Nucleotide variations, intra- and interspecific diversity
The length of the amplified trn T-trn L region withinC. lanatus ranged from 951-954 bp. No parsimony-informative site was found within C. lanatus , but 3 indels were found at positions 242, 295, and 296. The amplified ndh F-rpl 32 region ranged from 650-652 bp in the species, also with no parsimony-informative site, though 5 indels were found at positions 970, 1028, 1143, 1178, and 1198 (Tables S1). The combined length of the two cpDNA regions was found equal to 1601-1605 bp and included 1 SNP (position 1399) and 1 microsatellite (position 366); but no polymorphisms were parsimony-informative. In total, the sampled accessions of this species comprise 12 distinct haplotypes, among which 10 were singletons, with an overall haplotype diversity of 0.5656 (Table 2).
Sequence lengths within C. mucosospermus were similar, with the combined length of the two regions spanning by 1601-1604 bp. One SNP (non-parsimony informative) was identified in thendh F-rpl 32 region (position 1397), as well as two indels in trn T-trn L region (positions 242 and 296). Of the 5 haplotypes found among the sampled accessions of this species, three were singletons; and overall haplotype diversity is 0.5333.
The combined sequence length in C. amarus ranged between 1602-1604 bp (950-953 bp in trn T-trn L and 651-653 bp inndh F-rpl 32) and contained ten polymorphic sites. Of those, 4 indels were observed in trn T-L (positions 295, 296, 297, 405) and 1 in ndh F-rpl 32 (positions 1198). Four SNPs were found at positions 918, 1149, 1397, and 1526; and there is a microsatellite at position 1149. C. amarus was characterized by eight haplotypes, among which six were private; and overall haplotype diversity is 0.81.
C. colocynthis was characterized by a combined sequence length of 1599-1605 bp (948-954 bp for trn T-trn L and 650-653 bp forndh F-rpl 32) that features 10 SNPs (positions 406, 455, 487, 882, 918, 949, 1111, 1286, 1397, and 1526) and 3 microsatellites (positions 366, 423, 1149). In addition, there were 11 indels (positions 199, 242, 295, 296, 297, 972, 1179, 1180, 1200, 1262, and 1530), 7 of which were parsimony informative (6 within trn T-trn L and 1 within ndh F-rpl 32). The collection of this species contains 16 haplotypes, all private, and has an overall haplotype diversity of 0.96.
Based on the 29 polymorphic sites detected within the two cpDNA regions, 38 haplotypes were detected among the sampled accessions (Table 3). The most ancient haplotype (H1), according to TCS analysis, is exclusive to the cultivated species C. lanatus and C. mucosospermus . Of the 26 singleton haplotypes detected, 13 (50%) were found withinC. colocynthis , indicating recent haplotype divergence in that species (Fig. 1).