Dual-color GISH evidence supports a chromosome breakage hypothesis for aneuploid karyotype formation in a newly discovered cytotype of Drosera rotundifolia (Droseraceae)

A new aneuploid cytotype (2n=22), morphologically resembling Drosera rotundifolia (2n=20), was found in an area coexisting with D. spatulata (2n=40) in Kyushu, Japan. This is the first report of aneuploidy in section Drosera in Japan, and all previously reported chromosome numbers in this section, including those in natural hybrids between D. rotundifolia and D. spatulata, have conformed strictly to polyploidy based on a basic number of x=10. The aneuploid cytotype had a karyotype formula of 18m+4s, consisting of 18 medium-sized chromosomes (m-chromosomes) and four small-sized chromosomes (s-chromosomes), whereas D. rotundifolia and D. spatulata are known to consist solely of m-chromosomes (20m) and s-chromosomes (40s), respectively. During meiosis, the cytotype consistently exhibited 11 bivalents (11II), with nine pairs of m-chromosomes and two pairs of s-chromosomes. In comparison, D. spatulata from the same area always displayed 20II. These cytogenetic features led us to propose two hypotheses for the origin of this aneuploid karyotype: (1) chromosome breakage of one pair of m-chromosomes in D. rotundifolia, and (2) chromosomal insertion via hybrid formation involving D. spatulata. Genome size estimation by flow cytometry showed that the 2C value of the cytotype (2,318 Mbp) was nearly identical to that of D. rotundifolia (2,333 Mbp) and more than twice that of D. spatulata (1,131 Mbp). Chemical profiling using high-performance liquid chromatography demonstrated that the major phenolic fraction pattern of the aneuploid cytotype completely matched that of D. rotundifolia. Genomic in situ hybridization (GISH) analysis further showed that the four s-chromosomes did not hybridize with the D. spatulata genome probe, indicating that they were not derived through interspecific chromosomal insertion. This study provides GISH-supported cytogenetic evidence for an aneuploid karyotype in D. rotundifolia and suggests a possible pathway for the stabilization of structurally altered chromosomes during early karyotype evolution.