Abstract
Eukaryotic genomes can acquire bacterial DNA via lateral gene transfer (LGT).1 A prominent source of LGT is Wolbachia,2 a widespread endosymbiont of arthropods and nematodes that is transmitted maternally through female germline cells.3,4 The DNA transfer from the Wolbachia endosymbiont wAna to Drosophila ananassae is extensive5-7 and has been localized to chromosome 4, contributing to chromosome expansion in this lineage.6 As has happened frequently with claims of bacteria-to-eukaryote LGT, the contribution of wAna transfers to the expanded size of D. ananassae chromosome 4 has been specifically contested8 owing to an assembly where Wolbachia sequences were classified as contaminants and removed.9 Here, long-read sequencing with DNA from a Wolbachia-cured line enabled assembly of 4.9 Mbp of nuclear Wolbachia transfers (nuwts) in D. ananassae and a 24-kbp nuclear mitochondrial transfer. The nuwts are <8,000 years old in at least two locations in chromosome 4 with at least one whole-genome integration followed by rapid extensive duplication of most of the genome with regions that have up to 10 copies. The genes in nuwts are accumulating small indels and mobile element insertions. Among the highly duplicated genes are cifA and cifB, two genes associated with Wolbachia-mediated Drosophila cytoplasmic incompatibility. The wAna strain that was the source of nuwts was subsequently replaced by a different wAna endosymbiont. Direct RNA Nanopore sequencing of Wolbachia-cured lines identified nuwt transcripts, including spliced transcripts, but functionality, if any, remains elusive.