Macroinvertebrate Bio-Assessment of Camp and Pine Creeks, Zumwalt Prairie Preserve 2019

Macroinvertebrate Bio-Assessment of Camp and Pine creeks, Zumwalt Prairie Preserve 2019

c) a)

a) Suwallia (stonefly) adult: troutnut.com b) Suwallia nymph: bugguide.net c) Chironomidae (midge) adult: Wikipedia.com d) Chironomidae larvae: Alamy.com e) Limnephilus (caddisfly) adult: bugguide.net f) Limnephilus larvae: bugguide.net

Prepared for: The Nature Conservancy NE Oregon Field Office Enterprise, OR 97828

Prepared by: David Wooster and Sandra DeBano Hermiston Agricultural Research and Extension Center Oregon State University Hermiston, OR 97838

Summary

Macroinvertebrates were sampled at 16 reaches on Camp Creek and seven reaches on Pine Creek in May 2019. Macroinvertebrates have been sampled at Camp Creek since 2006 and at Pine Creek since 2007. 2019 was the fourth post-restoration period of sampling on Camp Creek and the fifth on Pine Creek. One hundred and two aquatic macroinvertebrate taxa have been identified from the preserve. Despite having sampled macroinvertebrate for seven years on the preserve, we continue to find new taxa. In 2019, five new taxa were identified.

Precipitation at the Zumwalt prairie has varied across the sampling years, with the highest amounts of precipitation occurring in post-restoration years. This pattern of precipitation confounds the effects of restoration with rainfall. This confounding was supported by ordination analyses which revealed that the amount of precipitation prior to sampling was an important driver of macroinvertebrate assemblage composition.

Have Camp Creek and Pine Creek improved in condition? The main focus of our sampling was to determine whether conditions in Camp Creek and Pine Creek have improved through time based upon macroinvertebrate assemblages. To control for the confounding effect of precipitation we conducted mixed models that used precipitation as a covariate. Response variables were 12 macroinvertebrate metrics related to stream reach condition. Mixed models using time period as a categorical variable (before versus after restoration) revealed that the relative abundance of EPT (Ephemeroptera-Plecoptera-Trichoptera) and sensitive taxa were greater after restoration than before at Camp Creek, indicating an improvement in stream reach conditions after restoration. At Pine Creek, functional feeding group evenness increased after restoration also indicating an improvement in conditions.

Ecosystems often show a lag time between changes in land management and response. In addition, the response of ecosystems can be gradual, increasing through time. Therefore, a consideration of time as a continuous variable is an additional method of considering restoration impacts, and a second set of mixed models were conducted with year as a continuous variable. Macroinvertebrate metrics were more sensitive to this analysis, with five metrics indicating an improvement in stream reach condition through time at Camp Creek and three metrics indicating positive changes at Pine Creek. In addition to the mixed models, we tracked the distribution of specific sensitive taxa and found that three of six taxa in Camp Creek have increased their distribution through time and the only sensitive taxa with appropriate data has increased its distribution through time at Pine Creek. Taken all together these results strongly indicate an improvement in the conditions at Camp Creek and Pine Creek and this is likely the result of restoration actions taken in 2010.

Are there specific reaches that are consistently the highest quality? Based on macroinvertebrate metrics, five reaches at Camp Creek and two reaches at Pine Creek were consistently in the highest quality of all reaches sampled. At both creeks, these reaches were characterized by a substrate that was low in silt and high in cobble. We examined whether these high quality reaches were high in the drainage area. Streams tend to accumulate upstream anthropogenic effects, resulting in downstream gradient of increasingly worse conditions. However, in direct contrast to this, the five best-condition reaches at Camp Creek were all low in

the drainage area sampled. A likely explanation is that the downstream reaches tend to have higher discharge, resulting in a relatively silt-free substrate. This silt-free substrate is favored by many macroinvertebrate taxa that are indicative of good quality.

Do cattle/elk exclosures and riparian plantings have a positive impact on stream reach condition? Several reaches and their surrounding area at both Camp and Pine creeks are enclosed by cattle/elk fencing. An examination of two enclosed reaches at Camp Creek revealed that the macroinvertebrate community within these reaches does not appear to have changes in any consistent fashion, suggesting that the exclosures have not had an effect on the stream reaches. However, the stream environment within these two exclosures has changed since the exclosures were constructed. The amount of silty substrate decreased and the amount of riparian shade increased at the two exclosure reaches after exclosure construction. Riparian shade at both reaches has increased through time as the riparian trees planted start to mature. It is not clear why the macroinvertebrate assemblage did not respond to these changes in their environment. It is possible that there is a time lag between a change in the stream environment and a response by macroinvertebrates and that time lag has not passed yet. If this is true, we expect to see macroinvertebrate responses in the future. It is also possible that the exclosed reaches are simply too small to elicit a macroinvertebrate response.

Are macroinvertebrate assemblages responding to cattle grazing? Cattle grazing intensity had a negative impact on macroinvertebrate assemblages, particularly taxa richness. Our measure of grazing intensity was based on the number of years a pasture was grazed or not, it did not take into account the number of AUMs or the amount of access cattle had to the creek.

Continued macroinvertebrate monitoring will be important particularly at the exclosure reaches where riparian plantings are still maturing and influencing the stream environment. In addition, obtaining more detailed metrics of cattle grazing intensity would make our understanding of how the Nature Conservancy grazing regimen affects the Zumwalt Prairie streams.

Introduction

The aquatic macroinvertebrate assessment of the Camp and Pine Creek drainages described in this report is provided to The Nature Conservancy (TNC) partially through cooperation with Oregon State University and partially through a contract with David Wooster. In May 2019, macroinvertebrates were collected at 16 sites on Camp Creek and seven sites on Pine Creek within The Nature Conservancy’s Zumwalt Prairie Preserve, Wallowa County, Oregon (Figure 1).

The main objective of the ongoing macroinvertebrate assessment is to examine changes in stream reach condition across a period of time that spans restoration actions on both Camp Creek and Pine Creek. Restoration actions on Camp Creek were quite extensive and involved the removal of 11 earthen dams constructed by previous landowners to pond water for cattle, planting of over 10,000 native shrubs along the creek, improving the fencing around the creek, and changing cattle management in pastures along the creek (Fields 2011). Actions on Pine Creek were less extensive and included fencing the riparian corridor to keep cattle off the creek and planting ~7,000 native shrubs (Fields 2011). Restoration on Camp Creek occurred in 2010 and on Pine Creek in 2008.

Camp Creek was sampled for three years prior to restoration (2006, 2007, and 2008) and the sampling in 2019 represents the fourth post-restoration sampling session spanning eight years (2011, 2013, 2016, and 2019). The 2019 sampling on Pine Creek represents the fifth session of macroinvertebrate sampling in that drainage and the fourth session post-restoration (the other years were 2007, 2011, 2013, and 2016).

The analysis of aquatic macroinvertebrate assemblage structure provides a useful means of assessing stream reach condition and tracking changes in condition through time. Macroinvertebrates are particularly good candidates for assessing water quality and ecological condition for several reasons. First, a large amount of information exists regarding the sensitivity of different taxa to human impacts (e.g., Johnson et al. 1993, Barbour et al. 1999). Therefore, changes in the abundances of these taxa represent changes in stream reach condition. Second, the lifespan of most macroinvertebrates (one year) is long enough to incorporate impacts across multiple seasons, but short enough to make tracking population changes resulting from management actions feasible. Third, the home range size of many macroinvertebrates is small and thus populations might respond to local environmental conditions such as those resulting from stream restoration.

Figure 1. Macroinvertebrate sampling sites established on the Zumwalt Prairie Preserve at both Camp Creek (site names start with “CC”) and Pine Creek (site names start with “PC”). Map generated by H. Schmalz.

These factors make aquatic macroinvertebrates ideal for monitoring stream restoration projects. For example, the Oregon Department of Environmental Quality completed a bioassessment of restoration activities in McCoy Creek, Union County, Oregon in which aquatic macroinvertebrate community structure was important in revealing an improvement in water quality after restoration (Whitney 2007).

For this report, our objectives are to: 1) Provide an overview of the number of aquatic macroinvertebrate taxa collected on the Zumwalt preserve to date and give a brief description of the dominant taxa. 2) Examine whether three areas in which sampling has occurred differ in macroinvertebrate assemblage composition and determine environmental drivers that influence composition. 3) Determine whether the condition of Camp Creek and Pine Creek has improved through time. 4) Determine whether there are sites on both Camp Creek and Pine Creek that are consistently in the best condition based upon the macroinvertebrate assemblage and examine the environmental attributes of those sites. 5) Examine the impact that grazing exclosures and riparian plantings have on macroinvertebrate assemblages in Camp Creek and Pine Creek. 6) Examine the relationship between recent grazing history and stream reach condition. 7) Examine changes in levels of silt on the substrate through time.

Field Methods

Site Selection 2019 was the seventh year of sampling macroinvertebrates in Camp Creek. Sampling was conducted prior to restoration in 2006, 2007, and 2008, as well as after restoration in 2011, 2013, 2016, and 2019. During this period a total of 18 macroinvertebrate sites have been established

(Appendix 1). Most of these sites were selected as random subsets of the photopoint sites from Dingeldein et al. (2005, 2006). However, several sites were established during recent sampling years (2016 and 2019) to increase the number of cattle/elk exclosure sites sampled (these recent additions include CC18A, CC25A, CC38, and R2P9). In addition, two of the 18 sites were established in the canyon below summer camp in 2006 in consultation with Phil Shephard and Robert Taylor. Camp Creek in this canyon is characterized by a relatively high discharge, an abundance of woody riparian vegetation, and a substrate composed largely of silt-free gravel, cobble and boulder. This is in marked contrast to the creek at the16 sites upstream of the canyon, which is characterized by little to no woody riparian vegetation, an abundance of emergent vegetation (e.g., Juncus spp.), and an often silty substrate. In 2006, we hypothesized that the macroinvertebrate assemblages in the canyon might harbor sensitive taxa that would potentially colonize the upper area of Camp Creek if conditions improved through restoration. Therefore, we established study sites in the canyon to determine the composition of the macroinvertebrate assemblage there. Given the environmental differences between the canyon area and the upper prairie area, the sites in Camp Creek are referred to as the “prairie” sites (for the upper 16 sites) and the “Below” sites (for the sites established in the canyon). In 2019, we sampled 15 of the Camp Creek prairie sites and one Below site (Below1).

Fieldwork In 2019, sites at Camp Creek and Pine Creek were sampled from May 13th – May 23rd. Benthic macroinvertebrates were sampled following the protocols developed by the Oregon Department of Environmental Quality (outlined by the Oregon Plan for Salmon and Watersheds 1999). The sampling reach at each site was 50m long and the coordinates for each site represented the 0m mark (the sampling reach extended upstream from that point). Four sampling locations were randomly chosen in riffle/fast water habitat within each study reach. At each of these locations, a D-frame kick net was placed firmly against the substrate and the substrate in a standard area upstream of the net (0.09m2) was agitated for 30 seconds to a depth of approximately 10 cm. The four samples taken at each reach were composited into a single sample that was used to characterize the macroinvertebrate assemblage at the study reach. Composited samples were preserved in ethanol and shipped to the National Aquatic Monitoring Center at Utah State University in Logan, Utah for processing and macroinvertebrate identification. For each sample, invertebrates were picked from random subsamples until at least 500 macroinvertebrates had been found. These macroinvertebrates were identified to the lowest taxonomic category feasible (genus for most insect taxa and order or family for most non-insect invertebrates) using published keys (Thorp and Covich 1991, Merritt et al. 2008, Wiggins 2000).

In addition to collecting macroinvertebrates, we measured a suite of environmental parameters at each site. These physical and water quality parameters were measured at the same scale at which the invertebrates were collected (i.e., the reach scale) and, in other stream systems, have important effects on the reach-scale distribution of macroinvertebrates (Griffith et al. 2001, Johnson et al. 2003, Townsend et al. 2003). Environmental parameters measured at each site during all sampling years included wetted width, substrate composition, water depth, water velocity, and riparian vegetation cover.

Climate – Precipitation Our preliminary analysis of the 2019 data suggested that condition of the creeks varied between years, particularly in terms of wetted width. Flow in small streams can be highly dependent upon the amount of recent precipitation (Lake 2007), which in turn can influence the abundance of biota in streams. For example, high spring flows can scour streambeds resulting in very low abundances of macroinvertebrates (Power et al. 1988, Lake 2007). While we attempted to sample each year during similar climates, a large amount of variation existed among years in precipitation before sampling (Figure 2). Unfortunately, the three wettest years based upon 60 and 90 days prior to sampling all occurred after restoration on Camp Creek, 2011, 2013, and 2019 (Figure 2). The variation in precipitation 60 days and 90 days prior to sampling was significantly correlated to variability in wetted width of the sampling reaches on Camp Creek (Figure 3; Table 1). Thus, high precipitation influenced the physical nature of Camp Creek and led to a potential confounding of the effects of restoration with effects of high flows. However, for our analyses of changes in stream reach conditions through time (Objective 3) we controlled for differences among years in precipitation by using precipitation during the 60 days prior to sampling as a covariate.

18 16 14 12 10 8 6

4 Total Precipitation (cm) Precipitation Total 2 0 2005 2007 2009 2011 2013 2015 2017 2019 30 Days 60 Days 90 Days Figure 2. Total amount of precipitation for the sampling years. Total precipitation is shown for 30, 60, and 90 days prior to the initiation of sampling.

Table 1. Results of correlation analysis comparing mean wetted width of sampling reaches on Camp Creek to amount of precipitation for different time periods prior to sampling. Data for each sampling year were used (n=7). Time Period Pearson r p-value Day of sampling 0.164 >0.25 7 Days prior to sampling 0.130 >0.25 30 Days prior 0.405 >0.10 60 Days prior 0.716 0.03 90 Days prior 0.707 0.03

450 400 350 2016 300 250 2011 200 2019 150 2013 2008 Wetted Width (cm) Width Wetted 100 2007 50 2006 0 0 2 4 6 8 10 12 14 60 Days Precipitation (cm)

Figure 3. The relationship between wetted width of stream reaches and the total amount of precipitation for 60 days prior to the start of sampling. Points represent the mean wetted width for all Camp Creek prairie sites sampled in a given year and vertical lines are the standard errors.

Objective 1. The Macroinvertebrates As of the 2019 sampling, we have identified102 distinct taxa of macroinvertebrates from Camp and Pine Creeks (Appendix 2). Despite sampling for seven years, we continue to find new taxa each year (Table 2). For example, in 2019 five taxa that had not been previously identified from the preserve were collected. This included a new family and genus of stonefly (family Perlidae, genus Calineuria), a new genus of mayfly (family Baetidae, genus Callibaetis), a relatively sensitive sub-family of midges (Diamesinae), the first specimens of soldier fly (family Stratiomyidae, genus Caloparyphus_Euparyphus), and the first specimens of net-winged midges (family Blephariceridae).

Table 2. Increases in the number of aquatic macroinvertebrate taxa identified each year. “# of Reaches” indicates the number of stream reaches sampled that year. # of Total Taxa Number of Cumulative Number Year Reaches Richness “New” Taxa of Taxa Identified 2006 12 43 43 2007 26 65 29* 72 2008 11 44 7 79 2011 14 49 8 87 2013 14 52 4 91 2016 28 49 6 97 2019 23 55 5 102 * Both Pine Creek and the “Below” sites were first sampled in 2007 and this, in part, resulted in the large number of new taxa that year.

At the reach level and for all locations and years, stream reaches averaged 14.4 taxa. Average taxa richness varied among the areas sampled on the preserve, Camp Creek prairie reaches averaged 14.2 taxa, Pine Creek 12.8, and Camp Creek in the downstream canyon (the “Below” sites) averaged 22.9 taxa. For the Camp Creek prairie reaches and Pine Creek this level of richness is somewhat lower than that found for intermittent reaches draining the Blue Mountain foothills in Umatilla County, Oregon (mean richness of 11 intermittent, headwater reaches was 20.1; Wooster and DeBano 2018).

Camp Creek in the downstream canyon has an abundance of woody riparian vegetation, fast flowing water, and relatively silt-free substrate. The macroinvertebrate assemblage in this area of Camp Creek reflects these relatively good conditions, as it is comprised of a greater richness of sensitive taxa. The “Below” reaches averaged 6.6 sensitive taxa per sample and a total of 21 sensitive taxa were collected at the “Below” reaches through all years. In contrast, the Camp Creek prairie reaches averaged only 1.2 sensitive taxa per reach and a total of 15 sensitive taxa were found across all years. The values for Pine Creek were also relatively low, reaches averaged 1.4 sensitive taxa and a total of 7 sensitive taxa were collected from all of the Pine Creek samples.

Across the entire preserve and all sampling years, several diptera taxa (true flies) dominated the stream assemblages. In particular, the midge sub-families Orthocladiinae and Chironominae and the black fly genus Simulium were dominant taxa throughout the sampling years and across all areas (Appendix 3). Midges are small, non-biting flies that superficially resemble mosquitoes as adults. Midge larvae are very common and often numerically dominant members of river and stream macroinvertebrate assemblages throughout the world (Ferrington et al. 2008). They are moderately tolerant to human disturbances, Orthocladiinae have a tolerance value of 4.7 and the tolerance value of Chironominae is 6.4. Both sub-families are considered collector-gatherers which are invertebrates that feed on deposited organic matter found on the streambed (Figure 4a). Simulium is a widespread genus of black flies that, like midges, is moderately tolerant of human disturbances (its tolerance value is 6.1). Black fly larvae cling to the substrate in fast- flowing areas of streams and use modified labral “fans” to filter small (often <1µm) pieces of organic matter from the water (Figure 4b). Given this feeding mode, black flies are considered collector-filterers. In Camp and Pine creeks these larvae can often be found clinging to submerged stems of Juncus and sedges where the current is fast.

Other dominant taxa include several genera of mayflies. In particular, the genera Baetis and Centroptilum were dominant taxa found in Camp Creek (at both the prairie sites and the Below sites) (Appendix 3). These two genera belong to the same family, Baetidae, which are commonly referred to as “minnow flies” or “minnow mayflies.” The common name most likely derives from the torpedo-shaped bodies and strong swimming abilities of the nymphs (Figure 4c). These mayflies are more sensitive than the dipterans described above, but still would not be considered highly sensitive. The tolerance values are 3.4 and 3.5 for Baetis and Centroptilum, respectively. The nymphs feed both as collector-gatherers and as scrapers. Scrapers are invertebrates that graze periphyton from stone surfaces. These taxa can be observed throughout Camp and Pine creeks, particularly where there is a stony substrate that is reasonably free of silt. When disturbed they swim away in short, rapid bursts.

Several non-insect invertebrates were also common. The most abundant was the segmented worms (Class Clitellata) (Appendix 3). These worms belong to the same class as earthworms and the aquatic species collected look very similar to the terrestrial species. These are also collector- gatherers, feeding on organic matter on the substrate. Clitellata are tolerant of many types of human disturbance and are often considered indicators of poor water quality (tolerance value of 7.9). While not a dominant taxa based on relative abundance, another common non-insect taxa (based on its distribution, see Appendix 3) was water mites (order Trombidiformes in the Class Arachnida) (Figure 4d). As with midges, aquatic mites are very widespread and can be extremely abundant (Smith et al. 2010). Their collection and identification is difficult and while widespread (found throughout the world) their distributions within geographic areas are not well known. Part of this likely stems from their size. Because they are very small (about 1mm long) they fall right at a size that is often used as a point separating macroinvertebrates from microinvertebrates. Therefore, they show up in some macroinvertebrate reports and not in others. As a group, Trombidiformes are moderately tolerant (tolerance value of 4.3) and aquatic taxa are often parasites as juveniles and either parasites or predators as adults.

a) b)

c) d)

Figure 4. Common aquatic invertebrates in the Zumwalt Prairie streams. a) Midge larvae belonging to the Diptera subfamily Orthcladiinae (photo: landcareresearch.co.nz); b) Simulium black fly larva, the specialized labral fans used for filtering organic matter from the water can be seen (photo: Warren Photographic; c) Baetis mayfly nymph (photo: Alamy Stock Photo); d) water mite (Trombidiformes, photo: cfb.nhu.edu).

These dominant taxa are typical of intermittent streams throughout Oregon. Particularly the Orthocladiinae and Chironominae midges, Simulim black flies, Baetidae mayflies, and aquatic mites, which are also dominant taxa in other headwater systems, including the Blue Mountain foothills in Umatilla County, Oregon (Wooster and DeBano 2018) and throughout western Oregon (Herlihy et al. 2005).

Objective 2. Do Camp Creek prairie sites, Pine Creek, and the “Below” sites differ in macroinvertebrate assemblage composition and what are the environmental drivers of composition?

Analyses The “Below” sites on Camp Creek have a higher taxa richness and more sensitive taxa than either the Camp Creek prairie sites or Pine Creek. However, the Below sites also share many taxa with both the prairie sites and Pine Creek. The Camp Creek prairie sites and Pine Creek have different histories in terms of restoration activities and are the headwaters of different basins. To determine whether these three areas differed in macroinvertebrate assemblage composition, we ran non-metric multidimensional scaling (NMS) ordination to visualize differences in macroinvertebrate assemblage structure. A multi-response permutation procedure (MRPP) was conducted to test for significant differences in assemblage composition. NMS is a robust ordination technique that, in contrast to many other ordination techniques, avoids assumptions regarding linear relationships among variables (McCune and Grace 2002). In the ordinations conducted in this report, samples are placed in a multi-dimensional “taxa space,” such that samples closer together in the ordination are more similar in their assemblages than samples farther removed. Distance or dissimilarity among samples was calculated using the Sorenson distance metric which calculates distance between sites in a “city block” fashion, essentially summing the distances across all axes (as opposed to Euclidean or straight-line distance). The Sorenson distance measure is considered to be one of the most reliable for community data (McCune and Grace 2002). To determine how close the final ordination solution is to the true distances among sites, a stress statistic is calculated. Stress values less than 20 indicate a good ordination solution that should reflect important trends in similarity among sites (McCune and Grace 2002).

MRPP is a method for comparing two or more groups in multi-dimensional space with the null hypothesis that the amount of heterogeneity within groups is no smaller than that predicted by chance. The test uses a randomization procedure to determine whether groups are significantly different from each other. MRPP also provides an effect size value (“A”) which compares the observed homogeneity within groups to the expected homogeneity. “A” values range from 0 to 1, with larger values indicating greater effect size (i.e., members of groups are increasingly more similar to each other than to members of other groups). In MRPP it is important to consider both the effect size and the p-value when deciding whether groups are different in an ecologically meaningful way. As a benchmark, we used “A” values of ≥0.10 (McCune and Grace 2002) and a significant p-value (≤0.05) as indicators of ecologically meaningful differences among groups.

We also conducted separate NMS ordinations for the Camp Creek prairie sites and Pine Creek. These ordinations were conducted to examine environmental drivers of macroinvertebrate assemblage structure. The two drainages were examined separately because differences in restoration actions taken in the two drainages might influence the environmental drivers of macroinvertebrate assemblage composition. Environmental drivers of assemblage composition were examined using ordination bi-plots. Bi-plots produce vectors for environmental variables that are correlated with at least one ordination axis. The cut-off r2 value for the vectors was 0.20 (i.e., environmental variables with r2 values less than 0.20 were not considered important). Environmental variables examined in the ordinations included substrate characteristics (the amount of silt, gravel/cobble/boulder, and emergent vegetation), stream reach size characteristics (wetted width, depth), flow velocity, variability in depth and flow velocity (measured as the coefficients of variation), amount of riparian cover, and elevation.

All ordinations, the MRPP test, and the bi-plots were conducted in PC-Ord v. 7.0 (McCune and Mefford 2016).

Results and Discussion A 3-dimensional solution was found for the ordination that included all samples and was designed to determine whether the three areas (Camp Creek prairie sites, Camp Creek Below sites, and Pine Creek) had different macroinvertebrate assemblages (Figure 5). This ordination had a low stress value (13.96) indicating a robust solution. Axis 1 of the ordination explained 58.7% of the variation in macroinvertebrate assemblage structure and axes 2 and 3 explained an additional 16% and 12%, respectively. The Below sites appeared to separate out from both the Camp Creek prairie sites and Pine Creek (Figure 5). However, MRPP analysis indicated that this separation, while significant (p < 0.001), was not strong (A=0.04). While these analyses indicate that the three areas do not strongly diverge in macroinvertebrate assemblage structure, the Below sites have the greatest average taxa richness per sample as well as the greatest richness in sensitive taxa (see above under Objective 1). The lack of separation of the Below samples from Camp Creek prairie and Pine Creek samples stems in part from the fact that most common taxa are found at all three areas, while taxa unique to the Below sites are generally low abundance (and thus have relatively small effects on the ordination).

Figure 5. NMS ordination solution for all macroinvertebrate data collected across all sites and sampling years (n=128). Each point represents one site during one sampling year. Points closer together are more similar in macroinvertebrate assemblage composition than points farther apart. Polygons encompass all samples taken from each “area,” Camp Creek prairie (“C”), Pine Creek (“P”), and Camp Creek in the canyon below Summer Camp (“B”). A “+”indicates the centroid for each area.

The ordination for the Camp Creek prairie sites also generated a three-dimensional solution (Figure 6). Axis 1 explained 60% of the variation among samples in macroinvertebrate composition, and axis 2 and 3 explained 19% and 10%, respectively. This ordination had a relatively low stress values (13.59) indicating a robust solution. The bi-plots indicated that axis 2 represented a gradient in wetted width and year, with later sampling years and larger wetted widths at lower values of axis 2. In addition, axis 3 represented a gradient in elevation and substrate composition. Samples with larger axis 3 values had less silt and emergent vegetation and had more gravel/cobble/boulder substrate. Larger axis 3 values also indicated lower

elevations. While axis 1 explained the greatest amount of variation in the macroinvertebrate assemblages, no environmental variables were associated with axis 1. Instead, six samples were outliers along axis 1 (Figure 6). These samples were different enough in assemblage composition that they likely drove the high level of variation explained on axis 1. These six samples were all from 2016 and were found throughout the drainage. The samples were from reaches CC1, CC2, CC18A, CC19, CC32, and CC38. To further illustrate that these six samples were outliers, we ranked the Axis 1 scores and plotted them in Figure 7.

These six outlier samples were different from all other prairie site samples in that they had a remarkably low taxa richness. The outlier reaches varied from having only 2-5 taxa, with a mean of 4. The other nine Camp Creek reaches sampled during 2016 had an average of 14 taxa. In addition, the outlier reaches had very low densities of macroinvertebrates, with an average density of 202 individuals/m2. In contrast, the average density for the other nine Camp Creek reaches sampled in 2016 was 2075/m2.

It is not clear why these sites had so few taxa and at such low densities. High flows, such as spring floods, can lead to low abundances of benthic macroinvertebrates (Power et al. 1988). However, 2016 had less precipitation prior to sampling than either 2011 or 2019 (Figure 2) and particularly low abundances were not recorded for either of those years. Our field notes indicate that it rained most of the sampling days in 2016. If the ground is saturated with water, even relatively short periods of rain can cause high flows in small streams. We examined whether this might account for the low abundances and richness values of the six 2016 outlier reaches by comparing the amount of rain on the sampling date for the outlier reaches to the amount of rain on the sampling dates for the other nine Camp Creek prairie reaches (Figure 8). The figure illustrates that sampling at the outlier reaches occurred on days with much higher rainfall than on sampling days for the non-outlier reaches. It is likely that high rainfall on the days that the outlier reaches were sampled led to rapidly increasing discharge at those sites which either flushed benthic macroinvertebrate downstream or caused them to seek refuge in areas that were not sampled (near-shore areas and under large substrate; Rempel et al. 1999).

Figure 6. NMS ordination solution for Camp Creek prairie sites. a) the solution for axes 1 and 2, and b) the solution for axes 1 and 3. Each point represents one reach during one sampling year. Each sampling year has a unique symbol and the centroids for each year are labeled and marked with a “+.” Bi-plot vectors are labeled with the environmental variable and are illustrated in red.

1.5

1.0

0.5

0.0 0 10 20 30 40 50 60 70 80 -0.5

-1.0

Axis Value 1 Axis -1.5

-2.0 2016 Sites -2.5

-3.0 Axis 1 Values Ranked from Highest to Lowest

Figure 7. Axis 1 values for the Camp Creek prairie ordination. Values are ranked from highest to lowest on the x-axis. The ellipse encompasses the six 2016 outlier values.

2.5

2.0

1.5

1.0

0.5 Precipitation CI) cm Precipitation (95% in

0.0 Camp Creek CC Outliers Fig. 8. The average amount of precipitation on the Zumwalt Prairie on the day of sampling for each Camp Creek prairie site in 2016. The “CC Outliers” are the reaches that were considered outliers based on the ordination. “Camp Creek” indicates the mean precipitation for all other non-outlier reaches sampled in 2016. Vertical lines encompass the 95% confidence intervals.

Given that the 2016 outliers appeared to have a strong influence on the ordination solution, we ran the Camp Creek prairie ordination again after removing the six outlier samples. This ordination also produced a three-dimensional solution with a final stress of 16.0 (Figure 9). Axis 1 explained 25% of the variation in the macroinvertebrate assemblages, axis 2 explained 34% and axis 3 explained 21%. Environmental gradients were associated with both the dominant axis (axis 2) and axis 3. Year, stream width, and the amount of precipitation were all positively

correlated with axis 2. Axis 3 was a substrate and elevation gradient, samples with higher axis 3 values tended to be at higher elevations and have more emergent vegetation and silt as their substrate (Figure 9).

The environmental correlations with axis 2 reveal the confounding of precipitation and sampling year. Therefore, it is difficult to determine whether increasing ordination values along axis 2 represent a trajectory of change in the macroinvertebrate assemblage resulting from changes through time or whether they reflect the effects of high precipitation years on assemblage composition. In the next section, we used mixed models to assess changes to macroinvertebrate metrics through time while holding the effects of precipitation constant. That substrate composition formed an environmental gradient on one of the ordination axes (axis 3) was not surprising as it is well established that substrate composition has important effects on macroinvertebrate assemblages in rivers and streams (Minshall 1984).

The ordination of the Pine Creek samples produced a three-dimensional solution with a final stress of 11.5 (Figure 10). Axis 1 explained 54% of the variation in assemblage structure and axes 2 and 3 explained 21% and 13%, respectively. Environmental gradients were found on each axis. Axis 1 was a gradient in velocity, width, and year. Axis 2 was a gradient in substrate composition and water depth, and axis 3 was a gradient in the amount of precipitation and year (Figure 10).

As with the ordination of Camp Creek, the relationship of year to assemblage structure in Pine Creek was confounded with the amount of precipitation (on axis 3) and wetted width (on axis 1). Therefore, based on results of the ordination it is difficult to determine whether stream reach conditions are changing through time (and possibly based upon restoration activities) or based upon relatively high levels of precipitation in the later sampling years. The correlation of year with axis 1 was driven by the separation of years 2007 and 2016 along the axis. This difference between 2007 and 2016 stems largely from differences in density. At Pine Creek, macroinvertebrate density was highest in 2007 (average of 13,211/m2) and lowest in 2016 (average of 485/m2). In contrast to Camp Creek, 2016 did not produce a subset of Pine Creek samples that were outliers. Instead, all of the 2016 Pine Creek samples had low densities and all samples grouped out on axis 1. Similar to Camp Creek, the low densities at Pine Creek in 2016 likely reflect recent rain events. While it did not rain on any of the dates that sampling occurred on Pine Creek an examination of rainfall records indicates that in 2016, rainstorms occurred more recently (prior to sampling) than most other years, were of longer duration than most other years, and produced more rainfall than any other year (Figure 11).

Figure 9. Ordination solution for the Camp Creek prairie sites without the six 2016 sites that were outliers. Environmental gradients are shown in red. A “+” indicates the centroid for each year. Year, precipitation, and width were all positively correlated with axis 2.

Figure 10. Ordination solution for Pine Creek. Environmental gradients are shown in red. A “+” indicates the centroid for each year. Year, velocity, and wetted width are all negatively correlated with axis 1.

In conclusion, the ordination analyses revealed several important features of the streams on the Zumwalt. First, the three “areas” sampled – Camp Creek prairie, Camp Creek below, and Pine Creek – had similar macroinvertebrate assemblages despite differences in physical nature and history of restoration. Second, the analyses revealed that recent precipitation plays an important role in the density and taxa richness of the creeks. It is not clear how quickly the reaches recover after large spring rain events, but this is an important consideration for future sampling. Third, the assemblages in both Camp Creek and Pine Creek respond to gradients in substrate composition, moving from reaches with high levels of silt and emergent vegetation to

reaches with a dominance of gravel/cobble/boulder. Finally, the assemblages changed through time; however, it is not clear whether this represents a change resulting from restoration or from high precipitation years.

a) 20 b) 6 c) 3.0 18 5 2.5 16

14 4 2.0 12 10 3 1.5 8 2 1.0

6 Total Precipitation (cm) Precipitation Total Days Since Last Rainfall Last Since Days 4 1 0.5

2 (Days) Rainfall Lastof Duration 0 0 0.0 2007 2011 2013 2016 2019 2007 2011 2013 2016 2019 2007 2011 2013 2016 2019 Figure 11. Rainfall at Pine Creek during each sampling year. a) The number of days since the last rainfall event before sampling. b) The duration (number of days) of the last rainfall event. c) The total precipitation recorded at the Zumwalt Prairie from the last rainfall event. Bars show the mean values and vertical lines are the 95% confidence intervals.

Objective 3. Is the condition of Camp Creek and Pine Creek improving through time?

Methods The major objective of the macroinvertebrate monitoring on the Zumwalt Prairie preserve is to determine whether stream reach conditions at both Camp Creek and Pine Creek are improving through time in response to restoration. Stream reach condition was examined using 12 macroinvertebrate metrics of stream reach condition (Table 3) and using the density and distribution of specific sensitive taxa.

Changes in reach condition through time were addressed in three fashions. First, we conducted mixed models to compare macroinvertebrate metrics for the time period before restoration to the time period after restoration. In these models, time is a categorical variable (before vs after restoration). Because the amount of precipitation was confounded with time, we used the amount of precipitation for 60 days prior to sampling as a covariate. Since reaches were sampled multiple times over the years, reach was used as the random variable. We first attempted to fit “full” models to each macroinvertebrate metric. The full models incorporated both random slopes and random intercepts:

Metric ~ Time Period+Precipitation+(Random Slope(Time Period)+Random Intercept|Reach)

Where “Metric” stands for each macroinvertebrate metric; “Time Period” is either before or after restoration; “Precipitation” is the amount of precipitation during the 60 days prior to sampling, and “Reach” is the random effect. For each reach, both random slopes across years and random intercepts were estimated. While full models are the preferred form of a mixed model because they can account for the greatest amount of structuring in the random effect, they can also suffer from low statistical power and can produce poor results when working with small sample sizes or highly variable data (Matuschek et al. 2017). Therefore, in cases where a solution with the full model was not appropriate, we used a simpler model that estimated random intercepts only. However, mixed models with only random intercepts can inflate Type I error rates (Barr et al. 2013, Matuschek 2017) and therefore results must be interpreted cautiously.

Table 3. Metrics describing macroinvertebrate community structure and the predicted direction of change with restoration. Additional information on these metrics can be found in Barbour et al. (1999), Johnson et al. (1993), and Karr and Chu (1999). Metric Description Predicted Change with Restoration Taxa Richness Count of the number of taxa present Increase Taxa Diversity Shannon’s H’ Increase Taxonomic Evenness Diversity scaled to the mathematical maximum Increase EPT1 Richness Count of the number of mayfly, stonefly, and Increase caddisfly taxa EPT Abundance The density of all individuals belonging to EPT Increase taxa EPT Relative Abundance The percent abundance of individuals belonging Increase to EPT taxa Sensitive Taxa Richness The number of taxa considered sensitive. Increase Sensitivity is based on tolerance values from Whittier and van Sickle (2010). Taxa with a tolerance value of ≤ 2.5 are considered sensitive. Sensitive Taxa The density of all individuals belonging to Increase Abundance sensitive taxa. Sensitive Taxa Relative The percent abundance of individuals belonging Increase Abundance to sensitive taxa. FFG2 Diversity Shannon’s H for feeding groups Increase FFG Evenness FFG diversity scaled to the mathematical Increase maximum Assemblage Tolerance Summation of the relative abundance of each Decrease Index (ATI) taxa multiplied by its tolerance value

1 – EPT refers to Ephemeroptera (mayfly), Plecoptera (stonefly), and Trichoptera (caddisfly) taxa. These taxa are generally sensitive to human disturbances. 2 – FFG refers to Functional Feeding Group. Macroinvertebrates were placed into functional feeding groups based upon information from Merritt et al. (2008).

The second method for addressing Objective 3 also involved examining changes in the macroinvertebrate metrics through time, but here time was used as a continuous variable. While time is easily split into two periods – before restoration and after restoration – ecosystem responses to restoration can take years (Meals et al. 2010) and testing for a continuous improvement in metric values through the years might be a more appropriate fashion to assess restoration effects. For these analyses we followed the same analytical path as the models using time as a categorical variable, but considered time as a continuous variable instead. First, we tried to fit a full model that estimated random slopes and intercepts:

Metric ~ Year + Precipitation + (Random Slope (Years) + Random Intercept |Site)

If the data did not fit a full model, then the random slope term was dropped and a reduced model that estimated random intercepts was used.

Analyses were conducted with data from reaches that had at least four points (i.e., they had been sampled at least four years), which is considered a minimum number of data points for a mixed model to accurately predict a slope and intercept (Arnqvist 2020). Ten Camp Creek prairie sites had at least four data points and were used in the mixed models (see Fig 13g). Sampling in Pine Creek has been less intensive and four reaches fit the minimum criteria of four samples (see Fig. 14c).

The third approach used to assess changes in stream reach condition involved examining the mean abundance and distribution of specific sensitive taxa. If conditions in both creeks are improving, we predict that both the abundance and/or distribution of sensitive taxa will increase through time. Mean abundance was calculated by averaging the site abundances (including 0 values) across all sampled sites for each year. Distribution was calculated as the percentage of sites a sensitive taxon was collected from during a sampling year. For these analyses, we used simple linear correlations. Sensitive taxa were defined as those taxa with tolerance values ≤ 2.5. Here we only examined sensitive taxa that were collected for at least four of the sampling years (i.e., they generated at least four non-zero data points).

Results and Discussion At Camp Creek, the relative abundance of both EPT individuals and individuals belonging to sensitive taxa was greater after restoration than before (Table 4, Figure 12a,b). Increases in the dominance of these two groups indicates an improvement in stream condition after restoration. On average, the relative abundance of EPT taxa before restoration was only 6% and increased to 34% after restoration. For sensitive taxa, the relative abundance was 1.4% before restoration and 3.5% after. None of the other 10 metrics were significantly different between the two time periods.

Only one metric changed between the time periods on Pine Creek, FFG evenness was higher after restoration than before, This indicates a more equal distribution of individuals making up the different FFGs (Table 4, Figure 12c). Despite this increase in evenness, the macroinvertebrate assemblage at Pine Creek continued to be dominated by collector-gatherers. In 2007, before restoration, collector-gatherers made up 82% of all individuals. In 2019, this relative abundance was 70%. The drop in the relative abundance of collector-gatherers in 2019

was balanced by an increase in the relative abundance of collector-filterers (from 2% in 2007 to 13% in 2019) and shredders (from 3.5% in 2007 to 7% in 2019).

Table 4. Results of significant mixed models that examined macroinvertebrate metrics before versus after restoration on Camp Creek and Pine Creek. Results of all mixed models are in Appendix 4. Creek Metric t-value p-value Camp EPT Relative Abundance 2.83 0.006 Sensitive Taxa Relative Abundance 3.36 0.004 Pine FFG Evennness 2.62 0.021

a) 1.0 b) 0.35 0.9 0.30 0.8 0.7 0.25 0.6 0.20 0.5

0.4 Taxa 0.15 0.3 0.10 0.2

0.1 0.05 Relative Abundance Sensitive Abundance Sensitive Relative 0.0 0.00 Relative Taxa Abundance of EPT Relative 0.5 1 1.5 2 2.5 Before Restoration After Restoration Before Restoration After Restoration

c) 0.9 0.8

0.7

0.6

0.5

0.4

FFG Evenness FFG 0.3

0.2

0.1

0.0 Before Restoration After Restoration Figure 12. Significant differences in macroinvertebrate metrics between two time periods, before restoration versus after restoration. a) Relative abundance of EPT taxa at Camp Creek. b) Relative abundance of sensitive taxa at Camp Creek. c) Functional feeding group evenness at Pine Creek. ○ = raw data values, ■ = means.

When time was treated as a continuous variable, additional significant relationships emerged. At Camp Creek, EPT relative abundance and EPT richness as well as sensitive taxa density, relative abundance and richness all significantly increased through time (Table 5, Figure 13). A significant correlation also existed between EPT relative abundance and richness and the amount of precipitation (Table 5, Figure 13). Since precipitation was used as a covariate in the mixed models, the positive year effect reflects an increase in these metrics through time while holding the effects of precipitation constant.

Increases in the EPT metrics through time were dramatic at Camp Creek. EPT relative abundance went from a low in 2006 of 1.5% of all individuals collected, to over 20% of all individuals during the years 2011-2016, and then to a high of ~45% of all individuals in 2019 (Figure 13a). EPT richness followed a similar trend with an average of 3.2 taxa per site in 2006 to a doubling of that in 2019 with an average of 6.4 taxa per site (Figure 13c). Increases in sensitive taxa metrics were not as dramatic, and they remained relatively low throughout the sampling period. In 2006, the relative abundance of sensitive taxa was 0.2%. The highest year for sensitive taxa relative abundance was 2013 when 6.8% of all individuals collected belonged to sensitive taxa (Figure 13f). The richness of sensitive taxa was also consistently low. In 2006 and 2007, the average number of sensitive taxa per site was 0.8 and 0.9, respectively. Starting in 2013 and continuing through 2019 the average number of sensitive taxa per site increased to ~1.5 (Figure 13g). Despite the relatively small changes in sensitive taxa metrics, the positive response through time as well as the increases in EPT relative abundance and richness through time all indicate that the condition of Camp Creek is improving.

At Pine Creek, and similar to Camp Creek, the relative abundance of EPT taxa as well as richness of sensitive taxa increased through time indicating an improvement in conditions (Table 5, Figure 14). The increase in the relative abundance of EPT taxa through time occurred mainly because of the low relative abundance in 2007 (15%) and the consistently high relative abundances in all of the subsequent sampling years (ranging from 40% to 79%, Figure 14a). As with Camp Creek, despite the richness of sensitive taxa in Pine Creek increasing through time, it has remained low throughout all sampling years. In 2007, the average richness of sensitive taxa was 1.3 and by 2019 sensitive richness had increased to an average of 2 (Figure 14b). In addition, at Pine Creek, functional feeding group evenness increased through time also indicating an improvement in condition (Table 5, Figure 14c). An increase in FFG evenness indicates a more equal distribution of individuals making up the different FFGs.

Overall, these results are encouraging, particularly the increase in dominance and richness of EPT taxa. That these taxa have become the dominant taxa at many of the sites in both drainages strongly indicates an improvement in the condition of both Camp and Pine creeks. EPT taxa density, relative abundance, and richness have a long history as an indicator of good quality in stream reach conditions (Barbour et al. 1999, Karr and Chu 1999).

The third method of examining whether Camp and Pine creek have improved in condition through time involved determining whether the abundance and distribution of specific sensitive taxa increased over the sampling years. Sensitive taxa were defined as having a tolerance value of ≤ 2.5 and were used in the analysis only if they had been collected at least four years. At Camp Creek, six taxa fitting these criteria were collected. Of these six, three showed significant

positive changes through time in abundance and/or distribution (Table 6). The caddisfly Lepidostoma increased in both mean abundance and distribution (Figure 15a,b). This genus was not collected at any prairie sites in 2006 and 2007 (however, it was collected at site Below1 both years). For the next four sampling years (2008, 2011, 2013, and 2016), Lepidostoma larvae were collected from a single prairie site, CC14. In 2019, its range expanded to include five Camp Creek prairie sites. Another sensitive caddisfly, Rhyacophila, also increased its range through time at the prairie sites (Fig. 15c). This genus was not collected in 2006, but by 2013 it occupied ~33% of the sites and continued to occupy that proportion of sites through 2019. The last taxa from Camp Creek that showed a positive response with time was the stonefly genus, Suwallia. This stonefly increased its site occupancy from 20% in 2006 to over 50% in 2019 (Figure 15d). All three of these genera are regularly collected from the Below sites and, if the population at the Camp Creek prairie sites is not reproducing in large enough numbers to spread, the Below sites are a likely source for colonists.

In contrast to these three taxa, the sensitive black fly Prosimulium decreased in mean density over time at Camp Creek and has not been collected from the prairie sites since 2011 (Figure 16). It is not clear why this decline occurred. However, the more abundant and tolerant black fly, Simulium (its tolerance value is 6.1, while the tolerance value of Prosimulium is 2.3), has increased in relative abundance through time (Figure 16). It is possible that the drop in Prosimulium is the result of competition with Simulium. While there is some speculation regarding whether different species of black flies compete strongly for resources (Colbo 1979, Lake and Burger 1983), we are unaware of any work that has shown one species of black fly driving another species to local extinction, but that is a possibility.

In Pine Creek only one sensitive taxa has been collected for at least four years, the stonefly Suwallia. While the average abundance of Suwallia has not significantly changed through time, its distribution has increased from a low of 50% of reaches in 2007 to 100% or reaches in 2019 (Figure 17; n = 5, Pearson r = 0.94, p = 0.015).

Table 5. Results of significant mixed model analyses with time as a continuous variable. Models examined the effect of sampling year and the amount of precipitation 60 days prior to sampling as fixed effects. Site was the random factor. “RA” stands for relative abundance. In all cases, “Intercepts” only models were run, as the models were unable to estimate slopes. Results of all mixed models are in Appendix 4. Creek Metric Estimate t-value p-value Camp EPT RA Year: 0.033 4.536 <0.001 Precip: 0.024 2.607 0.012 EPT Richness Year: 0.038 3.309 0.002 Precip: 0.032 2.188 0.033 Sensitive Year: 0.063 2.275 0.027 Density Precip: -0.032 -0.926 0.359 Sensitive RA Year: 0.009 3.303 0.002 Precip: -0.007 -1.987 0.052 Sensitive Year: 0.051 3.152 0.003 Richness Precip: -0.021 -1.024 0.311 Pine EPT RA Year: 0.041 2.271 0.041 Precip: 0.019 0.962 0.353 Sensitive Year: 0.073 2.789 0.015 Richness Precip: -0.032 -1.102 0.290 FFG Evenness Year: 0.024 3.597 0.003 Precip: -0.014 -1.975 0.070

a) 1.0 b) 1.0 0.9 0.9 0.8 0.8 0.7 0.7 0.6 0.6 0.5 0.5 0.4 0.4 0.3 0.3 0.2

EPT Relative Abundance Relative EPT 0.1

EPT Relative Abundance Relative EPT 0.2 0.1 0.0 0 2 4 6 8 10 12 14 0.0 Sixty Days Precip (cm) 2004 2006 2008 2010 2012 2014 2016 2018 2020 c) 14 d) 14

12 12

10 10 8 8 6 6 4

4 of Taxa Number EPT

2 Number of Taxa Number EPT

2 0 0 2 4 6 8 10 12 14 0 Sixty Day Precipitation 2004 2006 2008 2010 2012 2014 2016 2018 2020 Figure 13. Significant relationships between macroinvertebate metrics and sampling year or amount of precipitation for Camp Creek. a) EPT relative abundance versus sampling year, b) EPT relative abundance versus amount of precipitation for 60 days prior to sampling, c) EPT taxa richness versus sampling year, d) EPT taxa richness versus amount of precipitation. Figure continued on next page. Each site is coded with a unique color and linear trendlines are drawn for each site. The legend for site colors is in panel g.

e) 3.5 f) 0.35

3.0 0.30

2.5 0.25

2.0 0.20

1.5 0.15 Abundance

1.0 0.10 Sensitive Taxa Relative Relative Taxa Sensitive

Ln(Sensitive Density) Taxa Ln(Sensitive 0.5 0.05

0.0 0.00 2004 2006 2008 2010 2012 2014 2016 2018 2020 2004 2006 2008 2010 2012 2014 2016 2018 2020 g) 7 6

Sensitive Richness Taxa Sensitive 1

0 2004 2006 2008 2010 2012 2014 2016 2018 2020

CC1 CC11 CC13 CC14 CC16 CC17 CC19 CC22 CC24 CC32

Figure 13, continued. Significant relationships between macroinvertebate metrics and sampling year for Camp Creek. e) sensitive taxa density (natural log transformed) f) sensitive taxa relative abundance, and g) sensitive taxa richness. Each site is coded with a unique color and linear trendlines are drawn for each site. The legend for site colors is in panel g.

a) 1 b) 3.0 0.9 2.5 0.8 0.7 2.0 0.6 0.5 1.5 0.4 1.0

0.3 Sensitive Richness Taxa Sensitive

EPT Relative Abundance Relative EPT 0.2 0.5 0.1 0 0.0 2006 2008 2010 2012 2014 2016 2018 2020 2006 2008 2010 2012 2014 2016 2018 2020 c) 0.9 0.8 0.7 0.6 0.5 0.4

0.3 FFG Evenness FFG 0.2 0.1 0 2006 2008 2010 2012 2014 2016 2018 2020

PC12 PC14 PC18 PC23

Figure 14. Significant relationships between macroinvertebrate metrics and sampling year for Pine Creek. a) EPT relative abundance, b) sensitive taxa richness, and c) functional feeding group evenness. Each site is coded with a unique color and linear trendlines are drawn for each site. The legend for site colors is in panel c.

Table 6. Correlation results of the relationships between year and mean density and year and the proportion of sites a taxa was collected from for sensitive taxa collected from Camp Creek. The number of years, out of seven, that a taxon was collected from Camp Creek is shown in the last column. Order Family/Genus Mean Density Percent of Sites # of Pearson r p-value Pearson r p-value Years Collected Trichoptera Apatania -0.185 >0.25 0.580 0.10 4 Lepidostoma 0.783 0.023 0.867 0.006 5 Rhyacophila 0.535 0.11 0.859 0.007 6 Diptera Clinocera -0.412 0.20 -0.150 >0.25 5 Prosimulium -0.758 0.025 -0.633 0.06 4 Plecoptera Suwallia 0.063 >0.25 0.848 0.008 7

Lepidostoma Lepidostoma 25 35

30 20 25

15 20

10 15 % of of % Sites

Mean Density Mean 10 5 5

0 0 2004 2006 2008 2010 2012 2014 2016 2018 2020 2004 2006 2008 2010 2012 2014 2016 2018 2020 Rhyacophila Suwalia 40 60 35 50 30 40 25

20 30 % of of % Sites

15 of % Sites 20 10 10 5

0 0 2004 2006 2008 2010 2012 2014 2016 2018 2020 2004 2006 2008 2010 2012 2014 2016 2018 2020 Fig. 15. Sensitive taxa that significantly increased in either average density or the proportion of sites they occupied over time. a) and b) Increases in the average density and proportion of sites for the caddisfly Lepidostoma. c) Increase through time in the proportion of sites occupied by the caddisfly Rhyacophila. d) Increase through time in the proportion of sites occupied by the stonefly Suwallia.

35 0.6

) 30 2 0.5

25 0.4 20

Density Density (#/m 0.3

15 Relative Abundance Relative 0.2 10 Prosimulium 0.1

Prosimulium 5 Simulium Simulium Simulium

0 0 2005 2010 2015 2020 Fig. 16. The decline in average density of the sensitive black fly Prosimulium through time and the increase in the relative abundance of the more tolerant black fly Simulium.

Suwallia 100

40 % of of % Sites

0 2006 2008 2010 2012 2014 2016 2018 2020 Figure 17. The percentage of sites the stonefly genus Suwallia was collected from in Pine Creek over the sampling years.

The results of the abundance and distribution of sensitive taxa indicate that the condition of both Camp Creek and Pine Creek are improving. However, there are still relatively few sensitive taxa that are consistently found in either drainage, particularly Pine Creek. In both drainages the stonefly Suwallia has increased its distribution through time. These stoneflies are the most sensitive taxa found at the Camp Creek prairie sites and Pine Creek (tolerance value of 0.9). They are relatively small predators (8-10 mm, Figure 18) that preferentially consume chironomids and black flies (Stewart and Stark 2002) which are abundant in both Camp Creek and Pine Creek.

Figure 18. The stonefly Suwallia (family: Chloroperlidae), a sensitive taxa found in both Camp Creek and Pine Creek. The aquatic larvae (left picture from BugGuide.net) are predators feeding on black flies and chironomids. The adults (right picture from DiscoverLife.org) are commonly referred to as green stoneflies.

Objective 4. Are there reaches that are consistently considered the highest quality?

Within each year, reaches varied in macroinvertebrate metrics such that some were considered in better condition than other reaches. However, individual reaches also varied across years in macroinvertebrate metric values. Thus, it’s not clear whether certain reaches are consistently the highest quality reaches (of all the reaches sampled that year) or whether the reaches considered in the highest quality change each year. We examined the macroinvertebrate metrics at each site in Camp Creek and Pine Creek across all years to determine whether sites existed that were consistently considered the highest quality. We then looked at the environmental characteristics of those reaches, and their position in the drainage. Stream reaches can accumulate effects of upstream land use with greater accumulation at reaches lower in a watershed, often resulting in a gradient of decreasing stream reach quality with decreasing elevation (Allan 2004).

Methods To determine which reaches were in the best condition of the Camp Creek prairie reaches, we ranked each reach by each macroinvertebrate metric for each year of sampling. Within each year, sites that were ranked in the top three for a metric were considered in the “best” condition based on that metric. We considered a site to be “consistent” if the site was in the top three for a given metric for at least four of the seven sampling years.

Sampling in Pine Creek was not as extensive as in Camp Creek. Twelve macroinvertebrate sites were established on Pine Creek. However, all twelve were sampled only in 2007 and 2016. We used these two years to rank reaches based on the macroinvertebrate metrics. In contrast to Camp Creek, in Pine Creek the highest scores for the metrics were spread across a large proportion of the reaches. In 2007, 10 (of 12) sites ranked in the highest three for at least one metric and in 2016 all 12 sites ranked in the highest three for at least one metric. Because of this pattern we considered the best condition sites in Pine Creek as those that were ranked in the top 3 for at least six metrics during both 2007 and 2016.

To examine whether the sites considered in the best condition differed environmentally from all other sites, we calculated the mean and 95% confidence intervals for all environmental parameters for the high quality sites as one group and all other sites as another group. Means were calculated across all years and differences between high quality sites and all other sites were considered significant if the 95% confidence intervals for one group did not overlap the mean of the other group. We used elevation as a measure of position in the drainage.

Results and Discussion At Camp Creek, five reaches fit the criteria of being in the top three reaches for a metric for at least four years (Table 7). At Pine Creek reaches PC17 and PC18 were considered to be in the best condition based upon being ranked in the top three for at least six metrics during 2007 and 2016 (Table 8).

Environmental differences between the best condition sites and all other sites are shown in figures 19 and 20. Generally, stream reaches are predicted to accumulate upstream impacts resulting in a pattern of declining stream reach quality with distance downstream in a drainage (Li et al. 1994, Lindberg et al. 2011). In contrast to this general pattern, we found that the highest quality reaches in Camp Creek were low in the drainage (Figure 19a). Indeed, sites CC11, CC13, and CC14 are the three lowest prairie sites in Camp Creek and sites CC17 and CC19 are also low in the drainage. In Pine Creek, the highest quality reaches were approximately in the middle of the sampling area, resulting in no difference in elevation between the high quality reaches and all other reaches (Figure 20a).

Substrate composition at high quality sites in both drainages differed from all other sites in a similar fashion, high quality sites had greater gravel/cobble/boulder and less silt and emergent vegetation (Figure 19b and 20b). Many sensitive macroinvertebrate taxa avoid areas of high silt (Barbour et al. 1999, Zweig and Rabeni 2001), and thus the relationship between high quality reaches and low silt levels is not surprising. The only other environmental variables that were different between high quality reaches and all other reaches were depth and water velocity. In Camp Creek, high quality sites had higher velocities, but similar depths compared to other sites (Figure 19c). In contrast, at Pine Creek high quality sites had greater depths, but similar velocities compared to other sites (Figure 20c).

While the result that the highest quality reaches in Camp Creek are low in the drainage was not expected, we believe that this result reflects the relatively high discharge and water velocities found at these reaches. High water velocities at a reach prevent silt from settling, resulting in substrate that is preferred by many sensitive taxa. This factor appears to override any cumulative

effects that these reaches are subject to from upstream. In addition, at Camp Creek the highest quality reaches had less emergent vegetation than the other reaches (Figure 19b). Emergent vegetation tends to slow water velocities enabling silt to settle, resulting in conditions that are low quality based on macroinvertebrate assemblages. In contrast to Camp Creek, it is not clear why the highest quality reaches on Pine Creek had low silt levels. These reaches did not have higher water velocities than all other reaches; in addition, these reaches did not have less emergent vegetation than all other sites. It is possible that the low levels of silt at the high quality reaches result from little local inputs of sediment. In addition, sediment from upstream might settle out before entering these reaches if there are slow water areas just upstream.

Table 7. The sites considered in the “best” condition for the Camp Creek prairie sites. Numbers indicate the number of years that a site ranked in the top 3 for a metric. Sites are only considered to be representative of best condition sites if, for a given metric, they were ranked in the top 3 sites for at least 4 years. All sites were sampled each of the seven years except for CC11, which was sampled for only six years (it was not sampled in 2006). Site Metric CC11 CC13 CC14 CC17 CC19 Total Diversity 5 4 Total Evenness 4 Total Richness 6 EPT Density 6 5 EPT Relative Abundance 5 4 EPT Richness 5 5 6 4 Sensitive Taxa Density 6 5 Sensitive Taxa Relative Abundance 4 7 6 Sensitive Taxa Richness 6 5 6 Functional Feeding Group Diversity 4 Functional Feeding Group Evenness 4 ATI 6 5 4

Table 8. The best condition sites at Pine Creek and the metrics they were ranked in the top 3 for (indicated by an “X”). 2007 2016 Metric PC17 PC18 PC17 PC18 Total Diversity Total Evenness Total Richness X EPT Density X X X EPT Relative Abundance X X X EPT Richness X X X Sensitive Taxa Density X X X X Sensitive Taxa Relative Abundance X X X Sensitive Taxa Richness X X X X Functional Feeding Group Diversity Functional Feeding Group Evenness ATI X X X X

a) 1400 b) 100

1350 95% CI 95%

95% CI 95% 60

1300 40

20 1250

Mean % Substrate % Mean Substrate 0

Mean Elevation Elevation Mean (m) Silt GrCoBo EVeg 1200 HighQuality Other HighQuality Others c) 35 d) 140 30 120

25 100 95% CI 95%

20 80

15 60 95% CI 95%

10 40

5 20 Velocity (cm/sec) Velocity Mean Depth Depth Mean Mean and (cm) 0 Mean Coefficient of Variation Variation of Coefficient Mean 0 Depth Velocity CVDepth CVVelocity

HighQuality Others HighQuality Rest e) 300 f) 9 8 250 7

200 6 95% CI 95%

5 150

95% CI95% 4

100 3

2 Mean Wetted Width (cm) Width Wetted Mean 50 % Mean Cover 1

0 0 HighQuality Others HighQuality Rest Figure 19. Mean and 95% confidence intervals of environmental variables for Camp Creek prairie sites. The highest quality sites are compared to all other sites. a) Elevation in meters (note that the y-axis does not start at 0); b) substrate types (GrCoBo indicates a mixture of gravel, cobble, and boulder; EVeg indicates emergent vegetation); c) water depth; d) reach-level variability in depth and velocity measured as the coefficient of variation, e) wetted width, and f) the percent cover resulting from riparian and/or nearby vegetation.

1400 a) b) 100

1380 80

95% CI 95%

95% 95% CI 1360 60

40 1340

Elevation (m) Elevation 1320 Mean % Substrate % Mean Substrate 0 Silt GrCoBo EVeg 1300 HighQuality Other High Others c) 45 d) 140 40 120 35 100

95%CI 30

25 80

20 60

15 95%CI 40 10 20

5 Velocity (cm/sec) Velocity

Mean Depth Depth Mean Mean and (cm) 0 0 Mean Coefficient of Variability Variability of Coefficient Mean Depth Velocity CVDepth CVVelocity High Others High Others e) 300 f) 16 14 250

12 95%CI

200

95%CI 10

150 8

6 100 4

50 %Cover Mean 2

0 0 Mean Wetted Width (cm) Width Wetted Mean HighQuality Other HighQuality Other Figure 20. Mean and 95% confidence intervals of environmental variables for Pine Creek sites considered high quality based upon macroinvertebrate metrics and all other sites. Means are calculated across all sampling years. Mean values are not considered different if they overlap the confidence interval of the other group. a) Elevation in meters (note that the y-axis does not start at 0), b) substrate types (GrCoBo indicates a mixture of gravel, cobble, and boulder; EVeg indicates emergent vegetation), c) water depth and velocity, d) reach-level variability in depth and velocity measured as the coefficient of variation, e) wetted width, and f) the percent cover resulting from riparian and/or nearby vegetation.

Objective 5. Do grazing exclosures have an effect on macroinvertebrate assemblages and environmental parameters?

Grazing exclosures with riparian plantings of woody vegetation were established by The Nature Conservancy on a number of reaches in Camp Creek in 2010. These reaches are expected to improve in quality to a greater extent than non-exclosure sites through at least two mechanisms. First, grazing exclosures reduce sediment input derived from bank trampling by large herbivores. Second, the protected woody riparian plantings will eventually shade stream reaches, keeping water temperature down, and increase habitat complexity within the stream (through inputs of large woody debris and exposed root wads) (Gregory et al. 1991). We predicted that stream reaches within exclosures would be in better condition than stream reaches outside of exclosures.

Methods At both Camp Creek and Pine Creek, macroinvertebrate metrics were compared between exclosure reaches and non-exclosure reaches. For the Camp Creek data, t-tests were conducted to compare the two groups for 2016 and 2019. We used these years because the greatest number of exclosure sites were sampled then. In 2016, four reaches in exclosures were compared to six reaches outside of exclosures. In 2019, six exclosure reaches were compared to nine non- exclosure reaches. Sampling on Pine Creek was more limited. Only one exclosure site was sampled in 2016 (PC14) and only two in 2019 (PC14 and PC26). For the Pine Creek data, we calculated the mean and 95% confidence intervals for all non-exclosure reaches and examined whether the metric values for the exclosure reaches fell outside of the confidence intervals.

For the Camp Creek data, we also used a second method to determine whether macroinvertebrate metrics in exclosures differed from those at non-exclosure reaches. Here we did not examine absolute values of macroinvertebrate metrics, but instead considered how much reaches within exclosures changed through time relative to reaches outside of exclosures. For this analysis, we examined the change in stream condition for two exclosure sites (CC13 and CC16) for which we had data both before exclosure construction and after exclosure construction. From this data, we calculated a ratio for each metric that was the mean metric value (averaged across years) after construction of exclosures divided by the mean metric value before exclosure construction. Values greater than one indicate that the metric has increased after exclosure construction, and values less than one indicate a decline in the metric. The ratios for CC13 and CC16 were compared to mean values generated from all non-exclosure reaches in which pre-restoration and post-restoration data were available. For the non-exclosure reaches, mean and 95% confidence intervals were calculated. If the ratio value for an exclosure reach fell outside the 95% confidence interval of the non-exclosure reaches, the exclosure reach was considered to have changed (based on that metric) to a greater degree than the rest of the drainage.

Finally, we examined whether the stream environments at the Camp Creek exclosure reaches CC13 and CC16 changed through time. We predicted that in the absence of bank trampling from cattle or elk and the multiple effects of planted riparian vegetation, the stream environments would change. Specifically, we predicted a decline in silt on the substrate, an increase in

environmental complexity, and an increase in riparian cover at the two sites in exclosures. In contrast, the environment in reaches outside of exclosures was predicted to either not change or change very little. To examine this we calculated the mean and 95% confidence intervals for environmental factors before exclosure construction and compared that to the mean and 95% confidence intervals after construction. These means were compared to the mean values for two neighboring non-exclosure reaches, CC14 and CC17.

Results and Discussion No significant differences in any of the macroinvertebrate metrics between exclosure and non-exclosure reaches emerged from the Camp Creek data (p-values for all t-tests >0.05) (Figure 21). Several metrics appeared to differ between exclosure and non-exclosure reaches at Pine Creek (Figure 22). However, there was no consistency in the response of metrics to exclosures. Total taxa richness was higher at reach PC14 (an exclosure reach) than non-exclosure reaches in 2016, but lower in 2019 (Figure 22e, f). In 2019, EPT density was lower at PC14 than non- exclosure reaches, but higher at exclosure reach PC26 (Figure 22h).

The lack of an apparent response by macroinvertebrates to reaches within exclosures was somewhat surprising. However, it is possible that the condition a stream reach is in before exclosures are constructed has an effect on whether and how quickly the reach improves. In this case, we might expect that a better metric of an exclosure effect is to compare the change in condition of a given stream reach before versus after exclosure construction. This possibility was addressed on Camp Creek by examining the ratios of metrics after exclosure construction to metric values before exclosure construction for two exclosure reaches, CC13 and CC16. Both exclosure reaches improved in condition at a faster rate than non-exclosure sites based on a subset of macroinvertebrate metrics. However, the two exclosure sites differed in the metrics that indicated an improvement (Figure 23). CC13 improved at a greater rate than non-exclosure sites based on sensitive taxa density and sensitive taxa richness (Figure 23). In contrast, CC16 improved at a greater rate than non-exclosure sites based on total diversity, total evenness, EPT relative abundance, sensitive taxa relative abundance, functional feeding group diversity, and functional feeding group evenness (Figure 23).

Several metrics indicated that CC13 and CC16 decreased in condition after exclosure construction. The ATI score at site CC13 increased after exclosure construction indicating a decline in the dominance of sensitive taxa (ATI is the only metric in which an increase in value indicates a decline in condition). At CC16 the ratios for EPT density and EPT richness indicated that this site did not improve to the same extent as non-exclosure sites based upon these metrics (Figure 23).

Despite an apparent lack of any consistent effects of exclosures on macroinvertebrates, several important environmental differences were found. Substrate composition changed as predicted. At the two exclosure sites, CC13 and CC16, the amount of silt decreased and the amount of gravel/cobble/boulder substrate increased after exclosure construction (Figure 24). In contrast, the two control sites, CC14 and CC17, showed no change in substrate composition between the two time periods (Figure 24).

a) 2.5 2016 b) 2.5 2019

2.0 2.0

1.5 1.5

1.0 1.0

Scores Scores

0.5 0.5 Mean Diversity Diversity Mean Evenness and

Mean Diversity Diversity Mean Evenness and 0.0 0.0 Shannon H Evenness FFG H FFG Evenness Shannon H Evenness FFG H FFG Evenness

Exclosure No Exc Exclosure No Exc c) 1200 d) 12000

1000 10000

)

) 2 2 800 8000

600 6000

400 4000

Density Density (#/m Density Density (#/m

200 2000

0 0 EPT Dens Sensitive Dens EPT Dens Sensitive Dens

Exclosure No Exc Exclosure No Exc e) 80 f) 0.7 70 0.6 60 0.5 50 0.4 40 0.3 30

20 0.2 Relative Abundance (%) Relative 10 Abundance (%) Relative 0.1

0 0.0 EPT RA Sensitive RA EPT RA Sensitive RA

Exclosure No Exc Exclosure No Exc g) 20 h) 25

16 20

12 15

8 10

(# of Taxa) of (# (# of Taxa) of (#

4 5

ATI Scores and Richness Scores Richness and ATI ATI Scores and Richness Scores Richness and ATI

0 0 ATI Total Richness Sensitive Rich EPT Richness ATI Total Richness Sensitive Rich EPT Richness Exclosure No Exc Exclosure No Exc Figure 21. The twelve macroinvertebrate metrics comparing the average value for sites within grazing exlcosures (gray bars) to the average values for sites with no exclosures (open bars) at Camp Creek. Metric values from 2016 are shown in the panels in the left column and 2019 metrics are illustrated in the right column. Vertical lines encompass the 95% confidence intervals and an asterisk indicates a significant difference (i.e., the mean values for each group do not fall within the 95% confidence interval of the other group).

a) 2016 b) 2019 1.6 2.5 1.4 2.0 1.2

1.0 1.5

0.8 Scores

0.6 Scores 1.0 0.4 0.5 0.2

Mean Diversity Diversity Mean Evenness and 0.0

Mean Diversity Diversity Mean Evenness and 0.0 Shannon H Evenness FFG H FFG Evenness Shannon H Evenness FFG H FFG Evenness

PC14 NonExc PC14 PC26 NonExc

c) d) 4500 200 4000

3500

)

) 2 150 2 3000 2500 100 2000

1500

Density Density (#/m Density Density (#/m 50 1000 500 0 0 EPT Density Sensitive Density EPT Dens Sensitive Dens

PC14 NonExc PC14 PC26 NonExc

e) 70 f) 100 60 80 50

40 60

30 40 20

20 Relative Abundance (%) Relative

10 Abundance (%) Relative

0 0 EPT RA Sensitive RA EPT RA Sensitive RA

PC14 NonExc PC14 PC26 NonExc g) 12 h) 25 10 20

8 15 6 10

(# of Taxa) of (# (# of Taxa) of (# 5

ATI Scores and Richness Scores Richness and ATI ATI Scores and Richness Scores Richness and ATI 0 0 ATI Total Richness Sensitive EPT Richness ATI Total Richness Sensitive EPT Richness Richness Richness PC14 NonExc PC14 PC26 NonExc Figure 22. The twelve macroinvertebrate metrics comparing values the exclosure sites, PC14 (2016 and 2019) and PC26 (2019 only) to the average values for all non-exclosure sites at Pine Creek. An asterisk indicates that the exclosure site fell outside of the 95% confidence interval for the non-exclosure sites. Confidence intervals are illustrated by the vertical lines.

Total Richness Total Diversity Total Evenness 1.4 1.4 * 1.4 * 1.2 1.2 1.2

1.0 1.0 1.0 Restoration - 0.8 0.8 0.8

0.6 0.6 0.6

0.4 0.4 0.4 Restoration/Pre

- 0.2 0.2 0.2

Post 0.0 0.0 0.0 NonExc CC13 CC16 NonExc CC13 CC16 NonExc CC13 CC16

EPT Density EPT Relative EPT Richness 8 Abundance 2.0 50 7 * 45 6 40 1.5

Restoration 35

- 5 30 * 4 1.0 25 3 20 2 15 0.5

10 Restoration/Pre - 1 * 5

Post 0 0 0.0 NonExc CC13 CC16 NonExc CC13 CC16 NonExc CC13 CC16

Sensitive Density Sensitive Relative Sensitive Richness Abundance 25 6 * * 50 * 5 20 40

4 Restoration

- 15 30 3 10 20 2

5 10

Restoration/Pre 1 -

Post 0 0 0 NonExc CC13 CC16 NonExc CC13 CC16 NonExc CC13 CC16

ATI FFG Diversity FFG Evenness 1.2 1.6 * 1.4 * * 1.4 1.2 1.0 1.2 1.0 0.8 Restoration 1 - 0.8 0.6 0.8 0.6 0.6 0.4 0.4 0.4

Restoration/Pre 0.2 0.2 - 0.2

Post 0.0 0 0.0 NonExc CC13 CC16 NonExc CC13 CC16 NonExc CC13 CC16 Figure 23. The change in macroinvertebrate metrics from pre-restoration to post-restoration time periods. Values greater than 1.0 indicate that the metric was higher post-restoration than pre- restoration, and values less than 1.0 indicate a drop in the metric value during the post- restoration years. “NonExc” indicates the mean value for the eight non-exclosure reaches and the vertical line encompasses the 95% confidence interval. Proportions are shown for the two exclosure reaches, CC13 and CC16, that had macroinvertebrate data for both the pre-restoration and post-restoration periods. The values for CC13 and CC16 are considered different from the non-exclosure reaches if their values fall outside of the 95% confidence interval for the non- exclosure reaches (this is indicated by an asterisk).

Riparian cover also increased at the two exclosure reaches after exclosures were built while there was no change at the control sites (Figure 24c). While the average amount of cover increased after restoration at the exclosure sites, there was a large amount of variation (Figure 24c). This variation can be accounted for by examining how cover changed over the post- restoration years (Figure 24d). Exclosures were constructed and riparian vegetation planted in 2010. Initially, no cover was recorded at either of the exclosure sites after construction. By 2016 riparian vegetation started to shade both CC13 and CC16. The vegetation continued to grow and increase its shading of the stream reaches such that by 2019 the average amount of cover at site CC13 was 53% and the average cover at CC16 was 21% (Figure 24d). In contrast, the control sites had low riparian cover that varied little between years and showed no directional change through time (Figure 24d).

In contrast to changes in substrate and riparian cover there appeared to be no consistent changes in habitat complexity as measured by the coefficient of variation for both stream depth and velocity (Figure 24b).

The change in the stream environment within exclosures indicates that a lack of environmental changes is not the reason behind the lack of response by macroinvertebrates. It is possible that there simply has not been enough time for macroinvertebrates to respond to the changed exclosure reaches. In their review of studies that measured time lags in aquatic organisms to implementation of best management practices, Meals et al. (2010) report lags from two years (for fish responding to habitat restoration in first order streams) to 25 years (for fish responding to Conservation Reserve Program buffers in a large watershed). As of the 2019 sampling, exclosures on Camp Creek were in place for nine years. This amount of time falls right in the middle of the range reported by Meals et al. (2010). Another possibility is that the exclosures might be too small to have an effect on stream biota. For example, both Wang et al. (1997) and Dauwalter et al. (2018) found localized effects of grazing exclosures on riparian vegetation and streambank condition, but little to no effect on macroinvertebrates. The authors of both studies concluded that biotic assemblages, such as macroinvertebrates, most likely respond to larger scale processes occurring at the entire drainage/watershed. However, other research has indicated that exclosures can influence stream biota. For example, Bayley and Li (2008) found increased densities of juvenile redband trout (Oncorhynchus mykiss) in cattle exclosures in northeastern Oregon.

Currently it is not clear why macroinvertebrates are not responding to exclosures on Camp Creek. However, continued monitoring of these stream reaches is important as our results show that the riparian vegetation is still maturing and has not yet had as large an effect on the environment of the stream reaches as it likely will in the future.

a) 120 b) 350 300 100 250 80 200 60 150 40

100 % of Transects of %

20 50 Coefficient Variation Coefficient of 0 0 Before After Before After Before After Before After Before After Before After Before After Before After CC13 CC14 CC16 CC17 CC13 CC14 CC16 CC17 Silt Gr/Co/Bo E Veg CV Depth CV Velocity c) 50 d) 60

50 40

40 30

30 % Cover %

% Cover % 20 20

10 10

0 0 Before After Before After Before After Before After 11 13 16 19 11 13 16 19 11 13 16 19 11 13 16 19 CC13 CC14 CC16 CC17 CC13 CC14 CC16 CC17 Figure 24. Mean and 95% confidence intervals for environmental variables measured at exclosure sites CC13 and CC16 on Camp Creek and nearby control sites CC14 and CC17. “Before” indicates the mean value before exclosures were constructed and “After” indicates mean values after exclosures were constructed. a) Percent of transects that a substrate type was considered dominant. “GrCoBo” stands for gravel, cobble, and boulder substrate. “E Veg” stands for emergent vegetation. b) Mean coefficient of variation in depth and velocity. d) Mean percent cover of riparian vegetation. e) this panel deviates from the others and shows the percent cover at each reach for each year after exclosure construction. Numbers indicate the year (e.g., “11” is 2011 and “13” is 2013).

Objective 6. Does recent grazing history influence stream reach condition?

The last objective was to examine whether the condition of a stream reach was related to the intensity of grazing in the pasture that a reach flowed through. We examined the potential impact of grazing intensity on 2019 macroinvertebrate metric values. For this analysis, we used Camp Creek data and excluded sites in cattle/elk exclosures (since those wouldn’t be strongly impacted by cattle grazing). We considered the number of years a pasture had been grazed as our metric of grazing intensity (Table 9). The number of years grazed for each reach was calculated for eight different time periods. The first time period was the most recent prior year (2018) only, the

second period was 2017 to 2018, the third was the three years prior to 2019, and so on with the last time period being 2011 to 2018 (Table 10). We restricted our analysis to only the post- restoration years (2011 – 2019) to avoid confounding the effects of cattle grazing with changes in land use due to restoration. Simple linear regressions between grazing intensity and 2019 metric values were conducted for each metric and each time period.

Table 9. Macroinvertebrate sampling and livestock grazing history for Camp Creek during the post-restoration years. “Y” indicates that the site was sampled or in a grazed pasture that year. “-“ indicates no sampling or grazing occurred. Sites were not used if they were not sampled in 2019 or if they were within a cattle/elk exclosure. Macroinvertebrate Sampling Livestock Grazing Site 2011 2013 2016 2019 N 2011 2012 2013 2014 2015 2016 2017 2018 N CC1 Y Y Y Y 4 - - - Y - - Y Y 3 CC11 Y Y Y Y 4 Y Y Y - Y Y Y - 6 CC14 Y Y Y Y 4 ------0 CC17 Y Y Y Y 4 Y - - - - Y - - 2 CC19 Y Y - Y 3 Y - - - - - Y - 2 CC22 Y Y - Y 3 Y ------1 CC24 Y Y Y Y 4 Y - - - - Y - - 2 CC32 Y - Y Y 3 Y Y Y Y Y Y - Y 7

Six of the 12 macroinvertebrate metrics were significantly related to the intensity of grazing for at least one time period (Table 10, Figure 25). The relationship between grazing intensity and these macroinvertebrate metrics was negative in all cases, indicating that as grazing intensity increased stream reach condition decreased. Five of the six metrics were significantly related to grazing intensity during only one or two time periods (Table 10). The three metrics associated with sensitive taxa (sensitive taxa density, relative abundance, and richness) were all related to whether grazing occurred in 2018. This suggests that the metrics associated with sensitive taxa respond rapidly to grazing intensity, but also recover rapidly (i.e., with one exception, there was no relationship with longer time periods). In contrast, the other three metrics were related to grazing intensity only over longer time periods (at least two years). In contrast, total taxa richness was negatively related to grazing intensity in seven of the eight time periods, with only the most recent time period not significant (Table 10). This indicates a very robust relationship between macroinvertebrate taxa richness and grazing intensity. The results from other work on cattle management and macroinvertebrate richness have come to varied conclusions. Herbst et al. (2012) found that macroinvertebrate taxa richness responded positively to the removal of cattle in meadows of the Sierra Nevada mountains. In contrast, McIver and McInnis (2007) did not find any effect of excluding grazing on macroinvertebrate richness in a Blue Mountain stream in northeastern Oregon.

Table 10. Results of linear regressions for macroinvertebrate metrics that were significantly related to grazing intensity during at least one of the time periods. Significant relationships are in bold. For each cell, the first number is the r2 value and the second number is the p-value. All significant relationships were negative, as grazing intensity increased the metric value (and stream reach condition) decreased. Metric Total Shannon EPT Sensitive Sensitive Sensitive Richness Diversity Richness Density Relative Richness Years Abundance 2018 0.33, 0.13 0.11, 0.41 0.44, 0.07 0.90, <0.01 0.74, <0.01 0.61, 0.02 2017-2018 0.65, 0.01 0.55, 0.04 0.26, 0.20 0.38, 0.10 0.22, 0.24 0.38, 0.10 2016-2018 0.82, <0.01 0.46, 0.06 0.58, 0.03 0.43, 0.08 0.21, 0.25 0.40, 0.09 2015-2018 0.66, 0.01 0.30, 0.16 0.43, 0.08 0.32, 0.14 0.15, 0.34 0.24, 0.22 2014-2018 0.67, 0.01 0.28, 0.17 0.52, 0.04 0.55, 0.04 0.32, 0.14 0.39, 0.10 2013-2018 0.59, 0.03 0.22, 0.24 0.42, 0.08 0.43, 0.07 0.24, 0.21 0.27, 0.18 2012-2018 0.52, 0.04 0.18, 0.30 0.35, 0.12 0.36, 0.12 0.19, 0.28 0.20, 0.26 2011-2018 0.50, 0.05 0.17, 0.31 0.37, 0.11 0.28, 0.17 0.14, 0.36 0.15, 0.34

30 2.5 a) CC14 b) CC14 25 CC22 2.0 CC24 20 CC17, CC22, CC32 1.5 & CC24 CC17 15 CC19 CC19 CC11 CC32 1.0 10 CC1 CC11

Number Taxa of Number CC1 Shannon Diversity Shannon 5 0.5

0 0.0 0 1 2 3 4 5 6 7 8 0 1 2 Number of Years Grazed Number of Years Grazed 12 c) CC14 d) 600

CC14 )

10 2 CC22 500

8 400 CC11 CC19 & CC24 6 300 CC19 CC17 4 CC1 200 CC11 CC32 CC17

Number of Taxa Number EPT 2 100 CC22 CC24 CC1 CC32

0 Density (#/m Taxa Sensitive 0 0 1 2 3 4 5 0 1 2 3 4 5 Number of Years Grazed Number of Years Grazed 6 e) 0.05 f) CC22 CC11, CC14, 5 0.04 CC17, & CC19 4 CC11 0.03 3 CC14 0.02 Abundance CC22 2 CC24 0.01 CC1 & CC1 &

Sensitive Taxa Relative Relative Taxa Sensitive 1

CC32 of Taxa Number Sensitive CC17 & CC32 CC24 CC19 0.00 0 0 1 0 1 Number of Years Grazed Number of Years Grazed Figure 25. The relationship between grazing intensity and macroinvertebrate metric values. X- axis values indicate the time period examined. a) taxa richness, b) macroinvertebrate diversity, c) EPT taxa richness, d) sensitive taxa density, e) sensitive taxa relative abundance and f) sensitive taxa richness. 47

Objective 7. Has the amount of silt on the substrate changed through time?

Our results indicate that substrate composition, particularly the amount of silt and the amount of relatively silt-free gravel, cobble, and boulder, is an important driver of macroinvertebrate assemblage composition at both Camp Creek and Pine Creek. This result is not surprising as substrate composition is known to be an important environmental driver of stream macroinvertebrate composition (Minshall 1984). Silt is a particularly important component of stream substrates that can result in macroinvertebrate assembalges indicative of poor stream reach quality (Barbour et al. 1999; Karr and Chu 1999).

Given that the macroinvertebrate assemblages of both creeks indicate an improvement in stream reach quality through time, we expected that this is likely the result of decreases in the amount of silty substrate through time. To test this we conducted simple Pearson correlations between the amount of silt and the amount of gravel, cobble, and boulder substrate versus year. In contrast to our expectations, there was no relationship between either the amount of silt or the amount of cobble through time on either creek (Table 11, Figure 26).

When we graphed individual sites, all possible relationships between time and amount of silt were observed. Sites on both Camp Creek and Pine Creek showed decreases in silt through time, no clear change through time, and increases through time (Figures 27 and 28). The cause of this inter-site variability is not clear. In addition, macroinvertebrate metrics indicated an improvement in stream reach condition through time and most of the metrics used are sensitive to levels of silt (e.g., metrics associated with EPT taxa). However, the lack of consistent change through time in the levels of silt on the substrate suggests that the macroinvertebrate assemblages are responding to environmental variables other than silt. What this environmental factor might be is still unclear.

Table 11. Results of Pearson correlations between year and the dominance of both silty substrate and silt-free gravel/cobble/boulder substrate. Creek Substrate Pearson r p-value Camp Silt -0.10 ~0.40 Cobble 0.04 >0.50 Pine Silt -0.12 ~0.50 Cobble 0.07 >0.50

a) 100

20 % Transects Dominant Transects %

0 2006 2008 2010 2012 2014 2016 2018

Silt GrCoBo

b) 100

40 % of Transects of % 20

0 2006 2008 2010 2012 2014 2016 2018 2020

Silt GrCoBo

Figure 26. The dominance of silt and gravel/cobble/boulder (GrCoBo) substrate at a) Camp Creek and b) Pine Creek. “% of Transects” is the percent of transects where the substrate was considered one of the dominant substrates. Data points are mean values and vertical lines are standard deviations.

100

40 % of Transects of % 20 CC14

0 2004 2006 2008 2010 2012 2014 2016 2018 2020

CC1 CC11 CC13 CC14 CC16

100

80 CC24 60

CC17

40 % of Transects of % 20

0 2004 2006 2008 2010 2012 2014 2016 2018 2020

CC17 CC19 CC22 CC24 CC32

Figure 27. The percent of transects in which silt was a dominant substrate For reaches at Camp Creek. The data is split into two panels for clarity. Linear trendlines are labeled with the reach number.

100

40 % of Transects of % 20

0 2006 2008 2010 2012 2014 2016 2018 2020

PC12 PC14 PC18 PC23 PC8 PC9

100 PC25

PC22

40 % of Transects of % 20

0 2006 2008 2010 2012 2014 2016 2018 2020

PC11 PC16 PC17 PC19 PC22 PC25

Figure 28. The percent of transects in which silt was a dominant substrate for reaches at Pine Creek. The data is split into two panels for clarity. Linear trendlines are labeled with the reach number.

Conclusions

Our results indicate that the condition of both Camp Creek and Pine Creek has improved through time. Mixed models revealed that at Camp Creek the relative abundance and richness of EPT taxa as well as the abundance, relative abundance and richness of sensitive taxa have significantly increased through time. At Pine Creek the relative abundance of EPT taxa, the richness of sensitive taxa and the evenness of the functional feeding group assemblage all increased through time. In addition, an examination of specific sensitive taxa revealed that three of the six taxa that have been collected multiple years at Camp Creek have significantly increased their distribution through the drainage. At Pine Creek only one sensitive taxa has been collected for multiple years and, as in Camp Creek, it has increased its distribution through time.

While these results are encouraging, our study design lacks true control reaches and it is difficult to tell whether increases in macroinvertebrate metrics and sensitive taxa distributions are the result of management actions on the Zumwalt preserve or reflect some larger-scale environmental trends. One trend we were able to statistically control for in the mixed models was higher amounts of precipitation in years after restoration.

One of the main factors that influences macroinvertebrate assemblages at both Camp and Pine creeks is substrate composition. The importance of substrate in influencing macroinvertebrate assemblages was revealed in the ordination bi-plots and in the relationship of substrate to sites consistently in best condition. Substrate at two reaches within exclosures at Camp Creek has changed from a dominance of silt to a dominance of silt-free cobble since construction of the exclosures, indicating that exclosures are successful in changing the stream environment.

While exclosures are changing the stream and riparian environment in positive ways, the macroinvertebrate assemblage has not responded to these changes as of 2019. This likely represents a lag time in the response of macroinvertebrates, but it might also be the case that exclosures are simply too small to generate a response from macroinvertebrates. However, it might also be that the exclosures are simply too small to generate a response from macroinvertebrates as others have concluded (Wang et al. 1997, Dauwalter et al. 2018). It is also possible that the macroinvertebrates in the exclosures will respond sometime in the future. The riparian vegetation in the exclosures is continuing to grow and the amount of shading of the stream reach is increasing. At some point this change might generate a response by the macroinvertebrate assemblage. We recommend a continued monitoring of these reaches.

We found that there are specific reaches at both Camp Creek and Pine Creek that are consistently in the best condition. At Camp Creek these reaches were low in the drainage. This was a somewhat surprising finding as streams tend to accumulate impacts as water moves downstream resulting in poor condition reaches at low elevations. However, the reasonably high discharge and silt-free substrate of the downstream reaches appear to override any cumulative upstream impacts. The best condition sites at Pine Creek are approximately in the middle of the sampling area. Like Camp Creek, the best condition sites at Pine Creek had relatively silt-free substrates.

Grazing intensity negatively influenced macroinvertebrate taxa richness. Our measure of grazing intensity was somewhat coarse as we only considered whether a pasture was grazed or not each year. A more detailed study of grazing intensity that examines the number of cattle, the timing of grazing, the length of time grazing occurs, and access to the stream would provide a much better picture of how grazing influences stream reach condition on the Zumwalt.

An examination of substrate composition through time revealed that, despite the restoration efforts and the increase in stream reach quality based on macroinvertebrate metrics, there has been little overall change. We expected that restoration actions would lead to a lower level of silt on the substrate of the stream reaches and an increase in the amount of cobble substrate. However, this pattern did not emerge.

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Appendices

Appendix 1. Macroinvertebrate sampling sites on Camp Creek and Pine Creek, their UTM locations, and the years that they were sampled. UTM (NAD 83, Zone 11) Year

Drainage Site Easting Northing ‘06 ‘07 ‘08 ‘11 ‘13 ‘16 ‘19 Camp Ck Below1 503464 5049396 X X X X X X Below2 503317 5049837 X X X CC1 500530 5045293 X X X X X X CC2 500578 5045575 X X X X X CC11 502924 5047969 X X X X X X CC13 502871 5047398 X X X X X X X CC14 502754 5047167 X X X X X X X CC16 502523 5046689 X X X X X X CC17 502396 5046270 X X X X X X X CC18A 502294 5045935 X X CC19 502248 5045670 X X X X X X X CC20 502091 5045500 X X X CC22 502376 5045043 X X X X X X X CC24 502687 5044562 X X X X X X X CC25A 502652 5044220 X X CC32 502076 5041974 X X X X X X CC38 502406 5042480 X X R2P9 502769 5043924 X X Pine Ck PC8 497672 5045399 X X X PC9 497901 5045718 X X PC11 498203 5046299 X X PC12 498492 5046600 X X X X X PC14 499125 5047202 X X X X X PC16 499558 5047527 X X PC17 499742 5047736 X X PC18 499939 5047929 X X X X PC19 500248 5048255 X X PC22 500482 5049101 X X X PC23 500280 5049832 X X X X X PC25 499990 5050109 X X X PC26 499069 5047056 X

Appendix 2. Aquatic macroinvertebrate taxa list. The three areas sampled on the Zumwalt prairie are shown. An “X” in the cell indicates the taxon was collected from that area. An “*” in the “Genus” column indicates a sub-family or tribe. Camp Pine Camp Creek Creek Creek Class Order Family Genus Prairie “Below” Insecta Ephemeroptera Ameletidae Ameletus X Baetidae Acentrella X X X Baetis X X X Callibaetis X Centroptilum X X X Diphetor X X X Fallceon X X Pseudocloeon X Ephemerellidae Attenella X Ephemerella X Seratella X Heptageniidae Cinygmula X Ecdyonurus X Epeorus X Ironodes X Leptophlebiidae Paraleptophlebia X X X Plecoptera Capniidae X X X Chloroperlidae Suwallia X X X Leuctridae X Nemouridae Malenka X Zapada X Perlidae Calineuria X Trichoptera Apataniidae Apatania X X X Hydropsychidae Cheumatopsyche X Parapsyche X Hydroptilidae Hydroptila X X Lepidostomatiidae Lepidostoma X X Limnephillidae Dicosmoecus X Ecclisomyia X Grammataulius X Hesperophylax X X Limnephilus X X X Philopotamidae X Polycentropodidae Polycentropus X Rhyacophilidae Rhyacophila X X X Uenoidae Neophylax X Diptera Blephariceridae X Chironomidae Chironominae* X X X Diamesinae* X Orthocladiinae* X X X Tanypodinae* X X X Ceratopogonidae X X X Forcipomyiinae* X Dixidae Dixa X X Dixella X Meringodixa X X

Appendix 2. continued. Camp Pine Camp Creek Creek Creek Class Order Family Genus Prairie “Below” Insecta Diptera Chaoboridae Chaoborus X X Dolichopodidae X X Empidiidae Clinocera X Neoplasta X Ephydridae X X Muscidae X Phoridae X Psychodidae Pericoma X X Sciomyzidae X Simuliidae Prosimulium X X Simulium X X X Stratiomyidae Caloparyphus_ X Euparyphus Tabanidae Tabanus X X Tipulidae Antocha X X Dicranota X X X Gonomyia X Limonia X Molophilus X X X Rhabdomastix X Tipula X X Coleoptera Curculionidae X Dytiscidae Acilus X X Agabus X X X Dystiscus X Hydroporinae* X X X Elmidae Celptelmis X X X Narpus X Optioservus X X X Ordobrevia X X Zaitzevia X X Haliplidae Haliplus X X X Peltodytes X X Hydraenidae Hydraena X X X Ochthebius X Hydrophilidae Anacaena X X Helophorus X X X Hydrobius X Tropisternus X X Megaloptera Sialidae Sialis X Odonata Aeshnidae Aeshna X Coenagrionidae Argia X Enallagma X X Libellulidae X X Gastropoda Physidae Physa X X X Planorbidae X X X Lymnaeidae X X X

Appendix 2. continued. Camp Pine Camp Creek Creek Creek Class Order Family Genus Prairie “Below” Gastropoda Pisidiidae X X X Hydrobiidae X X Malacostraca Amphipoda X X X Isopoda X X Branchiopoda Cladocera X Arachnida Trombidiformes X X X Clitellata X X X Subclass: X X X Hirudinea Turbellaria X X Phylum: X X X Nematoda

Appendix 3. The five dominant taxa based upon relative abundance (Tables A3-1, A3-2, and A3-3) and based upon distribution (Tables A3-4 and A3-5) for the three areas sampled on the Zumwalt prairie. Empty cells indicate that a taxa was not considered dominant that year, they do not indicate, necessarily, that the taxa was not collected at all that year. Yellow highlighting indicates a taxon that was dominant in that area and not dominant in either of the other two areas. Dominance based upon distribution was not determined for the “Below” sites because in most years only a single site was sampled.

Table A3-1. Dominant taxa at the Camp Creek Prairie sites for each year based on relative abundance. Values are the relative abundance calculated based on all individuals collected at all prairie sites for that year. Taxa 2006 2007 2008 2011 2013 2016 2019 Ephemeroptera Baetis 0.12 0.14 0.03 Centroptilum 0.09 0.05 0.09 0.15 0.10 Fallceon 0.16 Paraleptophlebia 0.06 0.08 0.09 Diptera Orthocladiinae 0.58 0.27 0.23 0.13 0.19 0.07 0.07 Chironominae 0.05 0.08 0.04 004 Simulium 0.06 0.26 0.29 0.42 0.48 0.56 0.42 Non-Insects Clitellata 0.06 0.07 0.25 0.06 0.09

Table A3-2. Dominant taxa at the “Below” sites on Camp Creek for each year based on relative abundance. Values are the relative abundance calculated based on all individuals collected at all prairie sites for that year. Taxa 2006 2007 2008 2011 2013 2016 2019 Ephemeroptera Baetis 0.08 0.04 0.25 Centroptilum 0.13 Ameletus 0.08 0.07 Paraleptophlebia 0.10 0.18 Epeourus 0.12 Seratella 0.09 Plecoptera Suwalia 0.06 0.03 0.12 0.05 0.13 Diptera Orthocladiinae 0.44 0.14 0.56 0.55 0.26 Chironominae 0.11 Simulium 0.09 0.16 Coleoptera Helophorus 0.10 Ordobrevia 0.08 0.10 0.11 0.08 Zaitzevia 0.10 0.13 0.13 Non-Insects Amphipoda 0.08 Isopoda 0.16 Pisidiidae 0.25 Turbellaria 0.07

Table A3-3. Dominant taxa at the Pine Creek sites for each year based on relative abundance. Values are the relative abundance calculated based on all individuals collected at all prairie sites for that year. Taxa 2007 2011 2013 2016 2019 Ephemeroptera Centroptilum 0.11 0.20 0.41 0.25 0.35 Paraleptophlebia 0.29 0.12 Plecoptera Suwalia 0.04 0.11 Trichoptera Limnephilus 0.08 0.08 Diptera Orthocladiinae 0.54 0.04 0.18 0.07 Chironominae 0.04 0.05 Simulium 0.30 0.11 0.10 Non-Insects Clitellata 0.13 0.11 0.44 Nematoda 0.05 Pisidiidae 0.07

Table A3-4. Dominant taxa based on distribution for the Camp Creek prairie sites. Values are % of sites occupied for each year. A taxon was considered dominant if it was found in at least 75% of the sites sampled in a given year. Note that a blank cell does not indicate a taxon was absent that year, just that it did not occur in at least 75% of the sites that year. Taxa 2006 2007 2008 2011 2013 2016 2019 Ephemeroptera Centroptilum 100 100 100 89 100 100 Fallceon 87 Paraleptophlebia 80 89 Trichoptera Limnephilus 92 89 100 93 Diptera Orthocladiinae 100 92 100 100 100 87 100 Chironominae 100 100 100 100 100 80 93 Ceratopogonidae 80 92 78 Simulium 80 92 100 100 89 80 100 Coleoptera Agabus 90 100 89 80 Non-Insects Clitellata 100 100 89 Nematoda 90 92 Trombidiformes 80 90 89 89 93

Table A3-4. Dominant taxa based on distribution for the Pine Creek sites. Values are % of sites occupied for each year. A taxon was considered dominant if it was found in at least 75% of the sites sampled in a given year. Note that a blank cell does not indicate a taxon was absent that year, just that it did not occur in at least 75% of the sites that year Taxa 2007 2011 2013 2016 2019 Ephemeroptera Baetis 75 86 Fallceon 86 Centroptilum 100 75 100 92 100 Paraleptophlebia 100 100 Plecoptera Suwallia 75 83 100 Trichoptera Limnephilus 83 100 75 86 Rhyacophila 86 Diptera Orthocladiinae 100 100 100 100 100 Chironominae 100 75 Ceratopogonidae 83 75 Simulium 100 100 83 86 Dicranota 75 Coleoptera Hydroporinae 75 Non-Insects Clitellata 83 75 75 100 Nematoda 92 Trombidiformes 75 75 100

Appendix 4. The following tables present the results of all mixed models, both significant and non-significant. Table A4-1 and A4-2 show the results of mixed models that categorized time into two periods, before restoration and after restoration, for the Camp Creek prairie sites and Pine Creek, respectively. Table A4- 3 and A4-4 show the results of mixed models in which time was a continuous variable for Camp Creek prairie sites and Pine Creek, respectively.

Table A4-1. Mixed model results for Camp Creek prairie sites with time categorized as two periods, before restoration and after restoration. Metrics with a significant effect of period are in bold. “EPT” stands for Ephemeroptera, Plecoptera, Trichoptera; “RA” stands for relative abundance, and “FFG” stands for functional feeding group. Metric Model Type Estimate t-value p-value Total Richness Intercept Period: -0.120 -0.704 0.486 Precip: 0.014 0.628 0.533 Total Diversity Intercept Period: 0.194 1.400 0.167 Precip: 0.019 0.967 0.338 Evenness Intercept Period: 0.086 1.818 0.074 Precip: 0.007 0.956 0.343 EPT Density Intercept Period: -0.086 -0.43 0.672 Precip: 0.058 2.23 0.030 EPT RA Intercept Period: -0.208 -2.83 0.006 Precip: 0.033 3.18 0.002 EPT Richness Intercept Period: -0.206 -1.81 0.076 Precip: 0.045 2.84 0.006 ATI Intercepts+Slopes Period: 0.037 0.168 0.869 Precip:-0.004 -0.145 0.885 Sensitive Intercepts Period: -0.280 -1.056 0.295 Density Precip: -0.006 -0.158 0.875 Sensitive RA Intercepts+Slopes Period: -0.096 -3.358 0.004 Precip:-0.008 -2.404 0.021 Sensitive Intercepts Period: -0.298 -1.875 0.066 Richness Precip:-0.006 -0.266 0.791 FFG Diversity Intercepts Period: 0.001 0.012 0.990 Precip: 0.008 0.608 0.546 FFG Evenness Intercepts+Slopes Period: 0.035 0.571 0.572 Precip: 0.004 0.491 0.626

Table A4-2. Mixed model results for Camp Creek prairie sites with time categorized as two periods, before restoration and after restoration. Metrics with a significant effect of period are in bold. “EPT” stands for Ephemeroptera, Plecoptera, Trichoptera; “RA” stands for relative abundance, and “FFG” stands for functional feeding group. Metric Model Type Estimate t-value p-value Total Richness Intercept Period: 0.662 1.639 0.121 Precip: 0.051 1.122 0.278 Total Diversity Intercept Period: 0.050 0.184 0.957 Precip: -0.003 -0.085 0.933 Evenness Intercept Period: -0.061 -0.801 0.438 Precip: -0.007 -0.851 0.410 EPT Density Intercept+Slope Period: 1.160 1.004 0.330 Precip: 0.074 0.571 0.576 EPT RA Intercept Period: -0.332 -1.550 0.161 Precip: 0.020 0.931 0.369 EPT Richness Intercept Period: -0.146 -0.529 0.604 Precip: 0.025 0.802 0.434 ATI Intercepts+Slopes Period: 0.157 0.323 0.752 Precip: -0.045 -0.820 0.427 Sensitive Intercepts Period: -0.745 -0.475 0.643 Density Precip: 0.016 0.088 0.931 Sensitive RA Intercepts+Slopes Period: -0.151 -1.629 0.131 Precip: -0.008 -0.800 0.438 Sensitive Intercepts Period: -0.401 -1.215 0.246 Richness Precip: -0.017 -0.471 0.646 FFG Diversity Intercepts Period: -0.305 -1.798 0.121 Precip: -0.023 -1.476 0.164 FFG Evenness Intercepts+Slopes Period: -0.204 -2.619 0.021 Precip: -0.014 -1.644 0.124

Table A4-3. Results of significant mixed model analyses for Camp Creek prairie sites. Models examined the effect of sampling year and the amount of precipitation 60 days prior to sampling as fixed effects. Year was treated as a continuous variable. Site was the random factor. “Model Type” indicates whether the full model (“Intercepts+Slopes”) or the reduced model (“Intercepts” only) fit the data. Significant variables are in bold. “RA” stands for relative abundance. Metric Model Type Estimate t-value p-value Total Richness Intercepts Year: 0.030 1.710 0.093 Precip:-0.00004 -0.002 0.998 Total Diversity Intercepts Year: -0.018 -1.179 0.243 Precip: 0.016 0.810 0.422 Evenness Intercepts Year: -0.010 -1.939 0.057 Precip: 0.007 1.016 0.313 EPT Density Intercepts Year: 0.032 1.589 0.118 Precip: 0.039 1.471 0.147 EPT RA Intercepts Year: 0.033 4.536 <0.001 Precip: 0.024 2.607 0.012 EPT Richness Intercepts Year: 0.038 3.309 0.002 Precip: 0.032 2.188 0.033 ATI Intercepts Year: -0.013 -0.660 0.512 Precip: 0.007 0.256 0.799 Sensitive Intercepts Year: 0.063 2.275 0.027 Density Precip: -0.032 -0.926 0.359 Sensitive RA Intercepts Year: 0.009 3.303 0.002 Precip: -0.007 -1.987 0.052 Sensitive Intercepts Year: 0.051 3.152 0.003 Richness Precip: -0.021 -1.024 0.311 FFG Diversity Intercepts+Slope Year: 0.009 0.920 0.368 Precip:-0.0006 -0.050 0.960 FFG Evenness Intercepts+Slope Year: 0.00007 0.103 0.919 Precip:-0.00002 -0.003 0.998

Table A4-4. Results of mixed model analyses for Pine Creek. Models examined the effect of sampling year and the amount of precipitation 60 days prior to sampling as fixed effects. Year was treated as a continuous variable. Site was the random factor. “Model Type” indicates whether the full model (“Intercepts+Slopes”) or the reduced model (“Intercepts” only) fit the data. Significant variables are in bold. “RA” stands for relative abundance. Metric Model Type Estimate t-value p-value Richness Intercepts Year: -0.026 -0.632 0.537 Precip: 0.021 0.461 0.651 EPT Density Intercepts Year: -0.086 -0.772 0.451 Precip: 0.046 0.372 0.715 EPT RA Intercepts Year: 0.041 2.271 0.041 Precip: 0.019 0.962 0.353 EPT Richness Intercepts Year: 0.039 1.606 0.132 Precip: 0.011 0.420 0.681 ATI Intercepts Year: -0.034 -0.741 0.472 Precip: -0.036 -0.718 0.486 Sensitive Intercepts Year: 0.155 1.069 0.304 Density Precip: -0.022 -0.140 0.891 Sensitive RA Intercepts Year: 0.016 1.865 0.085 Precip: -0.007 -0.748 0.468 Sensitive Intercepts Year: 0.073 2.789 0.015 Richness Precip: -0.032 -1.102 0.290 FFG Diversity Intercepts+Slope Year:0.029 1.394 0.251 Precip: -0.024 -2.071 0.059 FFG Evenness Intercepts Year: 0.024 3.597 0.003 Precip: -0.014 -1.975 0.070 Diversity Intercepts Year: 0.016 0.627 0.542 Precip: -0.015 -0.546 0.595 Evenness Intercepts Year: 0.010 1.471 0.166 Precip: -0.009 -1.183 0.259