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].