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substitutions. The standard errors were obtained by 1000 bootstrap replicates. To test whether
positive selection was operating at each locus we compared the relative abundance of
synonymous and nonsynonymous substitutions using a Z-test at the 5% level (Tamura et al.,
2007). PBR and non-PBR were identified assuming homology with predictions made for
human MHC molecules (Brown et al., 1993; Bjorkman et al., 1987).
Mismatch distribution analyses (Figueroa et al., 2000; Go et al., 2002; Suárez et al.,
2006) were used to detect convergence in each locus and demographic history of the baiji and
finless porpoise. The sudden expansion model (Rogers & Harpending, 1992) and goodness-
of-fit tests (sum of squared deviations, SSD; Harpending's raggedness index, R; Schneider &
Excoffier, 1999) of the observed to the estimated mismatch distributions were computed in
ARLEQUIN version 3.0 (Excoffier et al., 2005). The program GENEVONV version 1.81
(Sawyer, 1999) was employed to find the most likely candidate alleles for intragenic
recombination/gene conversion events in the baiji and finless porpoise. This method uses
pairwise comparison of sequences in the alignment to find blocks of sequence pairs that are
more similar than would be expected by chance. GENEVONV finds and ranks the highest-
scoring fragments globally for the entire alignment. Global permutation test P-values of <0.05
(derived from BLAST-like global scores using 10,000 replicates) were considered as
evidence of intragenic recombination. Phylogenetic trees employing the neighbor-joining
(NJ) method were constructed according to Kimura 2-parameter (or K2P) nucleotide
distances in MEGA version 4 (Tamura et al., 2007) in order to reveal relationships between
alleles of the baiji and finless porpoise, and alleles from some other cetacean species.
Bootstrap confidence intervals were obtained from 1000 replicates.
R ESULTS
Allelic Diversity of the Finless Porpoise at Three MHC Genes
DRA
A total of six unique sequences were detected for the DRA gene from all the samples. All
individuals tested had no more than two sequences, suggesting that the DRA primers used in
this study amplified a single locus. Allele Neph-DRA*06 had a stop codon in the middle part
of the sequence, representing most likely a pseudogene, and it was excluded from further
analyses. The remaining allele sequences have been submitted to GenBank with accession
numbers DQ843609-DQ843613. Sequence polymorphism analysis showed that the overall
variation was very low, with 2.1% variability at the nucleotide level (four polymorphic sites
of 189 base pairs sequenced) and 4.8% at the amino acid level (three polymorphic sites of 63
amino acid residues) (Figure 1a). On the other hand, the similarity between DRA alleles was
extremely high, with each pair containing only a maximum of two different nucleotides. For
the deduced amino acid sequences, three of the four polymorphic sites represented non-
synonymous substitutions (Figure 1a) but none were found in the α1 domain which encodes
the putative MHC class II PBR (Brown et al. 1993; Stern et al. 1994). Of all the alleles
identified, Neph-DRA*01 had the highest frequency while the other alleles only appeared
once or twice.
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