Data from: Effects of pasture and forest microclimatic conditions on the foraging activity of leaf-cutting ants

Study site This study was carried out in a 13.4-ha remnant of tropical dry forest (Holdridge et al. 1971, GEMA 1998) and its surrounding matrix of managed cattle pasture at the Alejandría Farm (4°51´ North, 75°52´ West), a private property located in the Cauca Valley (Risaralda, Colombia) between the Central and Oriental Andean Cordilleras at 900-1000 m asl. The site receives ~1700 mm of rainfall annually (8-year mean, 1993-2000) concentrated in two rainy seasons (March-May and September-November). Sunrise is at 5:00 hours and sunset at 17:00 hours throughout the year, and the average temperature is approximately 24 °C. The forest vegetation forms a closed, diverse canopy, the height of which varies from 20 to 40 m, and the dominant tree species include Oxandra panamensis, Trophis caucana Berg., Chamaedorea linearis Mart., Clarisia biflora Ruiz., and Gustavia speciosa Kunth. This forest patch is one of the last remnants of the original continuous dry forest along the central Cauca River Valley (Ramírez et al. 2002), which has undergone large-scale deforestation to less than 1% of its original area due to intensive agriculture (sorghum, soybean, sugarcane and cotton) and cattle-ranching since 1920 (GEMA 1998). Study species Atta cephalotes (Hymenoptera: Formicidae) is considered a “woodland species” and is commonly found in mature, well-conserved forest (Rockwood 1973, Correa et al. 2005). Surprisingly, the occurrence of A. cephalotes in Colombia is not restricted to closed forests, and the species exhibits more disturbance tolerance than indicated by published observations. In fact, it is common throughout almost the entire country from sea level to 2500 m asl (Fernández et al. 2015). Although the Cauca Valley has been extensively converted from forest to pasture and sugarcane crops, A. cephalotes is broadly distributed and easily found, even in parks within cities such as Cali and Pereira (Montoya-Lerma et al. 2012) and in the grasslands, croplands and forests of the region (Armbrecht et al. 2001, Chacon et al. 2012). Study colonies We systematically surveyed A. cephalotes colonies in 13 ha of forest and in a similar area of the surrounding pasture. After a preliminary 15-day monitoring period of their foraging activity and general health status, we choose 12 active mature colonies as study colonies, six within the forest and six within the pasture. The minimum distance of each colony from the edge of its habitat was 50 m, a distance that should have excluded edge effects (Meyer et al. 2009). Because nest size can affect foraging activity (Lewis et al. 1974a), the size of the colonies was calculated as the minimum elliptical area circumscribing the central nest mound (Hernández et al. 1999), and although mature (e.g., presence of soldiers and well-established foraging trails), the colonies were consistently smaller in the pasture than in the forest, with mean nest sizes of 27.39 m2 ± 22.19 and 109.3 m2 ± 65.35, respectively (Welch two-sample t-test: t = -2.90, df = 6.14, N = 12, P = 0.026). Measurements were taken on the main foraging trail of each nest, which was identified as the trail with the highest number of ants at the time of the measurement. The studied foraging trails represented cleared, persistent trunk trails as typically described for Atta ants (Kost et al. 2005). Microclimate Measurements To characterize the effects of microclimatic conditions on the main foraging trails of A. cephalotes, we assessed the following parameters for each nest: (1) air temperature, (2) RH, (3) maximum surface temperature, and (4) minimum surface temperature. The air temperature and RH were measured using a HOBO Pro Temperature/Relative Humidity sensor (Onset Computer Corp., Bourne, MA, USA ®) equipped with internal data loggers. The sensor was located 5 cm above the soil surface on the main foraging trail near the entrance hole of each colony nest, and it recorded measurements at hourly intervals from the 18th of May to the 12th of December 2013. The maximum and minimum surface temperature were recorded for all nests across the two habitats during twelve field work periods of eight hours: four from 6:00 to 14:00, four from 14:00 to 20:00, and four from 20:00 to 6:00 (totaling 96 hours of data at each nest). The surface temperatures were measured by walking slowly from the nest entrance to the end of the foraging trail while registering the maximum and minimum temperatures observed using an infrared thermometer (Fluke 62 MAX). Foraging Activity To describe and compare the foraging activity of A. cephalotes between the two habitats and to examine relationships with microclimatic factors, the activity rates of all above ground workers involved in foraging-related tasks were assessed on the foraging trails. Particularly, we counted the number of outgoing and incoming unladen and laden ants at each nest. Outgoing ants are those that leave the colony to search and harvest resources for fungal substrate, to maintain the trail, e.g., by reinforcing the pheromone trail (Moser 1967), or to protect the foragers, e.g., by clearing foraged leaves to control parasites such as phorid flies (Yackulic & Lewis 2007). Incoming unladen ants are foragers that have lost their loads in the route or workers returning to the nest after being involved in other above ground tasks. Laden ants directly reflect the efficiency of foraging as they consist of ants that are transporting harvested leaves, flowers, seeds or other materials to feed the fungus garden. Foraging rates were calculated as the number of ants passing by a fixed point on the main foraging trail near the main hole of each nest in 5 minutes (Wirth et al. 2003) and were measured at the same time as the maximum and minimum surface temperatures. We used a video camera (Canon PowerShot A490) fixed to a tripod to count the ants, and the total number of ants, both incoming and outgoing, were determined using AntCounter V.1.0 software, which counts the number of ants that pass through a given point in two directions with respect to the nest entrance (Bustamante & Amarillo-Suárez 2016). Because this software does not distinguish between laden and unladen ants, the number of laden ants was counted manually by playing the recorded videos in slow motion. To control for differences in nest size when comparing the foraging rates between forest and pasture, we used the relative foraging activity (i.e., the hourly foraging activity divided by the maximum foraging activity observed of a given nest). Data analysis Differences in microclimatic conditions between habitats and hours were determined for each microclimate parameter by a two-factorial ANOVA with repeated measures on one factor, where habitat and time were fixed factors. “Habitat” had two levels, pasture and forest, and “hour” had 24 levels representing each hour of the day. “Nest” corresponded to each of the subjects with repeated measures each hour and with each nest located either in the forest or the pasture. Additionally, to identify differences in hourly foraging between habitats, we performed a nested ANOVA with colony as a random factor nested within habitat (fixed factor) for each microclimatic condition and hour. Because there were 24 ANOVAs, one for each hour, we used an alpha of 0.01 instead of 0.05 to reduce the probability of a type II statistical error (Zar 2010). Differences in foraging activity between habitats and hours were analyzed for each foraging variable (unladen and laden incoming and outgoing ants) in the same way as for the microclimate variables. To identify differences in foraging activity between habitats, we performed a nested ANOVA with the same parameters as with the microclimatic variables for each foraging activity variable and for each hour. The assumptions of normality and homoscedasticity were met in many, though not all, cases, even after the data were log or arcsine transformed. However, we decided to use nested ANOVA because there is no equivalent non-parametric approach (Dytham 2011) and it is robust against deviations from normality and homoscedasticity (Sokal & Rohlf 1995, Zar 2010). Due to unequal sample sizes, type III sums of squares are reported. Because the numbers of outgoing and unladen and laden incoming ants were expressed as percentages of the maximum flow, they were arcsine-transformed prior to ANOVAs (Doncaster & Davey 2007). To determine the relationships between foraging activity and microclimatic variables, we performed simple linear regressions between each microclimatic variable (air temperature, RH, and maximum and minimum temperature along the foraging trail) and the response variables (number of unladen or laden incoming or outgoing ants). All analyses were carried out using R v. 3.1.0 (R Core Team 2015).

研究样地
本研究在哥伦比亚考卡山谷（里萨拉尔达省）亚历杭德里亚农场（北纬4°51′，西经75°52′）的一片13.4公顷的热带旱林残片（Holdridge等，1971；GEMA，1998）及其周边的人工牧牛牧场基质中开展，该私有林地位于安第斯山脉中部与东部科迪勒拉之间，海拔900~1000米。样地年降雨量约1700毫米（1993-2000年8年平均值），降雨集中在两个雨季（3-5月和9-11月）。全年日出时间为5:00，日落时间为17:00，平均气温约24℃。该森林植被形成封闭且多样的冠层，冠层高度介于20~40米，优势乔木物种包括巴拿马安纳木（Oxandra panamensis）、考卡刺桑（Trophis caucana Berg.）、线形竹节椰（Chamaedorea linearis Mart.）、双花克拉里西亚木（Clarisia biflora Ruiz.）以及美丽古斯塔维亚木（Gustavia speciosa Kunth）。该林块是沿考卡河谷中部现存的原始连续旱林残片之一（Ramírez等，2002），自1920年以来，由于集约化农业（高粱、大豆、甘蔗与棉花种植）和牧牛业的开发，原始旱林已被大规模砍伐至不足原始面积的1%（GEMA，1998）。

研究物种
切叶蚁（Atta cephalotes，膜翅目：蚁科）被视为“林地物种”，通常见于成熟且保存完好的森林中（Rockwood，1973；Correa等，2005）。令人意外的是，哥伦比亚境内的A. cephalotes分布并不局限于封闭森林，该物种对干扰的耐受能力超出已有文献记载的水平。事实上，该蚁几乎遍布哥伦比亚全境，分布海拔从海平面至2500米（Fernández等，2015）。尽管考卡河谷已被大量改造为牧场与甘蔗种植园，但A. cephalotes仍广泛分布且极易被发现，甚至在卡利和佩雷拉等城市的公园中（Montoya-Lerma等，2012），以及该区域的草原、农田与森林中均有分布（Armbrecht等，2001；Chacón等，2012）。

研究蚁群
我们系统调查了森林区域13公顷范围内及周边牧场中相似面积内的A. cephalotes蚁群。在对其觅食活动与整体健康状况进行为期15天的初步监测后，我们选取12个活跃成熟蚁群作为研究对象，其中6个位于森林内，6个位于牧场中。每个蚁群距离其生境边缘的最小距离为50米，该距离可排除边缘效应的影响（Meyer等，2009）。由于蚁群规模会影响觅食活动（Lewis等，1974a），我们以环绕核心蚁丘的最小椭圆面积来计算蚁群规模（Hernández等，1999）。尽管所有蚁群均处于成熟阶段（例如存在兵蚁且觅食通路稳定），但牧场内的蚁群规模始终小于森林中的蚁群，两者的平均巢区面积分别为27.39 m²±22.19与109.3 m²±65.35（Welch两样本t检验：t=-2.90，df=6.14，N=12，P=0.026）。测量在每个蚁群的主觅食通路上进行，该通路被定义为测量时段内蚂蚁数量最多的通路。本研究涉及的觅食通路为切叶蚁典型的清理后持久的树干通路（Kost等，2005）。

微气候测量
为表征微气候条件对A. cephalotes主觅食通路的影响，我们针对每个蚁群评估了以下参数：(1) 气温；(2) 相对湿度（Relative Humidity）；(3) 地表最高温度；(4) 地表最低温度。气温与相对湿度采用HOBO Pro温湿度传感器（HOBO Pro Temperature/Relative Humidity sensor，Onset Computer Corp.，美国马萨诸塞州伯恩，USA ®）进行测量，该传感器内置数据记录仪。传感器安装于每个蚁群巢入口附近的主觅食通路上，距土壤表面5厘米处，于2013年5月18日至12月12日期间以每小时1次的频率记录数据。地表最高与最低温度的测量在两个生境的所有蚁群中开展，共进行12个为期8小时的野外工作时段：4个时段为6:00至14:00，4个为14:00至20:00，剩余4个为20:00至6:00（每个蚁群累计获得96小时的测量数据）。地表温度通过红外测温仪（Fluke 62 MAX）进行记录：测量人员缓慢从蚁巢入口行进至觅食通路末端，同时记录沿途观测到的最高与最低温度。

觅食活动
为描述并比较两个生境中A. cephalotes的觅食活动，并探究其与微气候因子的关联，我们对参与觅食相关任务的所有地表工蚁的活动速率进行了评估，统计了每个蚁巢主觅食通路上的空载与负载工蚁的出巢与入巢数量。出巢工蚁指离开蚁巢以搜寻和采集真菌基质所需资源、维持信息素痕迹（例如强化信息素通路，Moser，1967）或保护觅食者（例如清理采集到的叶片以控制蚤蝇（phorid flies）等寄生虫，Yackulic & Lewis，2007）的个体。入巢空载工蚁指在途中丢失负载物的觅食者，或完成其他地表任务后返回蚁巢的工蚁。负载工蚁直接反映觅食效率，其携带采集到的叶片、花朵、种子或其他材料以饲喂真菌花园。觅食速率以5分钟内经过每个蚁巢主入口附近主觅食通路上固定点的蚂蚁数量计算（Wirth等，2003），测量时间与地表最高、最低温度的测量时间一致。我们使用固定于三脚架上的摄像机（佳能PowerShot A490）对蚂蚁进行计数，并通过AntCounter V.1.0软件统计工蚁总数（包括出巢与入巢个体），该软件可统计相对于蚁巢入口两个方向上经过指定点的蚂蚁数量（Bustamante & Amarillo-Suárez，2016）。由于该软件无法区分负载与空载工蚁，负载工蚁的数量需通过慢放录制视频的方式人工计数。为在比较森林与牧场的觅食速率时控制蚁群规模的差异，我们采用相对觅食活动（即每小时觅食活动量除以对应蚁群观测到的最大觅食活动量）进行分析。

数据分析
针对每个微气候参数，我们采用双因素重复测量方差分析（two-factorial repeated measures ANOVA）确定不同生境与时段间的微气候差异，其中生境与时间为固定因子：“生境”包含牧场与森林两个水平，“时间”包含代表每日各小时的24个水平；“蚁群”为重复测量的受试者，每个蚁群均位于森林或牧场生境中。此外，为识别不同生境间的逐时觅食差异，我们针对每种微气候条件与每个小时，采用嵌套方差分析（nested ANOVA），其中蚁群为嵌套于生境（固定因子）内的随机因子。由于需针对每日24个小时分别开展方差分析，我们将显著性水平α设为0.01而非0.05，以降低II类统计错误的发生概率（Zar，2010）。针对每个觅食变量（空载与负载工蚁的出巢、入巢数量），我们采用与微气候变量相同的分析方法，比较不同生境与时段间的觅食活动差异。为进一步识别不同生境间的觅食活动差异，我们针对每个觅食活动变量与每个小时，采用与微气候变量一致的参数开展嵌套方差分析。尽管在部分情况下，即使经对数转换或反正弦转换后，数据仍未满足正态性与同方差性假设，但我们仍选择使用嵌套方差分析，原因在于目前尚无等效的非参数方法（Dytham，2011），且该方法对正态性与同方差性偏离具有较好的鲁棒性（Sokal & Rohlf，1995；Zar，2010）。由于样本量不均，我们报告了III型平方和（Type III sums of squares）。由于出巢、入巢空载与负载工蚁的数量均以最大流量的百分比形式表示，我们在开展方差分析前对其进行了反正弦转换（arcsine transformation，Doncaster & Davey，2007）。为探究觅食活动与微气候变量间的关联，我们针对每个微气候变量（觅食通路的气温、相对湿度、地表最高与最低温度）与响应变量（入巢/出巢的空载或负载工蚁数量）开展简单线性回归分析（simple linear regression）。所有分析均使用R v.3.1.0软件完成（R核心团队，2015）。