The Short Term Responses of Benthic Macroinvertebrates to the Removal of Riparian Rhododendron in Southern Appalachian Streams

Kevin M Eliason

Thesis submitted to the faculty of the Virginia Polytechnic Institute and State University in partial fulfillment of requirements for the degree of

Master of Science

Biological Sciences

E. F. Benfield, Chair

John E. Barrett

Bryan L. Brown

Scott M. Salom

May 3rd, 2017

Blacksburg, Virginia

Keywords: Benthic macroinvertebrates, Riparian disturbance, Rhododendron

The Short Term Responses of Benthic Macroinvertebrates to the Removal of Riparian Rhododendron in Southern Appalachian Streams

Kevin M Eliason

ABSTRACT (Academic)

The southern Appalachian forests of the United States are undergoing changes due to the death of hemlock trees attacked by the hemlock wooly adelgid. This paper addresses the management impacts of Rhododendron maximum removal in the riparian and upslope areas previously occupied by hemlock.

This study measured the consequences macroinvertebrates faced due to riparian Rhododendron removal from 300 m reaches of two low order streams. Two additional low order streams served as reference sites for the experiment. The stream macroinvertebrate communities were assessed using a before-after controlled impact model comparing communities between fall 2014 to those from fall 2015 and from spring 2015 to spring 2016. Macroinvertebrate collections consisted of 288 samples with a total of 61,056 individuals. There was a significant increase in collector-gathers in both removal sites, mostly from increases in Ephemerellidae and Chironomidae. There was also a significant decrease in filter feeding organisms in the removal reaches. Traits analysis also revealed that several traits that are shared by collector gathers also increased, e.g., short life cycles that are related to the increase in

Chironomidae. Using Nonmetric Multidimensional Scaling (NMDS) and permutational MANOVA significant annual differences in macroinvertebrates were found in all of the stream reaches during both seasons. However, the trait based NMDS and permutational MANOVA found significant change only in one removal site between fall collections based on traits. These finding are consistent with findings from logging and other riparian removal projects; suggesting that the short-term impacts of selective

Rhododendron removal on benthic macroinvertebrates are comparable to that of logging activity.

The Short Term Responses of Benthic Macroinvertebrates to the Removal of Riparian Rhododendron in Southern Appalachian Streams

Kevin M Eliason

Abstract (General Audience)

The southern Appalachian forests of the United States are undergoing changes due to the death of hemlock trees attacked by the hemlock wooly adelgid. This paper addresses the management of

Rhododendron removal in the riparian and upslope areas previously occupied by hemlock. This study investigates the consequences stream insects face due to near stream Rhododendron removal from 300 m section of two small streams. Two similarly sized streams served as reference sites for the experiment.

The stream insect communities were assessed using a before-after controlled impact model comparing collections from the fall 2014 to those from fall 2015 and from spring 2015 to spring 2016. Insect collections consisted of 288 samples with a total of 61,056 individual insects. There was a significant increase in generalist feeding insects in both removal sites, mostly from increases in common mayflies and small non-biting midges. Analysis of insect attributes also revealed that attributes shared by generalist feeding insects also increase, e.g., short life cycles that are related to the increase in non- biting midges. Looking at the insect communities we found annual changes in all of the stream reaches during both seasons. However, analysis of insect attributes found significant change only in one removal site between the fall collections. These finding are consistent with findings from logging and other riparian removal projects; suggesting that the short-term impacts of selective Rhododendron removal on stream insects are comparable to that of near stream logging activity.

ACKNOWLEDGEMENTS

I would like to thank my advisor Dr. Fred Benfield, my committee, and my graduate colleagues for helping me through my graduate program at Virginia Tech. I would like to thank Fred, first for giving me the opportunity and the support to attend graduate school at Virginia tech. Also, for slogging around in the Rhododendron hell that were my field sites.

Special thanks to Dr. Bryan Brown for fielding many of my statistical questions, often without much warning and being an active contributor to my learning at Virginia Tech. My inclusion to your lab meetings was of great benefit to my time; myself being the only member of my lab, it was enjoyable and helpful to be part of your lab group.

In addition, I would like to thank my peer graduate students within STREAM Team. I appreciate the constant aids to my morale and helpful actions in navigating the more benign parts of graduate school. I would like to thank Matt Hedin, my colleague and friend, who aided me with several of my sampling trips; sticking with me through getting caught in downpours, and freezing conditions, I am grateful for his help over the past 2 years. I also would like to thank my undergraduates who put in plenty of hours on the microscopes and sorting samples. Without you all this project would not have been possible.

TABLE OF CONTENTS

Abstract (Academic) ii

Abstract (General Audience) iii

Acknowledgments iv

List of figures vi

List of tables vii

Introduction 1

Methods 3

Results 6

Discussion 11

References 15

Figures and Tables 20

Appendix A 34

LIST OF FIGURES

Fig. 1: Study area and sampling sites located in Nantahala National Forest ...... 4

Fig. 2: Plot of means of density by season ...... 20

Fig. 3: Plot of means of richness by season ...... 21

Fig. 4: Functional Feeding groups (% of population) for pre-post fall samples ...... 26

Fig. 5: Functional Feeding groups (% of population) for pre-post spring samples . . . 27

Fig. 6: NMDS of community pre-post fall samples for each reach ...... 28

Fig. 7: NMDS of community pre-post spring samples for each reach ...... 29

Fig. 8: NMDS of traits pre-post fall samples for each reach ...... 30

Fig. 9: NMDS of traits pre-post spring samples for each reach ...... 31

LIST OF TABLES

Table 1: Family level count data for Removal 1 ...... 22

Table 2: Family level count data for Removal 2 ...... 23

Table 3: Family level count data for Reference 2 ...... 24

Table 4: Family level count data for Reference 1 ...... 25

Table 5: List of traits with significant change from fall 2014 – fall 2015 ...... 32

Table 6: List of traits with significant change from spring 2015 – spring 2016 ...... 33

Table 7: Supplementary data for macroinvertebrates ...... 34

vii

INTRODUCTION

Forests in the eastern United States are undergoing a change as eastern hemlock, a once abundant riparian tree, is being eliminated by the hemlock wooly adelgid (Adelges tsugae) (Webster et al. 2012). In the southern Appalachians the loss of hemlock has created openings in the canopy allowing

Rhododendron (Rhododendron maximum) to rapidly expand (Brantley et al. 2013). Rhododendron is a strong competitor that prevents new riparian hardwood recruitment. Hemlock is the only species that effectively recruits within Rhododendron thickets and with the loss of hemlock, Rhododendron faces little competition as it expands (Mcleod 1988). Management and control of Rhododendron is now a concern of the USDA forest service and land managers in the southern Appalachians (Webster et al.

2012, Miniat et al. 2014).

Riparian vegetation is important to stream ecosystems because it strongly influences stream hydroclimate and produces conditions that influence community assembly and composition in streams

(Vannote et al. 1980, Allan et al. 1997, Allan and Arbor 2004). Riparian vegetation influences stream habitats by producing shade, regulating nutrient and sediment inputs, controlling runoff , and contributing allochthonous organic matter in the form of leaves and wood (Allan and Arbor 2004).

Biodiversity in streams is also linked to associated riparian vegetation through energy inputs and hydroclimate control. These links mean that changes in the riparian vegetation can have significant effects on the stream channel and its associated habitat (Brinson et al. 2002, Death and Collier 2010).

The southern Appalachian region of North America has experienced drastic changes over the past 200 years including at least two full scale timber harvests (Yarnell 1998). This historical logging activity has strong lasting effects on stream macroinvertebrate community through increased nutrient fluxes of dissolved organic nitrogen and dissolved organic carbon (Nislow and Lowe 2006, Qualls et al.

2014) Another historical ecological change was loss of the American chestnut to blight (Cryphonectria

1 parasitica (murr.)Barr) in just two decades early in the 20th century (Nelson 1955). Although almost extant, Chestnut still made up a significant portion of large wood in southern Appalachian streams 70 years after the blight (Wallace et al. 2001). The impacts of riparian hemlock loss will likely result in similar long-term effects on streams in the region.

Rhododendron-dominated areas are often characterized by poor recruitment of tree species

(Mcleod 1988, Baker and Van Lear 1998). This poor recruitment could lead to a future reduction in leaf litter quality in streams. Rhododendron leaves are similar to hemlock in that both have a waxy cuticle and contain polyphenolic compounds that result in significantly slower breakdown rates than hardwoods (Webster and Benfield 1986). Rhododendron leaves, like those of hemlock, have very low preference by shredders in feeding experiments (Wallace 1970). Rhododendron and hemlock are evergreens and produce leaf litter year round. Although poor in food quality, both leaf types may serve as an important food source for semi-voltine shredders when other more preferable food sources run out (Huryn and Wallace 1987, Sakai et al. 2016).

Rhododendron is a persistent riparian plant, leading managers to seek methods to control its impacts near stream habitats. Previous studies that removed Rhododendron found an influx of nutrients entering the stream when the plants were uprooted, but found relatively small increases when the roots where left intact (Yeakley et al. 1994, 2003). Rhododendron is resistant to fire control methods because the plants are readily able to sprout from roots and have moderate fire resistance. Small

Rhododendron plants suffer high mortality in fire but plants with stems over 3 inches diameter breast hight have relatively high survivability (Hooper 1969). While Rhododendron readily sprouts after being top killed by fire these sprouts are more susceptible to herbicide (Romancier 1971).

Stream benthic macroinvertebrates rely on allochthonous organic matter as the main source of energy in forested headwater streams (Vannote et al. 1980). Reliance on leaf litter means that the

2 expansion of Rhododendron in riparian areas could result in future reduction in litter quality available for associated streams which could cause a significant change in southern Appalachian stream energetics (Webster et al. 2012). Macroinvertebrate taxa respond to changes in their environment in different ways. The Environmental Protection Agency (EPA) has used stream benthic macroinvertebrates to inform stream health analysis and changes to stream systems (Barbour et al. 1999). In recent years the analysis of functional traits has become more commonplace in the analysis of macroinvertebrate communities, as it allows insights into changes in function across the community versus only changes in taxa (Poff et al. 2006, Cummins 2016).

The purpose of this study was to explore potential effects of Rhododendron removal from riparian areas on macroinvertebrate communities using both taxonomic and trait based approaches. It is predicted that like many logging studies we will see an increase in collector gatherers (e,g., Gurtz and

Wallace 1984, Boggs et al. 2016). However, unlike these logging studies there is no expected decrease in shredders due to removal of riparian Rhododendron as the main source of leaf litter, the hardwood trees, remain intact.

METHODS

Study Sites and treatments

The study was conducted in the in the Wine Spring area of the Nantahala National Forest in western

North Carolina. Four streams with dense Rhododendron riparian corridors were selected for study: two treatment and two reference. The streams are all first order tributaries of White Oak Creek and part of the greater Little Tennessee River watershed (Fig.1). The study reaches were 300 meters long and extended 50 meters on each side of the stream, providing a roughly 30,000 square meters (3 hectares)

of treatment area. Three treatments were proposed and two were carried out by the United States

Forest Service:

1. Removal 1: Rhododendron was cut using chainsaws in the Holloway Branch (HB) sub-watershed

(Fig1 (#1)). The cut limbs were placed in piles along the stream reach.

2. Removal 2: Rhododendron along Split White Oak Branch (SWO) was cut and the limbs were left

were they fell. The limbs were subsequently burned in march 2016 prior to our spring

macroinvertebrate collections (Fig1 (#2).

3. Reference 1: Rocky Bald Branch (RBB) received no manipulation and serves as a reference site

(Fig 1. (#3)).

4. Reference 2: Kit Spring Branch (KSB) received no manipulation and serves as a second reference

site. (Fig1 (#4)).

[Grab your reader’s attention with a great quote from the document or use this space to emphasize a

key point. To place this text box anywhere on the page, just drag it.]

2. 1.

1. Holloway Branch (Rem. 1) 2. Split White Oak Branch (Rem. 2)

3. Rocky Bald Branch (Ref. 1) 4. Kit Spring Branch (Ref. 2)

Figure 1. Map of the study sites located in the White Oak Creek watershed within the Nantahala National Forest. Numbers correspond to stream names.

Macroinvertebrate Sampling

Macroinvertebrates were collected in fall 2014 and 2015 and spring 2015 and 2016. Three kick samples were taken at 50 meter intervals along the 300 meter reach making eighteen samples per stream each date. A total of 288 macroinvertebrate samples were collected comprised of 61,056 specimens. Sampling was accomplished using a 250 micron net and a 0.5 m2 frame (Harding et al.

1998). Samples were stored individually in plastic bags, preserved in 80% ethanol, and transferred to the lab for processing. In the lab, macroinvertebrates were sorted from debris by elutriation, collected in a 250 micron sieve, and stored in 80% ethanol. Macroinvertebrates were Identified to the lowest practical taxonomic level, usually genus using Merritt et al. (2008). Chirononmids were identified based on functional feeding group as Tanypodinae (predator) and non-Tanypodinae (collector gatherer).

Capniidae and Luctridae were not differentiated due to loss of pigment, small size at collection, and general condition from storage they are refered collectively as Luctridae.

Data Analysis

The macroinvertebrate data were analyzed using the before-after controlled impact (BACI) design comparing pre-and post-treatment data to evaluate the impacts of the treatment (Smith 2002).

Fall and spring samples were analyzed separately due to seasonal differences in community composition and in rate of collection due to size(Linke et al. 1999, Šporka et al. 2006). Holloway Branch and Split

White Oak were analyzed as removal sites and Kit Spring Branch and Rocky Bald Branch were analyzed as reference sites.

Statistical tests were performed using open source R statistical software (R Core team 2015).

Macroinvertebrate density and richness were calculated based on the eighteen ½ m2 samples taken within each reach during each sampling season. T-tests and comparisons of means were produced using the Rcmdr package with significance being determined when p ≤ 0.05 (Fox and Bouchet-Valat 2015).

Transformations of data were not conducted to gain normality in T-tests do to having balance in sample design, this is in accordance with Box’s findings (Box 1954a, 1954b). Nonmetric multidimensional scaling

(NMDS) was performed using the (vegan) and (vegetarian) packages using the Bray Curtis abundance- weighted distance metric. NMDS analysis were conducted on subsets of data by reach and season. The community NMDS were analyzed using the betadisper function within the vegan package to detect differences in the variance between samples (Oksanen et al. 2017). Permutational MANOVA’s were conducted using the adonis function within vegan to determine if there were significant differences between sample years (Oksanen et al. 2017). The NMDS of functional traits, like the community data, were also analyzed by reach and season. Functional traits NMDS analysis were also conducted using the

Bray Curtis metric. The functional traits were assigned using a modified list from the metrics developed by Poff (Poff et al. 2006).

RESULTS

Density and Richness

There were no significant differences in macroinvertebrate density in any sites between fall 2014 and fall 2015 (Fig 2). Macroinvertebrate density significantly increased in both removal streams between spring 2015 and spring 2016; Removal 1 (p = 0.0074) and Removal 2 (p = 0.0005). Neither Reference 1 nor Reference 2 had significant change in macroinvertebrate density between spring samples. Richness significantly increased at Removal 1 between fall 2014 and fall 2015 (p < 0.001)(Fig. 3). Richness

6 significantly decreased in Reference 2 during the same period (p = 0.009). Neither Removal 2 nor

Reference 1 showed significant difference in richness between the fall 2014 and fall 2015 sampling.

Between spring 2015 and spring 2016 none of the stream reaches showed significant change in richness.

Family level differences

Fall 2014 vs. Fall 2015

The order of each family is denoted by: Ephemeroptera(E), Plecoptera(P), Trichoptera(T), Coleptera

(C), and Diptera(D). In Removal 1 there were significant increases in density of the families

Heptigenidae(E), Ephemerellidae(E), and Ameletidae(E) (Table 1). Removal 1 also had significant decreases individuals in the following families: Leptophlebiidae(E), Perlodidae(P), Rhyacophilidae(T), and

Hydropsychidae(T). Removal 2 showed an increase in individuals in the several families: Heptigenidae(E),

Ephemerellidae(E), Baetidae(E), Leptophlebiidae(E), Tipulidae(D), and Limniphilidae(T) (Table 2.)

Removal 2 had a significant decrease in the numbers of Hydropsychidae(T), and Philopotamidae(T).

Reference 1 (Table 4) had an increase in the number Baetidae(E) and Limnephilidae(T). Reference 1 showed a significant decrease in the number of: Heptigenidae(E), Perlodidae(P), Rhycophillidae(T), and

Elmidae(C). The second reference site, Reference 2 (Table 3) showed a significant increase in

Heptagenidae(E) and Ephemerellidae(E). Reference 2 showed a decrease in: Leptophlebiidae(E),

Perlodidae(P), Peltoperlidae(P), Leuctridae(P), Rhycophilidae(T), Hydropsychidae(T), Non-

Tanypodinae(D), and Perlidae(P).

Spring 2015 vs. Spring 2016

Removal 1 (Table 1) showed a significant increase in the density of: Ephemerellidae(E), Baetidae(E),

Peltoperlidae(P), Elmidae(C), Tipulidae(D), Tanypodinae(D), and non-Tanypodinae(D). Removal 1

7 showed a significant decrease in Lepidostomidae(T) and Limnephilidae(T). Removal 2 (Table 2) showed a significant increase in Heptigenidae(E), Ephemerellidae(E), Baetidae(E), Leptophlebiidae(E),

Perlodidae(P), Peltoperlidae(P), Elmidae(C), Non-Tanypodinae(D), and Perlidae(P). The only significant change in families in the Reference 1 reach was an increase in Peltoperlidae(P) (Table 4). Reference 2 had a significant increases in Baetidae(E), Peltoperlidae(P), and Perlidae(P) (Table 3). Reference 2 also had significant decreases in Leuctridae(P), Rhycophilidae(T), and non-Tanypodinae(D).

Functional Feeding Groups

Fall 2014 vs. Fall 2015

The macroinvertebrate communities in Removal 1 showed a significant increase in the proportions collector gatherers and grazers in the community between fall 2014 and fall 2015 (Fig 4). Removal 1 also showed a significant decrease in the proportions of filter feeders and predators. The treatment site,

Removal 2, also showed an increase in proportion of collector gatherers, but no change in that of grazers. Removal 2 also had a significant decrease in proportions of filter feeders and predators.

Reference 1 showed a significant increase in the proportion of collector gathers and a significant decrease in the proportion of predators. Reference 2, showed a significant increase in proportion of collector gathers and grazers. Reference 2 also showed a significant decrease in the proportion of filter feeders, predators, and shredders.

Spring 2015 vs. Spring 2016

In Removal 1 there was a significant increase in the proportion of collector gathers and a significant decrease in filter feeders, grazers, and shredders between the two spring samples, but no significant change in the proportion of predators (Fig 4). Removal 2 had a significant increase in the proportion of collector gathers, and a decrease in the proportion of filter feeders and predators. At

Reference 1 the proportion of shredders significantly increased and collector gathers decreased

8 between the two springs samples. Reference 2 also had an increase in the proportion of shredders, and a decrease in the proportion of collector gatherers and predators.

Community Composition

Fall 2014 vs Fall 2015

Between fall 2014 and fall 2015 permutational MANOVA revealed significant change in community composition in all four reaches (Fig. 6). In Removal 1 there were a significant difference between sampling years, but no significant difference in variance between sites. In Removal 2 there was significant differences in the community, but no significant difference in variance between samples.

Reference 1 had significant difference in community between samples and no significant difference in variation between samples. Reference 2 had significant community differences and lacked significant variation between samples.

Spring 2015 vs Spring 2016

Between spring 2015 and spring 2016 permutational MANOVA showed differences between spring

2015 and spring 2016 in all four streams (Fig. 7). Removal 1 had significant differences between spring samples within the community and a reduction in variation between samples. Removal 2 had significant community differences and significant reduction in variation between samples. Reference 1 had a significant community difference and a significant reduction in variance between sites. Reference 2 had a significant community difference, but did not have significant change in variation between sites.

Functional Traits

Fall 2014 vs. Fall 2015

Between fall 2014 and fall 2015 the removal and reference sites had several changes in macroinvertebrate traits (Table 5). Removal 1 had an increase in insects with very short life cycles.

Removal 1 also had a decrease in the traits: sedimentary attachment, poor armor, erosional habitat and tegument breathing. Removal 2 had an increase in fast development, very short life cycle, common drifter, weak swimming, free ranging, generalist habitat, sprawler and swimmer activity. Removal 2 significantly decreased in only sedentary attachment. Neither of the reference sites, Reference 1 or

Reference 2, had significant increases in any trait. Reference 1 had a significant reduction in insects displaying the traits: semi-voltine, non-seasonal development, high crawling, sedentary attachment, poor armor, erosional habitat, and desiccation survivability. Reference 2 had significant reductions in the traits: poor synchronization, short lifespan, high dispersal, strong flyers, rare drifter, high crawling, very low crawling, non- swimmer, sedentary attachment, poor armor, erosional habitat, desiccation survival, small size, and tegument respiration.

Between spring 2015 to spring 2016 the removal and reference stream reaches had numerous significant changes in macroinvertebrate traits (see Table 6). Removal 1 had 29 traits that significantly increased and none that were significantly reduced. Removal 2 had 36 macroinvertebrate traits with significant increases, only climbers was significantly reduced. Reference 1 had ten significantly increased macroinvertebrate traits. Reference 2 had six traits with significant increases and erosional habitat was the only significantly decreased trait.

Community functional traits

Between fall 2014 and fall 2015 there were no significant changes in the variance of macroinvertebrate traits within the four stream reaches between sample dates (Fig. 8). Removal 2 was the only reach to have a significant difference in macroinvertebrate traits between fall 2014 and fall

2015. Removal 1, Reference 2, and Reference 1 did not exhibit significant differences in traits between fall samples.

Between spring 2015 and spring 2016 both Removal 1 and Removal 2 were found to be significantly different using permutational MANOVA. In contrast, both reference sites, Reference 1 and

Reference 2, did not show any significant differences between spring samples. Removal 2 was the only stream to show significant differences in variance in macroinvertebrate traits between spring samples.

Removal 1, Reference 2, and Reference 1 did not have significant differences in variation in macroinvertebrate traits between spring samples.

DISCUSSION

The results showed that macroinvertebrate density was significantly higher in both post-removal reaches between spring 2015 and spring 2016. The increases in Ephemerellidae and non-Tanypodinae accounted for the majority of this change in density (Table 1 & 2). These two taxa also accounted for the significant increase in the collector gatherer functional feeding group in both fall vs fall and spring vs spring samples. (Figs. 4 & 5). Both reference sites, Reference 1 and Reference 2 exhibited a significant increase in shredders between spring collections (Fig. 5), which is almost completely attributable to increases in Peltoperlidae (Table 3 & 4). During the same time Removal 1 actually had a reduction in shredders and Peltoperlidae. There no significant increase in grazers, but given more time the grazer functional feeding group may increase in response to additional light promoting algal growth in the

11 stream.

The changes in density we observed in the benthic macroinvertebrate community following the

Rhododendron treatments are similar to findings following a full timber harvest of similarly sized streams in a neighboring watershed (Gurtz and Wallace 1984). In that study, there were significant increases of collector-gathers in the treatment sites, most markedly in the Ephemerellidae. This pattern of increased macroinvertebrate density has been seen elsewhere outside the southern Appalachian mountain region. A timber harvest study in the piedmont region of North Carolina similarly found an increase of collector-gatherers in associated streams (Boggs et al. 2016), and a study at Hubbard Brook following a clearcut harvest also exhibited the same increase in collector-gatherers (Noel et al. 1986).

Outside of the east coast the response of increasing collector gatherers and rises in Chironomidae and

Ephemeroptera were also seen in the Pacific Northwest during riparian width manipulations (Kiffney et al. 2003). This pattern of macroinvertebrate change associated with logging studies from several areas of the country suggests that Rhododendron removal has a similar impact on stream macroinvertebrate community to that of logging activity. For example, observed decreases in filter feeding insects in the removal sites could be related to increased sediment loads caused by the removal.

The permutational MANOVA analysis allowed investigation into whether there were significant differences in the macroinvertebrate community between years in all of the sites in both fall and spring pre- and post-removal. Due to our findings of significant change in all four streams suggest that there are annual differences that are natural to the system. This is not surprising as macroinvertebrate communities are often influenced by variable hydrologic factors like rainfall and flood intensity (Battle et al. 2007). The traits T-tests also confirmed what we saw in the community counts (Table 5 & 6). The rise in taxa with short life spans are in a large part a result of the increases in Chironomidae. As multi-voltine insects, the Chironomidae would have several generations within the study time. This high reproductive rate allows for the multi-voltine species to rapidly expand and to rapidly recover from harmful

12 disturbance. The Ephemerellidae however, are univoltine and I propose that the large increases in

Ephemerellidae numbers is due to either increased activity causing higher capture rates or higher survivability over the year following the riparian manipulation. This survivability may be linked to the reduction in predators that was observed within the removal sites.

For both reference and removal streams there were no significant changes in the variation of community composition between sites when comparing fall 2014 to fall 2015. This could be due to the short time frame between the cuts and the fall 2015 sample or possibly the cuts didn’t affect the newly hatched juveniles as much as the fully formed adults in the spring. In contrast, between spring collections both the removal sites had a significant reduction in variation in community composition between sites. This means that the differences between the individual samples within the reach began to look more alike in composition than the pre-treatment sample. However, Reference 1 also showed a significant reduction in variation between sites. One possible explanation for this is during December

2015 there was a very hard rainstorm that resulted in close to 2’’ of precipitation in under an hour. This caused some large changes in Reference 1 and Reference 2 where substrate changes were observed in both streams. Reference 1 in particular had several pools and riffles shift position within the reach.

Due to the limitations in the study design, mainly in the lack of replicate stream treatments, we were unable to draw significant conclusions as to the differences between the two treatment options.

Both streams appeared to respond similarly to the Rhododendron removal. Piling cut debris along

Removal 1 may prove to have different results in time. Additionally, we were unable to determine any specific response to the burn treatment at Removal 2. The burn only happened about a month prior to the final spring sampling which may have not been enough time for an effect. Other issues with the treatments included a 3-month lag from the start to finish as the contractors had setbacks related to heavy snows and workforce restrictions. The burns that were meant to occur during the summer

13 following the cuts did not happen due to the delays in cutting. This lead to Removal 1 not being burned and the lateness of the burn on Removal 2.

Conclusion

From the data analyzed in this study, we can observe similarities in macroinvertebrate response between selectively removing Rhododendron and several logging studies. This suggests that removing riparian Rhododendron creates a similar response to that of logging, on the short term. Whether this similarity continues into long term is yet to be seen. The increase in collector gathers was also responsible for the bulk of the increase in density between the spring samples. Although the treatments were not carried out optimally, the responses seen between the pre-post removal collections still allow us to observe the short-term macroinvertebrate responses. Future sampling will reveal whether the responses will proceed to change further or if they will revert to precut levels.

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Figures and Tables

Figure 2.Mean macroinvertebrate density plotted by stream reach and sample season. Bars around mean depict standard error. Removal 1 (HB) and Removal 2 (SWO) are the removal sites, SWO was also burned between spring samples. Reference 2 (KSB) and Reference 1 (RBB) are reference sites. There are no significant differences between fall samples. Removal 1 and Removal 2 both had significantly increased macroinvertebrate density (p = 0.007)(p < 0.001) between spring 2015 and spring 2016.

Figure 3. Mean macroinvertebrate richness plotted by stream reach and sample season. Bars around each mean depict standard error. Removal 1 (HB) and Removal 2 (SWO) are the removal sites, Removal 2 (SWO) was also burned between spring samples. Reference 2 (KSB) and Reference 1 (RBB) are reference sites.

Removal 1 Fall 2014 Fall 2015 Spring 2015 Spring 2016 Heptigeniidae 23 109*** 62 91 Ephemerellidae 163 888*** 801 2477*** Baetidae 4 10 17 310*** Leptophlebidae 233 65* 291 225 Ameletidae 22 103** 156 77 Perlodidae 130 74* 138 153 Peltoperlidae 157 141 131 372*** Leuctridae 159 97 338 242 Rhyacophilidae 210 94* 204 241 Hydropsychidae 583 359* 376 274 Elmidae 311 217 274 526** Tipulidae 80 83 113 277** Tanypodinae 47 51 102 220*** Non-Tanypodinae 107 168 636 1185*** Others 1481 1273 1635 1553 Denotes significant reduction * P < 0.05 ** P < 0.01 *** P < 0.001

Table 1. Total abundance of macroinvertebrates for Removal 1 with the 14 most common families displayed and the 35 less common families are lumped into the others row.

Removal 2 Fall 2014 Fall 2015 Spring 2015 Spring 2016 Heptigeniidae 71 147** 135 217* Ephemerellidae 92 406*** 693 1971*** Baetidae 0 15** 28 193*** Leptophlebidae 68 125* 179 313* Ameletidae 26 64 159 58* Perlodidae 79 50 133 261** Peltoperlidae 218 207 283 1206*** Leuctridae 114 188 323 314 Rhyacophilidae 103 64 121 100 Hydropsychidae 486 298* 489 547 Elmidae 245 252 409 650* Tipulidae 52 115* 139 193* Tanypodinae 29 20 93 83 Non-Tanypodinae 88 166 684 1049** Others 1130 991 1775 2014 Denotes significant reduction * P < 0.05 ** P < 0.01 *** P < 0.001

Table 2. Total abundance of macroinvertebrates for Removal 2 with the 14 most common families displayed and the 35 less common families are lumped into the others row.

Reference 2 Fall 2014 Fall 2015 Spring 2015 Spring 2016 Heptigeniidae 87 165* 318 328 Ephemerellidae 229 427* 926 942 Baetidae 7 7 52 257*** Leptophlebidae 149 37* 303 258 Ameletidae 58 98 161 104 Perlodidae 108 52*** 170 135 Peltoperlidae 240 71* 278 1022** Leuctridae 253 155** 477 270* Rhyacophilidae 191 52** 179 129* Hydropsychidae 237 55** 119 119 Elmidae 108 62 286 216 Tipulidae 78 60 178 148 Tanypodinae 52 9 282 78 Non-Tanypodinae 103 45* 844 460* Others 772 383 1094 932 Denotes significant reduction * P < 0.05 ** P < 0.01 *** P < 0.001

Table 3 Total abundance of macroinvertebrates for Reference 2 with the 14 most common families displayed and the 35 less common families are lumped into the others row.

Reference 1 Fall 2014 Fall 2015 Spring 2015 Spring 2016 Heptigeniidae 146 345** 300 344 Ephemerellidae 1054 925 760 664 Baetidae 39 79* 59 80 Leptophlebidae 361 305 204 166 Ameletidae 67 77 72 53 Perlodidae 285 168* 133 155** Peltoperlidae 512 238 202 686 Leuctridae 253 384 298 361 Rhyacophilidae 268 113** 112 101 Hydropsychidae 197 161 78 57 Elmidae 169 53** 164 167 Tipulidae 89 105 147 117 Tanypodinae 61 20 157 37 Non-Tanypodinae 150 122 924 358 Others 815 674 705 697 Denotes significant reduction * P < 0.05 ** P < 0.01 *** P < 0.001

Table 4. Total abundance of macroinvertebrates for Reference 1 with the 14 most common families displayed and the 35 less common families are lumped into the others row.

Figure 4. Proportion of functional feeding groups collected at all four sites between fall 2014 and fall 2015. Removal 1(HB), Removal 2 (SWO) both underwent Rhododendron manipulation. Removal 2 also was burned prior to the Spring 2016 sample. Reference 1 (RBB), and Reference 2 (KSB) were unmanipulatied.

Figure 5. Proportion of functional feeding groups collected at all four sites between spring 2015 and spring 2016. Removal 1(HB), Removal 2 (SWO) both underwent Rhododendron manipulation. Removal 2 also was burned prior to the Spring 2016 sample. Reference 1 (RBB), and Reference 2 (KSB) were unmanipulatied.

Figure 6. NMDS plots of each stream reach from fall 2014 – fall 2015 based on genera level count data and bray Curtis dissimilarity. Each stream reach was analyzed independently. Removal 1 (HB) had a stress of 0.179. Removal 2 (SWO) had a stress of 0.171. Reference 2 (KSB) had a stress of 0.148. Reference 1 (RBB) had a stress of 0.111. All four stream reaches had permutational MANOVA p values of less then 0.001 between fall 2014 and fall 2015 sites.

Figure 7. NMDS plots of each stream reach from spring 2015 – spring 2016 based on genera level count data and bray Curtis dissimilarity. Each stream reach was analyzed independently. Removal 1 (HB) had a stress of 0.137. Removal 2 (SWO) had a stress of 0.085. Reference 2 (KSB) had a stress of 0.099. Reference 1 (RBB) had a stress of 0.134. All four reaches had significant permutational MANOVA p values of 0.008 or less between samples taken spring 2015 vs spring 2016.

Figure 8. NMDS plots of each stream reach from fall 2014 – fall 2015 based on Poff (2010) trait matrix with genera count data and bray Curtis dissimilarity. Each stream reach was analyzed independently. Removal 1 (HB) had a stress of 0.025. Removal 2 (SWO) had a stress of 0.019. Reference 2 (KSB) had a stress of 0.018. Reference 1 (RBB) had a stress of 0.029. SWO is significantly different between fall 2014 and 2015 (p = 0.048). HB, KSB, and RBB are not significantly different between fall samples. HB, SWO, KSB, and RBB failed to show significant differences in variation between samples.

Figure 9. NMDS plots of each stream reach from spring 2015 – spring 2016 based on Poff (2010) trait matrix with genera count data and bray Curtis dissimilarity. Each stream reach was analyzed independently. Removal 1 (HB) had a stress of 0.040. Removal 2 (SWO) had a stress of 0.033. Reference 2 (KSB) had a stress of 0.025. Reference 1 (RBB) had a stress of 0.020. Both removal sites, HB and SWO, showed a significant difference in macroinvertebrate traits between spring samples (p = 0.001). Both reference sites, KSB and RB, failed to show significant differences in macroinvertebrate traits. SWO was the only reach to show significant change in variance between samples (p = 0.007).

Traits Rem 1 Rem 2 Ref 1 Ref 2 Semi-Voltine n.s. n.s. * n.s. Fast Seasonal Development n.s. ** n.s. n.s. Non-seasonal Development n.s. n.s. ** n.s. Non-Synchronized Dispersal n.s. n.s. n.s. ** Long Life n.s. n.s. ** n.s. Short Life n.s. n.s. n.s. * Very Short Life * ** n.s. n.s. High Dispersal n.s. n.s. n.s. ** Low Dispersal n.s. n.s. n.s. n.s. Strong Flyer n.s. n.s. n.s. ** Common Drifter n.s. * n.s. n.s. Rare Drifter n.s. n.s. n.s. * High Crawling Activity n.s. n.s. * * Low Crawling Activity n.s. n.s. n.s. ** Non- Swimmer n.s. n.s. n.s. ** Weak Swimmer n.s. * n.s. n.s. Free Ranging n.s. ** n.s. n.s. Attached to Substrate ** * * ** Poor Armor * n.s. * ** Riffle and Pool Habitat n.s. ** n.s. n.s. Riffle Habitat * n.s. * ** Ability to Survive Desiccation n.s. n.s. * * Size Small n.s. n.s. n.s. * Sprawling Activity n.s. ** n.s. n.s. High Swimming n.s. ** n.s. n.s. Tegument Breathing * n.s. n.s. **

Denotes significant reduction * P < 0.05 ** P < 0.01 *** P < 0.001 Table 5. Fields of traits that were statistically significantly different from fall 2014 to fall 2015. Rhododendron was removed between sampling dates in Removal 1 (HB) and Removal 2 (SWO). Reference 1 (RBB) and Reference 2(KSB) were unmanipulated.

Traits Rem 1 Rem 2 Ref 1 Ref 2 Semi-Voltine * * n.s. n.s. Univoltine * ** n.s. n.s. Non- Seasonal Development * * n.s. n.s. Slow Seasonal Development ** *** n.s. n.s. Poor Synchronized Dispersal ** *** * n.s. Well Synchronized Dispersal * * n.s. n.s. Long Life * * n.s. n.s. Short Life n.s. * * n.s. Very Short Life ** *** n.s. n.s. High Dispersal *** ** *** ** Low Dispersal * ** n.s. n.s. Strong Flyer *** *** *** ** Weak Flyer * ** n.s. n.s. Non-emergent ** *** n.s. n.s. Common Drifter ** *** n.s. n.s. Rare Drifter n.s. ** * n.s. High Crawling Activity n.s. ** ** * Low Crawling Activity ** ** n.s. n.s. Very Low Crawling Activity * * n.s. n.s. Weak Swimmer ** *** n.s. n.s. Free Ranging ** *** n.s. n.s. No Armor ** ** n.s. n.s. Poor Armor ** *** * n.s. Riffle and Pool Habitat ** *** n.s. n.s. Riffle Habitat n.s. ** n.s. * No Desiccation Ability ** *** n.s. n.s. Desiccation Ability n.s. ** n.s. n.s. Non-Streamlined ** ** n.s. n.s. Streamlined * *** ** * Size Medium ** *** n.s. n.s. Climbing Activity n.s. * n.s. n.s. Clinging Activity ** *** n.s. n.s. Swimming Activity n.s. * n.s. n.s. Cold Water Habitat n.s. ** * * Cool-Warm Water Habitat ** *** n.s. n.s. Gilled Breathing ** *** n.s. n.s. Tegument Breathing n.s. *** * n.s.

Denotes significant reduction * P < 0.05 ** P < 0.01 *** P < 0.001 Table 6. Traits that were significantly different from spring 2015 to spring 2016. Rhododendron was removed between sampling dates in Removal 1 (HB) and Removal 2 (SWO). SWO also was burned prior to the spring 2016 sample. Reference 1 (RBB) and Reference 2 (KSB) were unmanipulated.

Appendix A

Stream HB HB HB HB SWO SWO SWO SWO KSB KSB KSB KSB RBB RBB RBB RBB Season Fall Spring Fall Spring Fall Spring Fall Spring Fall Spring Fall Spring Fall Spring Fall Spring Year 2014 2015 2015 2016 2014 2015 2015 2016 2014 2015 2015 2016 2014 2015 2015 2016 Macaffentium 12 18 12 57 50 49 32 107 45 190 34 214 80 153 45 133 Epeorus 10 44 97 34 21 86 107 82 42 128 129 65 66 147 300 133 Stenocron 0 0 0 0 0 0 8 28 0 0 2 49 0 0 0 78 Ephemerella 160 785 881 2437 89 675 406 1950 228 890 418 935 1054 674 920 649 Europhella 1 4 1 2 3 1 0 0 1 4 0 0 0 0 1 0 Saratella 0 12 6 38 0 17 0 21 0 32 9 7 0 86 4 15 Baetis 3 10 3 123 0 7 0 30 5 26 2 49 39 37 2 7 Heterocleon 1 7 7 187 0 21 15 163 2 26 5 208 0 22 77 73 Paraleptophlebia 229 290 65 225 68 179 125 313 149 302 37 258 361 204 305 165 Ameletus 20 156 103 77 26 159 64 58 58 161 98 104 67 72 77 53 Isoperla 79 74 39 53 50 45 18 84 62 90 10 45 157 48 53 40 Yugus 28 33 18 41 20 28 16 71 26 19 13 19 92 25 32 28 Malirekus 0 0 0 0 0 0 0 0 18 1 0 0 0 0 0 0 Remenus 17 27 17 41 7 41 14 70 1 52 29 47 34 59 83 76 Eccoptura 1 4 0 16 1 19 2 36 1 7 0 22 2 1 0 11 Hansonoperla 4 10 12 14 4 10 7 21 12 5 3 7 4 4 5 3 Agnetina 1 0 0 8 0 0 0 4 0 0 0 2 0 2 1 15 Perlesta 0 0 0 4 0 0 0 2 0 0 0 4 0 0 0 0 Tallaperla 155 131 141 372 218 283 207 1206 240 278 71 1022 512 202 238 686 Leuctridae 157 338 97 242 114 323 188 314 253 477 155 270 253 298 384 361 Chloroperlidae 16 5 0 0 2 11 5 0 10 7 0 1 0 2 3 2 Strophopteryx 1 74 152 0 0 15 20 0 1 9 24 0 0 3 39 1 Amphinemura 21 42 52 26 11 70 25 48 7 43 3 79 13 35 21 45 Micrasema 31 74 42 6 13 42 20 3 11 56 14 1 37 7 23 2 Ceraclea 0 0 0 5 0 0 0 0 1 0 0 1 0 1 5 0 Lepidostoma 5 45 9 11 9 62 14 14 15 39 17 13 16 14 18 19 Theliopsyche 0 4 0 0 0 9 0 0 0 0 0 0 0 0 1 0 Dolophilodes 66 15 38 18 99 46 33 73 68 37 12 34 73 12 19 24 Wormaldia 9 0 2 1 3 1 3 2 7 0 1 1 1 1 1 4 Rhyacophila 204 204 94 241 103 121 64 100 191 179 52 129 268 112 113 101 Neophylax 1 21 8 17 0 10 5 45 3 13 4 39 4 7 1 19 Diplectrona 435 292 260 146 407 397 267 491 137 32 13 73 17 2 2 1 Parapsyche 34 62 62 110 11 37 17 39 47 60 27 41 61 50 98 39 Arctopsyche 109 22 37 18 68 55 14 17 53 27 15 5 119 26 61 17 Pychnopsyche 0 19 23 7 1 62 25 13 2 1 27 6 0 3 34 12 Psuedostenophylax 0 2 1 0 1 2 2 0 1 2 2 0 0 0 0 0 Palaeagapetus celsus 1 1 3 0 0 1 3 0 0 1 0 0 0 1 1 0 Agarodes 0 0 0 0 0 0 0 13 0 0 0 2 0 0 0 0 Fattigia 0 0 9 11 0 0 3 5 0 0 0 4 0 7 4 2 Lanthus 27 24 16 27 6 8 1 6 14 10 8 4 6 5 5 4 Cordulegaster 1 3 1 5 6 6 2 1 0 1 0 0 0 0 0 0 Optioservus 161 131 94 228 31 95 41 65 36 33 8 24 78 42 22 51 Olimnious 98 81 41 210 51 108 45 299 38 167 11 139 38 70 5 62 Promorsia 51 62 48 88 163 206 166 286 34 86 43 53 53 52 25 54 Setnelmus 0 0 34 0 0 0 0 0 0 0 0 0 0 0 1 0 Ectopria 48 39 14 42 5 8 2 8 0 0 0 1 0 0 3 8 Tipula 30 35 26 108 6 24 7 27 24 14 7 30 20 5 19 11 Hexatoma 32 26 23 53 2 2 0 11 16 24 8 32 9 15 2 10 Antocha 12 41 26 80 17 41 37 55 22 97 12 29 13 88 33 25 Dicronata 5 11 8 36 27 72 71 100 16 43 33 57 47 39 51 71 Blepharicera 0 0 0 0 0 0 0 0 0 0 0 0 0 21 1 11 Bezzia 25 44 17 81 1 28 3 53 21 37 7 17 28 21 9 8 Pericoma 21 20 3 3 2 12 2 3 5 9 0 0 3 2 2 1 Limnophora 0 0 1 4 0 2 2 12 0 2 2 0 0 2 1 2 Dixella 15 19 7 10 7 14 3 7 10 8 4 7 18 5 5 2 Tanypodinae 46 102 51 220 29 93 20 83 52 282 9 78 61 157 20 37 Non-Tanypodinae 103 636 168 1185 88 684 166 1049 103 844 45 460 150 924 122 358 Simuliidae 0 202 37 7 0 14 5 7 0 28 4 6 2 40 28 13 Atherix 3 4 1 13 0 3 2 3 0 2 0 0 0 1 0 2 Isopoda 0 11 0 0 0 1 0 0 1 0 0 0 0 0 0 0 Decapoda 9 2 6 7 3 1 1 5 12 9 1 12 7 5 3 10 Colembola 31 31 4 43 36 34 14 75 20 50 2 50 17 26 13 28 Leech 19 0 0 2 0 0 8 24 10 2 0 15 9 1 1 5 Table 7. Macroinvertebrate genus level counts for each sample by reach, season, and year.