MOLECULAR PHYLOGENETICS AND EVOLUTION Molecular Phylogenetics and Evolution 25 (2002) 501–510 www.academicpress.com Molecular phylogeny and evolution of the extinct bovid Myotragus balearicus Carles Lalueza-Fox,a Beth Shapiro,b Pere Bover,c Josep Antoni Alcover,c and Jaume Bertranpetitd,* a Laboratori d’Antropologia, Facultat de Biologia, Universitat de Barcelona, Avda. Diagonal 645, Barcelona 08028, Spain b Ancient Biomolecules Centre, Department of Zoology, Oxford University, South Parks Road, Oxford OX1 3PS, UK c Institut Mediterrani d’Estudis Avanßcats (CSIC-UIB), Cta. de Valldemosa km 7.5, Ciutat de Mallorca 07071, Spain d Unitat de Biologia Evolutiva, Facultat de Ciencies de la Salut i de la Vida, Universitat Pompeu Fabra, Doctor Aiguader 80, Barcelona 08003, Spain Received 11 June 2001; received in revised form 6 June 2002 Abstract Myotragus balearicus was a dwarf artiodactyl endemic to the Eastern Balearic Islands, where it evolved in isolation for more than 5 million years before becoming extinct between 3640 and 2135 cal BC (calibrated years BC). Numerous unusual apomorphies obscure the relationship between Myotragus and the extant Caprinae. Therefore, genetic data for this species would significantly contribute to the clarification of its taxonomic position. In this study, we amplify, sequence, and clone a 338-base pair (bp) segment of the mito- chondrial cytochrome b (cyt b) gene from a >9Kyr Myotragus subfossil from la Cova des Gorgs (Mallorca). Our results confirm the phylogenetic affinity of Myotragus with the sheep (Ovis) and the takin (Budorcas). In each tree, the Myotragus branch is long in comparison with the other taxa, which may be evidence of a local change in the rate of evolution in cyt b. This rate change may be due to in part to an early age of first reproduction and short generation time in Myotragus, factors that are potentially related to the extreme reduction in size of the adult Myotragus as compared to the other Caprinae. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Myotragus balearicus; Molecular systematics; Cytochrome b; Generation time; Evolution 1. Introduction human role in the extinction of Myotragus (Alcover et al., 1999a,b). Myotragus balaericus (Bate, 1909) was a dwarf bovid Morphologically, Myotragus was quite distinct from endemic to the Eastern Balearic Islands or Gymnesics the other Caprinae (see Fig. 1). It is the smallest Cap- (Mallorca, Menorca, Cabrera, and sa Dragonera) in the rinae known: the largest adult specimens found would Western Mediterranean Sea (Alcover et al., 1981). The not have reached more than 45–50 cm from the ground ancestor of Myotragus probably colonized Mallorca to the shoulder, and probably weighed no more than 50– while the Mediterranean was desiccated, between 5.7 70 kg, while neonatal weight has been estimated to and 5.35 million years ago (Mya) during the Messinian around 700–900 g (Bover and Alcover, 1999a). In addi- (Clauzon et al., 1996). When the Gibraltar Strait re- tion to its small size, Myotragus had eyes in a frontal opened, the ancestor of Myotragus became trapped on position, a monophiodontic incisiform dentition with the islands, where it evolved in isolation until going constantly growing incisor in both jaws (Bover and Al- extinct between 3640 and 2135 cal years BC (Ramis and cover, 1999b), and modified limb bones that most likely Alcover, 2001). The timing of this extinction coincides restricted it to slow locomotion (Alcover et al., 1981; with the first records of human settlement on the island Quetglas and Bover, 1998; Sondaar, 1977). These and (Burleigh and Clutton-Brock, 1980), which suggests a other automorphies have obscured the relationship of Myotragus with the other Caprinae. * Corresponding author. Fax: +34-935422802. Traditionally, the subfamily Caprinae has been di- E-mail address: [email protected] (J. Bertranpetit). vided into four tribes: Rupicaprini (including the genera 1055-7903/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S1055-7903(02)00290-7 502 C. Lalueza-Fox et al. / Molecular Phylogenetics and Evolution 25 (2002) 501–510 Fig. 1. Reconstruction of Myotragus balearicus. Rupicapra, Oreamnos, Capricornis, and Nemorhaedus), Myotragus specimens. In an earlier study, we recovered Ovibovini (Ovibos and Budorcas), Caprini (Ovis, Capra, a short (55-bp) fragment of cyt b from a 4770–4400 cal Pseudois, Hemitragus, and Ammotragus), and Saigini BC 2r (UtC 5171) year old Myotragus individual found (Saiga and Pantholops) (Nowak, 1991; Simpson, 1945). in the cave site Cova Estreta (Pollencßa, Mallorca) (La- However, recent morphological and molecular studies lueza-Fox et al., 2000). Although the fragment was have questioned these relationships (Chikuni et al., 1995; short, the authenticity of the results was supported by Gatesy et al., 1997; Geist, 1987; Gentry, 1980, 1992; cloning of PCR products, phylogenetic analysis, and Groves and Shields, 1996; Hassanin et al., 1998a,b; independent replication, as is required in aDNAre- Hassanin and Douzery, 1999; Thomas, 1994). For ex- search (Cooper and Poinar, 2000; Hofreiter et al., 2001). ample, two recent molecular studies have suggested that Preliminary phylogenies generated using this 55-bp Saiga should actually be placed in the Antelopinae, rather fragment suggested a close relationship between Myo- than in the Caprinae (Gatesy et al., 1997; Hassanin et al., tragus and Budorcas, as has previously been suggested 1998a). The accuracy of the other tribal classifications has based on morphological evidence (Andrews, 1915). Like also remained a matter of debate. Most modern molecular studies (Groves and Shields, 1996, 1997; Hassanin and Table 1 Douzery, 1999) have found only three stable clades into Primer sequences used in the study the Caprinae: Capra and Hemitragus, Capricornis, Ovi- L-14,899 50-ATCCTAACAGGCCTATTCCT-30 bos,andNemorhaedus, and Ovis and Budorcas. H-14,955 50-ACCATAGTTTACATCTCGGC-30 Myotragus is most often placed in the tribe Rupi- L-14,942 50-CAACAACAGCATTYTCYTCTG-30 caprini, most closely related to Nemorhaedus and Cap- L-14,983 50-CTATGGCTGAATTATCCG-30 0 0 ricornis (Nowak, 1991; Simpson, 1945), however, this L-15,062 5 -CGAGGCCTGTACTACGGATC-3 H-15,071 50-CCGATGTTTCATGTTTCTAGGA-30 relationship has been challenged (Gentry, 1992) and H-15,238 50-AACTGAGAATCCGCCTCAG-30 remains problematic. To clarify the issue, we use ancient L and H refer to the light and heavy strands, respectively, and DNA(aDNA)techniques to recover small fragments of numbers refer to the 50 position in the Anderson et al. (1981) human mitochondrial DNA(mtDNA)from the remains of two mtDNAsequence. C. Lalueza-Fox et al. / Molecular Phylogenetics and Evolution 25 (2002) 501–510 503 other molecular studies, these results did not support the sample was then extracted using phenol/chloroform traditional tribal divisions within the Caprinae. Unfor- techniques and desalted with Centricon 30 microcon- tunately, however, the advanced state of decay of the centrators (Amicon). Extraction procedures were per- Myotragus specimen used in the analysis made it im- formed in an isolated pre-PCR area with positive air possible to generate any additional molecular data, pressure. Appropriate controls were used in each step of which would have been necessary to confirm the the analysis, adopting the standard precautions of aDNA phylogenetic placement of Myotragus. In this study, we studies (Cooper and Poinar, 2000; Handt et al., 1994). extract and amplify 338 bp of cyt b from a Myotragus PCR amplifications were carried out in 25 ll reac- sample not used in the previous study. We use these data tions with 1 ll of extract, 1 U EcoTaq and 1Â buffer to construct a molecular phylogeny for the Caprinae, (EcoGen), 2 mg/ml BSA, 2.5 mM MgCl2, 0.25 mM and to confirm the phylogenetic position of Myotragus dNTPs, and 1 lM primers. PCR products were visual- within the subfamily. ized in low melting point agarose gels, and bands rep- resenting positive results were excised, melted in 150– 200 ll of water, and reamplified. Primers used in the 2. Materials and methods study are listed in Table 1. The primer pairs L14,899/H15,071, L14,942/H15,071, 2.1. DNA extraction, amplification, cloning, and sequenc- L14,983/H15,071, and L15,062/H15,238 yielded positive ing amplifications and were sequenced on an ABI 377a DNA sequencer (Applied Biosystems). To detect nuclear copies Aleft tibia (Accession No. MNIB 60173) from Cova or errors potentially introduced by template damage, two des Gorgs (Escorca, Mallorca) was chosen for analysis fragments (L14,942/H15,071 and L15,062/H15,238) were based on its unusually good macroscopic preservation. cloned using a Sure Clone Ligation Kit (Pharmacia) ac- The medial portion of the sample was used for DNA cording to the manufacturer’s instructions. analysis, while the proximal portion was sent for ra- To ensure the authenticity of the result, DNAwas diocarbon dating. The remainder of the sample is cur- extracted, amplified, cloned, and sequenced from a rently held in the vertebrate collection in the Museu de separate bone sample from the same Myotragus speci- la Naturalesa de les Illes Balears (MNIB, Palma de men at the Ancient Biomolecules Centre in Oxford, Mallorca). Despite the superior preservation of the England. The procedure was similar to that described sample, the bone yielded a radiocarbon date of 7750– above (details can be found in Barnes et al., 2002) except 7585 cal BC 2r (Beta 143117), approximately 3000 years a high-fidelity enzyme (Platinum Taq Pfu Hi-Fi, Gibco- older than the bone used in the previous study. BRL) was used in the amplification. In Barcelona, DNAwas extracted from approxi- mately 1 g of bone. The sample was powdered and de- 2.2. Phylogenetic analyses calcified overnight in 10 ml of 0.5 M EDTA, followed by an overnight incubation in 1 ml of 10% SDS, 0.5 ml of Cyt b sequences were obtained from GenBank for all 1 M Tris–HCl, and 100 ll of 1 mg/ml proteinase K.