event some 38000-39500 years ago, wiping out many populations (Morales Pérez and Sanchis Serra, 2009). However, while our study con- firms that Sicilian and Corso-Sardinian T. h. hermanni are not clearly differentiated in the studied genetic markers, they are slightly distinct from peninsular Italian tortoises (and from native Spanish tortoises; Fig. 2). This pattern supports the survival of several local populations in the Western Mediterranean, in agreement with fossil evidence (Delfino, 2002; Morales Pérez and Sanchis Serra, 2009) for instance on the Italian peninsula and Sic- ily. 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