Fig 1.
Flow-cytometry analyses of relative nuclear DNA contents in the Ipomoea littoralis Blume accession.
Suspensions of intact nuclei were prepared from young leaf tissues, stained using DAPI and analyzed using a FACSCalibur™ flow cytometer. The diploid I. tenuissima was used as an external reference standard and its G1peak positioned at channel 200. The three sweetpotato clones were used as external hexaploid references.
Fig 2.
Illustration of two distinguishable forms of variations and four phylogenetic partitions among cDNA variants of some COSII genes in sweetpotato.
The two alignments A-1 and B in zoom-out variation-highlighting views were two concatenations (_C1 and _C2) of 16 and 4 alignment blocks, respectively, which included three cDNA variants of the 16 and 4 COSII genes from the sweetpotato SC1149-19 or Tanzania clones (combination of IB_ with_C1 or _C2), and their homologs from I. tenuissima (IT_), I. trifida (Itr_ and Itrk_ for two lines), I. littoralis (IL_) and I. nil (Inil_) in matching column-orders. Black bars highlight all sequence disagreements at various positions against the consensus in individual alignment blocks. The green and maroon triangles indicate sites of two distinguishable forms of variations in among cDNA variants of some COSII genes in sweetpotato. The three cDNA variants each of the 4 and 16 sweetpotato COSII genes carried differential non-random clusters of variations that are either well conserved in one of the reference homologs from I. trifida, I. tenuissima and I. littoralis (as in A), or unshared (_Hp4_ in B) at the common interspecific variable sites, and could be thus distinguished into four species-specified phylogenetic partitions, which were marked with _Itr/Itrk_, _Itn_, _Ils_ and _Hp4_, and shaded with blue, purple, red and pink colors, respectively. The A-2 and A-3 alignments are zoom-in views of the first four concatenated blocks, and the first 132-bp (consensus length) of the fourth block in the first concatenation, respectively. The A-3 alignment also illustrates detailed phylogenetic variation partitions of three cDNA variants of the sweetpotato COSII gene (IB_ and CIP_ for isotigs from the SC1149-19 and Tanzania clones, respectively) with corresponding cDNA homologs from the three relatives.
Fig 3.
Phylogenetic partitions of the cDNA variants of 811 COSII genes in sweetpotato.
Alignments of equal-consensus-length homologous regions from cDNA variants of 811 COSII genes from two sweetpotato clones (IB_ and CIP_ for those isotigs from the SC1149-19 and Tanzania) and from homologous COSII cDNAs from two I. trifida lines (Itr_ and Itrk_), one I. littoralis (IL_) and one I. tenuissima (IT_) accessions, one I. nil line (Inil_), and tomato (Solyc_) were used for the phylogenetic partitions of cDNA variants of the sweetpotato COSII genes. The Circos plot (A) summarized numbers and types of partitions or group that could be distinguished among cDNA variants of the COSII genes in sweetpotato, and total numbers of different COSII cDNAs that were identified in each of the four partitions and the Mix/Ind group. In 505 of the 811 alignments, single or multiple cDNA variants of a COSII gene from the two sweetpotato clones could be distinguished into only one (the orange-colored arcs) of the four variation partitions, the Itr/Itrk/(blue-colored), Itn/ (purple-colored) or Ils/(red-colored) partitions by their differentially conserved non-random clusters of interspecific variations with the corresponding reference homologs from the two I. trifida lines, I. tenuissima and I. littoralis accessions, respectively, and the Hp4/ (pink-colored) by their unshared non-random clusters of variations at the interspecific variable sites; or were placed into the Mix/Ind group (gray-colored) as their variations at the interspecific variable sites were of mixed (Mix/) or indistinguishable (Ind/) relatedness with those in the corresponding three reference homologs. In the rest of the 811 alignments, the cDNA variants of a COSII gene from the two sweetpotato clones were of two (brown-colored arcs) or three (green-colored arcs) types of interspecific variations, which could be differentiated into various combinations of partitions and the Mix/Ind group. The sub-alignments shown in B to G were two examples each for one, two and three partitions of cDNA variants of different sweetpotato COSII genes. In the alignments D, E and F, the gray-shaded variations between the two COSII cDNA variants from the same or different sweetpotato clones exemplified the intraspecific variations (alleles) between cDNA variants of the COSII gene in the same partition.
Fig 4.
Illustration of the Hp4/ partition of cDNA variants from four sweetpotato COSII genes.
In the zoom-out variation-highlighting views of four sub-alignments, an Hp4/ partition of the cDNA variant(s) from one or both of the two sweetpotato lines, which displayed distinct clusters of variations at interspecific variable sites as compared to those in all the homologs from the other four Ipomoea species and tomato, could be the only identifiable partition of cDNAs for a COSII gene (A), one of the two partitions of cDNA variants in combination with the Itn/ (B) or Itr/Itrk/ partition (C) for two COSII genes, and one of the three partitions of cDNA variants in combination with the Ils/ and Itr/Itrk/ partition for another COSII gene (D). All the prefixes marking the species source of cDNAs and color-coding of the partitions were the same as in previous figures.
Fig 5.
Illustration of four levels of variation differentiation of COSII-marker genomic amplicon variants among 729 sweetpotato COSII genes.
The panels show variation-highlighting views of alignments of, and haplotype trees for the four sets of COSII-marker amplicon variants (_a1 to _a5) from four COSII genes in two sweetpotato clones (IB-SC1149- and IB-LSP-), as compared to their respective reference cDNA (_amp) and genomic amplicons (_G-amp) from corresponding homologous COSII-marker regions from I. tenuissima (IT_). The color-coded highlighting bars represent sequence variation(s) against consensus at the positions in each alignment. The black lines (gaps) connecting two horizontal gray bars for representing reference cDNA amplicons in each alignment delineate introns in respective COSII-marker genomic amplicons. In the panel B, C and D, distinct cluster(s) of variations that were exclusively shared by some amplicon variants in each set, and those that were shared with or different from those in the reference COSII-marker amplicons, distinguished the three sets of COSII-marker amplicon variants into two or three phylogenetic variation partitions, which are separated by wider gaps in the alignments. Those variations between partitions of amplicon variants were most likely homoeoalleles, and labeled by green triangles. Additional variations between the amplicons variants in the same partition were most likely true alleles, and were marked by brown triangles.
Fig 6.
Two hybridization networks constructed from two concatenations of 16 (A) and 4 (B) individual alignments of partial cDNA homologs of 16 and 4 COSII genes from five Ipomoea species. The first concatenation (_C1) comprised three taxa (IB_Itr/Itrk_C1, IB_Itn_C1 and IB_Ils_C1) corresponding to 16 expressed COSII homoeolog triplets in the Itr/Itrk/, Itn/ and Ils/ partitions, and five taxa of corresponding COSII cDNA homologs from the two I. trifida lines (Itr_C1 and Itrk_C1), I. tenuissima (It_C1), I. littoralis (IL_C1), and I. nil (Inil_C1 as an outgroup). The second concatenation comprised three taxa (IB_Itr/Itrk_C2, IB_Hp4_C2 and IB_Ils_C2) corresponding to 4 expressed COSII homoeolog triplets in the Itr/Itrk/, Hp4/ and Ils/ partitions, and 5 taxa of corresponding COSII cDNA homologs from the two I. trifida lines (Itr_C2 and Itrk_C2), I. tenuissima (IT_C2) I. littoralis (IL_C2) and I. nil (Inil_C2, as an outgroup). The two hybridization networks were the HybridizationNetwork transformations of two split networks that were resolved from the LogDet distances between the 8 taxa each in the two concatenated alignment matrices by the SplitDecomposition method. The LSFit was the least squares fit between the pairwise distances of splits in the two hybridization networks and the taxa in the alignment matrices. The internal nodes that explicitly predict ancestors in the two networks were highlighted with red dots, and number labeled except the earliest one in the networks. The blue-colored splits indicated four detected hybridization events (H1 to H4).
Fig 7.
The most-parsimonious species tree embodying the two hybridization networks.
The gray-shaded outline represents the most parsimonious species tree that could be reconciled with the two hybridization networks. The two hybridization networks minus their outgroups were fitted in the gray-shaded species tree trunks, having matching partition-color-coded splits, numbered nodes and hybridization events (H1 to H4 in blue) as those in the two redrawn color-coded hybridization networks minus their outgroups at the bottom left and right for comparison. The three pairs of sweetpotato COSII lineages fitted in the species tree and their corresponding taxa in the two hybridization networks were labeled as br, bt, bl and b4 for trifida-like, tenuissima-like, littoralis-like and sweetpotato-specific COSII lineages, respectively, in black (first network) and green (second network) color circles. The reference COSII lineages in the three sweetpotato relatives and their corresponding taxa in the two hybridization networks were labeled as tr or tk, t, and L for those from the two I. trifida lines, I. tenuissima, and I. littoralis, respectively, with subscripted number 1 or 2 indicating the first and second network in respective partition-color circles. The tree sections predicting ancestral species (diploid Ip-N1, -N2, -N3 and -N7, tetraploid Ip-Tn, -Lt and -Hb) to sweetpotato were delimited by dashed lines. The H1 and H4 hybridizations were modeled as intraspecific introgression (IS) in I. trifida. The br (IB_Itr/Itrk_C2) and b4 (IB_Hp4_C2) lineages (light-blue and pink colored, respectively) in the second network were modeled as outcomes of incomplete lineage sorting (ILS) in the tree. The dotted light-purple lines mark an additional predicted COSII lineage (btr) in sweetpotato and the trifida and tenuissima-like one (Ltr) in I. littoralis, which would have descended from an ancestral linage branched at the node N1/1’. The three color-gradient blocks underneath the subgenome designations were used to emphasize that only parts of these diploid subgenomes were differentiated as represented by the differentiated multi-gene COSII lineages.