Comparison of Morphological Characters from Bornean Lowland Nepenthes Inflorescences: Case Study from Post-Mining Area of Sintang Regency

. The existence of inflorescence is an important aspect of Nepenthes conservation. However, it ’ s usually abandoned because they are rarely found in nature. This research aimed to compare the morphological characters of the Bornean lowland Nepenthes inflorescences based on species and sexes. It was conducted in the post-mining area of Sintang Regency, West Kalimantan Province, Indonesia, from October 2022 to February 2023. Inflorescence samples were collected from males and females of N. ampullaria , N. bicalcarata , N. gracillis , N. mirabilis , N. rafflesiana var. typica. Inflorescence morphometry characters consist of inflorescence length, peduncle length, rachilla length, flower length, tepal length, tepal width, and number of pistillate/staminate per inflorescence. The results showed that morphological characters of Bornean lowland Nepenthes inflorescence differ between species and sexes. N. ampullaria is characterised by having the highest number of flowers of any Nepenthes species. In contrast, N. bicalcarata is characterised by inflorescence length and peduncle length. Males and females have different morphological characteristics. Males have significantly longer and wider tepals and more flowers per inflorescence than females of Bornean lowland Nepenthes .


Introduction
Nepenthes (Nepenthaceae) is a tropical pitcher plant with leaf modifications into jug-shaped organs called pitcher [1].Nepenthes pitcher has a vital role in fulfilling nutritional needs.This plant lives in a habitat with poor macronutrients such as nitrogen and phosphorus [2].
The nutritional gap is filled by pitchers, which trap and extract arthropod prey (carnivorous plants), leaf litter (detritivorous plants), and others for alternative nutrition sources [4,5].
Nepenthes habitat is spread out on the palaeotropical area from Madagascar to New Caledonia, with the centre of its diversity on Southeast Asia [1].To this day, Nepenthes species consist of more than 140 species in the world and are mostly found in the Philippines (40 species), Sumatera (35 species), Borneo (34 species), and Peninsular Malaysia (19 species) [5].
Altitudes are one of the important roles in Nepenthes categorisation [2].If Nepenthes lives on a habitat altitude between 0-1100 m asl, it is categorised as lowland Nepenthes.Meanwhile, it will be categorised as highland Nepenthes if it lives on a habitat altitude of more than 1100 m asl [1].Based on [5], the highland of Borneo (including Kalimantan, Sarawak, Sabah and Brunei Darussalam) is an important habitat for Nepenthes because of 21 species (62%) compared to 13 species (38%) in the lowland habitat.However, Cross et al. state that lowland habitat faces more anthropogenic threats than highland habitats, which threaten Nepenthes' existence in this area [6].
Nepenthes breeding occurs as a result of fertilisation of male and female flowers.Nepenthes is a dioecious species.Males and females are separated into different individuals [7].This condition can be a conservation problem if the flowering time of Nepenthes is not in the same period.Post-mining area on Kalimantan Island is one of the suitable habitats for Nepenthes to live and breed [8].Macro nutrients are scarce in post-mining areas.Nepenthes in this habitat adapt by increasing their breeding function through inflorescences.
Morphological description of Nepenthes inflorescence is usually being part of the new species description.Nevertheless, the inflorescence description of well-known species is hard to find and is based on qualitative data from field observation or herbarium specimens.Based on [1], comparing Nepenthes inflorescences is an important clue to species identification but is rarely used because of its periodicity.Some researchers interested in the Nepenthes flower usually describe the condition of the flower qualitatively [8,10].The research on morphometrics of lowland Nepenthes inflorescences is an interesting topic to be done.This research aims to compare the morphological characters of Bornean lowland Nepenthes inflorescences based on species and sexes.The results of this research can be a good addition for Nepenthes conservation strategy.It will give a different perspective on Nepenthes conservation act which usually focuses on pitchers rather than dioecious flowers.

Materials and method
This research was done on the post-mining area in Sintang Regency West Kalimantan Province Indonesia (Coordinate 0°2'28,482" N and 9°28'57,396 E) (Fig. 1).[8] state that the post-mining area is ideal for lowland Nepenthes in the Sintang Regency.This area is a former heath forest (kerangas) converted to illegal gold mining operated by the local community.After the gold mine was not operating, it was then used as a sand and stone mining area for local community construction.Some areas that were not carried out sand and stone mining operations were then revegetated by several types of plants including Nepenthes species.The gold mine operated from the 1990s to the early 2000s.Then the sand and stone mining area is carried out since the gold mine has not been operating until now (in 2023).
This research was conducted from October 2022 to February 2023; it is a rainy season in the Sintang Regency area.[5] state that wet conditions (in rainy season or humid areas) can help Nepenthes to maintain the evaporation from its pitcher and help wet the peristome for prey capturing mechanism, which leads to Nepenthes flourishing after fulfilling the macronutrients.
The exploration method with purposive sampling was used to observe Nepenthes on flowering conditions.For the location of Nepenthes inflorescences used as research stations, the researchers applied three plots of 10 m x 10 m on three stations which separated 100 m of minimum distance to observe Nepenthes (Fig. 1).This plot size (10 m x 10 m) is an optimal minimum area on Nepenthes observation [8].The researchers used 122 males and 105 females on the morphometric data from five lowland Nepenthes species.We used a guidance book from [1] and other relevant sources to identify Nepenthes species found.Based on Handayani [9], males are more commonly found on N. mirabilis than females and can be a reference for other lowland Nepenthes which share the same habitat with this species.All the Nepenthes inflorescence used in this research are safe (not harmful), except one of each Nepenthes flower used as the photograph sampled (one flower per species).

Fig. 1. Map of study location in Sintang District, West Kalimantan Province, Indonesia
The samples used in this research consist of males and females of N. ampullaria; N. bicalcarata; N. gracilis; N. mirabilis, and N. rafflesiana var.typica, which can be seen in Fig. 2. We used 16 males and two females of N. ampullaria, seven males and four females of N. bicalcarata, 34 males and 34 females of N. gracilis, 32 males and 32 females of N. mirabilis, and 33 males and 33 females of N. rafflesiana var.typica.The number of flowers is varied on each species depending on its existence in nature.Qualitative characters of Nepenthes inflorescences, such as colour, odour, and hairiness, were also recorded.For all these data, a binary quantitative score was applied (0/1) [10].The binary code for colour was 0 for green and 1 for another colour, then green.Meanwhile, the binary code was defined as 0 for no odour and 1 for any odour that could be smelled from inflorescences.The binary code for hairiness was 0 for no hair found and 1 for any hair in Nepenthes inflorescences.
The differences in inflorescence morphometry data on each Nepenthes were analysed using Brown Forsythe and followed by the Games Howell test at the 0.05 level using SPSS 16.00.Morphometry data and qualitative data on flower morphology were analysed using PCA (PAST software version 4.03) to determine the comparison between inflorescences.

Results
The morphological characters of Bornean lowland Nepenthes inflorescences show differences in the character of each Nepenthes species.Morphological characters differ significantly between species, and parameters are distinguished by different notations (Fig. 4).
Inflorescence length data showed that all Nepenthes species have a significantly different average length (Fig. 4A).Nepenthes with the longest inflorescences are males of N. bicalcarata with an average of 72.46 cm, while the shortest are females of N. gracilis with 13.54 cm long.There are significant differences between sexes in N. ampullaria, N. gracilis, and N. mirabilis inflorescences, while there were no significant differences in N. bicalcarata and N. rafflesiana (Fig. 4A).
Tepal length characters show significant differences between Nepenthes species (Fig. 4E).The longest tepal length average is in males of N. rafflesiana; it is 9.77 mm.It is compared to females of N. gracilis which only have a 2.83 mm tepal length average.There are no significant differences in mean tepal length between the sexes in N. bicalcarata, while all other species have significant differences.In N. ampullaria, N. gracilis, N. mirabilis, and N. rafflesiana, the average tepal length of male flowers is longer than that of female flowers (Fig. 4E).Qualitative data of Nepenthes inflorescences consist of a variety of colors, the presence of odor and hair.The qualitative data of Nepenthes inflorescences can be seen in Table 1.All Bornean lowland Nepenthes flowers have a variety of colors (not only green).For example, N. ampullaria has yellowish-green flowers ranging from rachillas to tepals.N. bicalcarata flowers have light green on rachillas, while tepals are brownish-red.N. gracilis flowers have dark green on the rachilla, while tepals are brownish-red.N. mirabilis flowers are light green on the rachilla, while tepals are white to brownish red.N. rafflesiana is dark green on rachilla, while tepals are white to brownish-red (see Fig. 2).
Every Borneo lowland Nepenthes inflorescence has a distinctive odour.However, it generally can be observed in the morning, when the smell decreases during the day.N. ampullaria and N. rafflesiana have delicate hair on their rachilla, tepals, male column and female ovary.Meanwhile, N. bicalcarata, N. gracilis and N. mirabilis have no hairs on their flower.Furthermore, only hairiness data can be continued with PCA analysis because no colour and odour data variation exists (Fig. 5).This PCA results can explain 80% from all variables used.Based on PCA analysis, the morphological characteristic of males and females are different; it can be seen from the non-overlap grouping of male and female data.N. bicalcarata generally has a higher inflorescence, rachilla, and flower length than other species.Males and females of N. rafflesiana and males of N. mirabilis are characterised by higher peduncle length, number of flowers, petal length, and petal width.Meanwhile, males and females of N. gracilis, females of N. ampullaria, and females of N. mirabilis have opposite characters with lower values of all parameters.

Discussions
Bornean lowland Nepenthes with the most extended average inflorescences length are N. bicalcarata, followed by N. mirabilis, N. rafflesiana, N. ampullaria, and N. gracilis.N. bicalcarata is known to have an alternative nutrition source such as leaf litter and other sources which lead this species to adapt more with their habitat including the length of inflorescences [4].Inflorescence and peduncle length are suggested to have correlations to avoiding conflicts between prey and pollinators.This indication based on research by El-Sayed et al. [11] mention that the distance between the inflorescence and the nearest traps affects pollination's success in the genus Drosera's carnivorous plants.The farther the distance between the inflorescence and trap, can minimise the possibility of pollinating insects being caught as prey.
In addition to inflorescence length, flower color also affects the possibility of inflorescence being observed visually [9].All Bornean lowland Nepenthes flowers have various colors (other than green), contrasting them with the background (Table 1).Flower color is one of the characteristics that attract pollinators to visit and spread pollen to various types of flowers; insects carry out this pollination.[12] stated that the more flower color contrasts with the background, makes it easier to be found by pollinators.
Based on Kato [13], non-flying insects are very rarely able to help pollination because of the antibiotic substances' presence produced by metapleural glands that harm pollen.The flower length of Bornean lowland Nepenthes shows that N. bicalcarata has the longest flowers among other species.The flower length of N. bicalcarata aligns with the body length of N. bicalcarata, which is known as the largest among other lowland species [1].Moreover, the association between N. bicalcarata and the ant Camponotus schmitzi can be the reason for the length of the flowers compared to other species.C. szhmitzi may interfered N. bicalcarata pollen production to some degree.
Tepals are nectar producers in Nepenthes flowers.Tepal size indicates the amount of nectar produced by Nepenthes flowers [9].The larger the tepal of Nepenthes flowers, the greater the number of nectars produced.This nectar production is an attraction for pollinator insects so that they can spread the pollen.The largest tepal size (length and width) in Bornean Lowland Nepenthes is owned by N. rafflesiana.Males of N. rafflesiana have an average tepal length of 9.77 mm and width of 7.14 mm, while females have an average length of 8.26 mm and width of 5.12 mm.It is inversely proportional to the size of N. gracilis tepals, with the smallest size among other species.The average length of males of N. gracilis is 4.17 mm, and the width is 2.59 mm.While for females' average length is 2.83 mm, and the width is 1.49 mm.
Interestingly, all males' tepal sizes (length and width) of Bornean lowland Nepenthes are larger than females (Fig. 2, Fig. 4 E&F).The tepal size of males of N. ampullaria, N. gracilis, N. mirabilis, and N. rafflesiana is significantly larger than females.Based on [14] males can attract more visitors (pollinator, prey or herbivore) than females due to the larger tepal size, estimated nectar production, and other complex interactions of dioecious flowers in Eurya japonica.It is also supported by [13] who states that the amount of nectar produced by male tepals of N. gracilis is more than females, with a total of 85 μl compared to 26 μl each.However, this statement needs to be confirmed by further research.Besides, there has been no research that explains the preferences of visitors to Nepenthes' male and female flowers, so that it will be interesting for future research.
Nepenthes species are known to produce flowers in large numbers in support of the breeding process [1].The lowland Nepenthes that produced the most flowers were male flowers of N. ampullaria, with an average of 202.06 flowers per inflorescence, while females of N. gracilis owned the fewest with an average of 76.41 flowers per inflorescence.The results showed that males of all Bornean lowland Nepenthes were more abundant than females.It is due to the need for male flowers to spread pollen to be able to fertilise female flowers.When Nepenthes are able to produce more numbers and bigger sizes of males compared to females and females succeed in becoming fruit then the conservation act of Nepenthes can be categorised as successful.

7 BIO
Web of Conferences 91, 01012 (2024) ICGRC 2023 https://doi.org/10.1051/bioconf/20249101012Morphological characters of Bornean lowland Nepenthes inflorescences differ between species and sexes.N. ampullaria has the highest number of male flowers than others.N. bicalcarata is characterised by inflorescences and rachilla length.N. gracilis was characterised by the lowest measurement values for all characters compared to other species.Males and females have different morphological characteristics.Males have significantly longer and wider tepals and more flowers per inflorescence than females of Bornean lowland Nepenthes.Inflorescences are an important part of Bornean lowland Nepenthes conservation.