4.1 Genetic Diversity and Structure of Notholirion
Biogenetic diversity is the result of the long-term evolution of species and serves as an essential foundation for organisms to adapt to their environment and evolve. It is also a key factor in maintaining the stability and function of ecosystems. The higher the genetic diversity of a species, the better it can adapt to environmental changes. Conversely, when genetic diversity is low, a species is often vulnerable to environmental changes and may lose ITS distribution or even become extinct(Soltis & Soltis, 1991; Vranckx, Jacquemyn, Muys, & Honnay, 2012). Previous studies have reported that the average genetic diversity of angiosperms, as inferred from plastid gene data, is 0.67(Petit et al., 2005). Our analysis of both plastid and ITSdata sets revealed that the overall genetic diversity (HT value) of the Notholirion was significantly higher than this average (cpDNA: HT = 0.931; ITS : HT = 0.796), indicating a remarkably high level of genetic diversity of this group (Table 1). Through a series of analyses of the evolutionary aspects of the origin of the Notholirion , we suggested that two factors may have contributed the most to the higher genetic diversity ofNotholirion : (1) Notholirion is the earliest divergent clade of the tribe Lilieae, during the late Oligocene. During ITSlong evolutionary history, it experienced numerous geological and climatic events such as orogenic movements, monsoonal climatic events, and drought events, which led to the accumulation of a large amount of genetic variation to adapt to environmental changes; (2)Notholirion grows at high altitudes with an average altitude of about 3000 meters, mainly in the Himalayan-Hengduan Mountains. The area is influenced by frequent mountain-building movements in the Miocene, resulting in an intricate and complex topography with interlocking rows of high mountain valleys that form the ”sky islands”. The unique ”sky island” environment fragments habitat, leading to alpine plant populations that form relatively isolated and special ecosystems subject to different environmental selection and ecological niche differentiation. This increases the opportunity for genetic drift within populations, which results in a wealth of genetic variation. Thus, the plant taxa distributed in this environment often exhibit a high degree of genetic diversity and unique genotypic assemblages, adapted to the challenges of these extreme environments. Similar results have been found in other plants distributed in the HHM region, such asChamaesium (cpDNA:HT = 0.794; ITS : HT = 0.718), Notopterygium incisum (cpDNA:HT = 0.939; ITS : HT= 0.725) and Allium section Sikkimensia (cpDNA:HT = 0.974; ITS : HT = 0.988)(Shahzad et al., 2017; C. Xie et al., 2019; H. Y. Zheng et al., 2021).
In addition, AMOVA analyses were performed based on both ITS and cpDNA datasets. The results showed (Table2) that if Notholirionis considered as a whole, the genetic variation among the 31 populations was large (ITS : 91.45%; cpDNA: 98.85%) and within the populations was relatively small (ITS : 8.55%; cpDNA: 1.15%), which may be related to the following reasons: (1) field surveys revealed that although Notholirion species are distributed in four provinces of southwestern China, they have very specific habitat requirements. Apart from altitude requirements, this genus also necessitates specific heights of companion plants. For instance,N. macrophyllum and N. thomsonianum prefer to grow in scrub or tall grasses at around 3000m above sea level, where the plants are roughly the same height as their own height. Hence, almost all populations collected in the field have an extremely limited local distribution, and communication between flora and populations is rare(J. Li et al., 2022); (2) Through our field investigations, we also have observed that Notholirion species have very limited seed dispersal ability, and their seed production cycle is quite lengthy. It takes approximately 5 years from seedling to flowering, and after flowering, the primary bulbs of the roots wither, making it impossible for seed production to occur in the following year. Mature seed production is only attainable through newborn seeds and asexual reproduction of small bulbs, which take an additional 5 years to reach the mature stage. This situation with seeds also makes interflora and interpopulation communication very difficult; (3) The genetic differentiation of many plant taxa in the HHM region has been observed to be primarily influenced by the complex geological activities and climate changes that occur in the area. For example, orogenic movements can lead to fragmented habitats, which can reduce gene flow among populations and consequently increase genetic variation among them. Therefore, it is likely that the high genetic differentiation observed among Notholirion flora is due to the significant fluctuations in the external environment in the HHM region and ITS own specificity. Similar results were found in Chamaesium ,Allium section Sikkimensia and Polygonatum (Xia et al., 2022; C. Xie et al., 2019; H. Y. Zheng et al., 2021). However, AMOVA results of N. bulbuliferum based on ITS data showed a lower percentage of genetic variation among ITS populations (74.86%) compared to the results based on cpDNA data (93.73). The reason for this inconsistent genetic structure is hypothesized to be as follows: according to the ITS data, multiple haplotypes were detected in 27 N. bulbuliferum populations, of which N1 was a haplotype common to 15 populations, thus reducing genetic variation among populations.
Both cpDNA and ITS datasets showed high levels of haplotype diversity in the haplotype analysis (cpDNA: Hd = 0.801; ITS : Hd = 0.788). There were no shared haplotypes among the threeNotholirion species, and most haplotypes were restricted to a single population or between several geographically adjacent populations, which is similar to the results found inChamaesium (H. Y. Zheng et al., 2021). We speculate thatNotholirion , as a high-altitude distributed taxon, has different populations that remain isolated and lack communication with each other during ice ages and interglacial periods. This situation may be due to several environmental factors and the species ITS elf, including the inability of high-altitude plants to adapt to low-altitude environments, the fragmentation of habitats in the HHM region, and the formation of unique habitats such as sky islands that make communication between populations difficult, and the long seed production cycle and difficulty of dissemination of Notholirion species themselves that limit communication between populations.