DOI: 10.5061/dryad.kwh70rz06
title: Data from: Insect herbivory and herbivores of Ficus species along a rainforest elevational gradient in Papua New Guinea

description: Classic research on elevational gradients in plant-herbivore interactions
 holds that insect herbivore pressure is stronger under warmer climates of
 low elevations. However, recent work has questioned this paradigm, arguing
 that it oversimplifies the ecological complexity in which plant-insect
 herbivore interactions are embedded. Knowledge of antagonistic networks of
 plants and herbivores is however crucial for understanding the mechanisms
 that govern ecosystem functioning. We examined herbivore damage and insect
 herbivores of eight species of genus Ficus (105 saplings) and plant
 constitutive defensive traits of two of these species, along a rainforest
 elevational gradient of Mt. Wilhelm (200 – 2700 m a.s.l.), in tropical
 Papua New Guinea. We report overall herbivore damage 2.4% of leaf area,
 ranging from 0.03% in Ficus endochaete at 1700 m a.s.l. to 6.1% in F.
 hombroniana at 700 m a.s.l. Herbivore damage and herbivore abundances
 varied significantly with elevation, as well as among the tree species,
 and between the wet and dry season. Community-wide herbivore damage
 followed a hump-shaped pattern with the peak between 700 and 1200 m a.s.l.
 and this pattern corresponded with abundance of herbivores. For two tree
 species surveyed in detail, we observed decreasing and hump-shaped
 patterns in herbivory, in general matching the trends found in the set of
 plant defences measured here. Our results imply that vegetation growing at
 mid-elevations of the elevational gradient, i.e. at the climatically most
 favourable elevations where water is abundant, and temperatures still
 relatively warm, suffers the maximum amount of herbivorous damage which
 changes seasonally, reflecting the water availability.
description: We performed the study along the Mt. Wilhelm elevational gradient
 in the Central Range of Papua New Guinea. The surveyed part of the
 transect, ca. 25 km long, comprises six study sites spaced regularly at
 500 elevational metre intervals, from the lowest study site located within
 the lowland floodplains of the Ramu River at 200 m a.s.l. (05° 44′ S,
 145°20′ E) to the elevational limit of Ficus distribution at 2700 m a.s.l.
 (05°48′ S, 145°09′ E). Mean annual temperature (measured by data loggers
 Comet R3120 placed in forest understorey) decreases from 27.4°C at the
 lowland site to 8.37°C at the tree line at a constant rate of 0.54°C per
 100 elevational metres (see Sam et al. 2019 for more detail). Air humidity
 measured by the same dataloggers ranged between 90 and 100% at all
 elevations, however the rainfall was not evenly distributed during the
 year (see Sam et al. 2019 for more detail). Average annual precipitation
 is 3,288 mm (measured at local meteorological station, data provided by
 Phil Shearman) in the lowlands, rising to 4,400 mm at 2,700 m a.s.l., with
 a distinct condensation zone between 2,500 and 2,700 m a.s.l. (Sam et al.
 in prep).  Mean monthly precipitation along the gradient is 315 mm, and
 mean monthly precipitation between the two survey (i.e. wet season)
 periods was 398 mm. The elevational gradient, further description of study
 sites, climatic and habitat characteristics are published elsewhere
 (Tvardikova 2013, Sam & Koane 2014, Sam et al. 2015, Sam et al.
 2019). Unfortunately, no tree genus was distributed
 along the entire elevational gradient. Therefore, we selected species from
 the widely distributed genus Ficus (Moraceae) as a model system, which
 allowed us to work from the lowlands up to 2,700 m a.s.l. (05° 48′ S,
 145°09′ E, Table 1). Ficus is an exceptionally species-rich genus, with
 New Guinea as the center of its diversity (Ronsted et al. 2008). Ficus
 also has high diversity (at least 75 species) and abundance (typically
 &gt;5% of stems with DBH ≥1 cm) along the Mt. Wilhelm elevational
 gradient (L. Sam, unpubl. data). Ficus has an upper elevational limit at
 2,900 m a.s.l at Mt. Wilhelm, as well as elsewhere (Berg & Corner
 2005). Some species of Ficus (e.g. F. hahliana Diels, 1935) have
 particularly wide elevational ranges (Berg & Corner
 2005). We selected and tagged experimental saplings
 belonging to 4 – 6 evergreen Ficus species at each elevational study site
 (Table 2). As far as possible, we selected the locally most common species
 which had also broad elevational ranges. The selected plant species did
 not produce any exudates or sugar droplets attracting ants. We selected
 between 14 and 20 saplings per elevational study site, i.e. 3 – 5 per
 plant species and elevation or 105 saplings along the gradient (Table 2).
 For statistical independence, we allowed at least 80 m between any pair of
 individuals. We visually assessed saplings of the focal species and
 selected individuals that looked similar, had approximately 500 leaves
 growing within a well-developed crown 2.5 – 4 m above the ground. The
 saplings did not have any ant nests and did not have any abnormally high
 herbivory or fungal damage. Average leaf-sizes of the selected species
 ranged from 16.31 to 154.10 cm2, and two species (Ficus arfakensis King,
 1888 and F. endochaete Summerhayes, 1941) had significantly different
 sized leaves at some elevations (Table 2). At the
 beginning of the experiment, at the end of the dry season between
 31-Aug-2014 and 3-Nov-2014 (Table 1), we first collected all arthropods
 (described below). Then we counted all leaves present at the sapling (to
 be able to estimate the total leaf area of the sapling) and further we
 proceeded with collection of leaves for herbivory measurement. To assess
 herbivore damage, we randomly selected two branches (with ca. 30 leaves
 each; 57 ± 5.6 (mean ± SE) in total per sapling, i.e. up to ca. 15% of
 standing foliage) per sapling. We clipped these two branches and collected
 all leaves from them. This survey period denotes the first point
 measurement (t = 0) of herbivore damage. A branch clipping (i.e.
 simulation of an artificial herbivory by a vertebrate herbivore) from a
 grown sapling should have only short-term and non-significant effect on
 subsequent herbivory measurement and plant growth in our experiment
 (Strauss et al. 1996, Seldal et al. 2017).  Using a 50
 x 50 cm2 white backgrounds, we took photographs of all collected and
 flattened leaves from each sapling (i.e. we took as many photos as needed
 to photograph all collected leaves). Using Adobe Photoshop CS6 (Adobe
 Systems Inc., USA). We first outlined the missing edges on the
 photographed leaves based on the expected shape and whitened or blackened
 various damages or holes on the leaves. Using various guides, google image
 searches, and our previous expertise, we carefully distinguished leaf
 damage caused by chewing and mining herbivores (which was whitened), while
 we did not consider fungal damage or mechanical leaf damage (which we
 blackened). We then turned the photos to black and white pictures. We used
 ImageJ version 1.47 (National Institute of Health, USA) to calculate the
 remaining leaf area (a, in cm2), the extrapolated leaf area without any
 herbivore damage (b), and the area lost to herbivory (c = b - a). We then
 estimated the percentage of leaf–area loss as c/b x 100. We calculated the
 herbivory loss as loss in % of are per leaf and in cm2 per 100cm2. These
 two values were highly correlated, so we used only % of herbivory per leaf
 in analyses and figures. We conducted a second survey
 of herbivore damage, on the very same saplings as used in the first
 survey, approximately six months after the first survey (between
 21-Mar-2015 and 11-May-2015, Table 1) at the end of the rainy season of
 2014/2015. Similarly, to the first survey, we conducted the arthropod
 collection first. Then we collected all leaves from each sapling into a
 bag. We randomly selected 55-60 of leaves (to match the samples size of
 the first survey), photographed them on the same white background as
 earlier, and analysed them in the same way as in the first
 survey. After each survey, we obtained values for 57 ±
 5.6 (mean ± SE, min = 50, max = 95) leaves per sapling. We weighed all
 scanned and the remaining leaves to calculate the total leaf area for each
 sapling from the second survey. We used measured mean leaf size per
 sapling multiplied by the calculated number of leaves on sapling to obtain
 total leaf area of the sapling the beginning of the experiment (first
 survey)  The amount of herbivore damage was assessed blindly; viz. the
 field collector (BK) and the research assistant (AM) handling the leaves
 did not know that the study aimed to measure leaf damage, preventing any
 bias in leaf selection. We measured herbivory on at
 least three saplings of each plant species at each elevational study site
 (Table 2) and in average of 438 leaves per plant species and elevation (or
 114 ± 11.8 leaves per sapling), which is a number recommended by previous
 studies (Zhang et al. 2015, Kozlov & Zvereva 2017). By analysis of
 11,448 leaves, our study belongs to those larger (Bito et al. 2011,
 Metcalfe et al. 2014, Kozlov & Zvereva 2017). The number of
 individual saplings sampled per species and elevational study site seemed
 to be sufficient as their herbivory between individuals did not differ
 significantly in majority of cases (Table S1) and the model with tree
 number as a random effect differed only marginally from model without
 random effect (AIC = 2.12). We therefore decided to use mean herbivory per
 species at each elevation and survey in further analyses.
 Arthropod survey Arthropod censuses were
 performed destructively during both surveys at the same individuals that
 were used for herbivory measurements, just before we started measurements
 of herbivorous damage and leaf collections. We slowly lowered the trunk of
 the sapling above a mosquito net, wrapped it to the mosquito net and
 sprayed with fast knock-down insecticide (Mortein®). After a while, we
 shook foliage firmly, opened the net and collected all arthropods
 (&gt;1mm) and preserved them in vials filled with DNA grade ethanol.
 In laboratory, arthropods were counted, identified into three feeding
 groups (i.e. “chewing herbivores”, “predators”, “other arthropods” who
 have no relationship to herbivorous damage as we measured it and do not
 act as mesopredators – e.g. adult flies, pupae, adult Lepidoptera etc.,
 sap sucking herbivores). Abundances of chewing herbivorous arthropods was
 then calculated as number of individuals in feeding guild per m2 of leaf
 area. Plant defensive trait measurements
 We measured plant defensive traits for two plant species (out of
 the 8 surveyed for herbivory). For each of these two species at each
 elevation, we collected leaf discs from five individuals. Where possible,
 the same individuals were used for herbivory and insect survey and trait
 measurements, but up to two individuals per plant species and elevation
 were sampled from different individual. These were usually individuals
 with smaller amount of leaves than was acceptable for the herbivory and
 herbivore survey. We collected two 4.5 cm2 leaf discs per leaf from 20
 young, but fully expanded leaves for each individual, avoiding the central
 vein (1 g of dry weight in total on average). The discs were air-dried and
 stored in silica gel. Half of them were used for the analysis of
 triterpenes and half of them for measurement of physical traits.
 Physical Traits: We measured trichome density and specific leaf
 area (SLA). Trichome density and SLA are parameters of leaf morphology
 with a possible impact on leaf-chewing insects. The total number of
 trichomes per 10 mm2 and their average length was measured on five leaf
 discs per individual using ImageJ (ver.1.48). Values for dorsal and
 ventral sides of the discs were combined. Specific leaf area (SLA) was
 calculated as the area per unit mass of five dried leaf discs collected
 from five leaves of known diameter for each individual.
 Triterpene analysis: Dried powdered leaf tissue samples were
 ground with methanol in TissueLyser. Then they were centrifugated and
 aliquot was saved for further use. Terpenoids in aliquot were measured on
 a Dionex Ultimate 3000 LC system equipped with an Open XRS autosampler and
 coupled to a Q Exactive Plus Orbitrap mass spectrometer (Thermo Fisher
 Scientific, San Jose, CA, USA). A reversed phase Kinetex was used for
 separating the analytes. For further measurement details, refer to the
 Methods S1 in Supplementary information.