Biology Reference
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46 °C and 25 cycles at 56 °C), Ev96 (10 cycles at 46 °C and 25 cycles at 56 °C), MK5 (35
cycles at 56° C), PPHO137 (35 cycles at 57° C), KWM2a (35 cycles at 57 ° C), KWM2b (35
cycles at 54° C) and KWM12a (35 cycles at 55° C). The PCR amplification products were
run in denaturant 6 % polyacrilamide gels within a Hoefer SQ3 sequencer vertical chamber.
Gels migrated for 2-3 hours depending on marker sizes, and were then stained with AgNO 3
(silver nitrate). Every sixth line in the gel contained molecular markers (174 cut with Hind
III and Hinf I).
For the analysis of the mitochondrial control region gene (400 bp), the primers employed
were H16498 and TRO (LeDuc, unpublished data). The sequences of these primers are 5'
CCT GAA GTA AGA ACC AGA TG3' for H16498 and 5' CCT CCC TAA GAC TCA AGG
3' for TRO. The PCR reactions were undertaken in a final 25 μl volume with the following
conditions: 0.4 μM of each primer, 2.5 l of reaction buffer (1X), 3.0 l of MgCl 2 2.5 mM,
0,4 μM of each dNTP, one Gold Taq Polymerase (Promega) unit and 2 l of (25 and 50 ng
per l) of DNA. The amplifications were carried out in a BioRad thermocycler with the
following protocol: 95 ºC for 5 min, 30 cycles at 95 °C for 45 s, at 52 °C for 45 s and at 72°C
for 45 s and at 72°C for 10 min for final extension. All DNA fragments were sequenced in
both directions and in cases of mismatching, the PCR reaction was repeated and the product
sequenced again. Sequence alignments and editing were performed by using Sequencer 3.0
(Gene Codes Corp.), Clustal X (Thompson et al., 1997) and MEGA 4.0.
Population Genetics Demographic Analyses
Microsatellites
Kimmel's et al. test (1998) was the first procedure to detect possible demographic
changes in the evolutionary history of the pink river dolphins. This test is based on the
principle that two different estimates of  (= 4N e ) could be obtained [one is  v = V , with V
being the variance of the tandem repeat size, whose expression is V = (2  I = 1….n (X i - X) 2 )/(n
- 1), where n is the number of chromosomes analyzed, X i is the number of repeats of each
allele found and X is the average repeat number of all the alleles found in a microsatellite.
The second one is  Po = (1/(P o 2 - 1))/2 (estimator of the homozygosity), where P o = (n  k =
1…..n (p k 2 - 1))/(n -1), with p k being the allele frequency of the kth allele.] An imbalanced 
index could be defined as:
(t) = E( v )/E( Po ) = V(t)/[((1/ P o 2 ) - 1)/2 or by the expression: ln(t) = ln  v - ln  Po =
ln (V) - ln (((1/ P o 2 ) - 1)/2).
If a population is in equilibrium, has a constant demographic size, and isn't suffering an
expansion,  = 1 (ln  = 0). On the contrary, if a population has suffered an expansion coming
from a mutation-drift equilibrium situation (constant population size),  < 1 (ln  < 0). If a
population has experienced an expansion coming from a previous bottleneck ,  > 1 (ln  >
0). This last value will be present for a long time (several thousand generations) before
showing the signature of a population expansion ( < 1 (ln  < 0)). There is an exception to
this general rule, when a bottleneck is so intense the population becomes monomorphic
before the demographic expansion, in which case,  < 1 (ln  < 0) all the time. All these 
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