Genome Sequencing of up to 6,000-Year-Old Citrullus Seeds Reveals Use of a Bitter-Fleshed Species Prior to Watermelon Domestication

Abstract

Iconographic evidence from Egypt suggests that watermelon pulp was consumed there as a dessert by 4,360 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 6,000- and 3,300-year-old seeds from Libya and Sudan, and from worldwide herbarium collections made between 1824 and 2019, and analyzed these data together with resequenced genomes from important germplasm collections for a total of 131 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-type" watermelon (Citrullus mucosospermus) rather than domesticated pulp-use watermelon (Citrullus lanatus ssp. vulgaris); (2) the 6,000-year-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 (3) both ancient genomes showed admixture from C. mucosospermus, C. lanatus ssp. cordophanus, C. lanatus ssp. vulgaris, and even South African Citrullus amarus, and evident introgression between the Libyan seed (UMB-6) and populations of C. lanatus. An unexpected new insight is that Citrullus appears to have initially been collected or cultivated for its seeds, not its flesh, consistent with seed damage patterns induced by human teeth in the oldest Libyan material.

Keywords: C-14-dated African seeds; Neolithic settlements in Libya; ancient DNA; domestication; population genomics; watermelon.

Fig. 1.

Geographic origin of the ancient DNA samples (stars) and historical Citrullus accessions used in this study. We also show the geography of two key fruit traits: flesh color (red flesh: bold border; non-red flesh: thin border) and fruit bitterness (bitter: triangle; non-bitter: circle), based on diagnostic SNPs in the bitterness regulator ClBT and Lycopene cyclase (LYCB) genes (see Results and Discussion). The inset shows computed-tomography scans of two UMB-6 seeds from Uan Muhuggiag, Libya, radiocarbon dated to 6,182–6,001 calibrated years before present (from Wolcott et al. 2021), which based on morphology had been identified as Citrullus lanatus. The lower seed shows a breakage patterns characteristic of modern watermelon seeds cracked by human teeth.

Fig. 2.

Phylogeny and principal component analyses (PCAs) of 123 Citrullus samples. (A) Multispecies coalescence phylogeny for 123 accessions of Citrullus including 47 historical herbarium-derived genomes (collected between 1824 and 2019), ancient Libyan and Sudanese seeds (UMB-6, AS-814), and Schweinfurth’s leaf from the vicinity of Luxor dating from 1883 (see supplementary table S2, Supplementary Material online, for vouchers). The phylogeny was inferred from 1,431 non-overlapping nuclear 10 Kb blocks derived from 11 chromosomes (Methods). Numbers above branches represent posterior probability. The first column to the right of the phylogeny shows the age of the samples, the second admixture at K = 4 (see supplementary fig. S5, Supplementary Material online, for K = 2–8). (B) PCAs based on estimated nuclear genotype likelihoods of all samples. (C) A zoom in of the PCA displayed on A, focusing on a cluster containing C. lanatus ssp. vulgaris, C. lanatus ssp. cordophanus, C. mucosospermus, and the aDNA Libyan and Sudanese samples.

Fig. 3.

Relationships of the 6,000-year-old Libyan seed with historical and modern accessions of Citrullus as inferred by phylogenetic analyses and gene-tree and species-tree space analysis. (A) Maximum likelihood (ML) and Multispecies coalescent (MSC) species-tree space. Each minute dot represents an individual ML tree derived from non-overlapping nuclear 10 Kb alignments, with its color and shape referring to which chromosome block it is derived from. Larger symbols represent MSC trees inferred in ASTRAL from chromosomal ML gene trees (B) ASTRAL MSC phylogenies per chromosome, showing the placement of the ancient Libyan seed UMB-6. The larger symbols above trees represent the corresponding ASTRAL MSC phylogenies inferred for the chromosomal ML gene trees.

Fig. 4.

ABBA-BABA test of introgression among the watermelon and its close relatives including the ancient DNA samples. (A) Tests of introgression among C. lanatus ssp. vulgaris, C. lanatus ssp. cordophanus, C. amarus, and C. mucosospermus based on all informative sites. (B) Test of introgression among the 6,000-year-old Libyan seed (UMB-6), the 3,000-year-old Sudanese seed (AS-814), and C. lanatus ssp. vulgaris, C. lanatus ssp. cordophanus, and C. amarus based on all informative sites. (C) Number of ABBA-BABA sites indicating gene flow between the Libyan seed and C. amarus. (D) Number of ABBA-BABA sites indicating gene flow between the Libyan seed (UMB-6), C. lanatus ssp. vulgaris, and C. lanatus ssp. cordophanus. (E) Number of ABBA-BABA sites indicating gene flow between the Sudanese seed and C. amarus. The plots presented in C, D, and E are inferred from all sites or non-polymorphic sites only (D-values, Z-scores, and sample IDs used in all ABBA-BABA tests are provided on supplementary table S3, Supplementary Material online).

Fig. 5.

The fruits of the 6,000-year-old Libyan seeds were bitter and greenish-white fleshed. (A) Position and coverage of the bitterness regulator ClBT gene in UMB-6. (B) Position and coverage of the lycopene cyclase gene (LYCB) in UMB-6. See also supplementary fig. S6, Supplementary Material online.