A staged evidence chain identifies a recent horizontal transfer of a HET-domain gene into a Fusarium tricinctum strain from a lineage related to the Fusarium oxysporum species complex

Abstract

Genes encoding the heterokaryon incompatibility (HET) domain form fast-evolving, highly paralogous families, which makes horizontal gene transfer (HGT) among them hard to separate from duplication, loss and annotation artefacts. We describe a staged evidence chain for testing a single HGT candidate and apply it to the strongest signal in the HET-domain view of a 109-genome similarity graph of 8,815 HET-domain (PF06985) proteins. Each stage is a separate test with a decision rule fixed before it was run: (i) a gene tree with ultrafast-bootstrap support; (ii) a species-tree check that donor and recipient lie in different clades; (iii) a genome-wide identity background and a contamination screen of the recipient assembly; (iv) a sister-species control that removes genes conserved by vertical descent; and (v) a tblastn search of whole genome assemblies, so that absence is tested in the DNA rather than in the gene annotation. We identify a recent horizontal transfer of a HET-domain gene into one F. tricinctum strain (MPI-SDFR-AT-0068) from a lineage related to the F. oxysporum species complex (FOSC). The F. tricinctum copy is 98.4% identical to its best FOSC hit (local protein identity), against a genome-wide median of 83%, and its synonymous divergence from the FOSC copies (median dS 0.11) is lower than that of 99.6% of F. tricinctum genes from their best F. oxysporum hits and of 91% of F. tricinctum genes from their best hits in the sister species, which argues strongly against vertical descent, hidden paralogy and shared ancestral alleles. Protein gene trees group it with the FOSC copies (UFBoot 96-98), and a nucleotide tree of the gene region places it as sister to all sampled FOSC copies (UFBoot 98). F. tricinctum and the F. oxysporum strains belong to different species complexes (species-tree patristic distance 0.075, about 22 times the distance between F. oxysporum strains). The gene is absent from the assembly of F. tricinctum's sister species, F. avenaceum (best locus 49% identity), and from 14 of 15 further F. avenaceum genomes, from 23 of the 26 assemblies of Fusarium species outside the FOSC and the F. tricinctum complex, and no other sequenced genome of the recipient strain's own lineage carries it; close copies (at least 90% identity) occur only in 7 of 9 FOSC strains and in one metagenome-assembled Fusarium genome from neither complex. In F. tricinctum the gene sits in an approximately 6-kb strain-specific island on a putative accessory scaffold with F. oxysporum-affine gene content, whereas in the FOSC it occupies one conserved, subtelomeric locus of a core chromosome. Assembly contamination was excluded, and single long reads span the gene and its flanks. The donor lineage remains unresolved, and formal reconciliation is still needed. The evidence chain is cheap and uses only public assemblies; each stage targets a specific alternative explanation, and synonymous divergence against the genome background proved the most decisive test.