Analysis of phylogeny and genetic diversity of Paphiopedilum species from China

【Objective】This study aimed to analyze the phylogenetic relationship between native Paphiopedilum germplasms in China， clarify genetic distances and coefficients of genetic similarity， providing theoretical basis for formula-ting strategies of sustainable conservation of Paphiopedilum resources.【Method】Using 31 Paphiopedilum germplasms from China and two exotic Paphiopedilum germplasms conserved ex situ as the core experimental materials， and one Phragmipedium germplasm as the outgroup， iPBS and ISSR primers were selected to amplify the genomic DNA of the 34 germplasms. A binary raw data matrix was generated by manually scoring the amplification electrophoretograms， and multidimensional scaling analysis was performed using NTSYS-pc 2.1， with genetic diversity indexes calculated using PopGen 1.32.【Result】The combined iPBS and ISSR molecular marker-assisted phylogenetic analysis results revealed that when the coefficient of genetic similarity was 0.37， 33 Paphiopedilum germplasms were divided into five groups （I-V）. The Group I， Group II， Group III， and Group IV were corresponded to subg. Parvisepalum， subg. Brachypetalum， subg. Paphiopedilum sect. Paphiopedilum， subg. Paphiopedilum sect. Barbata in classical taxonomy， and Group V was a collection of subg. Paphiopedilum sect. Pardalopetalum and subg. Paphiopedilum sect. Cochlopetalum. The genetic diversity at species level of Paphiopedilum germplasms iPBS molecular marker: effective number of alleles （Ne） was 1.5969， expected heterozygosity （He） was 0.3492， Shannon diversity information index （I） was 0.5210； ISSR molecular marker: Ne was 1.5515， He was 0.3340， and I was 0.5068 was obviously higher than that at group level （iPBS molecular marker: Ne was 1.4274， He was 0.2730，and I was 0.4292； ISSR molecular marker: Ne was 1.3733， He was 0.2492， and I was 0.4027）. This revealed that Paphiopedilum plants showed marked genetic differentiation at species level， forming multiple distinct genetic groups， as subg. Parvisepalum， subg. Paphiopedilum sect. Paphiopedilum， and subg. Paphiopedilum sect. Barbata exhibiting the top three genetic diversity among groups. Genetic differentiation was found between groups of Paphiopedilum， but the overall genetic similarity remained high. Analysis results based on iPBS molecular markers showed that subg. Paphiopedilum sect. Paphiopedilum and subg. Paphiopedilum sect. Barbata shared the closest relationship， while subg. Brachypetalum and subg. Paphiopedilum sect. Barbata shared the farthest relationship； analysis results based on ISSR molecular markers showed that the collection of Paphiopedilum sect. Pardalopetalum and subg. Paphiopedilum sect. Cochlopetalum had the closest relationship with subg. Paphiopedilum sect. Barbata， while subg. Parvisepalum and subg. Brachypetalum shared the farthest relationship.【Conclusion】Paphiopedilum germplasms in China are primarily distributed across five groups: subg. Parvisepalum， subg. Brachypetalum， subg. Paphiopedilum sect. Paphiopedilum， subg. Paphiopedilum sect. Barbata， and subg. Paphiopedilum sect. Pardalopetalum， among which the three groups （subg. Parvisepalum， subg. Paphiopedilum sect. Paphiopedilum， and subg. Paphiopedilum sect. Barbata） show high genetic diversity level. Obvious genetic differentiation is found between Paphiopedilum groups， implying that the groups have undergone long-term genetic isolation or possess distinct regional adaptations.