Major Histocompatibility Complex (MHC) genes are one of the most polymorphic gene groups known in vertebrates. MHC genes also exhibit allelic variants that are shared among taxa, referred to as trans-specific polymorphism (TSP). The role that selection plays in maintaining such high diversity within species, as well as TSP, is an ongoing discussion in biology. In this study we used deep-sequencing techniques to characterize MHC class IIb gene diversity in three sympatric species of darters. We found at least 5 copies of the MHC gene in darters, with 126 genetic variants encoding 122 unique amino acid sequences. We identified four supertypes based on the binding properties of proteins encoded by the sequences. Although each species had a unique pool of variants, many variants were shared between species pairs and across all three species. Phylogenetic analysis showed that the variants did not group together monophyletically based on species identity or on supertype. An expanded phylogenetic analysis showed that some darter alleles grouped together with alleles from other percid fishes. Our findings show that TSP occurs in darters, which suggests that balancing selection is acting at the genotype level. Supertypes, however, are most likely evolving convergently, as evidenced by the fact that alleles do not form monophyletic groups based on supertype. Our research demonstrates that selection may be acting differently on MHC genes at the genotype and supertype levels, selecting for the maintenance of high genotypic diversity while driving the convergent evolution of similar MHC phenotypes across different species.

See Million and Lively 2021 for detailed methods.

DNA was extracted from fin clips of three species of darter (genus Etheostoma). Two rounds of PCR amplified a fragment of MHC class IIb exon 2 using custom primers. Sequencing was performed using Illumina MiSeq. After demultiplexing and quality control, genetic variants were identified using an Excel macro for genotyping MHC in fish. Verified genetic variants were assigned titles and stored in the attached .fasta file (DarterMHC.fasta).

DNA sequences were translated into amino acid sequences. Positively selected sites (PSS) were identified using the software MEGA. PSS codons are highlighted in the attached .csv file (PSSTN2.csv).

Amino acid physicochemical properties (Z1-Z5) were obtained using a published database. The properties were read into a matrix of positively selected codon sites in the sequences (AAproperties.csv).

MHCs were assigned to one of four functional supertypes via a discriminant analysis of principal components (DAPC) using the software adegenet in R (supertypeassignments.csv).