DOI: 10.5061/dryad.qrfj6q5ht
title: Data from: Do experimental pH increases alter the structure and function of a lowland tropical stream?

description: Disturbances can alter the structure and function of ecosystems. In stream
 ecosystems, changes in discharge and physicochemistry at short,
 intermediate, and long recurrence intervals can affect food webs and
 ecosystem processes. In this paper, we compare pH regimes in streams at La
 Selva Biological Station, Costa Rica, where episodic acidification
 frequency across the stream network varies widely due to buffering from
 inputs of bicarbonate-rich inter-basin groundwater. To examine the effects
 of acidification on ecosystem structure and function, we experimentally
 increased the buffering capacity of a headwater stream reach, and compared
 it to an unbuffered upstream reach. We compared these reaches to a
 naturally buffered and unbuffered reaches of a second headwater stream. We
 quantified ecosystem structural (macroinvertebrate assemblages on leaf
 litter and coarse woody debris), and functional responses (leaf litter and
 coarse woody debris decomposition rates, and growth rates of a focal
 insect taxon (Diptera: Chironomidae)). Non-metric multidimensional scaling
 and analysis of similarity revealed that macroinvertebrate assemblages
 were relatively homogenous across the four study reaches, although the
 naturally buffered reach was the most dissimilar. Ecosystem function, as
 measured by chironomid growth rates, was greater in the naturally buffered
 reach, while decomposition rates did not differ across the four reaches.
 Our results indicate that biological assemblages are adapted to pH regimes
 of frequently acidified stream reaches. Our experiment informs the effects
 on structure and function at short time scales in streams that experience
 moderate acidification, but larger magnitude acidification events in
 response to hydroclimatic change, as projected under climate change
 scenarios, may induce stronger responses in streams.
description: <em>Study site</em> This study was
 conducted at La Selva Biological Station (LSBS, 10° 26’ N, 84° 01’ W),
 Costa Rica (Figure 1). La Selva (1536 ha area) consists of predominantly
 primary and secondary tropical wet forest, and receives, on average, 4000
 mm of rainfall per year (Sanford et al. 1994). Streams at LSBS can be
 distinguished by two sources of groundwater inputs. Local groundwater
 inputs, which originate within the streams’ watershed, are present in all
 streams. Certain streams below ~50 meters above sea level receive inputs
 of inter-basin modified groundwater (IMG) (Pringle and Triska 1991,
 Pringle et al. 1993, Genereux et al. 2005). Streams with IMG inputs are
 characterized by higher solute concentrations, including soluble reactive
 phosphorus (SRP), cations, and carbonate species (Pringle et al. 1990,
 Pringle 1991, Oviedo‐Vargas et al. 2015). Streams with IMG exhibit faster
 microbial respiration (Rosemond et al. 2002, Ramírez et al. 2003), faster
 macroinvertebrate turnover (Ramírez and Pringle 2006), faster leaf litter
 decomposition rates (Ardón et al. 2006), and higher and more stable pH as
 a result of higher buffering capacity (Small et al. 2012).
 <em>Experimental design</em> We
 selected two headwater streams to test the effects of episodic
 acidification on both structure and function: Carapa and Arboleda Seep
 (hereafter ArbSeep) (Table 1). Carapa is a typical low-solute, headwater
 stream at LSBS, experiencing seasonal and episodic acidification (Small et
 al. 2012). ArbSeep is a headwater tributary to the Arboleda, and the
 downstream reach of ArbSeep receives IMG, resulting in different
 physicochemical characteristics between the upstream and downstream
 sections of ArbSeep. Due to logistical constraints, our replication was
 limited to two streams. We selected two 5-10 m reaches within each of the
 two streams, resulting in a total of four study reaches: two upstream low
 buffering capacity and frequently acidified reaches, one downstream high
 buffering capacity reach in ArbSeep (due to natural IMG inputs), and one
 downstream low-buffering capacity reach in Carapa that we chose for
 experimental carbonate amendments to simulate IMG inputs (Figure 1). The
 reaches in ArbSeep were separated by ~100 m, over which IMG inputs modify
 stream chemistry. In Carapa, study reaches were separated by ~15 m to aid
 in logistics in the field. The Carapa experiment occurred from 15 June
 2018 – 20 July 2018, and measurements in ArbSeep occurred from 18 June
 2018 – 23 July 2018. Rainfall data was collected from this period using a
 tipping bucket rain gauge located ~1 km from our streams and are available
 from the Organization for Tropical Studies (OTS,
 https://tropicalstudies.org/) <em>Buffering
 capacity addition</em> To mimic IMG inputs and prevent episodic
 acidification from occurring in the experimental downstream reach of
 Carapa, we continuously added buffering solution in the form of carbonate
 species in two phases for five weeks (15 June 2018 – 20 July 2018). For
 the first two weeks of the experiment, we dissolved 1,920 g of
 NaHCO<sub style="text-indent:0.5in;">3</sub> in a 220 L barrel of stream water
 and pumped this solution (~0.1 M HCO<sub style="text-indent:0.5in;">3</sub><sup style="text-indent:0.5in;">-</sup>) into the stream above the
 downstream reach at a rate of 25 mL min<sup style="text-indent:0.5in;">-1</sup>, refilling and amending the
 barrel with upstream water as needed. The NaHCO<sub style="text-indent:0.5in;">3</sub> pumping addition was not as
 effective as anticipated in preventing episodic acidification; therefore,
 for the remainder of the experiment, we added 500 g of solid
 CaCO<sub style="text-indent:0.5in;">3</sub> in coarse mesh bags directly in
 the stream immediately upstream of the downstream reach so it would
 dissolve in water. CaCO<sub style="text-indent:0.5in;">3</sub> bags were replaced every 2-3 days
 for the remaining three weeks of the experiment.
 <em>Stream physicochemistry</em>
 We deployed a YSI EXO1 multiprobe sonde (Xylem Inc., Yellow
 Springs, OH) in the two downstream reaches during the five-week
 experiment. Sondes measured temperature (°C) and pH every 15 minutes and
 were calibrated weekly. Due to sensor error, we only collected data in
 Carapa for the final three weeks of the experiment. As the sondes were
 deployed in the downstream reach during the experiment, we deployed the
 sondes in the upstream reaches for five weeks the following summer to
 collect the same data from the upstream reaches from August 13 - September
 24 2019, during which there was 514.3 mm of rainfall. While these dates do
 not overlap with the dates of the 2018 experiment, the 2019 dates fall
 within the wet season at LSBS; in 2019, 811 mm of rainfall fell from June
 15 - July 23 (Figure 2, a) Figure, while in 2018, over 1100 mm of rainfall
 fell during the measurement period (Figure 2, b). To examine the frequency
 of acidification, we used the Shapiro-Wilk test of normality to assess the
 distributions of pH data in each of the four study reaches, assuming that
 buffered reaches would exhibit normal distributions and upstream reaches
 would not. The STREAMS project at LSBS (a long term monitoring and
 biological research project) shows higher pH due to IMG inputs and skewed
 distributions of pH in streams with local groundwater only (Small et al.
 2012, Ardón et al. 2013; Figure 1, b, c). We measured
 discharge and dissolved nutrients weekly during the experiment. Discharge
 was measured in the downstream reaches using the cross-sectional area
 method with a velocity meter (Marsh-McBirney, Frederick, MA). Triplicate
 nutrient (NO<sub>3</sub><sup>-</sup>-N,
 NH<sub>4</sub><sup>+</sup>-N, and SRP) samples
 were collected in 60 mL bottles, filtered in the lab (0.45 µm), and
 frozen. Samples were transported to North Carolina State University
 frozen, where NH<sub>4</sub><sup>+</sup> and SRP
 were analyzed on an AA3 Segmented Flow Analyzer (Seal Analytical, Mequon,
 WI) and NO<sub>3</sub><sup>-</sup> was analyzed on
 a Metrohm 930 ion chromatograph (Metrohm, Ionenstrasse,
 Switzerland). <em>Individual
 organism response</em> We measured the growth of
 chironomid larvae (Diptera: Chironomidae) in the four reaches of the
 buffering experiment. Chironomids are the dominant detritivores in streams
 at La Selva and have comprised &gt;50% of invertebrate biomass in
 previous leaf litter decomposition experiments (Rosemond et al. 2001,
 Ardón et al. 2006). Coarse mesh bags filled with air-dried <em>Ficus
 insipida </em>Willd. leaves were incubated in each of the four
 reaches for 10 days, allowing for colonization by chironomids, and
 returned to the lab. Chironomid larvae were picked from the leaf packs and
 measured to the nearest 0.2 mm. We selected six larvae between 2 and 5 mm
 for growth experiments. Groups of six larvae were placed into six
 replicate plastic tea strainers (mesh size = 224 µm) with six
 <em>Ficus </em>leaf discs (10 mm diameter) and deployed into
 the respective stream reach for 48 hr. After deployment, we counted
 surviving individual chironomids and re-measured the length of each
 individual. We estimated chironomid biomass using length-mass
 relationships (Small et al. 2011) as the mean of the initial six
 individuals deployed and the mean of the surviving individuals as the
 final biomass, and calculated percent change in biomass and survivorship
 for each tea strainer using the mean of the surviving individual
 chironomids. <em>Macroinvertebrate assemblage and
 ecosystem function response</em> In the four
 stream reaches, we deployed coarse mesh (3 mm) and fine mesh (300 µm)
 litter bags to measure leaf litter (LL) and coarse woody debris (CWD)
 decomposition. Fine mesh litter bags were used to exclude
 macroinvertebrates and estimate microbial decomposition rates, whereas
 coarse mesh bags allow macroinvertebrates access and contribute to
 decomposition (Woodward et al. 2012). Fine mesh bags were largely
 ineffective in excluding macroinvertebrates but were included in the
 analysis to determine what attributable effects remained. Litter bags of
 each mesh size and organic matter type were deployed for each collection
 date and type of organic matter, and triplicate litter bags were collected
 following one and five weeks of deployment in the stream (total n of
 decomposition bags = 96). Leaves from <em>Luehea seemannii
 </em>Triana & Planch. (Tiliaceae)<em> </em>(5 g)
 were collected as recently abscised litter and air dried for at least 3
 days; CWD segments from <em>Pentaclethra macroloba
 </em>(Willd.) Kuntze (Fabaceae)<em> </em>(50 g) were
 collected from recently fallen trees and air dried for at least 3 days. In
 an attempt to standardize organic matter inputs across all decomposition
 bags, leaves of similar size and small sticks (circumference &lt; 10
 cm, length &lt; 25 cm) were added to each bag. On the pre-assigned
 collection date, including a replicate set of sacrificed leaves and wood
 for each reach and mesh size on the deployment date, litter bags were
 removed from the stream using a dip net, transferred to a 4 L plastic bag,
 and transported to the lab. In the lab, leaf and wood matter were washed
 over a 250 µm sieve to remove all sediment and biota. Leaf litter and CWD
 material were placed into paper bags, dried at 40 ˚C for 48 h, and
 subsamples placed in a muffle furnace at 500 ˚C for 1 h to calculate dry
 mass (DM) and ash-free dry mass (AFDM), respectively. We calculated the
 decomposition rate constant for LL and CWD, k<sub>LL </sub>or
 k<sub>CWD</sub>, using a negative exponential model on AFDM
 data. Material collected in the sieve was preserved in 95% ethanol for
 macroinvertebrate identification. Macroinvertebrates were identified to
 the family level, where possible.
 <em>Statistics</em> Chironomid
 survival and growth rates were evaluated across the four study reaches. We
 used a nested ANOVA to analyze percent survival, percent biomass changes,
 and final chironomid biomass to determine differences between stream
 reaches. Percent biomass change was non-normally distributed and was
 log<sub>10</sub> transformed. Decomposition
 rates for LL and CWD were analyzed using two-way nested ANOVA, which
 allows evaluation of differences between the two study streams, the two
 reaches in each stream, and the effect of bag mesh size. We used a
 post-hoc Tukey test to determine significant groups among the four
 reaches. Macroinvertebrate abundance from both fine and coarse mesh litter
 bags was measured after one and five weeks of deployment from the four
 reaches. Abundance data failed the normality assumptions for ANOVA;
 therefore we evaluated macroinvertebrate abundance across the four study
 reaches, mesh bag size, and organic matter type using the non-parametric
 Aligned Rank Transform test in the ARTool v0.10.8 (Kay and Wobbrock 2020)
 and post-hoc evaluation in the emmeans v1.5.3 R packages (Lenth 2020). To
 evaluate macroinvertebrate richness, we calculated the number of families
 in each litter bag and evaluated richness across the experimental factors
 using non-parametric Kruskal-Wallis rank sum test due to the non-normally
 distributed data. To quantitatively assess the
 diversity of the macroinvertebrate assemblages over time, we used analysis
 of similarity (ANOSIM) using the Bray-Curtis dissimilarity index and 999
 permutations for the treatments in the experiment. We used the ANOSIM R
 statistic to evaluate diversity across the experimental treatments
 (reaches, mesh size, organic matter), where R values closer to 0 indicate
 similarity versus values closer to 1 indicating dissimilarity. To document
 the diversity of the macroinvertebrate assemblage at the end of the
 experiment, we used non-metric multidimensional scaling (NMDS). In the
 NMDS, we used the Bray-Curtis dissimilarity index in three dimensions on
 log<sub>10</sub> transformed abundance data and visualized the
 ordination across our experimental treatments (study reaches, mesh bag
 size, and type of organic matter) to assess the influence of each
 treatment. Analysis of the macroinvertebrate assemblage was done in the
 vegan v2.5-7 R package (Oksanen et al. 2019). We
 calculated the effect size statistic Hedges’ g to compare the upstream and
 downstream reach in each stream, isolating the effect within each stream
 of how the metrics in our experiment change moving from more frequently
 disturbed reaches into less frequently disturbed reaches. In ArbSeep,
 where no manipulations occurred, we used Hedges’ g to examine of the
 naturally buffered downstream reach relative to the unbuffered upstream
 reach. Similarly, in Carapa, we used Hedges’ g to examine the effect of
 the experimental buffering treatment in the downstream reach to the
 unbuffered upstream reach. We calculated g for metrics at the organismal
 (chironomid growth rate), community (macroinvertebrate abundance and
 richness), and ecosystem scales (k<sub>LL</sub> and
 k<sub>CWD</sub>). Hedge’s g is the preferred metric for small
 sample sizes (n &lt; 20) and was calculated using the effsize v0.8.1 R
 package (Torchiano 2020).
description: The Rmd file contains all code to execute the analysis for this
 mansuscript, including pulling in data. Data are source from the same
 folder containing the Rmd file. The rendered file from the Rmd are also
 attached, both as .html and PDF files.