POPULATION STRUCTURE AND PHYLOGEOGRAPHY OF TUCUXI DOLPHINS (SOTALIA FLUVIATILIS) - Biology, Evolution and Conservation of River Dolphins Within South America and Asia - page 288

products using SAP (shrimp alkaline phosphatase) and ExoI (exonuclease I) (USB) and
directly sequenced in both directions using the standard protocols of Big Dye™ terminator
sequencing chemistry on an ABI 3100 automated capillary sequencer (Perkin Elmer).
Alternatively, Brazilian samples were analyzed at Universidade Federal de Minas Gerais
(UFMG) in Belo Horizonte, Brazil using a slightly different method: samples were amplified
following the previously described protocol, cleaned using 20% PEG (Polyethyleneglycol)
and sequenced using an ETDye terminator kit and run in a MegaBACE automated capillary
sequencer (Amersham Biosciences).
DNA extracted from bone, tooth or degraded skin was amplified in at least two separate
PCR reactions, including extraction controls, in order to prevent amplification of possible
contaminants. Free nucleotides and primers were removed from the PCR products using the
QIAquick PCR purification kit (QIAGEN). PCR products from two independent
amplifications were sequenced in both forward and reverse directions separately and
subsequently compared, in order to improve the confidence and accuracy of our results.
Table 1. Sampling locations and tissue type obtained for tucuxi dolphins. Numbers in
parenthesis before each sampling location corresponds to the number of this sampling
location in Figure 1.

Geographic region Sampling location Sample size and type

Peruvian Amazon
(PA)

(1) Curaray River
(2) Caballo Cocha
(Loreto province)

1 skin
1 bone
1 skin

(3) Patrullero Island
(Loreto province)

1 skin

Colombian Amazon (4) Caquetá River 2 bones

(CA) (5) Puerto Alegria

(Amazonas province)

1 skin

(6) Puerto Nariño
(Amazonas province)

2 skins
4 teeth

(7) Leticia
(Amazonas province)

1 skin
1 tooth

Brazilian Amazon unknown 1 DNA**

(BA) (8) Benjamin Constant

(Amazonas state)

1 skin

(9) Tefé
(Amazonas state)

7 skins

(10) Santarém
(Pará state)

1 bone

(11) Formoso Araguaia River 1 bone
**Sample donated as extracted DNA by the SWFSC: Southwest Fisheries Science Center (La Jolla,
CA, U.S.A)

D ATA A NALYSES

Sequence quality was evaluated using the program Phred v.020425 (Ewing & Green,
1998, Ewing et al., 1998). Sequences with Phred scores ≤ 20 (a base call having a probability