Table 3.

Comparing sequence polymorphism and strength of selection between MHC classes using both peptide binding domains

Species	MHC region	nalleles	Pi	dN/dS	dN-dS	SE	Z-score	P
Human	HLA-I exon 2 (α1)	83	0.087	3.160	0.203	0.081	0.728	0.233
	HLA-I exon 3 (α2)	83	0.065	2.447	0.123	0.076	0.052	0.479
	HLA-I exon 2 & 3 (α1α2)	83	0.076	2.809	0.161	0.054	1.565	0.059
	HLA-II DRA exon 2 (α1)	1	0.000	NA	0.000	0.000
	HLA-II DRB1 exon 2 (β1)	29	0.062	1.922	0.117	0.086
	HLA-II DR- A & B1 exon 2 (α1β1)	29	0.033	1.750	0.050	0.046
Common buzzard	MHC-I exon 2 (α1)	10	0.094	3.461	0.219	0.075	-1.147	0.126
	MHC-I exon 3 (α2)	8	0.065	2.635	0.121	0.059	-2.042	0.021
	MHC-I exon 2 & 3 (α1α2)	10	0.079	3.089	0.166	0.042	-0.143	0.443
	MHC-IIA exon 2 (α1)	3	0.008	NA	0.035	0.036
	MHC-IIB exon 2 (β1)	8	0.105	9.156	0.367	0.105
	MHC-II A & B exon 2 (α1β1)	8	0.056	7.481	0.175	0.047

To demonstrate how a false conclusion can be reached when comparing only one set of peptide binding domains between MHC classes, we collected and analyzed well-curated alleles from humans and full alleles of common buzzards. Human class I HLA-A, B, C, and class II HLA-DRB1 alleles from the European population classified as "common" in the Common and Well-Documented allele catalog 3.0.0 [70] as well as HLA-DRA*01:01 from the monomorphic DRA locus were downloaded from IPD-IMGT/HLA Database version 3.51 [16] (Additional file Table S5). Common buzzard alleles with full peptide-binding grooves were retrieved from genomic data (GenBank accession #s: OL311287, OL311290, OL311292, OL311294, OL311304, OL311305, OP490259, OQ414190-OQ414202, OQ428163-OQ428174). All analyses for the strength of selection (dN and dS) were conducted in MEGA X [71] using human peptide binding residues from [72] and the Nei-Gojobori model [73] with 1000 bootstraps for variance estimation. The Z-score was calculated with the formula (dN-dS_MHC-I—dN-dS_MHC-II)/$\surd$(SE²_MHC-I + SE²_MHC-II). We compared MHC-I exon 2 and 3 separately with MHC-IIB exon 2, and then combined against concatenated MHC-IIA & B exon 2 sequences. We tested if MHC-I had stronger selection than MHC-II in humans and if MHC-IIB had stronger selection than MHC-I in common buzzards. One-sided p-values were generated from the Z-scores. P-values show how comparing single exons between the classes does not represent comparisons across the full binding domains (italicized for emphasis; MHC-I: α1α2, MHC-II: α1β1). n_alleles: number of alleles, Pi: nucleotide diversity, SE: standard error of the mean. Significant p-values are highlighted in bold