FIGURE 4: Bayesian phylogenetic reconstruction of MHC class I
exon 3 of four species of Turdidae family. All the nodes are well
supported (PP>0.90%) unless indicated otherwise. AB268885
Gallus gallus was used as an outgroup.
DISCUSSION
In this study, we have for the first time characterize MHC Class I gene
in four species of Turdidae family in the order Passeriformes
from the wide geographical area of Northeast china. Analysis of MHC
class I sequences revealed a total of 77 distinct Haplotypes/alleles
including 47 putative functional alleles ever reported in passerine
species, a group which is reported to have surprising MHC diversity
[58, 59]. According to our findings based on MHC class I sequences,
the functional loci in an individual ranged from one to three in three
of the four species, which was consistent with findings from other
passerine species studied till now [60]. In addition, we detected a
large number of presumed pseudogene sequences in the sampled population
as it retains important information about the evolution of MHC. This is
not surprising, as it is consistent with the expectation of evolution by
birth-and-death [61]. We made a significant effort to characterize
the variation in regions of MHC class I exon 3 in our study population,
we find that the primers would make some unlikely bias in allelic
variations among individuals. Hence, MHC class I alleles variations per
individual should, largly be due to copy number of genes variation among
individuals, which has been confirmed in other birds [62]. Few MHC
class I alleles were shared between Turdus naumanni andTurdus eunomus as well as among individuals of Turdus
ruficollis and Turdus atrogularis is indicating allelic sharing
due to common ancestors or challenging common pathogens, as this event
is frequent in numerous avian species such as owls, ardeid birds,
penguins and passerines [63-65].
Generally, abundant variation in genetic material in a species is an
indicator of the capacity to adapt to numerous environmental changes by
that species. Rapidly evolving environmental pathogens would cause MHC
genes to exhibit enlarged genetic diversity in species [66, 67].
Collectively, in our study, we find elevated genetic diversity among
functional sequences and significant divergence, whereas pseudogene has
low genetic variation and limited divergence. Similar results also have
been described in other passerine species, including common yellow
throat [68], great reed warbler and the great tit [69]. The
allelic variation described in our study could be due to increased
immunological defense against the internal pathogen since these are
highly unlikely to adapt to novel, infrequent variant [15].
Recombination has been considered an important mechanism that influences
allelic diversity and driving evolution of the MHC gene [70, 71] We
only find one potential recombination event in Tuna-MHCI*PFA06 at
two recombinant breakpoints at position 148 and 253 identified with
recombination detection program. Similarly, single recombination was
significant in Tueu-MHCI*PFA07. Recombination pattern was also
restricted two out of seven tests; hence our finding indicate
recombination is unlikely to have any significant influence on tests for
PSs. Though we could not find any substantial recombination among other
alleles, qualitatively our result suggests a role for recombination
during the evolution of MHC class I in our species studied. Our finding
is consistent with, that micro-recombination is frequently observed in
MHC genes [57]. Further study of recombinant function in the future
will contribute to a detailed understanding of its role in the evolution
of the MHC gene.
Positive selection is the maintainer of alleles having the advantageous
mutation that maintain fitness of an individual. In our study, the
classical test of selection Tajima’s D, Fu & Li’s D* and Fu & Li’s F*
showed no deviation from neutral selection or balance selection.
Considering the level of variation, conventional methods used to find
selection are not influential [72]. As sites positively selected
are
likely to accumulate more non-synonymous than synonymous substitutions,
influencing amino acid variation to result in functional modifications
in proteins [73]. Our study revealed differential expression of
selection pattern in functional sequences on regions related with PBR
and non-PBR of the MHC class I gene. Codons involved in peptide binding
region revealed more non-synonymous substitution than synonymous
(dN/dS=1.99) in Turdus
atrogularis as compared to non- peptide binding region
(dN/dS=0.884), pattern was consistent
among all species tested, which might be enlightened that stronger
selection pressure from intracellular pathogens than extracellular
pathogens [74]. Evidence of positive selection at PBR of MHC has
been reported in the house sparrow (PBR
dN/dS = 1.55 vs non-PBR
dN/dS = 0.51) [75] and golden
pheasant (PBR dN/dS = 1.45 vs non-PBR
dN/dS =0.91) [76]. Of the 12 codons
in total among species tested exhibit positive selection with Likelihood
methods using PAML, 9,29, 64 and 88 match homologues codons found
positively selected in other passerine
species.
It should be noted that the pooling of all alleles across loci will
mostly reduce selection detection tests, so the outcomes might be
conservative, but will be less prone to false positives [77, 78].
Therefore, attention should be given while inferencing about the
detected diversity in MHC and the possible effects of selection on
individual loci. Our results suggested that α2 domain of MHC class I
exon 3 of all species are under positive selection pressure. Pronounced
positive selection at antigen-binding sites permits a species or
population to present a larger repertoire of peptides (antigens), thus
increase the defensive ability against parasitic and pathogenic
infections.
Finally, phylogenetic clustering of MHC class I data set of sampled
species when pooled with other passerine species produces a contrasting
pattern. In general, the MHC class I sequence of Turdidae family
clustered together with sequences from congeneric species. We found
increased sequences similarities between same species rather than within
species (trans specific likenesses), is usually described with trans
species polymorphism (TSP), which occurs due to alleles passage from
ancestral to the decedent via partial arrangement of lineages [79].
Although trans specific similarities can be described with convergent
evolution due to the results of similar environmental selective
pressure. Studies indicate that TSP is a primary mechanism responsible
for clustering of alleles at avian MHC class I [80].