After being introduced outside of their natural distribution area, invasive alien plant species (IAS) invade and threaten biological diversity, ecosystem, and human well-being in another region and the processes of this invasion are known as biological invasions (UN Environment Programme 2014). The rising biological invasion has a higher influence on biodiversity (Kohli et al. 2004), ecosystem functioning (Pejchar and Mooney 2009), agricultural productivity, health, and socioeconomic stability globally (Paini et al. 2016; Seebens et al. 2017). IAS are spreading into new areas that are not native to them due to human-induced factors, either purposely or accidentally (Ehrenfeld 2010), and this is happening more frequently in the changing world, having an impact on the environment, agriculture, and livelihoods (Paini et al. 2016; Pejchar and Mooney 2009; Simberloff et al. 2013). As a result, it is seen as a major contributor to environmental degradation which threats biological diversity and ecosystem services (Simoncini et al. 2019).

Along with climate change, several changes such as a rapid change in land use and land cover including the construction of roads, prolific growth in international trade, a steady increase of labor migration, and a flourishing tourism sector facilitate an increase in human mobility (Ghimire 2016), which helps in the spread of invasive species directly and indirectly (Pyšek and Richardson 2010). Short generation times, greater environmental tolerances, high fecundity, and strong dispersion abilities make invasive species highly suited to change (Bradley et al. 2009). Similarly, the trade of ornamental plants and seeds facilitates biological invasions (Pyšek and Richardson 2010). However, the intensity of biological invasions is likely to increase in the future with changing climate and increasing anthropogenic disturbances in the mountains (Petitpierre et al. 2016). Biological invasions and climate change act simultaneously, and the relationship between these two parameters in this changing world is considered a serious threat to biodiversity (Mainka and Howard 2010; Walther et al. 2009). The change in the climatic conditions and ecosystem services regulate the regional shifts, species distribution, and composition across landscapes (Intergovernmental Panel on Climate Change 2022). Similarly, humans also play a significant role in reshaping socio-ecological systems in desirable ways, altering the species composition and diversity (Biggs et al. 2022).

In Nepal, the changing climate has increased the invasion risk at a higher level. Since the majority of invasive species are native to tropical America, their distribution is also expanding in the southern lowland with tropical to subtropical climates (Shrestha 2016; Tiwari et al. 2005). Many of the species are introduced by gardeners, horticulturists, foresters and the returned British Gorkha soldiers which has become a severe problem since they are invading Nepal’s lands (Kunwar 2003). Many of these species spread rapidly, invading native plants and altering the biological community and ecological systems, such as nutrient cycling, energy flow, or the hydro-dynamic features of its recipient ecosystem (Huston 2004). The topography of Nepal is characterized by its extreme altitudinal variation, which leads to the formation of diverse climatic zones (Bhattarai et al. 2004), making it susceptible to the establishment of IAS from a wide range of their origins. Altogether, 183 species of naturalized flowering plants have been reported from Nepal and 28 of them are considered invasive (Shrestha et al. 2021; Shrestha and Shrestha 2021). The majority of these IAS are herbs (including herbaceous climbers) or shrubs; none of the alien tree species in Nepal is invasive (Shrestha 2016). Among these 28 IAS, five species are most common and troublesome in Nepal’s protected areas: Chromolaena odorata, Mikania micrantha, Lantana camara, Ipomoea carnea, and Parthenium hysterophorus (Bhatt et al. 2021).

The distribution patterns of invasive species in Nepal and their colonization shows that it may have a serious impact on the biodiversity, particularly in the tropical and subtropical region, if steps are not taken to control it immediately. In addition, altitude significantly influences the composition of plants inhabiting a specific ecosystem due to the variation in biotic and abiotic factors that occur along with elevation. As altitude rises, plants encounter drastic abiotic transformations including temperature reduction, oxygen depletion, atmospheric pressure drop, intensified irradiation, accelerated wind speeds, increased snow accumulation, dry and frozen soils, and others (Körner 2003). It has been suggested that the high elevation presents a diverse range of abrupt environmental transitions within relative proximity (Körner 2003; Peterson et al. 1997), and many non-native species tend to establish their distribution boundaries along these elevational gradients. Despite the growing concern about plant invasions, little research has been conducted on the invasion of high mountain systems. Moreover, the limited number of studies that have been published demonstrated the significant variability between mountain areas regarding the altitudinal distribution of alien plants. For instance, Western and Juvik (1983) reported a less number of both introduced and native plant species along altitudinal gradients in roadside habitats in Hawaii. Similarly, Tassin and Riviere (2003) observed a comparable pattern for introduced plant species in the higher regions of an altitudinal transect on the tropical island of La R_ѐunion. On the other hand, Johnston and Pickering (2001) documented a substantial presence of non-native plant species at subalpine elevations exceeding 1,500 m asl in the Australian Alps. Unfortunately, there is a lack of studies on the local and regional level about the distribution of invasive species along the altitudinal gradient in the mountain areas of Nepal.

Precise and reliable information concerning the presence and distribution of IAS is of utmost importance, as it provides a fundamental basis for evaluating the potential risks associated with them and facilitating the implementation of appropriate response strategies. This study has been conceptualized to attain the distribution of IAS along with elevation in Makawanpur district, central Nepal. We examined the diversity and distribution patterns of IAS across various elevation levels and habitat types. The objective of this study was to understand the diversity and distribution trends of IAS along the elevation gradient. Furthermore, we sought to identify the preferred habitat types for IAS distribution along the elevation gradient, specifically focusing on roadside areas, forests, and cultivated lands.

Study area

The study was conducted on roadsides along an elevation gradient from Hetauda (500 m asl) to Simbhangyang (2,400 m asl) (27°27’57.89”N 85° 2’34.22” E–27°36’11.35”N 85° 4’19.15” E) along the Tribhuvan highway of Nepal (Fig. 1). It is the oldest and the first highway in Nepal which was the first serviceable road connection with India operated in 1959. This route is the arterial road from the lowlands to the highland of the Makawanpur district. This study area comprises tropical, subtropical, and temperate climatic zones (District Development Committee 2015). The tropical area stretches up to an elevation of 1,000 m asl, characterized by hot and humid summers and mild winters. On the other hand, the subtropical region spans from 1,000 to 2,000 m asl, featuring hot and humid summers and mild to cool winters. Similarly, above 2,000 m asl, the climate is characterized by mild to warm summers and cool to cold winters. Our study sites represent a diverse vegetation composition concerning elevation gradient. The tropical zone is characterized by Sal and riverine forests at the southern lower belts where Shorea robusta, Terminalia chebula, T. bellirica, Adina cordifolia, Acacia catechu, Dalbergia sissoo, etc. thrive very well. The subtropical region is dominated by Schima-Castanopsis, Chir pine, and Alder forests, with dominant species including Schima wallichii, Castanopsis indica, C. stribuloides, Pinus roxburghii, and Alnus nepalensis. Similarly, temperate forests are dominated by Rhododendron arboreum, Myrica esculenta, Lyonia ovalifolia, Quercus lanata, and P. walliachiana. The average annual temperature in the study region is 11.5°C, and the average annual precipitation is 1,781 mm. However, the difference in precipitation between the driest and wettest seasons is 487 mm (Department Of Hydrology and Meteorology 2017).

Figure 1. Map of study area indicating roadside survey from Hetauda (500 m asl) to Simbhangyang (2,400 m asl).

Vegetation sampling

Study plots were systematically located at 100 m elevation intervals from 500–2,400 m asl. In each step of 100 m elevation, we placed three plots, one on the roadside, one in the forest, and one on cultivated land. On the roadside, starting from the end of the paved or other hard artificial surfaces of the road, plots were established. In the forest, sample plots were located 100 m horizontally away from the roadside, and nearby cultivated land (between 100–150 m from the road) was selected for sampling on cultivated land. A total of 60 rectangular plots, each measuring 10 m × 5 m, were sampled across 20 altitudinal points in the study area. The rectangle plots were fixed such that the long axis was placed parallel to the road. In each plot, we recorded all IAS. Sampling was conducted between October 2022 and December 2022. Identification of invasive species was carried out by following Adhikari et al. (2022) and also by comparing specimens deposited at National Herbarium and Plant Laboratories (KATH), Godawari Lalitpur. The elevation of each plot was determined and the slope was measured with a clinometer. The percent tree canopy cover was estimated by using a densitometer (Lemmon 1957).

Data analysis

Species richness was calculated using the total number of shrubs and herbs recorded in each 100 m wide elevation band. The classification of species as native or alien was based on the literature by Adhikari et al. (2022). All the alien plants mentioned in the above literature are categorized as IAS. We determined the elevational range for species present in different habitats. For each species, we plotted the elevation of the lowest and highest plot where the species occurred. To explore the altitudinal richness patterns of invasive alien plants in different land-use sites, we performed linear regression analyses to detect the relationships between species richness and elevation for IAS. Also, we performed linear regression analyses to detect the relationships between the density of IAS on different sites and elevations. Additionally, linear regression plots have been utilized to demonstrate the correlations between tree canopy cover and the richness and density of IAS. All the statistical analyses were accomplished using the Statistical Package for Social Sciences version 16.0 (SPSS Inc., Chicago, IL, USA) and graphs were prepared in OriginPro.

A total of 18 invasive plant species belonging to eight families have been recorded at 53 sampling plots (20 roadside, 17 forests, and 16 cultivated lands). Seven sampling plots, comprising three within the forest and four within the cultivated lands, were free from alien invasive species. Among eight families Asteraceae with nine species was found to be dominant, followed by Fabaceae with three species; while the remaining 6 families were represented by single species (Table 1). The life form pattern distribution showed that 67% (12 species) of the plant species were herbs, followed by 28% (5 species) shrubs and 5% (1 species) of climbers. Concerning the longevity of the plants, 61% of the plant species were annuals while 39 % were perennials. In terms of families, Asteraceae exhibited a higher number of alien species richness along the altitudinal gradient compared to other families, which were predominantly confined to elevations below 1,600 m (Table 1).

Table 1 . Characteristic information of the studied IAS in study area.

Species	Local name (common name)	Family	Origina	Life form
Ageratina adenophora (Spreng.) R.M.King & H.Rob	Kalo Banmara (crofton weed)	Asteraceae	Mexico	PS
Ageratum conyzoides L.	Gandhe (billygoat)	Asteraceae	C & S America	AH
Ageratum houstonianum Mill.	Nilo Gandhe (blue billygoat)	Asteraceae	Mexico & C America	AH
Amaranthus spinosus L.	Kande Lude (spiny pigweed)	Amaranthaceae	T America	AH
Argemone mexicana L.	Thakal (Mexican poppy)	Papaveraceae	T America	AH
Bidens pilosa L.	Kalo Kuro (black jack)	Asteraceae	T America	AH
Chromolaena odorata (L.) R.M.King & H. Rob	Seto Banmara (siam weed)	Asteraceae	Mexico, C & S America	PS
Galinsoga quadriradiata Ruiz & Pav.	Chiltange (shaggy soldier)	Asteraceae	Mexico	AH
Lantana camara L.	Kime Kanda (lantana)	Verbenaceae	C & S America	PH
Hyptis suaveolens (L.) Poit.	Tulsi Jhar (bush mint)	Lamiaceae	T America	AH
Mikania micrantha Kunth.	Lahare Banmara (mile-a-minute)	Asteraceae	C & S America	PC
Mimosa pudica L.	Lajjawati (sensitive plant)	Fabaceae	S America	PH
Oxalis latifolia Kunth.	Chari Amilo (purple wood sorrel)	Oxalidaceae	C & S America	PH
Parthenium hysterophorus L.	Pati Jhar (parthenium)	Asteraceae	S America	AH
Senna occidentalis (L.) Link	Panwar (coffee senna)	Fabaceae	T America	AS
Senna tora (L.) Roxb.	Tapre (sicklepod senna)	Fabaceae	C America	AH
Spermacoce alata Aubl.	Alu Pate Jhar (broadleaf buttonweed)	Rubiaceae	T America	PH
Xanthium strumarium L.	Bhede Kuro (cocklebur)	Asteraceae	T America	AH

IAS: invasive alien plant species; C: central; S: south; T: tropical; PS: perennial shrub; AH: annual herb; PH: perennial herb; PC: perennial climber; AS: annual shrub.

^aShrestha (2016); Tiwari et al. (2005).

The most widespread alien species was Bidens pilosa in the study sites which occurred in 66% of the plots followed by Ageratina adenophora, Parthenium hysterophorus, and Chromolaena odorata which occurred in 49%, 47%, and 36% of the study plots, respectively (Table 2). The IAS showed great variation in their density. The density of A. adenophora was found higher (48,733 plants ha-1) followed by A. houstonianum (22,933 plants ha-1) B. pilosa (20,500 plants ha-1) and C. odorata (16,133 plants ha-1) (Table 2).

Table 2 . Frequency and density of IAS in study area.

Species	Frequency (%)	Relative frequency	Density (plants ha–1)	Relative density
Ageratina adenophora	49	12.74	48,733	29.141
Ageratum conyzoides	6	1.47	1,967	1.176
Ageratum houstonianum	38	9.80	22,933	13.713
Amaranthus spinosus	2	0.49	100	0.060
Argemone mexicana	2	0.49	67	0.040
Bidens pilosa	66	17.15	20,500	12.258
Chromolaena odorata	36	9.31	16,133	9.647
Galinsoga quadriradiata	6	1.47	2,100	1.256
Hyptis suaveolens	8	1.96	2,300	1.375
Lantana camara	17	4.41	7,333	4.385
Mikania micrantha	11	2.94	1,133	0.678
Mimosa pudica	11	2.94	2,600	1.555
Oxalis latifolia	19	4.90	11,900	7.116
Parthenium hysterophorus	47	12.25	9,967	5.960
Senna occicentalis	9	2.45	2,067	1.236
Senna tora	17	4.41	2,833	1.694
Spermacoce alata	28	7.35	10,133	6.059
Xanthium strumarium	13	3.43	4,433	2.651

IAS: invasive alien plant species.

IAS were distributed all along the elevation gradient in the study area i.e. from 500 m to 2,400 m. The maximum richness of IAS was observed at 500 m elevation. After this, a gradual decline in their richness was observed. Among the 18 species, only 3 species such as A. adenophora, P. hysterophorus, and B. pilosa occurred above the 2,200 m (Fig. 2). Distribution of 6 species such as Galinsoga quadriradiata, Argemone mexicana, Amaranthus spinosus, Xanthium strumarium, Mikania micrantha, and Ageratum conyzoides was found restricted below 800 m. However, A. adenophora was the only species that did not occur below 1,200 m, it was found up to higher elevations. Species such as P. hysterophorus, and B. pilosa colonize a wide range of elevation gradients (Fig. 2).

Figure 2. Elevation range of invasive alien plant species in the study area.

Species richness

The distribution of IAS richness showed statistically significant with elevation. It decreased with increasing elevation (p < 0.0001: Fig. 3). Maximum species richness occurred at 500 m indicating that low-elevation regions had a significantly higher number of alien species as compared to high-elevation regions with different habitat types. The number of IAS per habitat also declined strongly with increasing elevation. For example, at 500 m, there were 11 species while only one species was recorded at 2,400 m at the roadside. Similarly, in the forest, there were seven species at 500 m while only one species was recorded at 2,200 m whereas above that no IAS occurred in the forest. In addition, eight species were recorded at 500 m altitude in cultivated land, but only one species was noted at 2,100 m. However, above 2,100 m no IAS occurred in cultivated land. In contrast, there was no difference in species richness between road and other sites, but there was a significant interaction with elevation (p < 0.0001; Fig. 3); as shown in the distinct slopes of the regression lines for forest and cultivated land (Fig. 3).

Figure 3. Altitudinal pattern of species richness of invasive alien plant species in different habitats (roadside, forest, and cultivated land) in the study area.

Density

The density of IAS at different habitat sites also showed a statistically significant relationship with elevation. It decreased with increasing elevation (p < 0.0001: Fig. 4). In all habitats, low-elevation regions had a significantly higher density of alien species as compared to high-elevation regions. The density of IAS was higher in the road site followed by the cultivated and forest site. This pattern occurred throughout the elevation range.

Figure 4. Relatioship between invasive alien plant species density and elevation in different habitats (roadside, forest, and cultivated land) in the study area.

The density and species richness of IAS showed a significant negative correlation with tree crown cover (Fig. 5A, B). IAS were found mostly in the open canopy, degraded forest, and open land where light penetration is high. The species richness and abundance of IAS were found higher in open canopy sites. Similarly, the species richness of IAS showed a significant positive correlation with density.

Figure 5. (A) Relationship between invasive alien plant species (IAS) density and crown cover. (B) Relationship between IAS richness and crown cover.

The present study area was found to be inhabited by a total of 18 IAS belonging to eight families. Among them, Asteraceae, with nine species, is the most dominant family followed by Fabaceae (3 species). Some previous studies on the floristic composition and diversity of IAS in tropical and Himalaya regions of India have also described Asteraceae as a dominant family (Kosaka et al. 2010; Reshi et al. 2017; Sekar 2012). In our case, it would be the obvious reason that Asteraceae is the most species-rich family with 10 IAS in Nepal (Shrestha and Shrestha 2021). Alternatively, the plant species belonging to this family can tolerate the anthropic disturbance regimes and also have the potential nitrogen-fixing capacity that would help to colonize easily in vulnerable ecosystems (Sekar 2012).

Among 18 IAS in the study sites, the highest frequency and wider distribution range of Bidens pilosa (500 m to 2,200 m) might be due to its high seed production capacity and effective seed dispersion mechanism via human and animal vectors (Budmajji and Soloman Raju 2018). Plants with rapid growth, high adaptability, and a large amount of seed production capacity spread quickly throughout the world (Yang et al. 2019). B. pilosa was found in all types of microhabitats particularly, with efficient colonized forms on cultivated land of our study area. Due to its high palatability among livestock, B. pilosa is not considered a troublesome plant by farmers in subsistence farming systems that integrate livestock rearing (Siwakoti et al. 2016). Along with B. pilosa, Parthenium hysterophorus also exhibited a wider distribution range in our study area, ranging from 500 m to 2,300 m. P. hysterophorus is considered one of the most problematic weeds globally because of its exceptional ability to reproduce (producing around 20,000 seeds per plant), quick germination, rapid growth, and its allelopathic properties (Adkins and Shabbir 2014). The seeds of this plant can germinate across a broad range of temperatures, with soil moisture content being the primary limiting factor for germination (Bajwa et al. 2018; Navie et al. 1998; Tamado et al. 2002). The alien plants that successfully reach high elevations are characterized by their ability to thrive across various climatic conditions, possess extensive altitudinal distributions, and initiate their colonization from lower elevation points of introduction (Alexander et al. 2011).

With increasing elevation in the study area, there was a decline of IAS richness and coverage in all habitats, for instance, roadside, forest areas, and cultivated land. Studies around the world consistently demonstrate a pattern of declining non-native species richness from a maximum at the lowest elevation, regardless of the specific broad bioclimatic region. This trend has been observed in all biomes of the world (Alexander et al. 2011; Seipel et al. 2012) and regional studies from cold arctic systems (Lembrechts et al. 2014), temperate mountains in North America (Andersen et al. 2015), Europe (Medvecká et al. 2014) and China (Zhang et al. 2015), Mediterranean mountains (Barros and Pickering 2014), and Himalaya region of India (Ahmad et al. 2018; Kosaka et al. 2010; Manish 2021). In low-elevation regions, more invasive species richness is expected due to favourable climates for colonization, low geographic and climate isolation, high human disturbance, easy road/transportation access, human population, less environmental heterogeneity, and less extreme habitats (Becker et al. 2005). Because the roads and trails are regarded as key pathways for invasion into mountains (Fuentes et al. 2010; Lembrechts et al. 2017), they can provide opportunities for non-native species establishment and dispersion (Becker et al. 2005). The decline of exotic species richness at high elevations can be further explained by the lack of pre-adaptations to survive in extreme high-altitude conditions, along with limited colonization of high-elevation regions (Alexander et al. 2016). Mountains serve as environmental filters, where non-native species initially take root in the lowlands and the only species adapted to lowland conditions can expand their presence to higher elevations (Haider et al. 2010). Environmental constraints (Alexander et al. 2011; Seipel et al. 2012), a lower propagule pressure (Quiroz et al. 2011), or decreased human land use (hence lower disturbance, Seipel et al. 2012), all contribute to an alien species’ gradual drop-out with increasing elevation. Further limiting the spread of invasive species in the highland include low temperatures, poor ecosystem productivity, short growing seasons, and low precipitation at high elevations (Manish et al. 2017). Our study area is no different, as the higher elevation range of the sampling site is situated in a temperate climatic zone and experiences extremely cold (even snowfalls) during winter. Furthermore, higher elevational sites exhibit limited human settlements and transportation infrastructure within the area which limit the occurrence of non-adapted plant species from tropical regions.

This study indicated that throughout the elevation range, the density of IAS was higher at the roadside followed by cultivated land and forest area. Studies revealed that the degree of anthropogenic disturbance is a key predictor of IAS richness (Jauni et al. 2015; Liu et al. 2005). Studies suggested that anthropogenic factors can influence the time, amount, and pathways of IAS, and environmental factors can affect how many species can persist when introduced to new locations (Dyer et al. 2017; Wang et al. 2020, 2022). We found that the distribution rate of IAS on roadsides and cultivated land was high, and presented a pattern of spreading from road to cultivated land. The areas of lower elevation and proximity to roads are highly populated, resulting in the most significant disruptions to cultivated land, road construction, and urban development. Furthermore, roads significantly contribute to the spread of non-native species by creating corridors for dispersal providing suitable habitats, and containing reservoirs of propagules for future episodes of invasion (Arianoutsou et al. 2013; Dimitrakopoulos et al. 2022; McDougall et al. 2018; Pauchard and Alaback 2004). Shrestha et al. (2019) provided evidence that road verges not only serve as suitable microhabitats for colonization but also function as dispersal corridors for IAS like Parthenium hysterophorus, which is one of the most prevalent species in our study sites. When talking about cultivated land, worker outmigration in search of better opportunities has left a considerable amount of farmland abandoned in hilly regions of Nepal, allowing for the natural regeneration of vegetation, including IAS (Jaquet et al. 2015). That might be the case in our study area as well because we observed plentiful abandoned cultivated land where IAS are efficiently colonized in the sampling sites. Agriculture fallow lands with rich nutrients are generally prone to invasion shortly after abandonment (McGrath et al. 2001).

IAS are predominantly found in the open canopy, degraded forest, and open land where light penetration is high (Ghimire et al. 2020). In this study, we also recorded higher species richness and abundance of IAS in open canopy sites. Forest areas with dense canopies serve as physical obstacles that impede the dispersal of IAS, while the prevailing light and moisture conditions within these forests create environmental barriers that hinder the establishment of such species (Mavimbela et al. 2018). A closed canopy restricts seed dispersal and hampers the growth of early successional species due to limited light penetration within the forest (Lawes et al. 2004). Our result agreed with previous research indicating that roads serve as conduits for IAS, facilitating their spread from disturbed areas to cultivated lands in temperate forests (Pauchard and Alaback 2004; Trombulak and Frissell 2000). Despite harsh environmental conditions for growth in higher elevations, some IAS can establish themselves if there is an ample supply of seeds. Furthermore, our findings align with existing evidence highlighting roadsides as the initial sites of colonization for IAS, offering insights into potential invasion ranges (Parendes and Jones 2000; Pauchard and Alaback 2004; Trombulak and Frissell 2000).

The study revealed that species richness and density of IAS declined along with increasing elevation. The majority of IAS are introduced at low elevations, and as climatic conditions worsen and anthropogenic pressure decreases along the elevation gradient, they are gradually filtered out. IAS were found mostly on the roadside than in other habitats, open canopy, degraded forest, and open land with abundant sunlight penetration. Roads significantly contribute to the spread of non-native species by creating corridors for dispersal and disturbed areas to infiltrate adjacent natural vegetation. Asteraceae had the higher IAS richness as compared to other families which were found restricted below 1,600 m. This study revealed that only Ageratina adenophora, Parthenium hysterophorus, and Bidens pilosa occurred above the 2,200 m asl. Parthenium hysterophorus and Bidens pilosa are two of these species that colonize a wide range of elevation gradients. Further research in other higher elevations is strongly encouraged to investigate the true extent of invasion patterns. Such studies would enhance our understanding of underlying mechanisms and enable us to effectively mitigate the impact of biological invasions. Nonetheless, early detection and continuous monitoring certainly be helpful in managing plant invasion within mountain systems.

IAS: Invasive alien plant species

We are grateful to Professor Dr. Bharat Babu Shrestha, Central Department of Botany Tribhivan University, Nepal for his constructive comments in the early version of this manuscript.

BG and DK conceptualized the research. DK and BP collected and analyzed the data and wrote the manuscript. BG critically commented and approved the final version of the manuscript. The authors read and approved the final version of the manuscript.

Not applicable.

The data that supports the findings of this study will be made available upon reasonable request.

Not applicable.

Not applicable.

The authors declare that they have no competing interests.