DOI: 10.5061/dryad.zs7h44jj0
title: Why do understorey Licuala palm fruits turn from red to white and then black when ripe?

description: Licuala ferruginea Becc., a tropical forest understorey palm, is observed
 to have fruits that appear red in colour when unripe, turning
 pink, then white, purple and finally black in colour as they
 ripen. We monitored 13 fruiting palms in rainforest fragments and recorded
 the consumption of fruits by animals via camera traps. We also
 documented the fruiting phenology of two palms in the nursery. In
 the rainforest fragments, a Cream-vented Bulbul (Pycnonotus simplex) was
 observed plucking a mature purple fruit from a L. ferruginea
 palm, before flying away with the fruit in its beak. This was the
 only bird that was observed feeding on the mature fruit. A range
 of mammals, dominated by edge species such as the Long-tailed
 Macaque and Wild Boar, were observed to consume L. ferruginea
 fruits indiscriminately across all five colour stages,
 thereby limiting the dispersal of the fruits. Forest bulbul gape
 sizes also matched the fruit size, suggesting that forest bulbuls
 are the likely dispersers of the palm in the original forest
 where edge species are not in high densities. We further posit
 that the initial phase of red fruits, with high contrasting red
 reflectance against a green foliage background, might be a form
 of early advertisement to birds. The fruit then turns pink and
 white, which have high green reflectance and is less contrasting, thereby
 reducing the conspicuity of the fruit. This allows the fruit to
 ripen with high fructose and glucose content, and turn purple and
 black, which are known visual cues for birds. This study provides
 indicative support for the dispersal syndrome hypothesis and
 highlights the potential effects of forest fragmentation on
 plant-frugivore interactions.
description: <strong>Study overview</strong>
 This study was conducted in the field and nursery as follows: (a)
 natural lowland primary and secondary rainforest fragments of Singapore
 and (b) growing plant house in the nursery with understorey-lighting
 conditions (Figure 1). Within the lowland rainforest fragments at Bukit
 Timah Nature Reserve (BTNR; between 1.3463 and 1.3627 latitude and
 103.7695 and 103.7876 longitude), Central Catchment Nature Reserve (CCNR;
 between 1.3355 and 1.4157 latitude and 103.7741 and 103.8380 longitude)
 and Singapore Botanic Gardens Rainforest (SBG; between 1.3111 and 1.3149
 latitude and 103.8151 and 103.8170 longitude), clusters of <em>L.
 ferruginea</em> palms were identified (Figures 2 I-II). A total of
 17 fruiting <em>L. ferruginea</em> palms were identified and
 monitored over two years, from 2021 to 2022, using camera traps. This
 sought to identify animals that were visiting and feeding on the fruits,
 and hence understand the frugivores that were dispersing the fruits
 of <em>L. ferruginea</em> palms. At the
 same time, two individual <em>L. ferruginea</em> palms
 collected from the wild were grown at the Pasir Panjang Nursery (PPN) in
 Singapore under partial shade to mimic understorey lighting conditions of
 natural habitat (Figure 2 I and III). The two palms were monitored
 continuously and their fruiting phenology were documented to gain insight
 into fruit colour and biochemical changes (i.e. sugar content) associated
 with the different stages of fruit development. This was necessary as it
 was difficult to monitor the complete fruiting phenology of the L.
 ferruginea palms in the wild due to intense predation pressure on the
 fruits. In the nursery, 16-16-16 NPK fertilizer was applied once every two
 weeks to the palms, thereby providing sufficient required nutrients to
 enable fruiting all year round, as opposed to one or fewer fruiting cycles
 per year as observed in the wild.
 <strong>Capturing animal visitation and feeding of
 fruiting Licula ferruginea palms via camera traps</strong>
 A total of 17 camera traps (Bushnell Trophy Cam HD Aggressor and
 Reconyx Hyperfire HC500) were set up between 5 May 2021 and 20 Jun 2022,
 one camera trap for each of the identified fruiting <em>L.
 ferruginea</em> palms in the BTNR, CCNR and SBG. Each camera trap
 was mounted 30-50cm above ground level and facing the infructescences of
 the fruiting palm, and left in the field until there were no longer any
 fruits. This resulted in a total accumulated effort of 1270 camera trap
 nights. For each animal appearance that triggered the camera traps, a
 10-to-15-second video was captured and later analysed to identify the
 animal. Observed animal behaviours in each captured video were categorised
 into two activities: (1) feeding, and (2) visiting, which refers to
 animals that were captured in the camera trap, but were not observed to be
 feeding on the fruit. Captured videos of the same animal with interval
 less than 15 minutes were considered as replicates and counted as one
 independent capture. In addition to the anmal visitation and feeding data
 collected from 17 <em>L. ferruginea</em> palms in the wild,
 additional opportunistic observations were also made on the
 two <em>L. ferruginea</em> palms cultivated from seed in
 PPN. <strong>Documentation of fruiting phenology,
 spectral study and chemical testing of two Licuala ferruginea palms grown
 in Pasir Panjang nursery</strong> <em>Photo
 documentation</em> The two <em>L.
 ferruginea </em>palms in the PPN were checked closely for any
 indication of flowering. As soon as the flower buds emerged, the palms
 were monitored weekly. RGB photos were taken weekly for the same palms
 from the same angle to document flowering and fruit development. When
 fruits were set, Bugdorm-2120 insect rearing tents were used to protect
 developing fruit bunches from being predated by frugivores (Figure
 3). <em>Sampling of fruits</em>
 One bunch of fruits from each of the <em>L.
 ferruginea</em> palms was randomly selected on the 4th, 5th, 8th,
 9th, 13th, 14th, 15th, 16th and 17th week after fruit set, for
 quantitative measurement of fruit sizes, spectral reflectance and sugar
 content. Due to limited number of fruit bunches produced by
 two <em>L. ferruginea</em> palms, only up to a total of three
 fruit bunches were sampled from each palm per fruiting cycle. The data
 over several fruiting cycles were pooled to obtain full dataset for
 complete fruit development with nine time points as indicated
 formerly. <em>Fruit size
 measurement</em> Collected fruits were measured
 to obtain width and length of fruits, which were then plotted
 using ggplot and geom_smooth in RStudio 2021.09.0 build 351 to study the
 changes of fruit length and width over time.
 <em>Colour of fruits</em> Relative
 reflectance UV-VIS range (250-700 nm). Relative reflectance spectra in
 250-700 nm range of collected fruits were measured using Ocean Insight
 Flame Miniature Spectrometer with attached Ocean Insight PX-2 Pulsed Xenon
 Light Source positioned at 45o of fruit surface. Average relative
 reflectance spectra for all fruits were calculated using Rstudio 2021.09.0
 build 351. Relative reflectance in VIS-NIR range
 (400-900 nm). Reflectance spectra in 400-900 nm range of the fruits
 attached on the individual palms were measured weekly using the handheld
 SPECIM IQ device from the 4th week until 20th week after fruit set. Pixel
 of fruits in collected his images were labelled using Scyven (Scyllarus
 Visualisation Environment) developed by National ICT Australia Limited
 (NICTA). Relative reflectance spectra of all labelled fruit pixels were
 extracted. Average relative reflectance spectra for all fruits were
 calculated using Rstudio 2021.09.0 build 351.
 <em>Qualitative colour change in response to
 pH</em> Anthocyanin has been reported to be the
 main colour pigment responsible for colour of many palm fruit species
 (Hazir et al., 2012, Martin et al., 2017). To further confirm the nature
 of colour pigments of<em> L. ferruginea</em>, pigments of
 collected ripe fruits were extracted using acidified ethanol (400 mL 92%
 ethanol : 75 mL acetic acid), with incubation time of 4 hours (Silva et
 al., 2019). Glacial acetic acid and 10% KOH solution were added separately
 to sample solutions of extracted pigments. RGB photos of colour of pigment
 solutions in different pH conditions were taken.
 <em>Fruit nutrient content</em>
 For each event of fruit sampling, flesh of collected fruits were
 separated from seeds and combined to make three 5-gram composite samples.
 The samples were sent to BV-AQ (Singapore) laboratory for testing of
 hexoses (fructose, galactose, glucose), disaccharides (lactose anhydrous,
 maltose, sucrose) and total sugar using gas chromatography.
description: # Why do understorey Licuala palm fruits turn from red to white and then
 black when ripe?
 [https://doi.org/10.5061/dryad.zs7h44jj0](https://doi.org/10.5061/dryad.zs7h44jj0) This study looks at the changes in Licuala ferruginea reflectance, sugar contents, and animal fruigvory/visitation across the different stages of ripeness of Licuala ferruginea fruits.  Files included in this repository: This dataset contains data on the relative reflectance of Licuala ferruginea fruits at various stages of ripening (**Licuala fruit relative reflectance (Handheld Specim IQ).csv** and **Licuala fruit relative reflectance (Ocean Insight PX-2).csv**), fructose, glucose and total sugar content of L. ferruginea fruits at various stages of ripening (**Licuala fruit sugar content.csv**) and camera trap data for L. ferruginea (**Licuala camera trap triggers.csv**). ## Description of the data and file structure Licuala fruit relative reflectance (Handheld Specim IQ).csv * Relative_Reflectance_Wavelength_nm: Wavelength of light at which relative reflectance values are collected * Week X ([colour]): Relative reflectance of fruit surface (= Actual reflectance of fruit surface / Actual reflectance of white teflon board tile against which fruit is tested), for fruits at X weeks after fruit set and their corresponding colour as it appears to unaided human eyes. Licuala fruit relative reflectance (Ocean Insight PX-2).csv * Relative_Reflectance_Wavelength_nm: Wavelength of light at which relative reflectance values are collected - Stage X ([colour]): Relative reflectance of fruit surface (= Actual reflectance of fruit surface / Actual reflectance of white teflon board tile against which fruit is tested), for fruits at various stages (X) of ripening (higher roman numerals corresponding to a more mature fruit) weeks after fruit set and their corresponding colour as it appears to unaided human eyes. (Note: the values at the extreme ends of the instrument's range, approximately &lt;230nm, may appear quite haphazard) Licuala fruit sugar content.csv * Week: Number of weeks since fruit set (this should match the week number in the fruit reflectance csv file) - Fructose_g/100g: Grams of fructose in fruit pulp, per 100g of fruit pulp sample, in wet weight * Glucose_g/100g: Grams of glucose in fruit pulp, per 100g of fruit pulp sample, in wet weight - Total Sugar_g/100g: Total weight in grams of hexose sugars (fructose, galactose, glucose) and disaccharides (lactose anhydrous, maltose, sucrose), per 100g of fruit pulp sample, in wet weight. Only fructose and glucose were detected in the fruit samples. Licuala camera trap triggers.csv * Species: Identity of species recorded - Abundance: No. of individuals recorded at that instance * ObservedBehaviour: Either Feeding or Visitation - Ripeness: Roman numerals indicate the ripeness stage of the Licuala ferruginea fruits when the animal was recorded, with corresponding colour of the fruits.