Sequence analysis and haplotype coding
For each chloroplast region, the forward and reverse sequences were
manually edited and combined into a single sequence using Geneious 5.5.6
(Kearse et al., 2012); and these merged
reads were submitted to NCBI GenBank to make them publicly available.
Following merging, three alignments were generated: 1) Species-pairwise
alignments of C. lanatus accessions with those of C.
mucosospermus, C. amarus , and C. colocynthis for the chloroplast
region trn T-L; 2) the same species-pairwise alignments for the
region ndh F-rpl 32; and 3) a combined alignment of all
species, containing both trn T-L and ndh F-rpl 32
regions, yielding a matrix of 1,611 aligned nucleotides. In the combined
alignment, for the purpose of constructing coherent and parsimonious
haplotypes, repeats and indels were re-coded as single bp polymorphisms.
In the trn T-L region: 1) a microsatellite ACATA at position 366
was coded as A (repeat presence) or a single gap ”-” (absence); 2) A
TATT indel at position 405 was coded as a T (presence) or a single gap
(absence); and 3) Another TTTATA microsatellite at position 423 was
coded as T (presence) or a single gap (absence). In thendh F-rpl 32 region: 1) a poly AT, usually six to eight
units (position 1149), was just replaced by a single gap for 6*(AT), A
for 7*(AT), and T for 8*(AT); and 6) A TGATT microsatellite at position
1198 was coded as a T (presence) or a single gap (absence).