Chao-Wen She

The Lycoris aurea (L’Hér.) Herb. complex—valued for its medicinal properties and horticultural applications—is characterized by extensive intraspecific dysploidy. However, a comprehensive molecular cytogenetic framework elucidating intraspecific karyotypic differentiation and the underlying evolutionary mechanisms has yet to be established. By integrating fluorochrome banding with 5S and 45S rDNA fluorescence in situ hybridization (FISH), we performed a comparative molecular cytogenetic karyotyping of 20 natural populations and synthesized these results with previously published cytogenetic data to conduct a rigorous cytogeographic analysis of this species complex. Three distinct cytotypes were identified: cytotype A (2n = 14), cytotype B (2n = 15), and cytotype C (2n = 16). Both multivariate karyomorphometric analyses and comparison of DAPI banding and rDNA FISH patterns revealed high inter-population karyotypic uniformity in cytotype A, yet pronounced intra-cytotypic karyotypic polymorphism in cytotype C. Integrated cytogenetic evidence supports a working hypothesis wherein cytotype C likely represents the ancestral karyotype, while cytotypes A and B appear to have originated from cytotype C through two and one Robertsonian fusion events, respectively—thereby implying two independent, parallel evolutionary pathways: C → A and C → B. The evolutionary biogeographic framework for this species complex—derived from hypothesis-generating integrative inference—proposes that Quaternary climate oscillations, topographic barriers, and ecological niche divergence may have acted synergistically to shape the current parapatric distribution pattern of the three cytotypes.

The Lycoris aurea (L’Hér.) Herb. complex—valued for its medicinal properties and horticultural applications—is characterized by extensive intraspecific dysploidy. However, a comprehensive molecular cytogenetic framework elucidating intraspecific karyotypic differentiation and the underlying evolutionary mechanisms has yet to be established. By integrating fluorochrome banding with 5S and 45S rDNA fluorescence in situ hybridization (FISH), we performed a comparative molecular cytogenetic karyotyping of 20 natural populations spanning 13 provinces in China and synthesized these results with previously published cytogenetic data to conduct a rigorous cytogeographic assessment of this species. Three distinct cytotypes were identified: cytotype A (2n = 14 = 8m + 6t/st), cytotype B (2n = 15 = 7m + 8t/st), and cytotype C (2n = 16 = 6m + 10t/st). Both multivariate karyomorphometric analyses and comparison of DAPI banding and rDNA FISH patterns revealed minimal inter-population karyotypic variation in cytotype A but pronounced intra-cytotypic karyotypic polymorphism in cytotype C. Integrated cytogenetic evidence supports Robertsonian translocation as the primary mechanism driving dysploidy in the L. aurea complex: cytotype C is inferred as the ancestral karyotype, from which cytotype A arose through double Robertsonian fusions and cytotype B through a single such fusion, establishing two parallel, evolutionarily independent lineages. The parapatric partitioning of the three cytotypes is interpreted as the outcome of synergistic effects among Quaternary climatic oscillations, topographic barriers, and ecological niche divergence.

The complexity of the classification of the genus Lilium necessitates further molecular cytogenetic research to clarify the karyotype differentiation and chromosome evolution among species, validate the existing classification, and help resolve ongoing classification disputes. In this study, the chromosomes of eleven species of Lilium, encompassing main medicinal and edible lilies in China as well as seven species endemic to southwest and central China, were characterized using fluorescence in situ hybridization (FISH) with 5S and 35S rDNA oligonucleotide probes. Quantitative molecular cytogenetic karyotypes were established using the dataset of chromosome measurements and FISH signals. Karyotype asymmetry indices were calculated to assess their karyotype asymmetry and infer their karyological relationships. Karyotype structures could be differentiated through the scatter plot of M_CA versus CV_CL. FISH results revealed 1–2 5S rDNA loci and 2–6 35S rDNA loci across the examined species, with most 35S loci associated with secondary constrictions. Comparative analysis of rDNA patterns, combined with PCoA based on x, 2n, TCL, M_CA, CV_CL and CV_CI provided insights into the evolutionary relationships among these species. Our findings provide cytogenetic support for the taxonomic validity of subset. Leucolirion 6a in the most recent revised classification of Lilium.

To characterize the chromosomes of the four species of Polygonatum Miller, 1754, used in traditional Chinese medicine, P. cyrtonema Hua, 1892, P. kingianum Collett et Hemsley, 1890, P. odoratum (Miller, 1768) Druce, 1906, and P. sibiricum Redouté, 1811, and have an insight into the karyotype variation of the genus Polygonatum, fluorescence in situ hybridization (FISH) with 5S and 45S rDNA oligonucleotide probes was applied to analyze the karyotypes of 9 populations of the four species. Detailed molecular cytogenetic karyotypes of the 9 populations were established for the first time using the dataset of chromosome measurements and FISH signals of 5S and 45S rDNA. Four karyotype asymmetry indices, CV_CI, CV_CL, M_CA and Stebbins’ category, were measured to elucidate the asymmetry of the karyotypes and karyological relationships among species. Comparison of their karyotypes revealed distinct variations in the karyotypic parameters and rDNA patterns among and within species. The basic chromosome numbers detected were x = 9, 11 and 13 for P. cyrtonema, x = 15 for P. kingianum, x = 10 and 11 for P. odoratum, and x = 12 for P. sibiricum. The original basic chromosome numbers of the four species were inferred on the basis of the data of this study and previous reports. All the 9 karyotypes were of moderate asymmetry and composed of metacentric, submetacentric and subtelocentric chromosomes or consisted of two of these types of chromosomes. Seven populations have one locus of 5S rDNA and two loci of 45S rDNA, and two populations added one 5S or 45S locus. The karyological relationships among the four species revealed by comparison of rDNA patterns and PCoA based on x, 2n, TCL, CV_CI, M_CA and CV_CL were basically accordant with the phylogenetic relationships revealed by molecular phylogenetic studies. The mechanisms of both intra- and inter-specific dysploidy in Polygonatum were discussed based on the data of this study and literature.

To have an insight into the karyotype variation of eight Cucurbitaceae crops including Cucumis sativus Linnaeus, 1753, Cucumis melo Linnaeus, 1753, Citrullus lanatus (Thunberg, 1794) Matsumura et Nakai, 1916, Benincasa hispida (Thunberg, 1784) Cogniaux, 1881, Momordica charantia Linnaeus, 1753, Luffa cylindrica (Linnaeus, 1753) Roemer, 1846, Lagenaria siceraria var. hispida (Thunberg, 1783) Hara, 1948 and Cucurbita moschata Duchesne ex Poiret, 1819, well morphologically differentiated mitotic metaphase chromosomes were prepared using the enzymatic maceration and flame-drying method, and the chromosomal distribution of heterochromatin and 18S-5.8S-26S rRNA genes (45S rDNA) was investigated using sequential combined PI and DAPI (CPD) staining and fluorescence in situ hybridization (FISH) with 45S rDNA probe. Detailed karyotypes were established using the dataset of chromosome measurements, fluorochrome bands and rDNA FISH signals. Four karyotype asymmetry indices, CV_CI, CV_CL, M_CA and Stebbins’ category, were measured to elucidate the karyological relationships among species. All the species studied had symmetrical karyotypes composed of metacentric and submetacentric or only metacentric chromosomes, but their karyotype structure can be discriminated by the scatter plot of M_CA vs. CV_CL. The karyological relationships among these species revealed by PCoA based on x, 2n, TCL, M_CA, CV_CL and CV_CI was basically in agreement with the phylogenetic relationships revealed by DNA sequences. CPD staining revealed all 45S rDNA sites in all species, (peri)centromeric GC-rich heterochromatin in C. sativus, C. melo, C. lanatus, M. charantia and L. cylindrica, terminal GC-rich heterochromatin in C. sativus. DAPI counterstaining after FISH revealed pericentromeric DAPI⁺ heterochromatin in C. moschata. rDNA FISH detected two 45S loci in five species and five 45S loci in three species. Among these 45S loci, most were located at the terminals of chromosome arms, and a few in the proximal regions. In C. sativus, individual chromosomes can be precisely distinguished by the CPD band and 45S rDNA signal patterns, providing an easy method for chromosome identification of cucumber. The genome differentiation among these species was discussed in terms of genome size, heterochromatin, 45S rDNA site, and karyotype asymmetry based on the data of this study and previous reports.

To extend our knowledge on karyotype variation of the genus Vigna Savi, 1824, the chromosomal organization of rRNA genes and fluorochrome banding patterns of five wild Vigna species were studied. Sequential combined PI (propidium iodide) and DAPI (4',6-diamidino-2-phenylindole) (CPD) staining and fluorescence in situ hybridization (FISH) with 5S and 45S rDNA probes were used to analyze the karyotypes of V. luteola (Jacquin, 1771) Bentham, 1959, V. vexillata (Linnaeus, 1753) A. Richard, 1845, V. minima (Roxburgh, 1832) Ohwi & H. Ohashi, 1969, V. trilobata (Linnaeus, 1753) Verdcourt, 1968, and V. caracalla (Linnaeus, 1753) Verdcourt,1970. For further phylogenetic analysis, genomic in situ hybridization (GISH) with the genomic DNA of V. umbellata (Thunberg, 1794) Ohwi & H.Ohashi, 1969 onto the chromosomes of five wild Vigna species was also performed. Detailed karyotypes were established for the first time using chromosome measurements, fluorochrome bands, and rDNA-FISH signals. All species had chromosome number 2n = 2x = 22, and symmetrical karyotypes that composed of only metacentric or metacentric and submetacentric chromosomes. CPD staining revealed all 45S rDNA sites in the five species analyzed, (peri)centromeric GC-rich heterochromatin in V. luteola, V. trilobata and V. caracalla, interstitial GC-rich and pericentromeric AT-rich heterochromatin in V. caracalla. rDNA-FISH revealed two 5S loci in V. caracalla and one 5S locus in the other four species; one 45S locus in V. luteola and V. caracalla, two 45S loci in V. vexillata and V. trilobata, and five 45S loci in V. minima. The karyotypes of the studied species could be clearly distinguished by the karyotypic parameters, and the patterns of the fluorochrome bands and the rDNA sites, which revealed high interspecific variation among the five species. The V. umbellata genomic DNA probe produced weak signals in all proximal regions of V. luteola and all (peri)centromeric regions of V. trilobata. The combined data demonstrate that distinct genome differentiation has occurred among the five species during evolution. The phylogenetic relationships between the five wild species and related cultivated species of Vigna are discussed based on our present and previous molecular cytogenetic data.

Chromosomes of four Miscanthus (Andersson, 1855) species including M. sinensis (Andersson, 1855), M. floridulus (Schumann & Lauterb, 1901), M. sacchariflorus (Hackel, 1882) and M. lutarioriparius (Chen & Renvoize, 2005) were analyzed using sequentially combined PI and DAPI (CPD) staining and fluorescence in situ hybridization (FISH) with 45S rDNA probe. To elucidate the phylogenetic relationship among the four Miscanthus species, the homology of repetitive sequences among the four species was analyzed by comparative genomic in situ hybridization (cGISH). Subsequently four Miscanthus species were clustered based on the internal transcribed spacer (ITS) of 45S rDNA. Molecular cytogenetic karyotypes of the four Miscanthus species were established for the first time using chromosome measurements, fluorochrome bands and 45S rDNA FISH signals, which will provide a cytogenetic tool for the identification of these four species. All the four have the karyotype formula of Miscanthus species, which is 2n = 2x = 38 = 34m(2SAT) + 4sm, and one pair of 45S rDNA sites. The latter were shown as strong red bands by CPD staining. A non-rDNA CPD band emerged in M. floridulus and some blue DAPI bands appeared in M. sinensis and M. floridulus. The hybridization signals of M. floridulus genomic DNA to the chromosomes of M. sinensis and M. lutarioriparius genomic DNA to the chromosomes of M. sacchariflorus were stronger and more evenly distributed than other combinations. Molecular phylogenetic trees showed that M. sinensis and M. floridulus were closest relatives, and M. sacchariflorus and M. lutarioriparius were also closely related. These findings were consistent with the phylogenetic relationships inferred from the cGISH patterns.

The two cultivated Canavalia (Adanson, 1763) species, Canavalia gladiata (N. J. von Jacquin, 1788) A. P. de Candolle, 1825 and Canavalia ensiformis (Linnaeus, 1753) A. P. de Candolle, 1825 are closely related based on morphological and molecular phylogenetic data. However, the similarities and differences in genome organization between them have not been evaluated at molecular cytogenetic level. Here, detailed karyotypes of both species were constructed using combined PI and DAPI (CPD) staining, rDNA-FISH and self-genomic in situ hybridization (sGISH). For further comparison, comparative genomic in situ hybridization (cGISH) and sequence analysis of 5S rDNA were applied. Their chromosomes were accurately identified by sGISH and rDNA-FISH signals. Both species had the karyotype formula 2n = 22 = 18m + 4m-SAT, but the karyotype of C. ensiformis was shorter and more asymmetric than that of C. gladiata. They displayed similar CPD bands at all 45S rDNA sites and centromeres. C. gladiata had ten centromeric 5S rDNA loci and two SC (secondary constriction)-associated 45S rDNA loci. C. ensiformis had nine centromeric and one interstitial 5S loci, two SC-associated and one proximal 45S loci. Their sGISH signal patterns displayed both basic similarities and distinct differences. Reciprocal cGISH generated prominent signals in all pericentromeric regions and 45S sites. There was lower level of sequence identity of the non-transcribed spacer between their 5S rDNA repeats. These data confirmed the evolutionary closeness between C. gladiata and C. ensiformis and demonstrated obvious differentiation between their genomes, and supported the opinion that C. ensiformis is more advanced in evolution than C. gladiata. The two cultivated Canavalia (Adanson, 1763) species, Canavalia gladiata (N. J. von Jacquin, 1788) A. P. de Candolle, 1825 and Canavalia ensiformis (Linnaeus, 1753) A. P. de Candolle, 1825 are closely related based on morphological and molecular phylogenetic data. However, the similarities and differences in genome organization between them have not been evaluated at molecular cytogenetic level. Here, detailed karyotypes of both species were constructed using combined PI and DAPI (CPD) staining, rDNA-FISH and self-genomic in situ hybridization (sGISH). For further comparison, comparative genomic in situ hybridization (cGISH) and sequence analysis of 5S rDNA were applied. Their chromosomes were accurately identified by sGISH and rDNA-FISH signals. Both species had the karyotype formula 2n = 22 = 18m + 4m-SAT, but the karyotype of C. ensiformis was shorter and more asymmetric than that of C. gladiata. They displayed similar CPD bands at all 45S rDNA sites and centromeres. C. gladiata had ten centromeric 5S rDNA loci and two SC (secondary constriction)-associated 45S rDNA loci. C. ensiformis had nine centromeric and one interstitial 5S loci, two SC-associated and one proximal 45S loci. Their sGISH signal patterns displayed both basic similarities and distinct differences. Reciprocal cGISH generated prominent signals in all pericentromeric regions and 45S sites. There was lower level of sequence identity of the non-transcribed spacer between their 5S rDNA repeats. These data confirmed the evolutionary closeness between C. gladiata and C. ensiformis and demonstrated obvious differentiation between their genomes, and supported the opinion that C. ensiformis is more advanced in evolution than C. gladiata.

The genomic organisation of the seven cultivated Vigna species, V. unguiculata, V. subterranea, V. angularis, V. umbellata, V. radiata, V. mungo and V. aconitifolia, was determined using sequential combined PI and DAPI (CPD) staining and dual‐colour fluorescence in situ hybridisation (FISH) with 5S and 45S rDNA probes. For phylogenetic analyses, comparative genomic in situ hybridisation (cGISH) onto somatic chromosomes and sequence analysis of the internal transcribed spacer (ITS) of 45S rDNA were used. Quantitative karyotypes were established using chromosome measurements, fluorochrome bands and rDNA FISH signals. All species had symmetrical karyotypes composed of only metacentric or metacentric and submetacentric chromosomes. Distinct heterochromatin differentiation was revealed by CPD staining and DAPI counterstaining after FISH. The rDNA sites among all species differed in their number, location and size. cGISH of V. umbellata genomic DNA to the chromosomes of all species produced strong signals in all centromeric regions of V. umbellata and V. angularis, weak signals in all pericentromeric regions of V. aconitifolia, and CPD‐banded proximal regions of V. mungo var. mungo. Molecular phylogenetic trees showed that V. angularis and V. umbellata were the closest relatives, and V. mungo and V. aconitifolia were relatively closely related; these species formed a group that was separated from another group comprising V. radiata, V. unguiculata ssp. sesquipedalis and V. subterranea. This result was consistent with the phylogenetic relationships inferred from the heterochromatin and cGISH patterns; thus, fluorochrome banding and cGISH are efficient tools for the phylogenetic analysis of Vigna species.