POPULATION STRUCTURE AND PHYLOGEOGRAPHY OF TUCUXI DOLPHINS (SOTALIA FLUVIATILIS) - Biology, Evolution and Conservation of River Dolphins Within South America and Asia - page 287

samples by (Baker et al . , 1994). DNA was extracted from bones following a silica-
guanidinium thiocyanate based protocol described by Pichler et al. (2001)
Figure 1. Map of the Amazon Region, showing the Amazon River and most of its main tributaries, as
well as geographic regions, sampling locations and sizes included in this study. We also indicate the
proposed genetic boundaries between Sotalia fluviatilis population units from the SAMOVA analysis
(three groups, dotted lines, grey numbers): I = West Amazon, II = Central Amazon, III = Eastern
Amazon.
PCR A MPLIFICATION AND S EQUENCING
Two mitochondrial genetic markers were analyzed; a 577 base pair (bp) portion of the
mitochondrial DNA control region (CR) and a 425 bp fragment of the cytochrome b (Cyt-b)
gene. Degradation of DNA or inhibition prevented clean amplification and sequencing of
Cyt- b from all teeth and bone samples (n = 10). These samples are represented only by partial
control region sequences. Genes were amplified via the Polymerase Chain Reaction (PCR)
using standard reaction conditions (Saiki et al., 1988, Palumbi, 1996). For the CR, we used
the primer combination t-Pro-whale (5'-TCACCCAAAGCTGRARTTCTA-3') and Dlp8 (5'-
CCATCGWGATGTCTTATTTAAGRGGAA-3') (Baker et al . , 1998). The primer
combination Dlp1.5t-Pro-whale (5'-TCACCCAAAGCTGRARTTCTA-3') and Dlp4 (5'-
GCGGGWTRYTGRTTTCACG-3') was used in the case of DNA extracted from bones or
degraded tissue samples, since these amplify a smaller region of approximately 400 bp (M.
Dalebout, pers. comm.). For Cyt- b , we used the primers Tglu (5'-TGACTTGAARAACCAY
CGTTG-3') and CB2 (3'-ACTCCTGTTTATAGTAAGAC-5') (Palumbi, 1996). The PCR
profile for all combinations of primer pairs used was as follows: an initial denaturation at
95C for 2 min, 36 cycles of 94C for 30 s, 55C for 1 min and 72C for 1.30 min, and a final
extension at 72C for 5 minutes. Free nucleotides and primers were removed from the PCR