Genome sequencing of up to 6,000-yr-old Citrullus seeds

Iconographic evidence from Egypt suggests that watermelon pulp was consumed there as a dessert by 4360 BP. Earlier archaeobotanical evidence comes from seeds from Neolithic settlements in Libya, but whether these were watermelons with sweet pulp or other forms is unknown. We generated genome sequences from 6000 and 3370 years old seeds from Libya and Sudan, respectively, and geographically widespread herbarium collections made between 1824 and 2019, and analyzed these data together with resequenced genomes from germplasm collections for a total of 131 worldwide accessions. Phylogenomic and population-genomic analyses reveal that 1. much of the nuclear genome of both ancient seeds is traceable to West African seed use "egusi" watermelon (C. mucosospermus) rather than domesticated pulp-use watermelon (C. lanatus subsp. vulgaris); ii. the 6,000 years old watermelon likely had bitter pulp and greenish-white flesh as today found in C. mucosospermus, given alleles in the bitterness regulators ClBT and in the red color marker LYCB; and iii. gene flow traceable to the ancient seeds occurred between the African landraces of domesticated watermelon, the East African sweet-pulped progenitor of domesticated watermelon, egusi watermelons, and even South African C. amarus. An unexpected new insight is that Citrullus appears to have initially been collected or cultivated for its seeds, consistent with seed damage patterns induced by human teeth in the oldest Libyan material as well as taxonomic identity and identified fruit traits.