Nutrient and metal concentrations in Nepenthes macfarlanei Hemsl. (Nepenthaceae) from a Malaysian montane forest

Palaeotropical pitcher plants (Nepenthes) are a fascinating evolutionary case but their nutrient relationships are poorly known. To determine nutrient and metal concentrations in Nepenthes macfarlanei from Peninsular Malaysia, and contribute to our understanding of nutrient relationships in this genus, plants were sampled from the Genting Highlands – a disturbed montane forest location. Whilst many foliar nutrients showed typical concentrations, the foliar N concentration was, surprisingly, greater than most lowland species although the N:P ratio (c. 24) indicated nitrogen limitation in line with other studies on Nepenthes. Of particular note was the variable but high (< 240 μg g) lead concentrations that have not been reported in carnivorous plants before. This data adds to our understanding of the nutrient relationships of carnivorous plants and shows that they may accumulate high concentrations of certain metals.


Introduction
The montane forests of Peninsular Malaysia are a rich source of plant diversity (Saw, 2010). One example is the Genting Highlands, well-studied due to its proximity to the capital city Kuala Lumpur (Piggot, 1977;Stone, 1981;Chua and Saw, 2001;Bedawi et al., 2009;Ng et al., 2012). Whilst much of the area has been converted to buildings to service the tourist industry, there are still fragments of disturbed and natural vegetation. Of the 14 species of Nepenthes found in Peninsular Malaysia (McPherson and Robinson, 2012;Ghazalli et al., 2020;Tamizi et al., 2020), three species, viz. Nepenthes macfarlanei Hemsl., Nepenthes ramispina Ridl. and Nepenthes sanguinea Lindl. (along with natural hybrids between them) are found here (Shivas, 1983;Chua and Saw, 2001;Bourke, 2003). These pitcher plants present a fascinating case of plant evolution whereby they have developed a container ('pitcher') at the end of their leaf blades to catch insects and other nutritious materials to supplement their soil-derived nutrients (Thorogood et al., 2018). Through examination of nutrient concentrations and nutrient ratios, it appears this genus of pitcher plants are nitrogen limited (Brearley and Mansur, 2012;Mansur et al., in revision). However, there is little data from montane Nepenthes species to further support this suggestion. Additionally, there is little data on metal concentrations in Nepenthes species (but see van der Ent et al., 2015) and the current development in this area may lead us to use this genus for biomonitoring of pollution in the future. The objective of this study was to determine AcademicPres Notulae Scientia Biologicae nutrient and metal concentrations in Nepenthes macfarlanei and contribute to our understanding of nutrient relationships in this genus.

Materials and Methods
Description of the study site The Genting Highlands are found at 3º26'N, 101º47E in Peninsular Malaysia on the Selangor-Pahang border at around 1750 m a.s.l. and based on a granitic geology (Whitmore and Burnham, 1969). Tropical montane forest is the dominant vegetation found in the area although around the summit has been heavily disturbed by roads and high-rise buildings for the development of the well-known tourist resort.
Sampling design Three plants of Nepenthes macfarlanei ( Figure 1) were sampled from around the summit of Gunung Ulu Kali (the Telecom Tower) in February 2011. A single leaf (all leaves were estimated to be less than one year old) was collected from each plant, dried in a forced air oven and stored dry until analysis. About 650 mg of leaf material was digested in 2.5 ml concentrated sulphuric acid with a lithium sulphate/selenium (100:1) catalyst at 375 °C for 4 hours, diluted with deionized water, and analysed for nutrient concentrations on a Dionex ICS-5000 Ion Chromatography System (for N) or a Thermo iCAP 6300 Duo Inductively Coupled Plasma Optical Emission Spectrometer (for all other elements).

Results and Discussion
The results for the macronutrients showed typical concentrations (Figure 2) although nitrogen was high when compared with other studies (Osunkoya et al., 2007;Brearley and Mansur, 2012), which is surprising given that nitrogen availability generally decreases with elevation in tropical regions (Tanner et al., 1998). High nitrogen concentrations at this location could be due to localised deposition from traffic and other sources.
However, the other macronutrients fell within the range for other Nepenthes species from Borneo (Osunkoya et al., 2007;Brearley and Mansur, 2012;van der Ent et al., 2015;Mansur et al., in revision). The nitrogen-tophosphorus ratio was around 24 suggestive of nitrogen limitation (using the criteria of Ellison, 2006) although was wider than other studies from lowland (Osunkoya et al., 2007;Brearley and Mansur, 2012) or submontane (Mansur et al., in revision) elevations -this was due to the high nitrogen rather than low phosphorus. There is very little data on micronutrients in Nepenthes species but, again, our data (Table 1) were all within the range for typical 'healthy' plants (Pugnaire, 2001) across a range of habitats and were similar to previous studies from peat swamp forest (Brearley and Mansur, 2012) but were lower than concentrations from plants growing in ultramafic soils where greater foliar nickel, chromium and cobalt were shown (van der Ent et al., 2015). Granitic soils do not present high concentrations of these metals, but the acidic montane soils can increase the availability to plants of many metals. However, Nepenthes seems to exclude these metals to a certain extent, possibly due to their small root systems that limits nutrient uptake (van der Ent et al., 2015). Metal concentrations within the Nepethens macfarlanei plants were variable and, interestingly, lead was much higher than other studies with c. 240 µg g -1 as a maximum concentration which is particularly high and could be due to the density of traffic at this location. Highly variable concentrations of these micronutrients suggest that uptake via roots may not be the main pathway of uptake and direct deposition via leaf surfaces or via the pitchers may be more important (Rotkittikhun et al., 2006). The high lead concentration from this study site contrasts markedly with a site in central Borneo where lead was below detection limits in the majority of samples but there was a single plant of Nepenthes albomarginata with 19.6 µg g -1 foliar lead (Brearley, unpubl. data). Adlassnig et al. (2009) have shown that pitcher plants other than Nepenthes take up the metallic elements iron and manganese, so the uptake from insects highly enriched in such metals could be another possibility but would need additional data on elemental concentration in insects to test this.

Conclusions
This paper adds another data point to our understanding of Nepenthes pitcher plant nutrient relationships and further suggests primary nitrogen limitation of this genus whilst noting additional micronutrient composition of the plants and the potential to take up metallic elements to unexpectedly high concentrations.