Biology Reference
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length and five meters of width) and the fishermen and the biologists captured the dolphins
inside the water after of a brief body to body struggle between dolphin and humans. The
individuals were brought on board (in a little wood ―canoa‖ in the Bolivian case) and I
personally biopsied the caudal fin of each dolphin captured. After the biopsy, the wound was
covered with an antibiotic. Later, the animals were measured for different biometric
characteristics and safety released after 5-8 minutes of manipulation. The animals were
marked to avoid any recapture and the biopsies were stored in absolute alcohol until DNA
extraction.
For the microsatellite analysis, a total of 180 individuals were geno-typified [69
individuals from the Mamoré, Tijamuchí, Iruyañez, Securé, Iténez (= Guaporé), and
Ipurupuru Rivers in the Bolivian Amazon, 42 individuals from the Colombian Putumayo
River, 33 individuals from the Napo-Curaray rivers, 13 individuals from the Marañón-
Samiria rivers and 18 individuals from the Ucayali-Tapiche-Canal del Puhinauva rivers (thus,
a total of 64 exemplars were analyzed in Peruvian rivers) and five individuals from the
Colombian-Peruvian frontier (between Puerto Nariño in Colombia and Caballococha in Perú)
in the Amazon river and tributaries]. The population sets studied for microsatellites were in
the Napo-Curaray rivers, Ucayali River, Marañón River, all the collective upper Amazon
(exemplars collected from the Peruvian rivers, the Colombian-Peruvian Amazon, and the
Putumayo River were analyzed together) and the Bolivian rivers.
For the mtDNA analysis, 207 exemplars were analyzed [57 individuals from the Bolivian
rivers, 125 individuals from the Amazon River and its tributaries (Peruvian rivers previously
cited, Amazon Colombian-Peruvian frontier, Putumayo and Caquetá rivers in Colombia, and
one specimen from the Negro River in the central Brazilian Amazon) and 25 individuals from
the Orinoco, Bita, Inirida, Arauca and Meta rivers within the Orinoco basin in Colombia and
Venezuela; the Orinoco animals were not studied in this work)]. The population sets used for
mtDNA analyses were the Napo-Curaray rivers, Ucayali River, Marañón River, all the
Amazon exemplars analyzed together (all the Peruvian rivers, the Colombian-Peruvian
Amazon individuals and the exemplars captured in the Putumayo and Caquetá rivers) and the
Bolivian rivers.
Molecular Procedures
DNA extraction from the caudal fin biopsies was performed by the phenol-chloroform
method (Sambrock et al., 1989). The ten microsatellite markers studied in the Inia samples
were Ev14, Ev37, Ev76, Ev94 and Ev96 (Valsecchi & Amos, 1996), MK5 (Krutzen et al.,
2001), PPHO137 (Rosel et al., 1999), KWM2a, KWM2b and KWM12a (Hoelzel et al., 1998,
2002). The reactions were completed in 25 μl with the following conditions: 10 pmol of
forward and reverse primers, 2.5 l of reaction buffer (10X), 3.0 l of MgCl 2 3 mM, 1 l of
dNTPs 1 μM, one Taq polymerase unit, 13.5 l of H 2 O, and 2 l between 25 and 50 ng per l
of DNA. The PCR conditions were 95 o C for 5 minutes, a number of determined cycles of 1
minute at 95 o C, 1 minute at the most accurate annealing temperature (see in the next
sentence), one minute at 72 o C, and 5 minutes at 72 o C. The number of cycles and the
annealing temperatures were as follows: Ev14 (35 cycles at 58° C), Ev37 (10 cycles at 46 °C
and 25 cycles at 56 °C), Ev76 (10 cycles at 46 °C and 25 cycles at 56 °C), Ev94 (10 cycles at
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