Results from this study demonstrate the fundamental importance of continuing taxonomic investigations …

• Abstract
• Introduction
• Materials and methods
• Results and discussion
• Acknowledgements
• References
• Figures
• Tables

Biology Articles » Conservation Biology » A cryptic taxon of Galápagos tortoise in conservation peril » Materials and methods

Materials and methods

- A cryptic taxon of Galápagos tortoise in conservation peril

2. Materials and methods

(a) Sample and data collection

Galápagos tortoise (G. nigra) blood samples were collected in three expeditions conducted over 7 years. All samples were obtained in accordance with local, national and international regulations. Population-level analyses were based on a total of 139 blood samples collected on Santa Cruz from the following populations: La Caseta (n=65), Cerro Fatal (n=71) and Cerro Montura (n=3). DNA was extracted using the Qiagen DNeasy Tissue Kit (Qiagen, Inc.) according to the manufacturer's protocol. DNA was sequenced for 697bp of the mtDNA control region (CR) and genotyped at nine microsatellite loci following previously published conditions (Ciofi et al. 2002; Beheregaray et al. 2003). Higher-level analyses were based on consensus mtDNA sequences for extinct and extant lineages of Galápagos tortoises from four gene regions: control region (CR) (697bp), 12s rDNA (12S; 425bp), 16s rDNA (16S; 548bp) and cytochrome b (cyt b; 416bp). Bone samples were obtained from museum specimens representing the extinct Floreana population (Museum of Comparative Zoology No. R-46606) and extinct western, domed San Cristóbal population (California Academy of Sciences No. 8133). DNA was extracted from the museum samples in a dedicated facility for ancient DNA work according to a modified protocol (available from lead author), following all necessary precautions to prevent contamination by extant specimens. DNA was sequenced in 14 overlapping fragments not exceeding 200bp in length across all four mtDNA gene regions (primer information available upon request). Sequences from the extinct lineages were combined with previously published consensus sequences of the extant G. nigra lineages (Caccone et al. 2002) and three out-group taxa (Geochelone chilensis, Geochelone carbonaria and Geochelone denticulata) for subsequent analyses. GenBank accession numbers for all DNA sequences used and generated over the course of this study are as follows: AY97476-AY098119, AF548216-AF548226 and AY956612-AY956623 (see Electronic Appendix A for details regarding population genetic and phylogenetic analyses).