Xiaoyong Dai

Rhododendron nymphaeoides is a plant belonging to the genus Rhododendron ( Rhododendron Linn.) and the subsection Fortunea . Although this species is clearly listed as endangered (with an endangered status of EN) by the International Union for Conservation of Nature (IUCN) Red List, the Red List of Rhododendrons, the Red List of China's Higher Plants, and the Threatened Species List of China's Higher Plants, the question of whether its mating system is self‐pollinating or cross‐pollinating remains a perplexing issue that hinders the conservation and management of wild resources. This study investigated the flowering phenology, floral characteristics, pollen traits, breeding system, and pollination biology of R. nymphaeoides . The results indicate that (1) the peak flowering period for R. nymphaeoides occurs in late April, with the flowering process of a single flower divided into five stages: bud stage, bud swelling stage, initial flowering stage, peak flowering stage, and wilting stage, exhibiting synchronous flowering; (2) the pollen of R. nymphaeoides is tetrahedral, arranged in a regular tetrahedral formation, with an average diameter of 56.47 μm. The individual pollen grains are nearly spherical with a surface covered in sticky threads, featuring three pores with indistinct ornamentation around the grooves, and the outer wall consists of two layers with a faint reticulate sculpture visible on the surface; (3) the pollen vitality of R. nymphaeoides is relatively strong throughout the flowering period, peaking at 11:00 a.m. on the day of flowering at 91.86% and maintaining a 36.67% vitality until the end of the flowering period (9 days after flowering), making it suitable for use as a parent in the hybrid breeding work; (4) the stigma receptivity of R. nymphaeoides lasts for a long time, with the optimal pollination period occurring during the peak flowering stage (days 3–6 after flowering); (5) the most suitable culture medium for pollen is 150 g·L ⁻¹ sucrose + 200 mg·L ⁻¹ H ₃ BO ₃ + 50 mg·L ⁻¹ CaCl ₂ , with the optimal storage temperature being −80°C; (6) the breeding system of R. nymphaeoides is classified as a facultative cross‐fertilization mixed mating system, relying on pollinators, with a low natural fruit set rate (43.33%). The fruit set rate for cross‐pollination between different individuals is 83.33%, indicating pollination limitations and weak reproductive assurance (RA); (7) effective pollinators of R. nymphaeoides include Papilio paris , Papilio bianor , Papilio polytes , Papilio protenor , Apis cerana , Vespula koreensis , Vespa velutina , Xylocopa appendiculata , and Xylocopa tranquebarorum , while insects such as Bombus eximius , Anthophora plumipes , Syrphus torvus , Helophilus eristaloidea , Eristalis arbustorum, and Nemoraea pellucida act as nectar robbers, and Osmia taurus serve as pollen thieves.

Rhododendron nymphaeoides belongs to the Fortunea subsection of the Rhododendron genus. Despite being officially listed as an endangered species (with the conservation status of EN) by the International Union for Conservation of Nature Red List, The Red List of Rhododendrons, Red List of China's Higher Plants, and the Threatened Species List of China's Higher Plants, little is known about this species' community characteristics and population dynamics. This lack of knowledge hampers the conservation and management of its wild resources. This study was conducted on all existing distribution points of R. nymphaeoides through field investigations, revealing the species composition and community structure of its populations. This included analyzing the diameter class structure, creating life tables and survival curves, and analyzing population dynamics indices to shed light on the dynamics of the R. nymphaeoides populations. The results showed: (1) the four communities collectively comprise 122 species of vascular plants belonging to 55 families and 79 genera, with tropical elements predominantly influencing the community structure and environmental formation. Community IV exhibited the highest species diversity, Community III had the most even distribution of species, and Community II had the lowest species diversity. (2) A total of 288 R. nymphaeoides individuals were recorded, showing a spatially clustered distribution. (3) The age structure of the HSC population exhibits a reverse J‐shape, indicating a growing population, but there are still constraints in the transition from seedlings to saplings; the LSK population has a substantial number of young individuals and the highest proportion of middle‐aged individuals, characterizing it as a stable population; whereas the LZH and HTS populations have few young individuals and a high proportion of middle‐aged individuals, with an age structure that is spindle‐shaped and trending toward aging, placing these populations at significant risk of decline. (4) The survival curves for all four populations were of the Deevey‐II type, characterized by high mortality rates among seedlings. (5) The primary reasons for the difficulty in population renewal and development are the extremely low natural germination rates of seeds, weak competitiveness of seedlings, growth restricted to limestone mountain areas above 1500 m with thin soil layers, and frequent human disturbances. Therefore, conservation strategies that integrate in situ conservation, ex situ conservation, and reintroduction are recommended. Emphasis should be placed on long‐term monitoring of R. nymphaeoides populations and communities, enhanced management during the sapling stage, and efforts in seedling propagation and population restoration to achieve population strengthening and the natural reintroduction of artificially cultivated populations, thereby maintaining the population in a healthy state.

Rhododendron nymphaeoides is explicitly listed as an endangered species in the “the International Union for Conservation of Nature’s Red List (IUCN)”, “The Red List of Rhododendrons”, “Red List of China’s Higher Plants” and “Threatened Species List of China’s Higher Plants”. It is also listed as a provincial-level key protected wild plant in Sichuan, with few individuals in the wild and significant conservation value. The genetic diversity and population structure have never been described, making it difficult to plan conservation strategies for this plant. This study utilized 15 pairs of microsatellite markers to examine the genetic diversity of 79 samples of R. nymphaeoides sourced from five different geographic populations. A total of 214 alleles were detected, with the average effective number of alleles (Ne) of 7.0324. The averages for the polymorphism information index (PIC) and expected heterozygosity (He) were 0.7832 and 0.8102, respectively, indicating that the R. nymphaeoides populations harbor a rich genetic information content, the genetic differentiation coefficients (FST) average was 1.2607. There was high genetic diversity among populations, with average observed heterozygosity (Ho) and expected heterozygosity (He) values of 0.6375 and 0.6663, respectively, suggesting a degree of inbreeding within populations. Mantel test results showed a significant positive correlation between geographic distance and genetic distance amongst populations (r = 0.8456, P = 0.0021), which conforms to the isolation-by-distance (IBD) model. Due to geographical barriers, there is also a high level of genetic differentiation among populations, with an average genetic differentiation coefficient (FST) of 0.2685. Analysis of molecular variance (AMOVA) indicated that the main source of molecular variance exists within populations (73%), rather than between populations (27%). There was higher historical gene flow (average = 1.0850) and lower contemporary gene flow (average = 1.2849), with seed and pollen dispersal being impeded. Under the Two-Phase Model (TPM) assumption, findings are consistent with the mutation-migration model, suggesting that there has been no genetic bottleneck. STRUCTURE analysis, principal coordinate analysis (PCoA), and UPGMA analysis all support the division of the five natural populations into three genetic clusters. This is the first comprehensive analysis of the genetic diversity and population structure of the endangered plant R. nymphaeoides using microsatellite markers. The study results indicate that this endangered plant’s natural populations maintain a high level of genetic diversity. Due to geographical barriers, there is also a high level of genetic differentiation, with the primary source of genetic variation originating within populations. There is higher historical gene flow and lower contemporary gene flow, with seed and pollen dispersal being obstructed. The five populations can be divided into three evolutionary units, for which corresponding conservation management units should be established. These findings will benefit the conservation and development of the species and provide a theoretical basis for further studies on its evolution and biogeography.

Soil microbes play an important role in the microbial circulation and energy flow of ecosystems. In order to understand the change in the rhizosphere soil microbial community structure and function in the heterogeneous karst habitats, the nutrient content and enzyme activity were analyzed, and Illumina MiSeq high-throughput sequencing technology was used to detect the composition, quantity and functional types of the rhizosphere soil microbial community in Rhododendron pudingense under three kinds of karst microhabitats (soil surface, rock gully and rock surface) in Wangmo Country (WM), Zhenning Country (ZN) and Qinglong Country (QL). The results showed that SS and RG microhabitats had a higher nutrient content and enzyme activity, while RS had the lowest. At the phylum level, Proteobacteria and Actinomycetes were dominant in terms of bacteria, while Ascomycota and Basidiomycotina were dominant in terms of fungi. There was no significant difference in microbial diversity among different karst microhabitats (p > 0.05). At the microbial genus level, there were some differences in species composition among the three karst microhabitats, which may lead to soil heterogeneity in karst microhabitats. WM was a little different from ZN and QL. The results of PCoA showed that the community composition of RG and RS was more similar to that of SS. There was no significant difference in microbial functional types among different microhabitats (p > 0.05). Only the abundance of pathothoph-symbiothoph fungi in RG was significantly higher than that in RS (p < 0.05). The main function of bacteria was metabolism, and saprophytic and symbiotic fungi were the dominant fungal group. In conclusion, soil organic carbon and alkaline phosphatase are important factors affecting the level distribution of microflora in different karst microhabitats. R. pudingense in the SS and RG microhabitats has better soil conditions, which may require protection for the plants in the RS microhabitat. The current study results can provide a theoretical basis for the adaptation mechanism of Rhododendron pudingense to the karst microhabitat.