Molecular Phylogenetics and Evolution Vol. 22, No. 2, February, pp. 174–183, 2002 doi:10.1006/mpev.2001.1052, available online at http://www.idealibrary.com on Phylogenetic Relationships among the Species of the Genus Testudo (Testudines: Testudinidae) Inferred from Mitochondrial 12S rRNA Gene Sequences Antoinette C. van der Kuyl,*,1 Donato L. Ph. Ballasina,† John T. Dekker,* Jolanda Maas,* Ronald E. Willemsen,† and Jaap Goudsmit* *Department of Human Retrovirology, Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands; and †Centro CARAPAX, CP 34, 58024 Massa Marittima (GR), Italy Received January 4, 2001, revised August 6, 2001 icas at present, but the fossil record is extensive in To test phylogenetic relationships within the genus North America, dating back to the early Eocene (Wil- Testudo (Testudines: Testudinidae), we have se- liams, 1950; Auffenberg, 1974; Bramble, 1982; Ernst quenced a fragment of the mitochondrial (mt) 12S and Barbour, 1989a; Alderton, 1993). Tortoise fossils of rRNA gene of 98 tortoise specimens belonging to the considerable age have also been found in Africa and genera Testudo, Indotestudo, and Geochelone. Maxi- Asia (Crumly, 1983). At present, a large diversity, mum likelihood and neighbor-joining methods iden- about half of the recognized tortoise species, is found in tify two main clades of Mediterranean tortoises, one Africa and the Mediterranean region. Of these, six composed of the species Testudo graeca, Testudo mar- species are currently recognized in the genus Testudo. ginata, and Testudo kleinmanni and a second of The spur-thighed tortoise Testudo graeca is most prom- Testudo hermanni, Testudo horsfieldii, and In- dotestudo elongata. The first clade, but not the second, inent in northern Africa, but is also present in south- was also supported by maximum parsimony analysis. east Europe and has been introduced at several other Together with the genus Geochelone, a star-like radi- locations, including Greece and southern Spain. Four ation of these clades was suggested, as a sister-group subspecies are recognized, of which T. g. graeca of relationship between the two Testudo clades could not North Africa; T. g. ibera of the Balkans, Greece, Tur- be confirmed. The intraspecies genetic variation was key, Iran, and Russia; and T. g. terrestris of Libya, examined by sequencing the mt 12S rRNA fragment Israel, Egypt, and Syria are the best described. Little is from 28 specimens of T. graeca and 49 specimens of T. known about the fourth subspecies, T. g. zarudnyi, hermanni from various geographic locations. Haplo- which is restricted to the Central Iranian Plateau and type diversity was found to be significantly larger in T. Afghanistan. A morphological study suggested that the graeca compared with T. hermanni, suggestive of re- first three subspecies should be elevated to full species duced genetic diversity in the latter species, perhaps level (Gmira, 1993). Furthermore, it was recently hy- due to Pleistocene glaciations affecting northern and pothesized that T. graeca of Algeria is a separate spe- middle Europe or other sources of lineage reduction. cies, Testudo whitei, or should even be classified as a No ancient mt 12S rRNA gene haplotypes were identi- separate genus, Furculachelys whitei (Highfield and fied in T. graeca and/or T. hermanni originating from islands in the Mediterranean Sea, suggesting that Martin, 1989). Coloration and patterning vary within these islands harbor tortoise populations introduced T. graeca subspecies and are not reliable for identifi- from the European and African mainland. © 2002 Elsevier cation (Lambert, 1995). It has been postulated that the Science (USA) Egyptian tortoise Testudo kleinmanni is related to T. Key Words: Testudinidae; Mediterranean tortoises; graeca (Loveridge and Williams, 1957). This very small 12S rRNA gene; phylogeny. tortoise species is found in northern Africa (Libya, Egypt, and Israel), where it is severely endangered. Of the European tortoise T. hermanni, two subspecies and INTRODUCTION recognized: T. h. hermanni, which is endemic in Italy, France, and Spain, and T. h. boettgeri of the Balkans Testudinids are found on every continent except and Greece. Differences in type can easily be observed Australia and Antarctica. Few species live in the Amer- among T. hermanni subspecies (Guyot and Devaux, 1 To whom correspondence and reprint requests should be ad- 1997). Testudo horsfieldii, the four-toed or Russian tor- dressed. Fax: 31-20-566-9064. E-mail: [email protected]. toise, sometimes known as Agrionemys horsfieldii 174 1055-7903/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved. MOLECULAR EVOLUTION OF THE GENUS Testudo 175 (Khozatsky and Mlynarski, 1966), ranges more east- Phylogenetic Analysis ward into central Asia (southeastern Russia, Iran, Af- Obtained sequences were aligned with Clustal-W ghanistan, and Pakistan). Testudo marginata [Greece (Thompson et al., 1994), and the alignment (Fig. 1) was and probably introduced by man into Italy around 200 checked by eye. Maximum parsimony (MP) analysis BC (Ballasina, 1995)] and Testudo weissingeri (Bour, was performed with PAUP* 4.0 (version 4.0.0d55 for 1996) from Greece are species with restricted habitats Unix) (Swofford, 1998), using a heuristic search with and doubtful phylogenetic placement. It has been pos- simple step-wise sequence addition and tree bisection tulated that T. weissingeri is a dwarf form of T. mar- reconnection branch swapping (TBR) and saving all ginata, the largest European tortoise species. In- optimal trees for subsequent branch-swapping steps dotestudo elongata, a species ranging from India to (MULPARS). Random addition of sequences did not Malaysia, is regarded as being only distantly related to change tree topology. Gaps were treated as uninforma- European and African tortoises. Formerly it was in- tive and excluded from the analysis, as were areas with cluded into the genus Geochelone, but has now been difficult alignment. ACCTRAN character state optimi- elevated to full genus level (Ernst and Barbour, 1989b). zation was always used, and 10,000 bootstrap repli- Using samples obtained from six tortoise species, cates were performed. Branches with bootstrap values including several subspecies, of the genus Testudo, and less than 50% were collapsed. from I. elongata, Geochelone sulcata, Geochelone Neighbor-joining (NJ) trees (Saitou and Nei, 1987) of pardalis, Geochelone (Chelonoides) carbonaria, Geoch- the sequences and reference sequences were con- elone (Chelonoides) denticulata, Cuora flavomarginata, structed using the NJ option in the MEGA package Emys orbicularis, and Trachemys scripta elegans, we (Kumar et al., 1993). The distance matrix was based sequenced part of the mitochondrial (mt) 12S rRNA upon the two-parameter method of Kimura (1980). In gene to analyze phylogenetic relationships in this sub- distance analysis, gaps introduced for optimal align- set of the family Testudinidae. This gene has previ- ment were treated as additional information and used ously been used to elucidate relationships of chelid in pair-wise comparison, except for areas with ambig- turtles (Seddon et al., 1997), turtle lineages in general uous alignment, which were excluded from the analy- (Shaffer et al., 1997), and of Madagascan tortoises sis. Treating gaps as uninformative did not signifi- (Caccone et al., 1999a). cantly alter the NJ trees. Finally, the data were analyzed using the maximum-likelihood (ML) method MATERIALS AND METHODS as implemented in PHYLIP (Felsenstein, 1994). The option FASTDNAML version 1.1.1a was used with the Amplification and Sequencing transition/transversion ratio set at 2.0. To calculate divergence times between tortoise Blood or saliva was obtained from 98 specimens be- clades based upon the 12S rRNA gene, we first checked longing to 16 species or subspecies of tortoises (Testu- rate constancy by the method of Takezaki et al. (1995). dines: Testudinidae), a yellow-margined box turtle Substitution rates of 0.25%/my (Avise et al., 1992), [Cuora flavomarginata (Testudines: Bataguridae)], 1%/my, 1.63%/my (Schubart et al., 1998), and 2%/my three individuals of the European terrapin [Emys or- were used to date divergence events in tortoises (Ta- bicularis (Testudines: Emydidae)], and nine individu- ble 2). als of the American red-eared slider turtle [Trachemys scripta elegans (Testudines: Emydidae)] (Table 1). DNA was extracted by a procedure using silica and RESULTS guanidine thiocyanate (Boom et al., 1990). Amplifica- tion of approximately 400 nucleotides of the mt 12S Phylogenetic Reconstitutions rRNA gene was done with the primer set 12S-L01091/ The 12S rRNA gene data set consisted of 404 total 12S-H01478 described by Kocher et al. (1989). PCR characters, 282 of which were constant and 71 of which primers were extended with T7 and SP6 promoter se- were parsimony informative. MP, ML, and NJ trees for quences, respectively, to facilitate direct sequencing of the tortoise 12S data set are shown in Fig. 2. Irrespec- the PCR product. Sequencing was performed in both tive of the method used, tortoises always formed a directions using a PE–Applied Biosystems 373 auto- monophyletic clade, as node A is present in all three mated sequencer, using the Dyenamic direct cycle se- trees. Also, there is support for the batagurids as their quencing kit and the Dyenamic energy transfer dye sister group in two of the three trees (Gaffney and primer set from Amersham Int. (UK), following the Meylan, 1988). Several nodes appear in all trees, e.g., manufacturer’s protocols. Sequences used in the anal- node B (clustering T. graeca, T. kleinmanni, and T. yses were deposited with GenBank (Accession Nos. marginata). Differences between the trees will be dis- AF175326–AF175341). cussed in more detail below. 176 VAN DER KUYL ET AL. FIG. 1. Alignment of 16 tortoise and turtle 12S rRNA gene fragments amplified with primers 12S–L01091 and 12S–H01478 (Kocher et al., 1989); numbering of the primers refers to their location in the human mtDNA. The T. h. hermanni and T. h. boettgeri sequences are from animals originating from Italy and Albania, respectively. MOLECULAR EVOLUTION OF THE GENUS Testudo 177 —Continued FIG.