Genetic diversity, gene flow, genetic differentiation, population genetic structure, and their influencing factors are the focus of conservation biology research13. In particular, studies of rare and endangered species are more relevant and can lay a theoretical foundation for putting forward appropriate suggestions for effective conservation. Biological characteristics, the environment, human interference, historical events, and natural disasters can all affect the genetic diversity, genetic structure, and gene flow in a species. Limited gene flow may be the most important factor in the formation of the population genetic structure in insect-borne plants and species with patchy distribution. Another factor is human disturbance, such as habitat fragmentation or degradation, and overharvesting. CM is one of the few orchids with important ornamental and medicinal value in north China. It is mainly distributed in north and northeast China [32], but CM is not found in the southwestern region, which is the diversity center of the genus Cypripedium. Thus, CM also has important ecological and scientific value in studying the adaptation and evolution of the genus. Due to overcollection, deceptive pollination, and other disturbances, the number of CM individuals has declined dramatically in recent years. The genetic diversity, genetic structure, and gene flow in CM population need to be urgently studied to put forward appropriate conservation measures. However, most current studies of CM only involve pollination, seed germination, and rhizosphere fungi in China [33]. In this study, we obtained 41,154 SNPs from 99 CM samples using Super-GBS, and the average sequencing depth of the samples was 23.2×. There are studies that have shown that the sequencing depth exceeds 5×, then the subsequent series of analysis results are accurate and reliable [34, 35].
Based on our experimental data, we found some evidence of genetic diversity in CM, several major indices of genetic diversity indicated that 99 CM individuals had a low level of genetic diversity (Table 1). Several factors influence the level of genetic diversity of plants, such as the reproductive characteristics of plants themselves, gene flow, human interference, habitat fragmentation, and chance events. First, although CM reproduces sexually and asexually, its deceptive pollination leads to a low seed setting rate. In addition, its seeds have no endosperm and the germination rate is very low. Moreover, asexual reproduction in CM mainly relies on rhizomes, and it is difficult the population to extend to new habitats far away by the asexual reproductive mode, thus reducing the level of genetic diversity of CM. One of the greatest threats to the loss of genetic diversity is a small number of individuals in scattered populations, and this situation is common to rare and endangered species18. Second, the limited gene flow reduces the level of genetic diversity, and the pollinators of CM are mainly bumblebees [36], whose feeding and flying range is about 4 km [37]. Thus, the insect-mediated pollen flow range is limited. Moreover, because of gravity settling and other plants, seed dispersal is shorter. Therefore, restricted gene flow between populations is one of the important reasons for the low genetic diversity of CM. Third, human overharvesting and occasional events (e.g., the Greater Khingan Range forest fire in 1987 that burned an area of 17,000 sq. km) can also lead to the reduction or disappearance of small populations, thereby reducing genetic diversity. There is a unified theory in conservation biology: populations with a large number of individuals tend to be stable and survive longer, whereas those with few individuals may shrink and become extinct, as was observed in this species. For example, five CM individuals were present in Baicaowa Forest Park, Luanping County, north China, in 2013, but our resource survey found that this population disappeared in 2021. Therefore, although some small populations are not included in nature reserves (e.g., the SLS population, consisting of only three individuals, located near Jinshui Lake), they still need special protection. Otherwise, these small populations may become extinct in a few years.
Each population was analyzed separately, and the BHS population of CM had the highest level of genetic diversity among all populations. This population is located in the Baihuashan National Nature Reserve, which was established in 1985. The number of conserved CM individuals was higher than the other five north China populations. In addition, based on the natural global distribution of CM and its preference for cold climate, the BHS population may be the population that CM spread from north to south and settled down in North China in history, and developed a local CM diversity center. In contrast, the XAS population is located at the Xing’an Temple scenic spot in the center of Yichun city, which is surrounded by the Yichun River and several national roads, forming a geographical barrier. Therefore, the low level of genetic diversity of this population was mainly caused by human interference and the gene flow barrier. Unexpectedly, although SLS population was the smallest population with only three individuals, it was not the lowest in genetic diversity. Considering that this population was located only 21.3 km away from the BHS population, both SLS and BHS populations may have belonged to the same large population historically, SLS populations were split out due to interference from human activities, historical events, and the reproductive characteristics of CM. Thus, it still retains some genetic information of the original large population. This finding is similar to that observed in a genetic diversity study of CM in Korea [23]. In addition, the SLS population is the southernmost population in CM geographical distribution and is close to the scenic spot, which has human disturbance and ecological edge effect to some extent; therefore, such a population needs more attention and protection.
From the perspective of large-scale geographical scope, the genetic structure analysis, PCA, and phylogenetic tree analysis showed that CM was divided into two groups. The first group consisted of six populations from north China and the second group included five populations from northeast China. These results were consistent with the geographical distribution of CM populations; these populations of north and northeast China were more than 1,000 km apart, separated by the Yanshan Mountains, the Qilaotu Mountains, and the Bohai Sea. On a finer scale, the north China populations were divided into three subgroups. Based on the geographical distribution pattern, these populations were isolated from each other and were scattered in several high mountains. Therefore, substructures were detected in all samples from north China. The distribution pattern of northeast China populations was different. Except for the PDS population, the distribution of most northeast China populations was in plain and hilly areas, and the gene flow barrier was small, especially the individuals of the XAS, HLHZ_HLHC, and JSTZB_JSTZ populations showed obvious genetic admixtures. Thus, the northeast China populations showed up as a whole. Interestingly, 16 samples of the YWS population were divided into different subgroups, suggesting that this population was formed as a result of the intermixing of several ancestors. On the whole, these results of the population structure were similar to those of the PCA and phylogenetic tree analysis, in which although there was some degree of admixture, all the samples still showed a distinct genetic structure.
We calculated genetic differentiation and gene flow among populations, and the levels of genetic differentiation were significantly different among populations, with high genetic differentiation among populations across regions. Based on the classification of genetic differentiation levels by Wright [31], moderate-to-high genetic differentiation (Fst = 0.0601–0.2684) was detected between northeast China populations and north China populations. In this study, the genetic differentiation level of CM was similar to that of the other plants of Cypripedium38. Although the level of genetic differentiation among populations was high, 11 populations had moderate-to-high gene flow with each other (Nm = 0.6814–78.0708). Gene flow levels varied greatly between populations, with local populations showing high gene flow. According to Wright [31], the intensity of gene flow can be divided into three Nm levels: ≥1.0 (strong), 0.250–0.99 (medium), and < 0.249 (low). Thus, genetic differentiation and gene flow between populations were high in this study. How to understand these two seemingly contradictory results? From the perspective of geographical distribution, some of these populations were more than 1,000 km apart. For subalpine and high-latitude plant species, pollen and seed-mediated gene flow were greatly influenced by environmental heterogeneity, and topographic and geomorphic conditions [39, 40]. Gene flow between these populations was almost impossible at present; thus, the detected gene flow was historical gene flow. It can be inferred that CM was widely and continuously distributed in north and northeast China in the past. All CM can be considered a huge population connected by large gene flow, and the historical gene flow occurred according to the stepping-stone model, short distance gene flow without interference can make continuously distributed population can reach a dynamic balance during a long evolutionary process, but once disturbance occurs, such as excessive collection, orogeny in geological history, extreme weather, accidental event and so on, it will accelerate the genetic differentiation and formation of genetic structure of species13. Genetic differentiation detected among CM populations is evidence that these populations have been subjected to extensive human disturbance and habitat fragmentation in recent times. Various exogenous disturbances can upset this balance and reduce the genetic diversity of species and aggravate genetic differentiation between populations [41]. The current distribution pattern of CM populations is the result of long-term population dispersal in the past and human disturbance in recent times.
We evaluated the direction of gene flow using Treemix software. Our data indicated that the direction of historical gene flow was mainly from northeast China populations to north China populations. It implied that the north China populations were formed by the gradual migration and settlement of northeast populations by seed-mediated gene flow, which is consistent with the dominant distribution of CM and its preference for cold climates. In the resource survey, northeast China populations were widely distributed, ranging from the plain at 200 m above sea level to the Greater Khingan Mountains at more than 1,200 m above sea level, whereas the north China populations were only distributed in the subalpine meadows of several mountains. In addition, the petals of CM individuals in northeast China were purplish red, pure white, and light pink, and CM individuals with white petals were also found in Rebun Island, Japan [42]. However, the petals of all CM individuals in north China are purplish red. These phenomena indicate that genetic variation in CM was more abundant at high latitudes and that its diversity center was located at high latitudes. In our comprehensive analysis, these populations from northeast China gradually spread southward in evolutionary history and may be the ancestral populations of the north China populations.
The Mantel test is often used to detect IBD in populations in order to understand the relationship between genetic and geographic distances between populations of different geographical origins [43].
Although the populations of north and northeast China were more than 1,000 km apart, no significant correlation was observed between genetic and geographic distances (Fig. 5). This result may be related to the moderate-to-high historical gene flow that was detected. Strong gene flow counteracted IBD between populations in long evolution history. However, external factors (over-collection, habitat fragmentation, accidental events) and internal factors (deceptive pollination and low seed germination rate) have accelerated the rapid decline in CM population size and distribution area in the past, weakened or even hindered recent gene flow, and finally led to the genetic differentiation of the population. The present distribution pattern and genetic diversity of CM have been formed due to these factors.