DOI: 10.5061/dryad.ncjsxkssq
title: Data from: Does mining waste concentration in the soil interfere with leaf selection by Acromyrmex subterraneus (Formicidae)

description: Revegetation programs are proposed to recover the soil and biodiversity of
 disturbed sites, this being the case of the Rio Doce basin, Brazil. This
 region was hugely affected by a mining waste dam disruption, whose leakage
 on the soil altered its chemical and physical characteristics, and
 consequently the physiology and performance of plants. The expected
 alterations of the plants can make them more attractive for leaf-cutting
 ants, as lower water content induces an increase of non-structural
 carbohydrates. In this context, we evaluated whether Acromyrmex
 subterraneus workers differentiate among plants grown on soil with
 different mining waste concentrations. Leaf discs from plants grown in
 soil containing 0, 25, 50, 75 and 100% of mining waste were simultaneously
 offered to ant colonies in a foraging arena. The number of transported
 discs from each mining waste concentration was recorded until all discs of
 any concentration had been transported. Leaf selection assays were
 repeated after 30 days due to the novelty effect phenomenon. Leaf
 thickness, water, starch and total soluble carbohydrate contents were
 determined. Leaf discs from plants grown in soil with 100% of mining waste
 concentration were preferentially selected in both selection assays. Leaf
 thickness and water content were significantly lower in plants from the
 aforementioned treatment, while starch and total soluble carbohydrates
 were higher. Results suggest that seedlings implanted in sites with high
 mining waste concentration are under high predation risk. Revegetation
 programs must measure the impact of leaf-cutting ants as both herbivorous
 and soil ecosystem engineers, for the best management of these insects.
description: 2.1 | Species of study and maintenance of colonies
 The experiment was conducted in MirmecoLab at Universidade
 Federal de Juiz de Fora (UFJF), Brazil, under controlled conditions of
 temperature and relative humidity (25 ± 1ºC and 75 ± 5% R.H.). We used 10
 colonies of A. subterraneus collected in December 2016, and kept in a
 closed system composed of three compartments interconnected by transparent
 plastic tubes, corresponding to: foraging arena, fungus chamber and waste
 chamber. The fungus chamber contained a 1 cm layer of plaster at the
 bottom to maintain the moisture for the symbiotic fungus. All colonies had
 approximately 200 cm³ of fungus garden. Acalypha wilkesiana Müll.Arg.
 (Euphorbiaceae) leaves were provided daily to the colonies. Colonies were
 leaf-deprived for 24 hours prior to the experimental assays.
 2.2 | Plant cultivation and leaf discs preparing
 Seeds of Bauhinia rufa (Bong.) Steud. (Fabaceae) from the Plant
 Physiology Laboratory at UFJF, were germinated in Carolina Soil®
 substrate, composed by Sphagnum peat moss, expanded vermiculite, dolomitic
 limestone, agricultural plaster and traces of NPK fertilizer. After
 germination, 25 seedlings, whose height was 30 cm, were transferred to
 pots (25 L) with eutrophic substrates at different concentrations. The
 substrate consisted of a mixture of soil (latosol type, topsoil layer) and
 sand (1.5:1 by volume). this mixture, it wTo as added a chemical
 fertilizer, Forth Frutas®, composed by 12% N, 5% P2O5, 15% K2O, 1% Ca, 1%
 Mg, 5% S, 0.2% Zn, 0.2% Fe, 0.05% Cu, 0.06% B, 0.08% Mn and 0,005% Mo (1 g
 fertilizer/L substrate) and an acidity corrector, AgroSilício®, composed
 by 36% CaO and 9% MgO (2 g acidity corrector/L substrate). Mining waste
 from Fundão dam was then mixed at different concentrations to the
 eutrophic substrate by volume: 0, 25, 50, 75 and 100%, using a concrete
 mixer. The mining waste was collected at the Risoleta Neves hydroelectric
 power plant’s dam, located in the Santa Cruz do Escalvado municipality and
 stored in a greenhouse at UFJF for two months before the seedlings were
 planted. About 10.5 million m3 of mining waste from Fundão dam was
 retained at the reservoir of this hydroelectric (RAMBOLL, 2019). Bauhinia
 rufa seedlings were kept in the greenhouse since its germination in August
 2017 and after 13 months, the 25 individuals were taken for the
 experimental assays. This species was chosen because it has the
 characteristics required for revegetation programs (Chaer, Resende,
 Campello, de Faria, & Boddey, 2011), and it was the one most
 attacked by leaf-cutting ants in an area where the revegetation program
 was in progress (P.H.P.Peixoto personal communication, Feb 1st,
 2016).  Leaves from plants grown at different
 concentrations of mining waste were collected for the experimental assays,
 and cut in 0.5 cm diameter discs using a metal punch avoiding the leaf’s
 veins. Each leaf disc was marked with a color corresponding to each
 concentration of mining waste, using permanent markers (Edding®) (Camargo,
 Forti, Lopes, Andrade, & Ottati, 2007). Both sides of the discs
 have been marked to allow their identification when transported.
 2.3 | Effect of mining waste concentration on leaf
 selection To verify whether the concentration of mining
 waste in the soil influences the selection of plants, leaf selection
 assays were performed. Each ant colony was connected by a glass bridge (50
 x 3 cm) to a foraging arena containing 10 leaf discs of one plant
 individual from each concentration of mining waste (total of 50 leaf
 discs). The discs were randomly arranged in a semicircle form and the
 assay was started by opening the connection between the colony and the
 bridge. We recorded how many discs were transported in each of the
 concentrations of mining waste, one by one, until one of the
 concentrations had all the discs transported. This procedure was repeated
 five times in each colony, thus each plant was tested only once by colony,
 and all plants were tested in all colonies. This constituted the first
 phase, called "Unknown", of the selection process’ evaluation,
 which lasted 17 days. In order to control for novelty
 effects (Roces & Hölldobler, 1994), we repeated the experiment in
 a second phase, named "Known", following the same experimental
 procedures adopted for the Unknown phase. It took place 30 days later and
 lasted 19 days. The time lapse between the Unknown and Known phases is
 related to the workers' estimated average lifetime of 90 days
 (Camargo et al., 2007). During these 30 days, colonies were treated with
 A. wilkesiana leaves. 2.4 | Leaf analysis
 The thickness of the B. rufa leaves was measured from plants kept
 in the greenhouse with a digital caliper gauge, using 3 fresh leaves from
 5 plants of each concentration of mining waste (total of 75 leaves). The
 thickness of the leaves was determined in points without veins.
 The water content of leaves was determined using 150 fresh leaf
 discs (0.5 cm diameter), being 10 discs from 3 plants of each
 concentration of mining waste. Leaf discs were immediately weighed, to
 determine the fresh weight (FW), then oven-dried at 80ºC for 48 hours and
 weighed again to determine the dry weight (DW). Leaf water content was
 calculated by taking the difference between the FW and DW, and dividing it
 by FW (Garnier & Laurent, 1994; Shipley, 1995). 
 Total soluble carbohydrates (TSC) and starch content were
 determined by spectrophotometry, using the method proposed by DuBois,
 Gilles, Hamilton, Rebers, and Smith (1956) and modified by Shimizu (2009).
 Thirteen fresh leaves were used, being 2 leaves from 3 plants of each
 concentration of mining waste. Extractions were performed using samples
 with 0.05 g FW, homogenized in ethanol 80%. The samples were centrifuged
 and had their residue separated from the supernatant. From the
 supernatant, the TSC was measured using the anthrone method (Shimizu,
 2009).  From the TSC extraction residue, the starch
 content was determined. The TSC extraction residue was re-suspended in
 perchloric acid (30%) for 30 min, followed by centrifugation. Starch
 content was also determined by the anthrone method (Shimizu, 2009). In
 both TSC and starch content analyses, a standard glucose curve was used
 for calibration and the absorbance was determined at 620 nm.  All extract
 concentrations were expressed as mg of substance per g of FW.
description: In the file Leaf_Selection_MiningWasteEffect, column labels refer
 to: colony: the ID number of each used ant colony; rep: the ID number of
 each assay; treatment: the mining waste concentration on the soil of the
 plant from which the leaves were taken; freq: the number of transported
 leaf discs; moment: the Phases Unknown (A) and Known (B) described in the
 methods; weight: total number of leaf discs transported in the respective
 assay. The file
 Water_Content_MiningWasteEffect column labels refer
 to: sample: The ID number of each
 sample, plant: the ID number of each
 plant, treatment: the mining waste concentration on the soil of the plant from which the leaves were taken; rep: the ID number of each assay; fresh weight: the leaf disc's weight in g; dry weight: the leaf disc's dry weight in g; water content: fresh weight - dry weight/fresh weight. The file Leaf_Thickness_MiningWasteEffect column labels refer to: treatment: the mining waste concentration on the soil of the plant from which the leaves were taken; plant: the ID number of each plant; rep: the ID number of each assay; thickness: the leaf disc's thickness in mm. The file TSC_Starch_MiningWasteEffect column labels refer to: treatment: the mining waste concentration on the soil of the plant from which the leaves were taken; plant: the ID number of each plant; starch: the leaf disc's starch content in mg of starch/g of fresh weight; TSC: the leaf disc's Total Soluble Carbohydrates content in mg of TSC/g of fresh weight.