4.2 Phylogenetic relationships of Notholirion
The phylogenetic analysis using both nuclear gene fragments and plastid fragments confirmed the monophyletic nature of Notholirion , which is consistent with previous studies(Huang et al., 2018; Kim & Kim, 2018; J. Li et al., 2022). Within the genus Notholirion , the species N. thomsonianum was found to be a sister taxon to the branches of N. macrophyllum and N. bulbuliferum , representing the earliest divergence event as supported by the BEAST divergence time estimates. Notably, some populations showed inconsistencies in their nuclear genetic makeup. The cpDNA tree revealed that all populations of the three Notholirion species formed distinct monophyletic branches. However, in the ITS tree, two populations of N. macrophyllum (MNC and MZT) clustered together with most populations of N. bulbuliferum , while N. bulbuliferum exhibited a population (BDY) clustered into a single branch with the remaining two populations of N. macrophyllum .
The phenomenon of inconsistency between nuclear gene trees and chloroplast gene trees is very common in plant phylogenetic analyses and is typically attributed to convergent evolution, incomplete lineage sorting, hybridization, or gene introgression(Y. Gao, Harris, Li, & Gao, 2020; Y. D. Gao, Harris, & He, 2015; Y. D. Gao, Harris, Zhou, & He, 2013; Huang et al., 2018; X. Liu, Wang, Shao, Ye, & Zhang, 2016). The selected gene fragments in this study are mostly evolutionarily conserved and less influenced by the environment, reducing the likelihood of convergent evolution. Therefore, the main factors considered are incomplete lineage sorting and hybridization. And in many studies on the phylogenetic relationships of plants in the family Liliaceae, both incomplete lineage sorting and hybridization have been mentioned as factors contributing to conflicts in the phylogenetic relationships(Y. Gao et al., 2020; Y. D. Gao et al., 2015; Y. D. Gao et al., 2013; Huang et al., 2018). Studies have shown that incomplete lineage sorting is more likely to occur when species rapidly expand or have large population sizes(Maddison, 1997). Due to the stochastic nature of the coalescent process, incomplete lineage sorting can lead to random patterns of gene trees among taxa, which may result in inconsistencies between different gene trees(Buckley, Cordeiro, Marshall, & Simon, 2006). Furthermore, lineage sorting in plants generally takes hundreds of millions of years(X. Liu et al., 2016; Pelser et al., 2010). Both N. macrophyllum and N. bulbuliferum are relatively young species that diverged in the last few million years, which is far from reaching the timescale of hundreds of millions of years. Therefore, we speculate that incomplete lineage sorting may be one of the reasons for the observed incongruence between nuclear and chloroplast genomes in Notholirion . We know that hybridization plays a significant role in biodiversity and speciation by either reinforcing or disrupting reproductive barriers(Les et al., 2015). Due to the maternal inheritance of organelle genes, gene flow resulting from biparental hybridization is usually manifested only in nuclear genes, leading to inconsistencies between the phylogenetic trees constructed based on nuclear genes and organellar genes(X. Liu et al., 2016). Hybridization is widely observed in the family Liliaceae, as evidenced by numerous reports in wild Lilium species andITS frequent use in horticulture for plant breeding purposes(Y. Gao et al., 2020; Y. D. Gao et al., 2015; Y. D. Gao et al., 2013; Pelkonen, Niittyvuopio, Pirttilä, Laine, & Hohtola, 2007). Furthermore, some researchs have suggested that hybridization may be a common phenomenon in Liliaceae plants(Douglas et al., 2011; Muratović, Robin, Bogunić, Šoljan, & Siljak-Yakovlev, 2010). In conclusion, we speculate that incomplete lineage sorting and hybridization may be the primary factors leading to the conflicting phylogenetic trees observed between the nuclear genes and chloroplast genes in Notholirion .