Among our analyzed 31 species, 28 species had 30 chromosomes (Fig. S1 and S2) except that I. hederifolia (Fig. S2 f1-f3), I. coccinea (Fig. S2 g1-g3) and I. pes-tigridis (Fig. S2 l1-l3) had 28 chromosomes. The probes of 7 − 1 (green)/7 − 2 (red) (Fig. 1a1-m1; Fig. S1a1-m1; Fig. 2a1-n1; Fig. S2a1-n1; Fig. 3a1-d1) and 15 − 1 (red)/15 − 2 (green) (Fig. 1a2-m2; Fig. S1a2-m2; Fig. 2a2-n2; Fig. S2a2-n2; Fig. 3a2-d2) hybridized to only a pair of chromosomes in all analyzed species but with different signal distribution patterns. The sites number of 45S (red) and 5S rDNA (green) showed a great difference among species (Fig. 1a3-m3; Fig. S1a3-m3; Fig. 2a3-n3; Fig. S2a3-n3; Fig. 3a3-d3). The detailed results were described as follows in the order of smaller clades in phylogenetic tree constructed by Eserman et al. [10]. For higher resolution, the chromosomes with signals in Fig. S1 and Fig. S2 were cut out and arranged at Fig. 1 and Fig. 2, respectively.
The Batatas clade includes I. setosa and all species in Ipomoea series Batatas. In I. setosa, the chromosomes had 7 − 1 (green)/7 − 2 (red) (Fig. 1a1) and 15 − 1 (red)/15 − 2 (green) (Fig. 1a2) signals at the distal regions, the middle regions remaining unpainted. In I. splendor-sylvae, the chromosomes also had 7 − 1 (green)/7 − 2 (red) signals at the distal regions (Fig. 1b1), whereas had 15 − 1 (red)/15 − 2 (green) signals along their entire lengths (Fig. 1b2). In other species in the series Batatas, the hybridization signals of 7 − 1 (green)/7 − 2 (red) (Fig. 1c1-m1) and 15 − 1 (red)/15 − 2 (green) (Fig. 1c2-m2) covered the whole lengths of the chromosomes and no difference was observed among species. FISH results with 5S and 45S rDNA as probes showed that all species in the Batatas clade had 5S rDNA (green) signals on two chromosomes, located intercalary near to the centromeres (Fig. 1a3-m3). However, these species showed a great difference of 45S rDNA (red) sites number (Fig. 1a3-m3). Four sites of 45S rDNA were observed in I. setosa (Fig. 1a3), I. splendor-sylvae (Fig. 1b3) and I. littoralis (Fig. 1c3), six in I. trifida (Fig. 1d3) and I. lacunose (Fig. 1e3), eight in I. triloba (Fig. 1f3), I. cordatotriloba (Fig. 1g3) and I. tiliacea (Fig. 1h3), ten in I. grandifolia (Fig. 1i3), I. cynanchifolia (Fig. 1j3) and I. tenuissima (Fig. 1k3), nine in I. leucantha (Fig. 1l3), and fourteen in I. ramosissima (Fig. 1m3). The 45S rDNA hybridization signals occurred at the distal end of the short chromosome arm in all species. Based on 45S rDNA distribution pattern, the homologous pairs of chromosomes with 45S rDNA signals could be identified and distinguished from each other in all species except for I. leucantha. In I. leucantha (Fig. 1l3), two extremely weak signals among nine 45S rDNA signals localized to morphologically different chromosomes and one chromosome was obviously longer than the other. The intensity of two 5S rDNA signals was also different in I. leucantha. This confirmed that I. leucantha was of hybrid origin as suggested before by Austin [34].
Three species of I. fistulosa, I. saintronanensis and I. platensis in the Murucoides clade had identical distribution patterns of 7 − 1 (green)/7 − 2 (red) (Fig. 2a1-c1) and 15 − 1 (red)/15 − 2 (green) (Fig. 2a2-c2) to I. setosa (Fig. 1a1, a2) in the Batatas clade with signals only at the distal regions of chromosomes, the middle regions of chromosomes remaining unpainted. In I. fistulosa (Fig. 2a3) and I. saintronanensis (Fig. 2b3), there were four chromosomes with 45S rDNA signals and two chromosomes with 5S rDNA signals, which was also identical to I. setosa in the Batatas clade (Fig. 1a3). Whereas in I. platensis (Fig. 2c3), six chromosomes had 45S rDNA signals and four chromosomes had 5S rDNA signals.
The two species in Pes-caprae clade showed different distribution patterns of 7 − 1/7 − 2 and 15 − 1/15 − 2. In I. gracilis, 7 − 1 (green)/7 − 2 (red) (Fig. 2d1) and 15 − 1 (red)/15 − 2 (green) (Fig. 2d2) signals only hybridized to the distal part of the chromosomes, the middle regions remaining unpainted, which was same to I. setosa (Fig. 1a1, a2) in the Batatas clade and all species (Fig. 2a1-c1, a2-c2) in the Murucoides clade. While in I. amnicola, 7 − 1 (green)/7 − 2 (red) (Fig. 2e1) and 15 − 1 (red)/15 − 2 (green) (Fig. 2e2) signals nearly covered whole chromosomes except 7 − 2 interrupted by the centromere regions without FISH signals, and minor 7 − 2 signals localized at same chromosome arm with 7 − 1 signals (Fig. 2e1). In addition, the two species differed from each other in 45S rDNA sites number. In I. gracilis, four chromosomes had 45S rDNA signals (Fig. 2d3). While in I. amnicola, six chromosomes had 45S rDNA signals (Fig. 2e3).
The analyzed four species in Quamoclit clade also showed different painting patterns. In I. hederifolia (Fig. 2f1, f2) and I. coccinea (Fig. 2g1, g2) with 28 chromosomes, 7 − 1 (green)/7 − 2 (red) and 15 − 1 (red)/15 − 2 (green) colocalized at same chromosome arm, respectively. Whereas in I. nil (Fig. 2h1, h2) and I. hederacea (Fig. 2i1, i2), the FISH signals of 7 − 1 (green)/7 − 2 (red) and 15 − 1 (red)/15 − 2 (green) uniformly covered whole chromosomes without unambiguous signal gaps. Moreover, both I. nil and I. hederacea had 45S rDNA signals on seven pairs of chromosomes (Fig. 2h3, i3). The only difference between I. nil and I. hederacea was that they had different 5S rDNA sites number (Fig. 2h3, i3). In I. nil, two chromosomes had 5S rDNA signals (Fig. 2h3). While in I. hederacea, four chromosomes had 5S rDNA signals and two 5S rDNA signals were located syntenic to the 45S rDNA signals (Fig. 2i3). In I. hederifolia, there were twelve chromosomes with 45S rDNA signals and six chromosomes with 5S rDNA signals, and two 5S rDNA signals were located syntenic to the 45S rDNA signals (Fig. 2f3). In I. coccinea, ten chromosomes had 45S rDNA signals and four chromosomes had 5S rDNA signals (Fig. 2g3).
I. cairica (Fig. 2j1, j2) and I. aquatica (Fig. 2k1, k2) in Cairica clade showed same distribution patterns of 7 − 1 (green)/7 − 2 (red) and 15 − 1 (red)/15 − 2 (green) signals to I. amnicola (Fig. 2e1, e2) in Pes-caprae clade, i.e., 7 − 1/7 − 2 and 15 − 1/15 − 2 signals nearly covered whole chromosomes except 7 − 2 interrupted by the centromere regions without FISH signals, and minor 7 − 2 signals located at same chromosome arm with 7 − 1. The difference between I. cairica and I. aquatica was that they had different 45S rDNA sites number. In I. cairica, six chromosomes had 45S rDNA signals (Fig. 2j3). While in I. aquatica, four chromosomes had 45S rDNA signals (Fig. 2k3).
The Argyreiinae has been divided into two smaller clades of Pes-tigridis and Obscura. In I. pes-tigridis with 28 chromosomes, 7 − 1 (green)/7 − 2 (red) (Fig. 2l1) and 15 − 1 (red)/15 − 2 (green) (Fig. 2l2) colocalized at same chromosome arm, respectively, which was identical to I. hederifolia (Fig. 2f1, f2) and I. coccinea (Fig. 2g1, g2) with 28 chromosomes in Quamoclit clade. Whereas, I. eriocarpa in the Pes-tigridis had same distribution patterns of 7 − 1 (green)/7 − 2 (red) (Fig. 2m1) and 15 − 1 (red)/15 − 2 (green) (Fig. 2m2) to I. obscura (Fig. 2n1, n2) in the Obscura clade, i.e., 7 − 1 and the major 7 − 2 signals only ccupied one arm of same chromosome and 7 − 2 interrupted by the centromere regions without FISH signals, and 15 − 1/15 − 2 hybridized to distal regions of the chromosomes, the middle regions remaining unpainted. The three species had different rDNA sites number. In I. pes-tigridis (Fig. 2l3) and I. eriocarpa (Fig. 2m3), six chromosomes had 45S rDNA signals. In I. eriocarpa (Fig. 2m3) and I. obscura (Fig. 2n3), four chromosomes had 5S rDNA signals.
The other four species, I. alba, I. albivenia, I. digitata and I. heterophylla, were also analyzed in this study. I. alba had similar painting pattern of 7 − 1 (green)/7 − 2 (red) (Fig. 3a1) and 15 − 1 (red)/15 − 2 (green) (Fig. 3a2) to I. nil (Fig. 2h1, h2) and I. hederacea (Fig. 2i1, i2). The difference among three species was that they had different 5S rDNA sites number. In I. alba, six chromosomes had 5S rDNA signals and four 5S rDNA signals were located syntenic to the 45S rDNA signals (Fig. 3a3). The I. albivenia and I. digitata showed same distribution patterns of 7 − 1 (green)/7 − 2 (red) (Fig. 3b1, c1) and 15 − 1 (red)/15 − 2 (green) (Fig. 3b2, c2) to the species in Cairica clade (Fig. 2j1, j2, k1, k2). Interestingly, the odd numbers of 45S rDNA arrays were observed in I. albivenia and I. digitata. In I. albivenia, three chromosomes had 45S rDNA signals (Fig. 3b3). While in I. digitata, seven chromosomes had 45S rDNA signals (Fig. 3c3). The I. heterophylla had identical distribution patterns of 7 − 1 (green)/7 − 2 (red) (Fig. 3d1) and 15 − 1 (red)/15 − 2 (green) (Fig. 3d2) to I. eriocarpa (Fig. 2m1, m2) and I. obscura (Fig. 2n1, n2) within the Argyreiinae. In I. heterophylla, only two chromosomes had 45S rDNA signals (Fig. 3d3). Thus, these four species were accurately assigned to different clades of Ipomoeeae tribe based on painting pattern of 7 − 1/7 − 2 and 15 − 1/15 − 2 probes.