Zanthoxylum armatum DC. (Rutaceae), commonly called Timur in Nepali (English: Nepal pepper or prickly ash), is an aromatic large shrub up to 6 m high. It is one of the 30 prioritized medicinal plants for economic development (DPR 2006). It is found in hot valleys of subtropical to temperate Himalayas (Kashmir to Bhutan), north-east India and Pakistan, Laos, Myanmar, Thailand, China, Bangladesh, Bhutan, Japan, North & South Korea, North Vietnam, Taiwan, Lesser Sunda Islands, Philippines, Malaya peninsula and Sumatra (Nair and Nayar 1997). In Nepal, it is distributed from west to east at an elevation range of 1,000 m to 2,500 m in open places or in forest undergrowth (DPR 2007). The plant grows well in open pastures, wastelands and secondary scrub forests with adequate rainfall. Moist areas with deep soils exposed to sun and degraded slopes, shrub lands, natural forests and wastelands are the suitable habitat for Z. armatum (Phuyal et al. 2019).

Z. armatum (Fig. 1) has been used extensively in traditional indigenous medicinal practices in Nepal by different ethnic communities. Several ethnobotanical studies have documented the various ethno-medicinal uses in different types of ailments. The different parts of the plants: leaves, fruits, stem, bark, seeds have been used as carminative, antipyretic, appetizer, stomachic, toothache, dyspepsia (Kala 2005; Manandhar, 2002; Singh et al. 2016). This plant species is not only used for pharmaceutical purposes, but also used in the flavoring and fragrance industries (Phuyal et al. 2020). Based on its varied industrial uses, its demand is constantly increasing both in domestic and international markets. Increased market demands, devious modes of collection and insufficient technical knowledge and proper skills of harvest and postharvest techniques have posed serious threats to the native populations attributing to a sharp decline of the species in the wild, due to which the regeneration of this species is adversely affected (Phuyal et al. 2019).

Figure 1. Zanthoxylum armatum.

The National Conservation Strategy (1988), Master Plan for the Forestry Sector (1988), Industrial Enterprises Act (GoN 1992), Forest Act (GoN 1993) and Regulations (GoN 1995), Herbs and Non-Timber Forest Products Development Policy (DPR 2004) have emphasized on the subsequent development and commercialization of the Non-timber Forest Products including Medicinal and Aromatic Plants (MAPs) for uplifting the livelihood of the rural communities through sustainable and wise use of these valuable resources. Trade Policy in Nepal (GoN 2009) has also prioritized Z. armatum as an important commodity for export.

There have been ample studies on the medicinal plants of Nepal regarding their botany, ecology, ethnobotany, population size, and distribution. A significant amount of research has also been carried out in Zanthoxylum in the Indian subcontinent. However, there have been very few studies in Z. armatum in Nepalese context and the study of relation of ecological factors on the distribution and population structure in Nepal is still meager (Phuyal et al. 2019). Understanding the ecology of individual species is important for conservation and for cultivation purposes. Unsustainable harvesting from the wild without proper management practices is the major threat to most of the MAPs including Z. armatum. So, understanding of ecology and biology of these valuable plants is very crucial for agro-technology development and commercial cultivation, which ensures the steady supply of raw materials without hampering their natural population. No study has been conducted previously regarding ecological status including the population, distribution, frequency, abundance, and regeneration status of Z. armatum from Nepal. The present study, therefore, aims to find the ecological status of Z. armatum in Salyan district regarding its distribution, density, frequency, abundance, diversity, and regeneration potentiality.

Field sampling

A preliminary filed survey was carried out in April 2017 to select the study site and sampling areas, gather general information about the study species viz. Z. armatum, and rapport building with the local people and concerned authorities. The principal visit was conducted during the months of May 2017 and October 2018. All necessary data and samples were collected during that period. The details of study sites (DFRS, 2018) and map of the study area are presented in Figure 2 and Table 1.

Table 1 . Details of sampling sites of Zanthoxylum armatum in the study area.

SN	Municipality	Total area (ha)	Forest area (ha)	Forest cover (%)	Study site	Altitude (m)	Latitude (N), Longitude (E)	Land use/Forest type
1	Kapurkot	11,875	7,542	63.5	Dhanwang	1,000–1,200	28.26875, 82.30842	Forest near village settlement
2					Kapurkot	1,200–1,400	28.2707, 82.35038	Near roadside on edges of farmyard
3					Rim	1,400–1,700	28.27611, 2.36361	Mixed Quercus forest
4	Baghchaur	16,251	8,453	52	Baghchaur	1,400–1,600	28.46694, 2.28139	Mixed forest
5	Bangad Kupinde	33,678	22,709	67.4	Kupinde	1,600–1,800	28.41319, 82.0935	Disturbed forest due to road construction
6	Chhatreshwori	15,011	9,841	65.6	Chhatreshwori	1,800–2,000	28.38611, 2.36361	Moist and dense forest

Source: 1. Forest cover and land cover: DFRS (2018), 2. Field survey.

Figure 2. Map of Nepal showing study area and sampling sites of Zanthoxylum armatum.

The study was mainly based on primary data collection. Necessary information was collected through extensive field observation of the area. The data was collected through physical measurement in the field and review of relevant literature on similar previous studies. Systematic random sampling design was applied in which plots were selected by a random or stratified random plan (Misra 1968). The sampling sites were selected from six localities to cover all the possible habitats and associated vegetation types of Z. armatum so that a comparative study can be done based on disturbance factors, altitudinal difference, etc.

Vegetation sampling was done along the elevation of 1,000 m to 2,000 m. In each locality, four transect lines were set up at 30–50 m in Z. armatum available sites. In each transect line five plots of 5 m × 5 m were laid down at a distance of 10 m. The number of individuals of Z. armatum and other tree and shrub species (excluding grasses) in the sample plot associated with Z. armatum were recorded.

Vegetation attributes, including frequency, density, and richness, were recorded, along with environmental coordinates such as latitude, longitude, and elevation of each sample plot using a global positioning system (Garmin model 2000) (Shaheen et al. 2011a).

Vegetation analysis

The species richness in this study was obtained by counting the number of species present in each 5 m × 5 m sample plot. In this study, species richness has been defined as, the number of species per plot and expressed as species/m².

Both this term refers to the number of species in a community. Abundance of any individual species is expressed as a percentage of the total number of species present in community and therefore it is a relative measure (Khan et al. 2014). In sampling the abundance of species, the individual of species is counted instead of just nothing their presence or absence was done while studying the frequency of a species.

Density and relative density were calculated using Yadav et al. (1987), whereas abundance was determined based on the formula of Kilewa and Rashid (2014).

Density (D) (plants/ha)

=Total no. individuals of a species in all quadratsTotal no. of plots studied×size of the plot m2 ×10,000

Relative Density (RD%)

=Density of individual species Total density of all species×100

Abundance (A)

=Total no. of individuals of a species in all quadratsToal no. of quadrats in which the species occured 

Relative Abundance (RA%)

=Total no. of a particular species Total no. of individuals of all species recorded×100

Occurrence of trees and shrub species within each major plots of the study area were recorded to assess their distribution pattern in Z. armatum occurring areas. Then, frequencies of these species were obtained by following formula (Yadav et al. 1987). Relative frequency is the frequency of a species in relation to other species.

Frequency (F) (%)

=No. of quadrats in which an individual species occuredTotal no. of quadrats studied×100

Relative Frequency (RF%)

=Frequency of individual speciesSum of frequencies of all species×100

Abundance and frequency taken together are of great importance in determining the structure of a community. High frequency and low abundance indicate regular distribution whereas the converse indicates contiguous distribution. The ratio of abundance to frequency (A/F) for different species was determined for eliciting the distribution pattern. Spatial distribution of plant species was determined following Whitford index WI (Singh and Singh, 1987) as

If value is < 0.025 = regular distribution, value lies between 0.025–0.05 = random distribution and value > 0.05 = clumped distribution (Whitford 1949).

Importance Value Index (IVI)

The IVI was calculated to understand the species’ share in the community (Curtis and Cottam 1956). Species with the highest importance value are the leading dominant species of the specified vegetation (Shibru and Balcha 2004). This considers density, frequency, and abundance of the species present in the community. For each species, the relative density, relative frequency, and relative abundance were calculated and summed. It gives the overall importance of each species in the community structure. The IVI for all the species was calculated by adding the sum of relative values of density, frequency and abundance. It was calculated following Bhadra and Pattanayak (2016) as

Importance Value Index (IVI) = Relative Density + Relative Frequency + Relative Abundance.

Regeneration

Population structure of naturally emerged seedlings of Z. armatum reported in each sample plot was studied. Density of all the individuals of seedlings, saplings and adult were determined. The size classes of individuals of Z. armatum were broadly defined according to plant height. Plant height less than 0.1 m were classified as seedlings. Plant height ranging from 0.1 m to 1.0 m were classified as saplings and plant height usually more than 1 m and also bearing reproductive structures were classified as adult (Schemske et al.1994).

Regeneration status was totally based on population size of seedlings and saplings (Khan et al. 1987; Saha et al. 2016). Good regeneration if seedlings > saplings > adults; fair regeneration, if seedlings > or ≤ saplings ≤ adults; poor regeneration, if the species survives only in sapling stage, but no seedlings (saplings may be or = adults). If a species is present only in an adult form it is considered as not regenerating. The status of natural regeneration was determined based on the values as shown in Table 2 (Bhuyan et al. 2003; Khumbongmayum et al. 2006).

Table 2 . Different regeneration status.

SN	Regeneration status	Seedling (Se)	Sapling (Sa)	Compare to adult
1	Good regeneration	Present	Present	Se > Sa > adults
2	Fair regeneration	Present	Present	Se > or < Sa; Sa ≤ adult
3	Poor regeneration	Absent	Present	Sa > or < or = adult
4	No regeneration	Absent	Absent	Only adult

As shown in the above table, ‘good regeneration’ is defined as the condition in which an ample or adequate number of seedlings and saplings contribute to the mature population, while ‘fair regeneration’ is defined as the condition in which there were a fair number of seedlings, but the percentage of saplings was either lower than or close to that of the mature trees. ‘Poor regeneration’ is the condition in which individuals were found at either the seedling or sapling stage only, in greater numbers than the mature trees. The fourth regeneration status is termed as ‘no regeneration,’ in which a species presented only at the mature stage and did not occur in either seedling or sapling stages.

Species diversity

Diversity in species refers to the combined effect of richness and evenness in species. While richness pertains to the number of species in each sampling unit, evenness implies to the distribution of individuals among the species. Species richness is a biologically appropriate measure of diversity and the total number of species in any ecological community, landscape, or region relative to the total number of all individuals in that community.

A diversity index can reveal the structure of biological community in terms of numerical value. It gives more information on community composition than simply species richness. Further, it offers insights into rarity and commonness of species in a community, thereby diversity index acts as important tool for biologists in the understanding of community structure (Muthulingam and Thangavel 2012). Several indices are used to quantify the species diversity of which Simpson’s index (Simpson 1949) and Shannon-Wiener’s index are the most commonly used. Shannon’s diversity index (H) and Simpson’s Index (1 − D) in terms of density for each plot were calculated using the following indices:

Where, pi = (n/N),

n = density of individual species in a plot

N = Total density of all species in a plot

ln = Natural logarithm values

The Shannon diversity index ranges typically from 1.5 to 3.5 and rarely reaches 4.5 (Gaines et al. 1999). The Simpson’s Index values range from 0 to 1. The closer the value of Simpson’s Index to 0, the more diverse the plot will be. A plot with only one species would have a Simpson’s Index value of 1. Trends are opposite to those found for Shannon Weaver values since Simpson’s Index values decrease with increased diversity (Reich et al. 2001). In practice, the values below 0.5 indicates a relatively even community, while high values are indicative of communities dominated by one or a few species.

Soil analysis

Soil samples were collected from the four corners and center of each sample plot from the depth of 5–10 cm. The subsamples were mixed thoroughly, and about 100 g soil were collected, air dried in shade (Yadav et al. 1987), kept in zipper plastic bags, properly labeled, and brought to the Laboratory for the analysis of pH, soil organic carbon, total nitrogen, available phosphorus, and soil potassium. Soil organic carbon was determined by following Walkley and Black (1934), total nitrogen (N) by Kjeldahl method (Tsuji and Ohnishi 2001), available phosphorous (P) by a modified Olsen’s method following Gupta (2000), soil potassium (Flame photometer method following Trivedy and Goel 1986 and pH using a digital pH meter with 1:5 soil water ratio (Gupta 2000).

Statistical analysis

All the analyses were carried out in R platform (R Core Team 2020). The normality (Shapiro–Wilk test) for all the parameters were tested prior to choosing a parametric or non-parametric tool to analyze. All the parameters were tested by Analysis of Variance (ANOVA), Tukey’s test for normal data, and non-parametric Kruskal–Wallis one-way ANOVA, Duncan multiple comparison test for non-normal data.

Species richness

Altogether fifty plant species (trees and shrubs) belonging to 44 genera under 34 families were found to be associated with Z. armatum in the study area. Rosaceae was the dominant family with seven species, followed by Fagaceae with five species and the families Berberideae, Fabaceae, Moraceae, Oleaceae, and Rutaceae had two species each. Quercus was the largest genera with 4 species while the genera Castanopsis and Prunus had 2 species each (Table 3).

Table 3 . List of plant species (trees and shrubs) recorded in the study area.

SN	Name of the species	Family	Local name	Habit
1	Albizzia procera (Roxb.) Benth.	Fabaceae	Sirish	Tree
2	Adhatoda vesica Nees	Acanthaceae	Asuro	Shrub
3	Aesculus indica (Wall. ex Cambess.) Hook.	Sapindaceae.	Lekh pangre	Tree
4	Alnus nepalensis D. Don	Betulaceae	Uttis	Tree
5	Bauhinia variegate L.	Fabaceae	Badahar	Tree
6	Benincasa hispida (Thunb.) Cogn.	Cucurbitaceae	Kubindo	Climber
7	Berberis aristata DC.	Berberidaceae	Chutro	Shrub
8	Castanopsis hystrix Hook. f. & Thomson ex A. DC.	Berberidaceae	Patle katush	Tree
9	Castanopsis indica (Roxb. ex Lindl.) A.DC.	Fagaceae	Daale katush	Tree
10	Clematis sp.	Ranunculaceae.	Sikari lahara	Climber
11	Colebrookea oppositifolia Smith	Lamiaceae	Dhusure	Shrub
12	Cotoneaster microphyllus Wall. ex Lindl.	Rosaceae	Khareto	Shrub
13	Daphne bholua Buch.-Ham. ex D.Don	Thymelaeaceae.	Lokata	Shrub
14	Ficus neriifolia Sm.	Moraceae	Dudhilo	Tree
15	Ficus semicordata Buch.-Ham. ex Sm.	Moraceae	khaniu	Tree
16	Fraxinus floribunda Wall.	Oleaceae	Laakuri	Tree
17	Grewia optiva J.R. Drumm. ex Burret	Malvaceae	Bhimal	Tree
18	Juglans regia L.	Juglandaceae	Okhar	Tree
19	Pistacia integerrima Stew. ex Brand	Anacardiaceae	Kakadsinghi	Shrub
20	Ligustrum confusum Decne.	Oleaceae	Kanike	Shrub
21	Lyonia ovalifolia (Wall.) Drude	Ericaceae	Angeri	Shrub
22	Maesa chisia Buch.-Ham.ex D. Don	Primulaceae	Bilaune	Shrub
23	Mallotus philippinensis Muell. Arg	Euphorbiaceae	Sindure	Tree
24	Murraya koenigii (L.) Spreng.	Rutaceae	Karipatta	Shrub
25	Myrica esculenta Buch.-Ham. ex D. Don	Myricaceae	Kaphal	Tree
26	Persea odoratissima (Nees) Kosterm.	Lauraceae	Kaulo	Tree
27	Pinus roxhburghii Sarg.	Pinaceae	Khote salla	Tree
28	Prinsepia utilis	Rosaceae	Dhatelo	Shrub
29	Prunus cerasoides D.Don	Rosaceae	Paiyun	Tree
30	Prunus persica (L.) Batsch	Rosaceae	Aaru	Tree
31	Pyracantha crenulata (D. Don) M. Roeme	Rosaceae	Ghangaroo	Shrub
32	Pyrus pashia Buch.-Ham. ex D.Don	Rosaceae	Mayal	Tree
33	Quercus leucotricophora A.Camus	Fagaceae	Sano banjh	Tree
34	Quercus glauca Thunb.	Fagaceae	Phalat	Tree
35	Quercus incana Roxb. Hort. Beng.	Fagaceae	Thulo banjh	Tree
36	Quercus semecarpifolia Smith in Rees	Fagaceae	Khasru	Tree
37	Reinwardtia indica Dumort.	Linaceae	Pyauli	Shrub
38	Rhododendron arboretum Sm.	Ericaceae	Lali gurans	Tree
39	Rhus javanica L.	Anacardiaceae	Bhaki amilo	Tree
40	Rubus ellipticus Sm.	Rosaceae	Ainselu	Shrub
41	Salix sp.	Salicaceae	Bains	Tree
42	Sapindus mukorossi Gaertn.	Sapindaceae	Rithha	Tree
43	Sapium insigne (Royle) Trimen	Euphorbiaceae	Khirro	Tree
44	Schima wallichii Choisy	Theaceae	Chilaune	Tree
45	Smilax sp.	Smilacaceae	Kukurdaino	Climber
46	Stephania sp.	Menispermaceae	Batulpate	Climber
47	Tinospora sinensis (Lour.) Merr.	Menispermaceae	Gurjo	Climber
48	Toona ciliate M.Roem.	Meliaceae	Tuni	Tree
49	Viburnum erubescens Wall.	Adoxaceae	Asare	Tree
50	Zanthoxylum armatum DC.	Rutaceae	Timur	Shrub/Small tree

The species richness for each plot was the number of species per plot. Significantly higher species richness (p < 0.001) was recorded at Rim (1,400–1,700 m) and Chhatreshwori (1,800–2,000 m) while the species richness was significantly lower at Kupinde (1,600–1,800 m) (Fig. 3, Tables S1–S6).

Figure 3. Species richness at different locality and elevation (m). Different letters above bars indicate statistically significant difference between different altitudes at p < 0.001.

Density

The mean population density of Z. armatum in the study area was found to be 913.33 individuals/ha. The density among the different localities did not vary significantly (Fig. 4a). Among the six localities studied, the total density of Z. armatum was maximum at Kupinde (1,100 individuals/ha), followed by Baghchaur (1,020 individuals/ha), Rim (1,000 individuals/ha), Dhanwang (840 individuals/ha), Kapurkot (780 individuals/ha) and the lowest at Chhatreshwori (740 individuals/ha). The highest relative density (15.45%) of Z. armatum was at Baghchaur and lowest (5.35%) at Chhatreshwori (Fig. 4b). It was found to be associated with different species at different localities and altitudes. Mostly it was found growing in the northern and northeastern slopes and had least occurrence on the south and southeastern slopes.

Figure 4. Density (α) and Relative density (β) of Z. armatum at different locality and elevation.

Among all the species, Murraya koenigii had the greatest density with 1,140 individuals/ha at Dhanwang, Berberis aristata at Rim with 980 individuals/ha and Daphne bholua at Chhatreshwori with with 1,100 individuals/ha. Similarly Z. armatum had greatest density at Kapurkot, Baghchaur, and Kupinde with total of 1,000, 1,100, and 1,020 individuals/ha respectively (Tables S1–S6). The species having lowest densities were Ligustrum confusum, Tinospora sinensis (200 individuals/ha at Dhanwang), Ficus semicordata (260 individuals/ha at Dhanwang), Clematis sp. (200 individuals/ha at Rim).

Frequency

The mean frequency and relative frequency of Z. armatum in the study area were 70.83% and 5.61%, respectively. The frequency at different locality and elevation did not vary significantly (Fig. 5a) with the highest (80%) at Baghchaur (1,400–1,500 m) and lowest (60%) at Chhatreshwori (1,800–2,000 m). The highest total relative frequency 8.02% was at Kupinde (1,600–1,800 m) and the lowest 3.82% at Chhatreshwori (1,800–2,000 m) as compared to its associates (Fig. 5b). The frequency and relative frequency of other associates are presented in Tables S1–S6.

Figure 5. Frequency (%) (α) and relative frequency (%) (β) of Z. armatum at different locality and elevation.

The most frequently occurring associates (with > 70% of occurrence) at different localities were Aesculus indica, Alnus nepalensis, Bauhinia variegata, Berberis asiatica, Castanopsis hystrix, Colebrookeaa oppositifolia, Daphne bholua, Ficus nerifolia, Fraxinus floribunda, Grewia optiva, Juglans regia, Lyonia ovalifolia, Maesa chisia, Murraya koenigii, Persea odoratissima, Pinus roxhburghii, Prinsepia utilis, Prunus cerasoides, Pyracantha crenulata, Pyrus pashia, Quercus glauca, Q. incana, Rhododendron arboreum, and Sapium insigne (Tables S1–S6).

Abundance

The abundance of Z. armatum was almost similar and did not vary significantly in all the localities of the study area, the values ranging from 3.00 at Dhanwang and Kapurkot to 3.40 at Baghchaur and 3.44 at Kupinde (Fig. 6a). Likewise, the highest total relative abundance (15.45%) was at Baghchaur and the lowest (5.35%) at Chhatreshwori (Fig. 6b). Abundance of Z. armatum was highest (3.44) at Kupinde and relative abundance was highest (15.45%) at Baghchaur.

Figure 6. Abundance (α) and relative abundance (%) (β) of Z. armatum at different locality & elevation.

Importance Value Index (IVI)

Based on IVI values, Z. armatum was most dominant at Baghchaur and Kupinde and least dominant at Chhatreshwori. The highest IVI value of Z. armatum was 38.35% at Baghchaur and the lowest value was 14.53% at Chhatreshwori (Fig. 7). Based on IVI values, Berberis asiatica (22.88) and Daphne bholua (20.06) were the dominant species at Kapurkot and Daphne bholua at Chhatreshwori (Tables S1–S6). Likewise, Zanthoxylum armatum was dominant at Baghchaur, Kupinde, Dhanwang, and Rim with the IVI values of 38.35, 29.42, 23.09, and 21.81 respectively (Fig. 7).

Figure 7. Importance Value Index (IVI) of Zanthoxylum armatum at different locality and elevation.

Regeneration status

Natural regeneration of Z. armatum varied at different elevation/localities. Both seedlings and saplings at Kapurkot (1,200–1,400 m) had higher density and the lowest density was at Chhatreshwori (1,800–2,000 m). The total seedlings and saplings densities were 200 individuals/ha and 140 individuals/ha at Kapurkot and 100 individuals/ha and 60 individuals/ha at Chhatreshwori, respectively (Table 4). Similarly, the total seedlings and saplings densities were 180 individuals/ha and 100 individuals/ha, Baghchaur (1,400–1,600 m), 160 individuals/ha and 100 individuals/ha at Dhanwang (1,000–1,200 m), 140 individuals/ha and 120 individuals/ha at Rim (1,400–1,700 m), and 120 individuals/ha and 80 individuals/ha at Kupide (1,600–1,800 m), respectively.

Table 4 . Seedling, sapling and adult total density of Z. armatum at different locality.

SN	Samplig sites	Density (pl/ha)
		Seedling	Sapling	Adult
1	Dhanwang (1,000–1,200 m)	160	100	840
2	Kapurkot (1,200–1,400 m)	200	140	780
3	Rim (1,400–1,700 m)	140	120	1,000
4	Bagchaur (1,400–1,600 m)	180	100	1,020
5	Kupinde (1,600–1,800 m)	120	80	1,100
6	Chhatreshwori (1,800–2,000 m)	100	60	740
Average		150	100	913.33

Distribution

The distribution pattern of Z. armatum in all the localities studied were almost similar. The A/F ratio of 0.04 and 0.05 (Fig. 8) in all the localities showed that Z. armatum has random distribution in the study area. It was found scattered in patches associated with other species. Pure stands of Z. armatum were not evident anywhere in the study area. There were substantial differences in the distribution of Z. armatum, likely resulting from differences in the degree of management and disturbance as well as from different ecological factors.

Figure 8. Distribution pattern (abundance/frequency ratio, A/F ratio) of Zanthoxylum armatum at different locality and elevation.

Species diversity

Diversity values according to the Shannon and Simpson indices were higher at Rim and lower at Kupinde. The mean Shannon-Weaver diversity index (H’) which measures the diversity of Z. armatum along with its associates ranged from 2.5 to 3.08. Among the six study sites the highest species diversity (H’ = 3.08) was recorded from Chhatreshwori whereas the lowest species diversity (H’ = 2.5) was recorded at Kupinde. The mean Simpson’s diversity index (1 − D) value ranged from 0.92 to 0.95 (Fig. 9).

Figure 9. Simson’s diversity index and Shannon–Weaver index for Z. armatum and its associates.

Soil nutrient analysis showed that soil organic carbon, soil nitrogen, and soil phosphorus were highest at Chhatreshwori than other localities (Table 5).

Table 5 . Variation on soil chemical properties at different locality and elevation.

SN	Locality	SOC (%)	N (%)	P (ppm)	K (ppm)	pH
1	Dhanwang (1,000–2,000)	3.09 ± 0.12	0.36 ± 0.05	31.11 ± 1.73	135.89 ± 1.43	6.23 ± 0.03
2	Kapurkot (1,200–1,400)	3.07 ± 0.07	0.31 ± 0.04	35.11 ± 2.57	212.27 ± 8.39	5.51 ± 0.07
3	Baghchaur (1,400–1,600)	2.73 ± 0.08	0.29 ± 0.10	30.91 ± 1.29	138.67 ± 3.46	5.75 ± 0.14
4	Rim (1,400–1,700)	3.53 ± 0.16	0.49 ± 0.08	41.65 ± 0.92	428.34 ± 15.22	5.33 ± 0.15
5	Kupinde (1,600–1,800)	2.58 ± 0.09	0.30 ± 0.15	21.03 ± 3.48	121.28 ± 1.92	5.4 ± 0.09
6	Chhatreshwori (1,800–2,000)	4.87 ± 0.04	0.61 ± 0.03	57.85 ± 1.83	346.84 ± 10.36	5.73 ± 0.1

Values are presented as mean ± standard error.

SOC, soil organic carbon; N, nitrogen; P, phosphorous.

Species richness

Species composition and species richness are important indicators for assessing the biodiversity (Husch et al. 2002) and may strongly depend and/or be influenced by the applied management practices. The listing of 50 shrub and tree species in the area shows the forest is rich in diversity (Table 3). This is comparable to other studies carried out in similar forest types of Bhutan, India, and Nepal. Wangda and Ohsawa (2006) listed 78 tree species in west central part of Bhutan and Buffum et al. (2008) reported 39 tree species from the eastern part of Bhutan. Sundriyal and Sharma (1996) recorded 81 tree species in the temperate forest in Sikkim, and Shrestha et al. (2013) recorded 31 and 37 plant species in the two sites within the elevation range of 2,650–2,800 m asl in Nepal.

The lower number of species recorded at Baghchaur (1,400–1,600 m) and Kupinde (1,600–1,800 m) (Fig. 3) is because of the the nearby settlement and agriculture zones as compared to the other sites. Bhuyan et al. (2003) reported only 16 species in highly disturbed site as compared to 47 species in the least disturbed site in the eastern part of India. Sunil et al. (2011) observed 34 tree species in the low disturbed sites against tree species of 14 in the highly disturbed sites in the southern part of India. The lowest was found in plantation forests, with only 9 species in Nepal (Webb and Sah 2003). All these studies attribute the differences in the results to the degree of disturbances caused by anthropogenic activities.

There is generally a linear relationship between vegetation attributes like species richness, diversity, and ecological factors like altitude, aspect, and distance of the site from disturbance stimuli (Schuster and Diekmann 2005). A monotonic decline in the number of species with increasing elevation has often been considered a general pattern (Brown 1988; Stevens 1992). Inverse correlation between altitude and species richness in Himalayan alpines have also been established in several studies (Kala and Mathur 2002; Panthi et al. 2007; Vetaas 2000). However, the present study did not follow the similar pattern; maximum number of species was recorded at 1,400–1,700 m and 1,800–2,000 m. The high species richness may be attributed to less anthropogenic activities, higher soil moisture and greater topographic variations in habitat conditions.

Density

Several factors as lower elevation, moist habitat, resource availability, disturbance levels, moderate fragmentation together with climatic variability, fluctuations to resources and dispersal limitation may influence the population structure (Shaheen et al. 2011a). The variation in the densities along the elevation gradient at different localities might be the result of the variations in the soil nutrients and other abiotic as well as climatic factors. A study in Indonesia showed that fully opened habitat with full sun exposure during daytime may not be the suitable habitat for the natural population of Zanthoxylum acanthopodium (Junaedi and Nurlaeni 2019).

Abundance and IVI

The density and frequency values of Z. armatum were also high at Baghchaur and Kupinde. Nkoa et al. (2015) stated that the abundance is related to number (density) or frequency. The higher density and frequency might have influenced the abundance positively in this study also. The IVI depicts the importance of the species in terms of its dominance and ecological success (Misra 1968). The change in IVI among the study sites can be attributed to the change in species composition and degree of disturbance and altitude (Saravanan et al. 2019).

Regeneration status

The average seedlings and saplings densities of Z. armatum in the study area were 150 individuals/ha and 100 individuals/ha. A study by Rawat and Chandhok (2009) reported saplings and seedlings layer densities ranging from 90 to 410 individuals/ha and 50 to 510 individuals/ha, respectively. In another study, the seedling and sapling layer density ranged from 340 to 1190 individuals/ha. for seedlings and 340 to 920 individuals/ha for saplings (Saha et al. 2016). The densities of seedlings and saplings in the present study did not show any specific pattern for elevation gradient. However, a study by Gairola et al. (2008) for different high altitude Himalayan forests showed maximum seedling density throughout the altitudinal strata suggesting that the slope and aspect favor regeneration of tree species. Similarly, significant difference was observed between top hill and bottom hill positions, with the highest amount of regeneration in the bottom and lowest amount of regeneration in the top hill (Nur et al. 2016).

Regeneration status of any species is determined by the number of saplings and seedlings (Dhar et al. 1997; Singh and Singh 1992). The seedlings and saplings densities of Z. armatum were comparatively very lower than the adult densities in the study area. One reason for this kind of pattern might be due to the prematurely harvesting of the fruits before even falling off of the seeds on the ground. This severely hinders the natural seed bank stock and thus affecting the seedling and sapling density. According to the regeneration status table (Table 4), it can be said that the regeneration status of Z. armatum in the study area is fair. However, among the six localities, Kapurkot had comparatively better regeneration status than other sites and Chhatreshwori had lower regeneration potential.

Natural regeneration is a key process for the continued existence of a species in a community. The three major components of successful regeneration are the ability of species to initiate new seedlings, their survival and growth (Saikia and Khan 2013). Presence of sufficient number of seedlings, saplings and young trees in a given population indicates successful regeneration (Saxena and Singh 1984), which is frequently influenced by the biotic interactions and anthropogenic disturbances. The future composition of the forests depends on the potential regenerative status of tree species within a forest stand in space and time (Henle et al. 2004). Generally, regeneration of a species is usually affected by anthropogenic and natural factors. Seed germination rate in Z. armatum is very low (Phuyal et al. 2018) and hindered by the presence of hard seed coat; the seeds undergo a strong dormancy and may take few months to years for germination (Chadha 1976). Furthermore, the solitary seeds in the fruit also limit the quantities of seed (Singh and Rawat 2017) and lower the rate of germination. Because of the high demand of Z. armatum, its commercial cultivation in the study area is escalating during the last few years. There is also a high demand of plantlets but the supply is minimum due to lack of nurseries. District Forest and Plant Resources offices at Salyan provided free plantlets to the interested farmers, still the supply is inadequate to meet the demand. This has put a high pressure on the naturally regenerating seedling and saplings in the natural forests as the villagers uproot the seedlings and saplings from the forest to plant them in their farmland, which greatly alters the regeneration status of Z. armatum naturally. Furthermore, the fruits are prematurely harvested from the wild, probably affecting the seedbank of Z. armatum, leading to lower production of seedlings.

Regeneration of any species is confined to a peculiar range of habitat conditions and the extent of those conditions is a major determinant of its geographic distribution (Grubb 1977) and the presence of saplings under the canopies of adult trees also indicates the future composition of a community (Pokhriyal et al. 2010). The natural regeneration of Z. armatum is adversely affected by physiological dormancy and high emptiness nature of seeds as a result the seed germination is extremely rare in wild.

The reason behind less density, frequency, abundance and regeneration at Chhatreshwori might be due to the overexploitation by the local people as they collect bigger trees for their own consumption and extra income.

Distribution

It was apparent that the natural distribution of this valuable species has been shrinking in the recent years due to anthropogenic disturbances, as a similar trend for other Himalayan medicinal plants as well (Vashistha et al. 2006). Increasing market demand and unsustainable harvesting are posing serious threat to the natural population of Z. armatum. It is one of the many other medicinal plants that is collected with high preference for market as well as local use (Kunwar et al. 2015).

Anthropogenic disturbances mainly harvesting from the wild without proper care and uprooting of the seedlings and saplings for transplanting them in farmyard were found to be the major cause affecting the natural distribution of Z. armatum in the study area. Being a thorny plant, disturbance from grazing was however not apparent for the species as for many other medicinal plants.

There was a huge discrimination in the harvesting pattern and collection of Z. armatum in the study area. During field visit at the fruiting season, it was a very common scene that the plants in farmers’ farmyard were overloaded with ripe fruits while those in the wild were harvested prematurely. Plants in the farmyard were considered private and those in the forest were public so whoever saw them first would harvest haphazardly. Though the farmers were aware of the enormous economic benefits of the species, there seems to be a lack of awareness towards the conservation and sustainable harvesting of the species from the wild.

Species diversity

The mean average species diversity (Shannon index, H) value of 2.8 recorded in the present study is comparable to the results of similar investigations in different Himalayan regions: 1.53–2.88 in the western Himalayas (Gaur and Joshi 2006; Samant et al. 1998), 2.39–4.63 in the Gharwal Himalayas (Nautiyal et al. 1999), 2.5–3.10 in the trans-Himalayan alpines of Nepal (Panthi et al. 2007) and 3.13 in the alpine pastures of Kashmir, Pakistan (Shaheen et al. 2011).

The higher value of the diversity indices is an obvious indication of high species diversity and abundance (Adekunle et al. 2013). This diversity index is comparable to that found in the tropical forest of Eastern Ghats ranging between 3.76–3.96 (Naidu and Kumar 2016). Forests with Shannon index greater than 2 are considered as medium to highly diverse in terms of species (Giliba et al. 2011). Thus from the findings of this study it can be said that the study area falls in the category of forests with high diversity.

The differences of diversity between different localities and altitudes of the study area could be because of variations in the soil type, rainfall trends, anthropogenic action, land use change, and so forth. Lowest diversity noted in Kupinde could be explained by the fact that the forest is totally degraded due to road construction and land slides. Road networks increase resource extraction and encroachment into the forest leading to a reduction in biodiversity (Hitimana et al. 2004; Sundriyal and Sharma 1996). Higher species diversity and species richness at Chhatreshwori could be because of relatively high soil nutrients. Several studies have established a direct relationship between soil nutrients and species diversity. Grime (1973), Tilman and Pacala (1993) demonstrated that soil fertility has a considerable impact on species diversity. Similarly, Loreau et al. (2001) also suggested that species diversity is usually related to soil fertility. The forest was comparatively dense and moist at Chhatreshwori. Moisture is also one of the important determinants of species richness and composition (Vetaas 2000). Soil organic matter, nutrients and moisture plays an important role in the vegetation composition of any area (Tang 1990).

There have been ample studies on the population size and distribution of other medicinal plants in Nepal. But no study has been conducted previously on the population, distribution, frequency, and abundance of this species so no comparable data is available from Nepal. However, a study from India recorded a low population size of Z. armatum from the villages of Chamoli district of Uttarakhand, India; the average density was 368.2 individuals/ha. Due to low population size, it has been placed in the International Union for Conservation of Nature (IUCN) vulnerable category (Kala 2010). The same study also recorded Berberis aristata, Ficus, Grewia optiva, Pyrus pashia, Pyracantha crenulata, Quercus, etc. as the major associates of Z. armatum (Tables S1–S6).

The market price of the fruits is very good so the extensive demand of this species has put an enormous pressure on the natural population. Though the farmers have already started commercial cultivation of Z. armatum, the collection from wild has not yet decreased. Since the fruits are difficult to harvest because of the thorns, destructive harvesting without taking proper care is a common practice, creating a tremendous pressure on its existing populations in the wild. Similar scenario also prevails in Uttarakhand, India where harvesting of the entire plant before setting even flowers along with the profuse invasion from woody weeds such as Lantana has negatively impacted the natural distribution of Z. armatum (Kala 2010). Threats to Z. armatum from invasive species was however not evident in the present study. Since the plant is thorny and aromatic, grazing by livestock is not a threat to the natural population of Z. armatum, instead it also provides shelter and protection to its associated species in the natural habitats by preventing from livestock grazing and browsing (Kala 2010).

Z. armatum has been prioritized by the government of Nepal as one of the important medicinal plants for economic development with a high emphasis on cultivation and agro-technology development (DPR 2006). Owing to its great economic potential, the Karnali Provincial government, which includes Salyan, has announced a Zanthoxylum Year Program in the province’s hilly districts and all the three levels of government: federal, provincial, and local, have prioritized Z. armatum farming. The federal government also announced to celebrate fiscal year 2019–2020 as the year of Z. armatum plantation. The market price of the fruits is also very good so the extensive demand of this species has put an enormous pressure on the natural population. Though the farmers have already started commercial cultivation, the collection from wild has not yet decreased.

Diversity and distribution patterns of species are greatly affected by various factors, including area, latitude, precipitation, and temperature (Zhang et al. 2011). The large scale pattern in species distribution and physiognomy is also governed by the climate. Climate can be characterized by different variables which mainly determines the distribution pattern of species distribution in any area (Bakkenes et al. 2002).

Overall, due to the increase in population size and the overexploitation of forests, change of land use among other factors; negative impacts can result on the forest ecology including reduction of plant stock, disruption of regeneration, and loss of nutrients in harvested materials (Murkherjee and Chaturvedi 2017). Other factors that may affect the sustainability of plants are collection of premature plants, grazing, and soil erosion. Therefore, deliberate efforts should be taken by all stakeholders to ensure that these plants are used in a sustainable way.

Species composition, density, distribution and regeneration status can be considered important factors to judge the status of a forest. Z. armatum was found to be distributed randomly in Salyan district and the lower number of seedlings and saplings indicates a fair regeneration pattern. Although the dependency on natural forests for the collection of berries is gradually being replaced by cultivation in private land in the recent years, unsustainable harvesting and collection of saplings from the wild has not been stopped completely. Anthropogenic disturbances including premature harvesting and digging up of saplings was found to severely affect the natural distribution and regeneration in the study area. Increasing market demand and unsustainable harvesting procedures are posing serious threat to the natural population of Z. armatum. Thus, effective conservation and management initiatives are most important for conserving the wild genetic diversity of Z. armatum in the study area. Establishment of high-tech nurseries and free distribution of saplings to the farmers could possibly reduce the pressure on natural population and also uplift the economic status of the marginalized and poor communities. Assessment of diversity and regeneration status of species is important for their sustainable utilization, management, and conservation. Therefore, a systematic management plan is required for the conservation and sustainable utilization of this valuable species.

Supplementary information accompanies this paper at https://doi.org/10.1186/jee.22.015

Table S1. Population density (in ha), frequency, abundance, and distribution of Z. armatum and its major associates at Dhanwang (1000–1200 m). Table S2. Population density (in ha), frequency, abundance, and distribution of Z. armatum and its major associates. Table S3. Population density (in ha), frequency, abundance, and distribution of Z. armatum and its major associates. Table S4. Population density (in ha), frequency, abundance, and distribution of Z. armatum and its major associates at Baghchaur (1400–1600 m asl). Table S5. Population density (in ha), frequency, abundance, and distribution of Z. armatum and its major associates at Kupinde (1600–1800 m asl). Table S6. Population density (in ha), frequency, abundance, and distribution of Z. armatum and its major associates at Chhatreshwori (1800–2000 m asl).

We are thankful to Mr. Krishna Pun from District Plant Resources Office, Salyan for his help during the field work. Special thanks to Prof. Dr. Ram Kailash Prasad Yadav, Head, Central Department of Botany, Tribhuvan University for his encouragement.

NTFPs: Non-timber Forest Products

MAPs: Medicinal and Aromatic Plants

RD: Relative Density

RF: Relative Frequency

RA: Relative Abundance

IVI: Importance Value Index

SOC: Soil organic carbon

PKJ and SR conceptualized and supervised the research. NP collected and analyzed the data and wrote the manuscript. PKJ, PPR, and SR critically commented and approved the final version of the manuscript. The authors read and approved the final version of the manuscript.

This study was supported by the Dabur Nepal CSR Fellowship (Late Sri Ashok Chand Burman) 01/2016’.

All data involved in this study are available from the corresponding authors upon request.

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The authors declare that they have no competing interests.