Introduction

Plants of the Caprifoliaceae family exhibit high ornamental value in their flowers, fruits, and leaves, making them excellent choices for landscaping. The genus Lonicera includes evergreen and deciduous shrubs, dwarf shrubs, and climbing species. Deciduous varieties are characterized by early budding and late leaf fall resulting in prolonged foliage retention, large and colorful flowers with extended blooming periods, and fruits that transition from green to black, blue, or red. Thus, Lonicera plants possess significant ornamental value in terms of overall form, leaves, flower shape, color, and fruit appearance. In September 2021, Lonicera oblata (Hao ex Hsu et H.J. Wang), a species of the Caprifoliaceae family, was listed in the National Key Protected Wild Plants Directory by the National Forestry and Grassland Administration as a nationally second-class protected wild plant. Lonicera oblata, endemic to North China, is a deciduous shrub with fleshy roots; its branch pith is white and solid. Winter buds feature two pairs of ovate, sharply pointed outer scales. Leaves are opposite, thickly papery. Inflorescences are axillary, with peduncles emerging from the leaf axils of current-year branches; bracts are subulate; adjacent calyx tubes are separate and glabrous; and the corolla is yellowish-white and bilaterally symmetrical. Flowers are paired, borne at the ends of peduncles in leaf axils, without fused leaves. Fruits are red and round; seeds are nearly round or ovate. Flowering occurs in May, and fruiting in July1,2. Literature and specimen records indicate that it is distributed only in Hebei, Beijing, and Shanxi. The status of wild populations in Hebei’s Neiqiu and Beijing’s Dongling Mountain is unclear, with only one individual found in Beijing’s Yanqing Songshan and a few individuals in Shanxi’s Wutai Mountain3. In June 2022, during a wild plant resource survey in the Taihang Mountains of Shanxi, the research team from Taiyuan Botanical Garden discovered approximately 100 individuals of Lonicera oblata, the largest population found in China to date. Subsequent specimen collection laid the foundation for further research. Due to the small population size, natural regeneration of this wild plant is challenging. Currently, there is limited research on Lonicera oblata domestically and internationally, with studies confined to basic descriptions of morphology and ecological habits4,5,6,7. No in-depth research specifically targeting Lonicera oblata has been reported, making systematic studies on its population status in Shanxi even more urgent.

To protect Lonicera oblata effectively, this study investigated its population status, habitat characteristics, and growth traits in Shanxi Province, including population size, companion plants, growth conditions, and flowering features, to establish a foundation for seedling propagation techniques and provide scientific theoretical support for its conservation and utilization.

Research methods

Survey period

The survey was conducted from March to October 2022, 2023.

Survey locations

The survey was conducted in the mountainous areas of Chafang Village, Dongshan Township, Fanshi County, Xinzhou City, and Shanxi Province, within the Wutai Mountain region.

Plant identification

The plants were identified by Dr. Ren Baoqing of the Taiyuan Botanical Garden and are currently stored in the Taiyuan Botanical Garden specimen collection(TYH).Voucher specimen ID:2,022,052,401.

Habitat survey

Altitude and coordinates were measured using GPS on mobile devices. Plant height, crown width, leaf length, leaf width, petiole length, and flower bud development were measured using a tape measure.

Soil physicochemical properties

Soil surface samples were collected during the flowering period of Lonicera oblata in each year (August 2, 2022, and July 23, 2023). Five plots were selected at each distribution point. When collecting samples, areas with uniform plant growth and flat terrain were preferred, and the edges of the plots were avoided. Sampling points were determined within the blocks. Six soil samples at a depth of 20 cm were collected from each community using a soil drill according to the “S” type sampling method. Then, these six soil samples were thoroughly mixed to form one representative composite soil sample. Stones, plastic films, and other soil intrusions were carefully removed, and the soil samples were transported to the laboratory.

The determination methods used for soil physical and chemical indicators strictly followed the third edition of 《Soil Agricultural Chemical Analysis》 edited by Bao Shidan8. The specific determination methods were: pH value was determined by potentiometry, with a water-to-soil ratio of 2.5:18; available nitrogen content was measured via the alkaline hydrolysis diffusion method9; total nitrogen content was determined by sulfuric acid digestion and the Kjeldahl method9; soil organic matter content was measured using potassium dichromate oxidation and the external heating method; total phosphorus content was determined by sodium hydroxide molybdenum antimony reverse spectrophotometry10; total potassium content was measured by NaOH fusion flame photometry; available phosphorus content was determined by sodium bicarbonate/sodium fluoride extraction and molybdenum antimony anti-colorimetry; and available potassium content was measured using ammonium acetate extraction and flame photometry.

Scanning electron microscopy (SEM) for pollen observation

At each sampling point, anthers were collected from ten vigorous plants, one flower from each plant, totaling ten flowers. The pollen from the ten anthers was then mixed. Observations at the same time, on three consecutive days, were performed for three repetitions. Fresh pollen was quickly immersed in SEM fixative, fixed at room temperature for 2 h, transferred to 0.1 M phosphate buffer (PB, pH 7.4), and rinsed three times for 15 min each. The pollen was fixed in 1% osmium tetroxide (prepared with 0.1 M PB) at room temperature in darkness for 1–2 h, followed by three rinses with 0.1 M PB for 15 min each. The samples were dehydrated in a graded ethanol series (30%, 50%, 70%, 80%, 90%, 95%, and 100%) for 15 min each and then treated with isoamyl acetate for 15 min. The samples were dried using a critical point dryer, mounted on conductive carbon tape, and sputter-coated with gold for approximately 30 s. Pollen morphology was observed under SEM. For each observation, the inner polar axis length, equatorial axis length, mesh diameter and density, germination pore length, and width of 15 pollen grains were measured repeatedly, and the average values were calculated.

Pollen morphological data measurement

Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA) was used to measure the polar axis length, equatorial axis length, pore diameter and density, and germination aperture length and width, with a 1k 50-µm scale as the standard.

Stigma receptivity test

One day prior to the experiment, the flowering time as well as the plant were observed and documented, identifying a total of 10 stigmas. Three observations were conducted simultaneously over three consecutive days, mainly focusing on the development of the stamens and pistils. Twenty vigorously growing plants were selected, from which five flower buds, at different developmental stages, were chosen from each plant. Anthers were collected from ten plants at each sampling point and categorized: P1 = stigma shorter than filaments, P2 = anthers with dispersed pollen and stigma equal in length to filaments, P3 = stigma slightly longer than perianth segments, P4 = stigma beginning to wilt, and P5 = perianth segments drying out with a swelling ovary. Stigma receptivity was assessed using the hydrogen peroxide method, employing a culture medium consisting of 3% hydrogen peroxide. The column head was placed in a culture dish containing this medium. Numerous dense bubbles forming around the column head within the culture medium indicated strong stigma receptivity. The intensity of bubble formation was utilized to evaluate vitality. The classification standard, based on the methodology established by Sun Jian et al.11, is defined: Grade 1 indicates few bubbles present, suggesting relatively weak receptivity of the stigma (marked as +), earning it one point; Grade 2 denotes many bubbles, indicating moderate receptivity (marked as ++), totaling two points; Grade 3 signifies abundant bubbles, reflecting strong receptivity (marked as +++), worth three points.

Seed anatomical structure

Plump and shriveled seeds were fixed in Formaldehyde-Acetic Acid-Alcohol(FAA) solution and processed through fixation, washing, dehydration, clearing, wax embedding, sectioning, staining, and sealing to prepare paraffin sections for observing seed anatomical structures.

Effects of different temperatures on seed germination

Large, fully mature seeds were selected, treated with 75% alcohol for 30 s, and rinsed under running water for 2 min. The seeds were soaked in boiling water for 3 to 4 s to achieve disinfection. Next, the seeds were immersed in warm water for a duration of 12 h. Subsequently, the seeds were placed in culture dishes lined with two layers of absorbent paper. Fifty seeds were introduced into each culture dish. The dishes were then placed in incubators set at 0, 5, 10, 15, 20, or 25℃. Three petri dishes were placed in each temperature-controlled incubator, for three repetitions. The experiments were conducted under a light/dark cycle of 12 h each. The cultivation was carried out within artificial incubators. The germination rates of the seeds were recorded seven days post-treatment.

Data treatment

The experimental data were organized using Excel software and analyzed through variance analysis with SPSS 20.0.

Results and analysis

Population status

Based on a systematic literature review and population ecology methods, the population size, geographic distribution, and companion plants of Lonicera oblata were investigated. Currently, the sizes of wild populations vary significantly. A total of 299 individuals were surveyed (Table 1; Fig. 1).Distribution Point Three (39°10′42.69″N, 113°36′57.51″E) had only six individuals, representing the smallest population. In contrast, Distribution Point One (39°10′6.24″N, 113°10′6.24″E) had the highest number with 244 individuals and encompassed the largest area. Distribution Points Two and Three are reported herein for the first time. The distribution range is narrow and confined to Wutai Mountain in Chafang Village within Dongshan Township of Fanzhi County in Xinzhou City, Shanxi Province. The elevations at these sites range from 1400 to 1650 m above sea level. The habitat primarily consists of exposed rocky areas at mountain summits with limited ecological diversity. Distribution Point Two (39°10′47.50″N, 113°37′13.61″E) is located near roads and grazing areas and is significantly impacted by human activities. At Distribution Point One, associated plant species belong to 15 families across 28 genera, dominated by Asteraceae, with seven species represented there. At Distribution Point Two, associated plants include representatives from 15 families across 21 genera, with Ranunculaceae, Caprifoliaceae, Amaryllidaceae, and Rosaceae being predominant groups; whereas Distribution Point Three contains six families comprising ten genera, predominantly represented by Rosaceae and Ranunculaceae. The survey results indicate that Lonicera oblata is distributed across three locations with a total count of 299 individual plants, highlighting an extremely rare status for its wild resources (Fig. 2).

Table 1 Information for each distribution point.
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Fig. 1
figure 1

Distribution of Lonicera oblata population. Note: 1 represents distribution point 1 (39°10′6.24″N, 113°10′6.24″E); 2 represents distribution point 2 (39°10′47.50″N, 113°37′13.61″E); 3 represents distribution point 3 (39°10′42.69″N, 113°36′57.51″E).

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Fig. 2
figure 2

The typical habitat of the Lonicera oblata population.

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Soil physicochemical properties

Soil samples collected from the distribution points of Lonicera oblata were analyzed to compare and assess the physicochemical properties across different locations (Table 2). The analysis of soil physicochemical properties at three distribution points, along with a control group, indicated that all soils were neutral (pH 6.97–7.10). Compared to the control group, the organic matter content, available nitrogen content, total phosphorus content, available phosphorus content, and available potassium content at the three distribution points were significantly higher; among these, Distribution Point Three exhibited the highest organic matter content (90.72 g/kg), which is 5.1 times that of the control group. There was no significant difference in total nitrogen and total potassium contents when compared to the control group. Based on these soil analysis results, it can be concluded that Lonicera oblata has high nutritional requirements for its growth in soil. Coupled with its habitat, characterized predominantly by rocky terrain, there is a lack of suitable soil conditions necessary for its development.

Table 2 Physical and chemical properties of soil at each distribution point.
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Botanical traits of Lonicera oblata

From March to October 2022 and 2023, plant traits were surveyed (Fig. 3). Young leaves measured 4.5–5.2 cm in length, 2.6–4.1 cm in width, with petioles 2–2.5 cm long; mature leaves measured 5.4–7.3 cm in length, 3.8–5.1 cm in width, with petioles 2.4–3.5 cm long. Flower bud dimensions were: length 1.2–1.85 cm, width 0.4–0.6 cm; stamens 0.45–1.0 cm long; pistils 0.4–0.6 cm long; lip length 0.75–0.85 cm, width 0.3–0.6 cm. Growth conditions are shown in Figs. 2 and 3.

Fig. 3
figure 3

Floral morphological characteristics of Lonicera oblata. Note: 1, 2, 4, Double-flowered inflorescence. 3, Fruits. 5, 6, Flowers are bisexual, with five stamens; the ovary is ovate, green, located at the corolla tube base. 7, Fruits are berries, round, initially green and turning red at maturity.

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Pollen morphology of Lonicera oblata

Pollen is spherical (0.88< P/E<1.14), medium-sized(25–50 μm), measuring 41.44 × 45.21 μm. Germination apertures are tricolporate, short, and shallow (length 11 μm, width 3.39 μm). The exine surface features spiny projections and a perforate tectum, with 0.13 spines per 225 μm², spine base diameter 0.78 μm, and spine length 1.13 μm (Figure 4). The pollen characteristics align with those of the Lonicera genus12, serving as taxonomic evidence.

Fig. 4
figure 4

SEM images of pollens of Lonicera oblata. Note: 1, pollen population view, scale bar: 100 μm. 2 and 3, pollen from different perspectives, scale bar: 30 μm. 4, The surface of the pollen exine is covered with spiky and perforated structures, scale bar:5 μm.

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Stigma receptivity of Lonicera oblata

Stigma receptivity varied significantly across stages(Fig. 5; Table 3). After anther dehiscence, when stigmas were shorter than filaments, only sporadic bubbles were observed, indicating weak receptivity. When stigma lengths equaled filament lengths, abundant bubbles were observed, indicating peak receptivity, which declined as stigmas wilted. At petal withering, there were minimal bubbles, indicating very low receptivity.

Table 3 Variation in stigma fertility of Lonicera oblata.
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Fig. 5
figure 5

Classification of stigma receptivity of Lonicera oblata. Note: P1. Grade 1, there are a few bubbles and the stigma receptivity is weak, marked as +, 1 point; P2, P3. Grade 2, with more bubbles and the stigma receptivity is medium, marked as ++, 2 point. P4, P5. Grade 1, with a few bubbles and the stigma receptivity is weak, marked as +, 1 points.

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Seeds of Lonicera oblata

Approximately 5000 seeds were collected, averaging 4.01 mm in length, 3.12 mm in width, and 5.181 g per 1000 seeds. The plump-to-shriveled seed ratio was 1.5:1. Anatomical sections showed well-developed embryos in plump seeds and underdeveloped embryos in shriveled seeds(Fig. 6).

Fig. 6
figure 6

The seed anatomical structure of Lonicera oblata. Note: A Plump seeds, B Shriveled seeds.

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Effects of temperature on seed germination

As shown in Table 4, the highest germination rate (53.3%) was achieved at a temperature of 20 °C, followed by 13.3% at 15 °C. No germination occurred at 0–10 °C, indicating that optimal temperature significantly promotes seed germination, with 20 °C being the most suitable.

Table 4 Effect of different temperature on Lonicera oblata seed germination.
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Discussion and conservation recommendations

Lonicera oblata, a nationally second-class protected plant listed as endangered, was assessed through two consecutive years of habitat surveys, which showed that the number of mature individuals in the wild population is fewer than 250.Based on the International Union for Conservation of Nature(IUCN) Red List of Threatened Species and its assessment criteria, this species qualifies as endangered (Category D). The currently documented population comprises 299 individuals, all growing on exposed rocky substrates at the summit of a limestone mountain. The habitat is characterized by extreme environmental conditions, including poor soil development and insufficient rainfall, which contribute significantly to the limited population size.

During field investigations, no seedlings from the previous growing season were observed. This absence suggests that seed germination may be a critical limiting factor for wild population persistence. The scarcity of seedlings poses a serious challenge to population recruitment and long-term sustainability, indicating difficulties in natural regeneration. To evaluate potential causes, seed viability was assessed, revealing a seed shriveling rate of 40%. This high rate is attributed to underdeveloped or structurally incomplete embryos, along with poorly developed endosperm and cotyledons, which fail to provide sufficient nutritional reserves necessary for successful germination. These physiological deficiencies directly impair germination capacity and are likely major contributors to the paucity of seedlings in the natural habitat. To protect Lonicera oblata and prevent population decline, the following measures are recommended:

Establish in situ conservation sites

In situ conservation is the most fundamental and effective method for plant protection13,14,15,16. No signage or management was observed at distribution sites. Field protection stations should be established, with enhanced patrols and regulations to prevent human interference.

Develop field trials in different locations

Based on soil research data, select appropriate soil substrates and conduct transplantation protection experiments to help maintain population size and slow down population decline.

Develop seedling propagation techniques

Tissue culture, a mature technology, can rapidly produce large numbers of seedlings. Research on Lonicera oblata propagation systems should commence promptly to achieve conservation goals.