The Pennsylvania State University

The Graduate School

School of Ecosystem and Science Management

ANALYSIS OF HISTORICAL AND CONTEMPORARY CONSUMPTION OF AQUATIC

MACROINVERTEBRATES IN DARTER SPECIES OF A HIGHLY DIVERSE STREAM

A Dissertation in

Wildlife and Fisheries Science

Casey Renee Bradshaw

2015 Casey R. Bradshaw

Submitted in Partial Fulfillment of the Requirements for the Degree of

Doctor of Philosophy

December 2015

The dissertation of Casey Bradshaw was reviewed and approved* by the following:

Jay R. Stauffer, Jr. Distinguished Professor of Ichthyology Dissertation Advisor Chair of Committee

Elizabeth W. Boyer Associate Professor of Water Resources

Michael A. Rutter Associate Professor of Statistics

Jeanette L. Schnars Director, Regional Science Consortium and Research Director of PA Sea Grant

Gregory Hoover Special Signatory, Department of Entomology

Michael G, Messina Head and Professor of the Department of Ecosystem Science and Management

*Signatures are on file in the Graduate School

iii

ABSTRACT

Darters are small, benthic, insectivorous fish that comprise majority portion of vertebrate communities within riffles of North American streams. The objectives of this study were first, to determine diet overlap among sympatric darter species at a site with high darter diversity (French

Creek at Venango) and a site with low diversity of darters (Woodcock Creek). The hypothesis for this objective is that darters living sympatrically in French Creek at Venango will partition their diet due to increased competition and therefore become more specialized in their food choices. The second objective was to compare diet of both common species of darters and currently listed species from historic collections in French Creek to contemporary collections and determine if diet has changed over time. The hypothesis is that diet will be similar from both collections given the French Creek watershed has remained relatively unaltered throughout history. I approached this objective by including currently listed species which were collected in the 1980's and early 1990's; before they were deemed either threatened or endangered. Those specimens were able to be dissected and food items analyzed. By comparing diets among common species from historic and present collections inferences were made about the diet of threatened and endangered species of darter in French Creek currently. Summary of major findings include dietary trends for historic to current darter collections have remained similar, indicating species under current conservation protection have a diet that has also remained relatively unchanged. There were obvious seasonal trends in dietary components for darters and differences in diet between Woodcock Creek and French Creek. Lastly, darters found in French

Creek, especially in late summer, had a broader diet than those from earlier seasons and from

Woodcock Creek, which indicates food is probably not a limited resource and therefore both intraspecific and interspecific competition is low. French Creek remains one of the few streams in the United States that holds such a high abundance and diversity of fish species, including

iv many under conservation protection. It remains an important refuge for species in the upper

Allegheny River, both terrestrial and aquatic. With undeniable threats to the biodiversity in

French Creek, including invasive species (i.e. round gobies), global climate change, riparian zone alterations and non-point source pollution, it is important to investigate aquatic community structures and ensure preservation of one of North America's last aquatic treasures.

TABLE OF CONTENTS

List of Figures ...... vi

List of Tables ...... viii

Acknowledgements ...... x

Chapter 1 Introduction To Darters ...... 1

Darter Communities in French Creek ...... 5

Chapter 2 Methods ...... 8

Site Selection: French Creek Watershed ...... 10 Field Collections ...... 16 Fish Processing ...... 17 Benthic Macroinvertebrate Processing ...... 18 Data Analysis ...... 18 Methods Used in Historic Fish Collections ...... 20

Chapter 3 Results ...... 21

Abiotic Measurements from the Stream ...... 21 Benthic Macroinvertebrate Collections from the Stream ...... 21 Electivity by Species ...... 26 Etheostoma blenniodes ...... 26 Etheostoma zonale...... 27 Etheostoma caeruleum ...... 28 Etheostoma flabellare ...... 29 Etheostoma nigrum ...... 30 Etheostoma variatum...... 30 Percina macrocephala ...... 31 Percina caprodes...... 31 Percina maculata, Etheostoma camurum and Etheostoma tippecanoe ...... 32 Diet Comparison Using Principle Component Analysis and ANOVA ...... 38

Chapter 4 Discussion ...... 51

Summary of Major Findings ...... 67

Literature Cited ...... 68

LIST OF FIGURES

Figure 1-1. Photograph of French Creek in Venango, PA (N 41○46.366'/W 80○ 06. 397') looking downstream. Historically this location has housed up to 14 species of darters within a 200meter stretch of stream……………………………………….. 9

Figure 2-2. Photograph of Woodcock Creek in Saegertown, PA (N 41○ 41.859'/W 80○06.401') looking upstream. This location was used for the low species richness site, normally housing four to six species of darters within a 200 meter stretch of stream ...... 9

Figure 2-3: Map of French Creek Watershed showing sub-watersheds and major tributaries of French Creek. © Chris Shaffer, Allegheny College GIS Manager ...... 10

Figure 2-4: Aerial view of Union City Dam on French Creek ...... 12

Figure 2-5: Aerial view of Woodcock Creek Lake Dam on Woodcock Creek, a tributary to French Creek ...... 12

Figure 2-6: Delineated map of the French Creek Watershed created by Allegheny College using ArcGIS Desktop version 10.3 from 2013 land use data. Land use percentages are as follows: Water = 1.33%, Developed Area = 7.46%; Forest = 51.50%; Grasslands = 1.33%, Agriculture = 32.95%; Wetlands 5.42% ...... 15

Figure 3-1: PCA scores plot showing the relationship between darter species in terms of PC 1 (x axis) versus PC 2 (y axis) from Woodcock Creek in early summer (A) and French Creek in early summer (B) (May to June). Darter species were plotted and color coded, diet was broken into taxonomic level of Order ...... 41

Figure 3-2: PCA scores plot showing the relationship between darter species in terms of PC 1 (x axis) versus PC 2 (y axis) from Woodcock Creek in early summer (A) and French Creek in early summer (B) (July to September). Darter species were plotted and color coded, diet was broken into taxonomic level of Order...... 42

Figure 3-3: PCA scores plot showing the relationship between diet contents in Etheostoma blenniodes in terms of PC 1 (x axis) versus PC 2 (y axis) from both Woodcock and French Creek and early to late summer. Seasons and sites were plotted and color coded, diet was broken into the taxonomic level of Order ...... 43

Figure 3-4: PCA scores plot showing the relationship between diet contents in Etheostoma blenniodes in terms of PC 1 (x axis) versus PC 2 (y axis) from both Woodcock and French Creek and early to late summer with historic collections included. Seasons and sites were plotted and color coded, diet was broken into the taxonomic level of Order ...... 44

Figure 3-5:PCA scores plot showing the relationship between diet contents in Etheostoma zonale in terms of PC 1 (x axis) versus PC 2 (y axis) from both Woodcock and

vii

French Creek and early to late summer. Seasons and sites were plotted and color coded, diet was broken into the taxonomic level of Order ...... 45

Figure 3-6:PCA scores plot showing the relationship between diet contents in Etheostoma zonale, including historic collections, in terms of PC 1 (x axis) versus PC 2 (y axis) from both Woodcock and French Creek and early to late summer. Seasons and sites were plotted and color coded, diet was broken into the taxonomic level of Order ...... 46

Figure 3-7: PCA scores plot showing the relationship between diet contents in Etheostoma caeruleumin terms of PC 1 (x axis) versus PC 2 (y axis) from both Woodcock Creek and early to late summer. Seasons and sites were plotted and color coded, diet was broken into the taxonomic level of Order ...... 47

Figure 3-8:PCA scores plot showing the relationship between diet contents in Etheostoma caeruleumin terms of PC 1 (x axis) versus PC 2 (y axis) from both Woodcock Creek and early to late summer with historic collections included. Seasons and sites were plotted and color coded, diet was broken into the taxonomic level of Order ...... 48

Figure 3-9: PCA scores plot showing the relationship between diet contents in Percina caprodesin terms of PC 1 (x axis) versus PC 2 (y axis) from Woodcock Creek and French Creek in late summer. Sites were plotted and color coded, diet was broken into the taxonomic level of Order ...... 49

Figure 3-10:PCA scores plot showing the relationship between diet contents in Percina caprodesin terms of PC 1 (x axis) versus PC 2 (y axis) from Woodcock Creek and French Creek in late summer with historic collections included. Sites were plotted and color coded, diet was broken into the taxonomic level of Order ...... 50

Figure 4-2. Photograph illustrating terminal mouth of Etheostoma flabellare collected from Woodcock Creek ...... 59

Figure 4-3. Photograph illustrating terminal mouth of Etheostoma flabellare collected from Woodcock Creek ...... 61

viii

LIST OF TABLES

Table 1-1. List of darter (Family: Percidae) species found in the French Creek Watershed with global (G) and state (S) conservation rankings. Species proposed for 2015 delisting are also noted. A. denotes genus Ammocrypta; E. denotes Etheostoma and P. denotes Percina ...... 6

Table 3-1: Complete benthic macroinvertebrate collection from Woodcock Creek in early summer broken down by taxa collected and associated percentages. If specimens could not be identified to genera, their order or family was included with the percentage 22

Table 3-2: Complete benthic macroinvertebrate collection from Woodcock Creek in late summer broken down by taxa collected and associated percentages. If specimens could not be identified to genera, their order or family was included with the percentage ...... 23

Table 3-3: Complete benthic macroinvertebrate collection from French Creek in early summer broken down by taxa collected and associated percentages. If specimens could not be identified to genera, their order or family was included with the percentage……………………………………………………………… ...... 24

Table 3-4: Complete benthic macroinvertebrate collection from French Creek in late summer broken down by taxa collected and associated percentages. If specimens could not be identified to genera, their order or family was included with the percentage ...... 25

Table 3-5: Electivity Index for darters collected at Woodcock Creek in early summer. Letters denote fish species while E. Ind. represents the electivity index value, ranging from -1 to +1. A = Etheostoma blenniodes; B = Etheostoma zonale; C = Etheostoma caeruleum; D = Etheostoma flabellare; E = Etheostoma nigrum; F = Percina macrocephala. Only benthic macroinvertebrates found in the stomach contents are shown in this table and used in the index. The index was calculated at the family level for benthic macroinvertebrates and could not be calculated if contents in the stomach were not collected in the stream ...... 33

Table 3-6: Electivity Index for darters collected at Woodcock Creek in late summer. Letters denote fish species while E. Ind. represents the electivity index value, ranging from -1 to +1. A = Etheostoma blenniodes; B = Etheostoma zonale; C = Etheostoma caeruleum; D = Etheostoma flabellare; G = Percina caprodes. Only benthic macroinvertebrates found in the stomach contents are shown in this table and used in the index. The index was calculated at the family level for benthic macroinvertebrates and could not be calculated if contents in the stomach were not collected in the stream...... 34

macrocephala; H = Etheostoma variatum. Only benthic macroinvertebrates found in the stomach contents are shown in this table and used in the index. The index was calculated at the family level for benthic macroinvertebrates and could not be calculated if contents in the stomach were not collected in the stream ...... 35

Table 3-8: Electivity Index for darters collected at French Creek in late summer. Letters denote fish species while E. Ind. represents the electivity index value, ranging from -1 to +1. A = Etheostoma blenniodes; B = Etheostoma zonale; C = Etheostoma caeruleum; G = Percina caprodes; H = Etheostoma variatum. Only benthic macroinvertebrates found in the stomach contents are shown in this table and used in the index. The index was calculated at the family level for benthic macroinvertebrates and could not be calculated if contents in the stomach were not collected in the stream ...... 36

ACKNOWLEDGEMENTS

I would first like to thank my major advisor, Jay Stauffer, for taking me on as a Ph.D. student and for supporting my decision to start a family in the middle of this journey. Without his flexibility and willingness to advise by many phone calls and emails, I would have never gotten through this.

I would also like to pay a special thanks to, Jeanette Schnars, who has not only been a mentor to me for many years before my Ph.D. started, but remains someone I can look to for advice both personally and professionally. I truly would not have had some of best opportunities in my life without your help and constant reminder that no matter what, "it will all be just fine."

I am grateful to Mike Rutter, who always found the time to help me with data analysis and fix my R codes when everything I tried spit errors at me. I truly appreciated the time you spent with me on working through the statistics.To Greg Hoover, for his endless knowledge on aquatic macroinvertebrates and for always pushing me to know more than I do; and to Beth Boyer for her willingness to serve on my committee and encouragement through the process.

I would most especially like to thank my husband, Tyler, who has had more patience with me than anyone I've ever met,our beautiful daughter for supplying endless laughter when I was stressed out, and the rest of our family for constant love and support.

Lastly, I would like to thank everyone that helped from the PSU lab in data collection, advice, and a place to stay when I was in State College. Also, to my department at Allegheny

College, who have been nothing but supportive as I finished my dissertation while working a full time job.

Chapter 1

Introduction to Darters

Streams in the eastern United States hold many species that comprise communities based on fishes in the same guild that are closely related (Hlohowskyj and White, 1983). Darters, particularly, are small benthic fishes (rarely exceeding 8 centimeters) in the perch family

(Percidae) that are endemic to and found throughout North America, exceptin the Pacific and

Arctic drainages (Page and Burr, 2011). In general, darters exhibit a relatively similar body design: small size and elongated profile compared to other fishes (Hood et al., 2003) and occur in highest densities through the Appalachian Mountains of the eastern United States. Often times, two or more darter species will coexist in the same habitats and consume prey that superficially appear similar (Matthews et al., 1982). There are three genera of darters, Percina, Etheostoma, and Ammocrypta. Most darters have no gas bladder, and “dart” along the bottom of streams

(Page and Burr, 2011), however, individuals in Percina are generally larger than other darters, have kept a small gas bladder and tend to spend more time swimming off the stream bottom than other darter genera, and have large mid-lateral scales or a naked belly (Page and Burr, 2011).

Ammocryptaspp., or “sand darters” have adapted to living in sandy streams, have long and cylindrical body forms (Guill et al., 2003) and remain buried with only eyes and snout protruding, while darters in the genus Etheostoma are the most diverse genera of fishes in North America

(Page and Burr, 2011). They comprise 148 species (Kuehne and Barbour, 1983; Page and Burr,

2011) and a huge portion of the biomass in streams, specifically in riffles (Stauffer et al., 1995;

Hlohowskyj and White, 1983; Pratt and Laurer, 2013). Included in this group are some of the rarest fishes, some of the most common fishes, and some of the most colorful fishes in North

2 America (Page and Burr, 2011). Most darters exhibit sexual dimorphism, with males having brightly colored bodies and fins, especially during the breeding season (Lachner et al., 1950).

Darters normally inhabit clean lotic systems that are moderate in size and have swift flowing water with gravel and cobble substrate (Lachner et al., 1950) and present high site fidelity (Roberts and Angermeier, 2007; Roberts et al., 2008). Some darter species have adapted to living in alternative habitats, (i.e. Mississippi River) or small headwater streams (Page and

Burr, 2011). There is considerable literature on the life history, food, and habits of many species of darters, but less in the areas of niche relationships between synoptic darter species (Matthews et al., 1982) although competition in diet amongspecies is predicted to be high (Stauffer et al.,

1995). In general, darters consume a widevariety of aquatic macroinvertebrates including ephemeropterans (mayflies), plecopterans (stoneflies), trichopterans (caddisflies), simuliids

(black flies), isopods (sowbugs), and chironomids (non-biting midges) (Paine et al., 1982; Alford and Beckett, 2007). Ephemeropterans and trichopterans may comprise large components of darter diet due to their relatively large size, but chironomids also play important role in the composition of diet because they are typically abundant in many lotic systems (Marin, 1984;

Alford and Beckett, 2007). Darters may not follow the normal optimal foraging theory and positive body size-prey size relationships, given their environment is patchy (Adamson and

Wissing, 1977). Unlike water column feeders, many darters feed from the substrate on the stream bottom and the complex habitat may not include all possible prey items. Darters use a variety of feeding strategies, including Logperch (Percina caprodes) which flip stones over and eat exposed insects from underneath. Eastern Sand Darters (Ammocrypta pellucida) bury themselves in the sand and ambush their prey, while other species such as Greensides Darters (E. blenniodes) and Fantail Darters (E. flabellare), which are active foragers in rock interstitial spaces (White and

Stauffer, 1992).

3 Lotic environments can be considered patchy on many levels based on spatial and temporal habitat heterogeneity (Pringle et al., 1988; Petty and Grossman, 1996). One of the main variations in habitat is the riffle-pool transition (Begon et al., 1996; Odum, 1969). Both invertebrate and fish assemblages differ within these two habitat types because pool sections of stream are interrupted by riffles, and riffles with pools (Lonzarich et al., 2000). Diets of habitat specific fishes, such as darters, may correspond to these types of habitat segregation, which has been observed in other fish communities (Zaret and Rand, 1971). Many darters exhibit small home ranges within a stream, and prey may be constrained by differences in habitat (Gillette,

2012; Roberts et al., 2008).

Niche theory, as described by Gause (1934) and also by Vandermeer (1972) predicts species segregate based on at least one variable in order to live in the same habitat and coexist, however, only looking at one variable may limit valid conclusions about community structure.

Also, physical habitat variables may not be independent of each other (Pratt and Laurer, 2013).

Analyzing how darters partition their resources is necessary to understand community structure and function (Paine et al., 1981) as well as the ecology of fish communities in general (Welsh and

Perry, 1998).

Research shows that animals, in general, partition their resources in three ways: habitat, food, and time (Schoener, 1974), with habitat being the most important (Gorman and Karr, 1978).

Many studies suggest that sympatric/syntopic darter species partition their niches along various axes (Welsh and Perry, 1998), such as substrate composition (Stiles, 1972, Hlohowskyj and

Wissing, 1986; Klesser and Thorp, 1993), and substrate availability which is used for protective cover from predators and changes in velocity, spawning and foraging (Pratt and Laurer, 2013), depth (Fisher and Pearson, 1987; Chipps et al., 1994), flow (Matthews, 1985; Fisher and Pearson,

1987), diet (Smart and Gee, 1979; Paine et al., 1982; Hlohowskyj and White, 1983), Martin,

1984) and temperature (Ingersoll and Clausen, 1984).Pratt and Laurer (2013) have also suggested

4 that the dynamics of a streams natural flow regime, ability to avoid predators and habitat disturbances are also ways in which niches may be segregated. Many studies have found extensive degrees of overlap amongspecies (Stauffer et al., 1995). Morphological differences among darter species relates directly back to feeding strategy and efficiency, then linking to increased performance in predator avoidance and reproductive success and consequently increased fitness (Guill et al., 2003).Because darters are considered visual, diurnal insectivorous fishes (Adamson and Wissing, 1977) they may also be able to coexist in streams through trophic segregation (van Snik Gray et al., 1997). Literature suggests that olfactory cues are not utilized in darter feeding, even when turbidity is high. In these turbid areas, Daugherty et al. (1976) suggests that olfactory cues may draw darters to a specific habitat, but visual cues are then used and initiate pecking responses. Diet studies have been conducted as well as competitive interactions, but few studies have investigated diet shifts within the same watershed of systems of high darter diversity and low diversity.

The trophic ecology of many benthic, insectivorous fishes, such as darters, is lacking.

Darters play important ecosystem roles. Despite their small size, they can have disproportionate influence on lotic systems trophic organization as well as indicate species diversity and biological integrity (van Snik Gray and Stauffer, 1999). Darters in the genus Etheostoma have been informative models in the past to help understand complex interactions in stream communities that indicate healthy ecosystems (Pratt and Lauer, 2013; Rogers and Allan, 2012; White and

Stauffer, 1992). Studies suggest that Etheostomaspecies may give valuable insight to negative consequences of habitat alterations and degradation, specifically when water depth, velocity and substrate are affected. In addition, the absence of the species could also indicate habitat alterations have already been made and actions should take place to implement restoration practices (Pratt and Laurer, 2013).

5 There has been a considerable amount of speciation in darters, specifically inthe genus

Etheostoma. Many Etheostoma spp.occur sympatrically (two or more species) as benthic communities in streams (Scott and Crossman, 1973; Stauffer et al., 1995). Although much research has been done on darters, relatively few studies have examined niche partitioning in diverse communities (more than four species), with the exceptions being seven species (Winn,

1958), 11 species (Stauffer et al., 1996) and 12 species (Greenburg, 1991). In general, there is a lack of information in the literature regarding resource partitioning in high diversity streams, where many sympatric fishes live closely with one another. The importance of understanding changes in realized niches among sympatric species can help identify changes that might occur when non-native species enter the system and begin to alter competitive interactions with not only native species, but the invader as well (van Snik Gray et al., 2005).

Darter Communities in French Creek

French Creek is home to 16 species of darters, manylivingwithin a single riffle. Six species are listed as either threatened or endangered in Pennsylvania, and Etheostoma maculatum

(Spotted darter) only occur in French Creek and the upper Allegheny River.

In French Creek, White and Stauffer (1992) found as many as 13 species of darter within a single riffle. This survey also found that all species native to the upper Allegheny River basin, except channel darters (Percina copelandi) are found in French Creek. While these species are found in areas throughout the Allegheny River system, French Creek is the place where many of the species occur together. Table 1 displays the darter species found in French Creek as well as their current conservation status.

6 Table 1-1.List of darter (Family: Percidae) species found in the French Creek Watershed with global (G) and state (S) conservation rankings. Species proposed for 2015 delisting are also noted. A. denotes genus Ammocrypta; E. denotes Etheostoma and P. denotes Percina. G S PA Status – Common Name Scientific Name Rank Rank 2015 2015 Changes Eastern Sand darter A. pellucida G4 S1 Endangered

Greenside darter E. blennioides G5 S5 Not listed

Rainbow darter E. caeruleum G5 S5 Not listed Proposed for delisting on Bluebreast darter E. camurum G4 S4S5 Threatened 20 Dec. 2014

Iowa darter E. exile G5 S2 Endangered

Fantail darter E. flabellare G5 S5 Not listed Proposed for delisting on Spotted darter E. maculatum G2G3 S4 Threatened 20 Dec. 2014

Johnny darter E. nigrum G5 S5 Not listed Proposed for delisting on Tippecanoe darter E. tippecanoe G3G4 S4 Threatened 20 Dec. 2014

Variegate darter E. variatum G5 S5 Not listed

Banded darter E. zonale G5 S5 Not listed Proposed for delisting on Gilt darter P. evides G4 S4 Threatened 20 Dec. 2014

Blackside darter P. maculata G5 S5 Not listed Formerly threatened in Longhead darter P. macrocephala G3 S4 Not listed PA Formerly threatened in Channel darter P. copelandi G4 S5 Not listed PA

Logperch P. caprodes G5 S5 Not listed

The objectives of this study are to:

 Determine diet overlap among sympatric darter species at a site with high darter

diversity (French Creek at Venango) and a site with low diversity of darters

(Woodcock Creek). The hypothesis for this objective is that darters living

sympatrically in French Creek at Venango will partition their diet due to

increased competition and therefore become more specialized in their food

choices.

 Compare diet of both common species of darters and currently listed species

from historic collections in French Creek to contemporary collections and

determine if diet has changed over time. The hypothesis is that diet will be

similar from both collections given the French Creek watershed has remained

relatively unaltered throughout history. I approached this objective by including

currently listed species that were collected in the 1980's and early 1990's; before

they were deemed either threatened or endangered. Those specimens were able

to be dissected and food items analyzed. By comparing diets among common

species from historic and present collections inferences were made about the diet

of threatened and endangered species of darter in French Creek currently.

Chapter 2

Methods

Two sites were chosen for this study based on species abundance and richness from previous collections housed in the Pennsylvania State University Fish Collection (PSUFC). Sites were also chosen based on accessibility and the presence of riffle-run habitat.

The first site chosen was on the mainstem of French Creek in Venango, Crawford

County, Pennsylvania (N 41○46.366'/W 80○ 06. 397'). At this location, French Creek is a 4th order stream and approximately 63m wide with riffles, runs and pool habitat within a 200 meter stretch of stream. Historically, this site has housed 13 species of darter within 200meter stretch of stream (pers. comm. Jay Stauffer, 2013; White and Stauffer, 1992), allowing this site to be used as the high darter diversity site during the study (Figure 2-1). Many of the darters found within this site are listed as either federally or state threatened or endangered, so while collections from this study may show less diversity in terms of the darter kept for diet analysis, the stream itself holds many darters that are living among the individuals likely collected and competing for resources.

Woodcock Creek, located in Saegertown, Crawford County, PA was the site chosen with low diversity in sympatric species of darters. This tributary of French Creek was nearly 10meter in width where samples were collected and is located downstream of Woodcock Creek Lake Dam

(N41○ 41.859'/W 80○06.401'). This site was chosen based on riffle, run and pool habitats within a

200meter stretch of stream, similar in proportion to the site at Venango and previous collections made by Dr. Jay Stauffer. This site consistently houses five to six species of darters, which is why it was deemed the low species diversity of sympatric species of darters (Figure 2-2).

Figure 2-1.Photograph of French Creek in Venango, PA (N 41○46.366'/W 80○ 06. 397') looking downstream. Historically this location has housed up to 14 species of darters within a 200meter stretch of stream.

Figure 2-2.Photograph of Woodcock Creek in Saegertown, PA (N 41○ 41.859'/W 80○06.401') looking upstream. This location was used for the low species richness site, normally housing four to six species of darters within a 200meter stretch of stream.

Site Selection: French Creek Watershed

French Creek in the upper Ohio River Watershed (Mississippi River Drainage) in northwestern Pennsylvania and southwestern New York originates in Chautauqua County of western New York. It forms the West Branch of French Creek, which flows southwest to Erie

County, Pennsylvania to its confluence with the main branch of French Creek(Figure 1-1).

Figure 2-3: Map of French Creek Watershed showing sub-watersheds and major tributaries of French Creek. © Chris Shaffer, Allegheny College GIS Manager.

11 The South Branch of French Creek begins in southeast Erie County near Corry, PA and flows west to meet the main branch of French Creek near Union City, PA. It then flows south through

Crawford County, the northeast corner of Mercer County and into Venango County where it joins the Allegheny River in Franklin, PA. The length of the main stem is nearly 188 kilometers long and the watershed includes nearly 3,199 square kilometers. Eleven percent of the Allegheny River drainage basin is drained by French Creek and its tributaries. There are 72 municipalities in the four counties that French Creek flows through in PA and portions of New York with a total population of nearly113,000 for the entire watershed. French Creek is an example of relatively intact, free flowing, and medium-sized (4th order) stream.

Currently, there are two dams within the watershed, the Union City dam and Woodcock

Creek Lake dam, both of which are maintained and operated by the Pittsburgh District of the US

Army Corp of Engineers. The Union City dam is located in Erie Co. PA and is the only dry-bed reservoir in the entire district (Figure 2-4). Essentially, this dam is constructed of a concrete conduit that runs through the base of the dam allowing for uncontrolled discharge of water into

French Creek. The dam stores and releases water during period of peak flow, normally during spring snow melts and when the amount of water entering the reservoir exceeds the dam’s capacity to discharge it through the drainage conduit a temporary lake is formed. Woodcock Creek Dam, however, is used to reduce downstream flooding as part of the French Creek flood control system, to improve downstream water quality and to provide a diversified array of general recreation activities (Figure 2-5)(US Army Corp of Engineers, 2015).

Figure 2-4: Aerial view of Union City Dam on French Creek.

Figure 2-5: Aerial view of Woodcock Creek Lake Dam on Woodcock Creek, a tributary to French Creek.

The major portion of the French Creek watershed (over 90%) lies within the Appalachian

Plateau physiographic province. Before glacial advances between 2,000,000 and 20,000 years ago, streams in northwestern Pennsylvania's drainage was mostly towards the north and to the

Atlantic Ocean by way of the St. Lawrence Valley (Shepps et al., 1959; French Creek

Conservation Plan, 2002). As ice progressed from the north, streams flowing northward were

13 ponded, forced to flow over low elevations between ridges, and were in many cases rerouted to flow southward. During this time, and over many glacial advancements, French Creek reversed it direction from flowing north to Lake Erie, to south into the Ohio Drainage. Continental ice sheets advanced at least 7 times into northwestern Pennsylvania during the Pleistocene Epoch which occurred between 2,000,000 and 20,000 years ago. This time period was comprised of at least two stages, the Illinoian (2 advancements between 196,000 and 128,000 years ago) and

Wisconsin (5 advancements between 22,000 and 17,000 years ago) stages (Shepps et al., 1959).

As the glaciers retreated, they left "glacial drift" in the form of clay, silt, sand and gravel throughout the landscape of now rounded hills and newly formed valleys (French Creek

Conservation Plan, 2002). Glaciers werealso responsible for formingmany wetland and glaciated lakes throughout the region. An incredible feature within the watershed are the 5 glaciated lakes and numerous wetlands, including rare fens and bogs (French Creek Conservation Plan, 2002).

In addition, many theories have indicated that aquatic species from the northern St. Lawrence, the

Genesee and Susquehanna systems mixed with species ("stream capturing") from the Ohio River drainage, which would explain the immense biodiversity found in French Creek and other parts of the Allegheny and Ohio Rivers. The French Creek Conservation Plan (2002) claimedthat the

Ohio River drainage contained all native species presently found in the French Creek watershed and that the Atlantic slope species not found in the French Creek drainage provides the evidence.

The reversal of flow, from north to south, contributed inland species to certain Atlantic slope drainages (French Creek Conservation Plan, 2002). Other sources claim that the St. Lawrence is an Atlantic Coastal stream and there were stream captures between the Susquehanna and the

Genesee Rivers, and the Susquehanna and Allegheny Rivers, through which Atlantic slope fishes reached the Ohio drainage (Hocutt and Wiley, 1986).

French Creek's biological diversity is rich not only the in state of Pennsylvania, but the entire Ohio River watershed (French Creek Conservation Plan, 2002). It holds more species of

14 fish and freshwater mussels than any other similar sized stream in the northeast United States.

Eighty-eight species of fish and 29 native mussel species are found within the watershed. This includes both the clubshell (Pleurobemaclava) and northern riffleshell

(Epioblasmatorulosarangiana), which are both federally endangered (White and Stauffer, 1992;

Smith and Crabtree, 2010) and four other species which are endangered in PA and have global status of imperiled or vulnerable, or are candidates for federal listing. There are 13 other mussel species within French Creek that are considered rare, threatened, endangered, or possibly extirpated in Pennsylvania(Smith and Crabtree, 2010).

Several fishes are found nowhere else in the state, such as Spotted darters (Etheostoma maculatum) which is only found in the upper Allegheny River and French Creek. Other species such as Mountain Madtom, Brindled Madtom and Northern Madtom catfishes

(Noturuseleutherus, N. miurus and N. stigmosus) are only found within the Allegheny River watershed. French Creek also houses the rare Eastern Sand Darter (Ammocrypta pellucida), which was rediscovered in 1991 after 14 years of no sightings. This stream serves as an important refuge for species in the upper Allegheny River and contains many sensitive species in unusually high abundance (White and Stauffer, 1992). Both game and non-game species thrive within the aquatic and terrestrial environments of the watershed. There are 379 bird species that use the watershed (Crossley, 1999), 63 known extant mammals (Merrit, 1987), and lastly, breeding populations of eastern hellbenders (Cryptobranchus alleganiensis alleganiensis), one of two extant giant salamander genera that exists today (French Creek Conservation Plan, 2002;

McKinstry et al., 1999). Today, most of the watershed is rural and mixed between forest and agriculture (Figure 2-6) (Yang, J, 2013).

Figure 2-6: Delineated map of the French Creek Watershed created by Allegheny College usingArcGIS Desktop version 10.3 from 2013 land use data. Land use percentages are as follows: Water = 1.33%, Developed Area = 7.46%; Forest = 51.50%; Grasslands = 1.33%, Agriculture = 32.95%; Wetlands = 5.42%.

Public awareness regarding the diversity in French Creek has grown over the years. Today, there are many organizations that focus on educating students and the public as a whole, as well as watershed conservation, obtaining land trusts and conducting basic and applied scientific research. Some of the organizations include The French Creek Conservancy, Western

16 Pennsylvania Nature Conservancy, Crawford County Conservation District, Allegheny College,

Creek Connections and select aquatic biologists from nearby colleges and universities

Field Collections

Fishes were collected using a 1.2m by 3m by 0.32cm mesh seine rather than backpack electroshocking. Because darters are small and many lack air bladders, seining ensures darters that are shocked do not sink to the bottom of the stream and become lodged among rocks. Also, it is essential to ensure stomach contents were not regurgitated upon galvanotaxis during diet analysis studies. Fishes were collected at both French Creek and Woodcock Creek in a 200 meter stretch of stream that contained primarily riffles, with runs and pools present as well. The entire

200 meters was seined by a team of researchers working from the upstream section of stream to downstream to avoid disturbance of fish and macroinvertebrates before collections and also to allow fish and macros to flow into the seine rather than away from it. Fishes collected were immediately identified to species, anesthetized in MS-222, and placed in 10% formalin to prevent digestion of stomach contents (IACUC # 35338). After two weeks, they were rinsed out of the formalin mixture and transferred to 70% ethanol and stored until processed.

Aquatic benthic macroinvertebrates were collected using a D-frame kicknet. Kick samples are effective in all stream habitats (riffles, runs and pools) and typically collect 95% of the macroinvertebrate taxa present in a stream (Frost, et al., 1970). Nine, twenty second kicks

(Tzilkowski and Stauffer, 2004) were collected at each site, specifically in areas where darters were collected. Darters were collected primarily in riffles, therefore six kick samples were taken from riffles and the remainder taken from runs. Kicknet samples were taken within one hour of collecting fish to provide current food sources available to fish. This method has been used in the

17 past (Frost et al., 1970; Tzilowski and Stauffer, 2004) and gives the best representation of environmental benthic macroinvertebrates within a stream system and what would be available for fishes to consume. After collection, substrate and aquatic benthic macroinvertebrates were immediately placed in 70% ethanol and stored for at least 12 hours before processing.

Abiotic factors were also collected each time fishes and benthic macroinvertebrates were collected in the stream. A single transect was constructed across the stream where the first riffle within the stretch of stream was located and water velocity (m/s) and depth (cm) were taken at multiple locations as well as temperature (degrees C).

Stomach contents from fish previously collected and held in the Penn State University

Fish Museum (PSUFM) were also analyzed. Darters were chosen from thePSUFMbased on the location they were collected from and species. Fishes selected for this study were collected from the mainstem of French Creek from localities as close to the Venango site as possible. The fish were processed in the same manner as those collected during 2013 and 2014 field season and were returned to the Penn State museum. The species chosen were consistent with those currently found in the mainstem of French Creek, but are now listed as either threatened or endangered. Common, unlisted species were also chosen as a reference to compare current collected fishes to those historically collected and stomach contents analyzed.

Fish Processing

Total length (to the nearest mm) was recorded for each individual fish using a digital caliper, and sexes (if possible, normally determined by the presence of eggs during the spawning season) were also collected. Gut contents that were posterior to the stomach were not included in this study because they were too digested to reliably count and identify (Cordes and Page, 1980).

18 Partially digested organisms were counted as a whole organism if head capsules were not present.

When head capsules were present, especially for chironomids, heads were counted as the number organisms rather than counting unattached body parts. Fish that had no identifiable stomach contents or empty stomachs were removed from the data set and therefore not included in data analysis.

Benthic Macroinvertebrate Processing

Substrate and benthic macroinvertebrates from kicknet samples were examined using a dissection microscope (Leica ModelEZ4),benthic macroinvertebrates were sorted, identified to the lowest possible taxa and enumerated.Stomach contents were identified to the taxonomic level of genus when possible, except for chironomids larvae which were identified to family.

Data Analysis

Diet analysis was conducted using Ivlev's electivity index, E = (ri-pi)/(ri + pi), where ri is the percent of a prey item in the diet, and pi is the percent of a prey item available in the environment (as estimated from kicknet samples). Values of the index ranged from -1 to +1, with positive values indicating that prey type of found in higher proportion in the diet than in the prey community. Negative values indicate that prey type was found in lower proportion ofthe diet than in the prey community (Stewart, 1988). In order for this index to be valuable, taxa from both kicknet samples and stomach contents had to be identified to the same taxonomic level. In many

19 cases, stomach contents could only be identified to family, although there were many specimens that could only be identified to order (i.e. ephemeropterans missing gills, legs or caudal filaments). Some of the orders were separated to family level, while some members of the family could be identified to genus in the stomach contents and the kicknet samples.

Principle component analysis (PCA) was conducted using the statistical program R version 3.2. PCA is a multivariate approach that assess relationships within a single set of interdependent variables, and does not account for relationships with outside variables

(McGarigal et al., 2000). It was used in this situation to construct a coordinate system to help explain ecological relationships (Ludwig and Reynolds, 1988), specifically, diet and diet overlap between darter species. Analysis was conducted for each species at both locations and seasons, when fishes were collected for those time periods. Historic darters were also compared to present collections by species. Analysis was also conducted by site for all darter species found. Diet items that had less than 10 individual prey items for each sample were excluded from the analysis. Fishes that had no food items or unidentifiable food items in their stomach were not included in this analysis.

In addition to PCR, analysis of variance (ANOVA) was conducted on the principal components. Since the principle components represented independent, linear combinations of the taxa observed, conducting each ANOVA separately was appropriate. Tukey tests were also conducted to further explain differences in diets among darter species and between sites and seasons. For each principal component, identification of the most important taxa in each component was determined by looking at the largest (in absolute value) coefficients of each principal component. The number of components to run ANOVAs on for each analysis was determined by choosing the first x components in which biological interpretations were easily made. In addition, Bonferronni corrections (alpha/x) were used to counteract problems of multiple comparisons.

Methods Used in Historic Fish Collections

Historic collections were conducted by Dr. Jay Stauffer's fisheries lab using the same methodology stated above. All specimens collected via seining were identified to species, preserved in 10% formalin and transferred to 70% ethanol which are stored in the PSUFM. To verify collection methods, sampling dates and locations, original field notes from those collections were observed and remain in Dr. Jay Stauffer's lab at Penn State University in State

College, Pennsylvania.

Chapter 3

Results

Abiotic Measurements from the Stream

On average, Woodcock Creek had a depth of 24.1 cm and mean water velocity of 0.35 m/s2 with a bank width of 15 m. French Creek, on average, had a depth of 29.9 cm and mean water velocity of 0.51 m/s2 and a bank width 63 m.

Benthic Macroinvertebrate Collections from the Stream

Chironomids were the most abundant taxa at Woodcock Creek in early summer (n =

2043), followed by hydropsychid caddisflies (n = 587) and elmid riffle beetles (n = 408) (Figure

4-1; Table 4-1 ). The number of chironomids at French Creek in early summer was lower than at

Woodcock Creek (n = 440), however, they were still the most abundant taxa, followed by hydropsychid caddisflies (n = 320) and again, elmid riffle beetles (n = 190). The total number of macroinvertebrates found at French Creek in early summer was1,468 individuals with 43 different families represented and an average population size of 34.9. At Woodcock Creek in early summer, there were 3,565 individuals collected with 34 families represented and an average size of 108 individuals/family. At Woodcock Creek in late summer, there wasa total of 5,168 individuals collected from 31 families and an average of 147 individuals/family. At French

Creek in late summer, 8,495 individuals were collected, representing 43 families with an average of 198 individuals/family (Table 3-2 ).In late summer, however, hydropsychid caddisflies in the

22 genera Cheumatopsyche and Hydropsychae constituted the highest percentage of taxa found in the stream collections.

Taxon Order Family Genus Percentage Amphipoda Gammaridae Gammarus 3.36% Coleoptera Elmidae Dubiraphia 0.06% Coleoptera Elmidae Optioservus 0.76% Coleoptera Elmidae Stenelmis 19.07% Coleoptera Elmidae -- 0.06% Coleoptera Psephenidae -- 0.06% Coleoptera Chrysomelidae -- 0.00% Diptera Chironomidae -- 47.28% Diptera Empididae -- 0.70% Diptera Simuliidae Prosimulium 0.82% Diptera Tabanidae -- 0.19% Diptera Tipulidae Antocha 0.44% Diptera Tipulidae Hexatoma 0.32% Diptera Tipulidae Tipula 0.95% Ephemeroptera Baetidae -- 0.02% Ephemeroptera Baetidae Baetis 0.04% Ephemeroptera Caenidae Caenis 4.25% Ephemeroptera Ephemerellidae Eurylophella 0.19% Ephemeroptera Heptageniidae Rhithrogena 0.01% Ephemeroptera Heptageniidae Stenacron 0.06% Ephemeroptera Heptageniidae Maccaffertium 1.03% Ephemeroptera Leptophlebiidae Habrophlebia 0.06% Ephemeroptera -- -- 1.58% Ephemeroptera Siphlonuridae Ameletus 0.06% Ephemeroptera Siphlonuridae Siphlonuris 0.06% Megaloptera Corydalidae Corydalus 0.38% Megaloptera Corydalidae Neohermes 0.19% Megaloptera Sialidae Sialis 0.19% Oligochaeta -- -- 0.32% Plecoptera Chloroperlidae -- 0.19% Plecoptera -- -- 0.06% Plecoptera Perlidae Acroneuria 0.32% Plecoptera Pteronarcyidae Pteronarcys 0.32% Trichoptera Glossosomatidae Glossosoma 0.06% Trichoptera Hydropsychidae Cheumatopsyche 3.11% Trichoptera Hydropsychidae Hydropsyche 3.80% Trichoptera Hydropsychidae -- 6.34% Trichoptera Hydroptilidae Leucotrichia 0.06% Trichoptera Leptoceridae Ceraclea 0.06% Trichoptera Leptoceridae Oecetis 0.63% Trichoptera -- -- 0.25% Trichoptera Philopotamidae Chimarra 1.65% Trichoptera Polycentropodidae Neureclipsis 0.44% Trichoptera Psychomyiidae Psychomyia 0.06%

23 Table 3-2: Complete benthic macroinvertebrate collection from Woodcock Creek in late summer broken down by taxa collected and associated percentages. If specimens could not be identified to genera, their order or family was included with the percentage.

Taxon Order Family Genus Percentage Amphipoda Gammaridae Gammarus 0.31% Amphipoda Talitridae Hyalella 0.12% Coleoptera Elmidae Dubiraphia 0.01% Coleoptera Elmidae Macronychus 0.05% Coleoptera Elmidae Optioservus 0.28% Coleoptera Elmidae Stenelmis 5.51% Coleoptera Gyrinidae Dineutus 0.51% Coleoptera Hydrophilidae -- 0.02% Coleoptera Psephenidae Psephenua 0.12% Diptera Chironomidae -- 35.44% Diptera Empididae -- 0.51% Diptera Ephydridae -- 0.04% Diptera Simuliidae Simulium 23.45% Diptera Tipulidae Antocha 0.18% Diptera Tipulidae Hexatoma 0.21% Diptera Tipulidae Tipula 0.04% Diptera Tipulidae -- 0.04% Ephemeroptera Baetidae -- 0.04% Ephemeroptera Baetidae Centroptilum 0.04% Ephemeroptera Caenidae Caenis 1.19% Ephemeroptera Heptageniidae -- 0.02% Ephemeroptera Heptageniidae Maccaffertium 0.88% Ephemeroptera -- -- 0.27% Ephemeroptera Isonychiidae Isonychia 0.02% Ephemeroptera Leptohyphidae Tricorythodes 0.02% Hemiptera Gerridae Neogerris 0.02% Hemiptera -- -- 0.04% Hirudinea -- -- 0.02% Isopoda Asellidae -- 0.02% Isopoda Asellidae Caecidotea 0.49% Isopoda -- -- 0.04% Megaloptera Corydalidae Corydalus 0.31% Megaloptera Corydalidae Nigronia 0.18% Odonata Gomphidae Stylogomphus 0.21% Oligochaeta -- -- 0.80% Plecoptera Pteronarcydiae Pteronarcus 0.06% Trichoptera Hydropsychidae Cheumatopsyche 18.58% Trichoptera Hydropsychidae Hydropsyche 8.83% Trichoptera Hydroptilidae -- 0.04% Trichoptera Leptoceridae Ceraclea 0.02% Trichoptera Leptoceridae Mystacides 0.04% Trichoptera Leptoceridae Oecetis 0.14% Trichoptera Philopotamidae Chimarra 0.14% Trichoptera Polycentropidae Neureclipsis 0.02% Trichoptera Polycentropodidae Polycentropus 0.04% Trichoptera Polycentropodidae -- Trichoptera Psychomyiidae Psychomyia 0.06% Turbellaria -- -- 0.64%

Taxon Order Family Genus Percentage Amphipoda Gammaridae Gammarus 2.28% Coleoptera Elmidae Stenelmis 8.51% Coleoptera Elmidae Optioservus 4.10% Coleoptera Elmidae Dubiraphia 0.46% Coleoptera Hydrophilidae Hydrophilus 0.08% Coleoptera Psephenidae Psephenus 1.29% Coleoptera Scritidae -- 0.08% Diptera Ceratopogonidae -- 0.15% Diptera Chironomidae -- 33.43% Diptera Simuliidae Simulium 8.81% Diptera Tipulidae Hexatoma 0.08% Ephemeroptera Ameletidae -- 0.15% Ephemeroptera Baetidae -- 0.15% Ephemeroptera Caenidae Caenis 0.46% Ephemeroptera Ephemerellidae Ephemerella 1.75% Ephemeroptera Ephemerellidae Euylophella 0.08% Ephemeroptera Ephemerellidae Serratella 0.23% Ephemeroptera Ephemeridae Ephemera 0.30% Ephemeroptera Heptageniidae Stenacron 0.08% Ephemeroptera Heptageniidae Maccaffertium 1.14% Ephemeroptera Leptophlebiidae Leptophlebia 0.08% Ephemeroptera Isonychiidae Isonychia 0.23% Ephemeroptera Leptohyphidae Tricorythodes 0.08% Ephemeroptera Siphlonuridae -- 0.00% Hemiptera Belostomatidae Lethocerus 0.08% Hirudinea -- -- 0.08% Odonata Calopterygidae Calopteryx 0.08% Odonata Coenagrionidae Argia 1.29% Odonata Cordulegastridae Cordulegaster 0.08% Odonata Gomphidae Gomphus 0.15% Oligochaeta -- -- 7.67% Plecoptera Chloroperlidae -- 0.08% Plecoptera Choloroperlidae Utaperla 0.15% Plecoptera Nemouridae Amphinemura 0.30% Plecoptera Perlidae Attaneuria 0.38% Plecoptera Perlidae Perlesta 0.38% Plecoptera Perlodidae -- 0.08% Plecoptera Pteronarcyidae Pteronarcys 0.15% Trichoptera Brachycentridae Brachycentrus 0.15% Trichoptera Helicopsychidae Helicopsyche 0.08% Trichoptera Hydropsychidae Cheumatopsyche 22.87% Trichoptera Hydropsychidae Hydropsyche 1.44% Trichoptera Limnephilidae Grammotalius 0.08% Trichoptera Philopotamidae Chimarra 0.30% Trichoptera Polycentropidae Neureclipsis 0.15%

25 Table 3-4: Complete benthic macroinvertebrate collection from French Creek in late summer broken down by taxa collected and associated percentages. If specimens could not be identified to genera, their order or family was included with the percentage. Kicknet Sample Taxon Order Family Genus Percentage Amphipoda Gammaridae Gammarus 1.64% Coleoptera Elmidae -- 2.96% Coleoptera Elmidae Ancyronyx 0.02% Coleoptera Elmidae Dubiraphia 0.52% Coleoptera Elmidae Macronychus 0.06% Coleoptera Elmidae Optioservus 1.56% Coleoptera Elmidae Stenelmis 5.51% Coleoptera Gyrinidae Dineutus 0.10% Coleoptera Gyrinidae Gyrinus 0.07% Coleoptera Psephenidae Psephenus 0.63% Diptera Anthericidae Antherix 1.89% Diptera Chironomidae -- 4.77% Diptera Empididae -- 0.02% Diptera Simuliidae Simulim 6.23% Diptera Tipluidae Antocha 0.04% Diptera Tipulidae Tipula 0.15% Diptera -- -- 0.06% Ephemeroptera Baetidae -- 2.36% Ephemeroptera Baetidae Centroptilum 0.52% Ephemeroptera Caenidae Caenis 0.55% Ephemeroptera Ephemerellidae Ephemerella 0.18% Ephemeroptera Ephemerellidae Serratella 0.82% Ephemeroptera Ephemeridae Ephemera 0.07% Ephemeroptera Heptageniidae -- 0.22% Ephemeroptera Heptageniidae Stenacron 0.08% Ephemeroptera Heptageniidae Maccaffertium 7.94% Ephemeroptera Leptophlebiidae Leptophlebia 0.01% Ephemeroptera Leptohyphidae Tricorythodes 4.83% Ephemeroptera Isonychiidae Isonychia 1.88% Ephemeroptera Polymitarcyidae Ephoron 0.67% Ephemeroptera Siphlonuridae Ameletus 0.04% Ephemeroptera Siphlonuridae Siphlonurus 0.08% Ephemeroptera -- -- 0.44% Hirudinea -- -- 0.02% Isopoda Asellidae Caecidotea 0.01% Megaloptera Corydalidae Corydalus 0.06% Megaloptera Corydalidae Nigronia 0.04% Megaloptera Sialidae Sialis 0.11% Odonata Calopterygidae Hetaerina 0.01% Odonata Coenagrionidae Argia 0.11% Odonata Gomphidae Gomphus 0.01% Odonata Gomphidae Lanthus 0.04% Odonata Gomphidae Ophiogomphus 0.04% Odonata -- -- 0.02% Oligochaeta -- -- 0.84% Planaria -- -- 0.07% Plecoptera Perlidae Agnetina 0.04% Plecoptera Perlidae Bleneuria 0.01% Plecoptera Pteronarcyidae Pteronarcys 0.30% Trichoptera Brachycentridae Brachycentrus 0.18% Trichoptera Brachycentridae Bracycentrus 0.06% Trichoptera Brachycentridae Micrasema 0.01% Trichoptera Helicopsychidae Helicopsyche 0.05% Trichoptera Hydropsychidae Cheumatopsyche 37.77% Trichoptera Hydropsychidae Hydropsyche 12.83% Trichoptera Hydropsychidae Macrostenum 0.01% Trichoptera Hydroptilidae -- 0.05% Trichoptera Leptoceridae Ceraclea 0.01% Trichoptera Leptoceridae Oecetis 0.02% Trichoptera Philopotamidae Chimarra 0.15% Trichoptera Polycentropodidae Neureclipsis 0.10% Trichoptera Polycentropodidae Nyctiophlax 0.05% Trichoptera Psychomyiidae Psychomyia 0.02% Turbellaria -- -- 0.02%

Electivity by Species

The following analysis of Ivlev's Electivity Index are broken down by species of darter.

Etheostoma blenniodes

At Woodcock Creek in early summer, results show that 94.7% of the stomach contents were chironomids (order Diptera; family Chironomidae). The electivity index value was0.3, indicating chironomids were consumed at a higher percentage compared to their abundance in the stream (Table 6). After chironomids, simuliid larvae comprisedthe next abundantdiet composition in E. blennioides (1.4%) with an electivity of 0.3. At this location and time, hydopsychidcaddisflies (order Trichoptera) were not an important food item (electivity value = -

0.9).

In late summer at Woodcock Creek, results show that, again, chironomids comprised the majority of the stomach contents (73.1%) with an electivity value of 0.3. Although 11.9% of the stomach contents were simuliids (order Diptera), the proportion found in the environment is higher during this sampling period than in the stomach contents (electivity value = -0.3). Again, hydropsychid caddisflies were not an important food item during this time (electivity value = -

0.4) (Table 4-2).

In French Creek during early summer, chironomids comprised the majority of stomach contents (95.2%) with an electivity value of 0.5. Simuliids comprised the other 4.4% with an electivity value of -0.3 (Table 4-3).

In late summer at French Creek, the majority of stomach contents was chironomids

(82%). The diet composition of chironomids was higher in the diet than was available in the

27 stream samples (electivity value = 0.9). They were also eating simuliids (electivity value = 0.1) while Heptageniidaemayflies and Hydropsychidae caddisflies were consumed at lower proportions than was available in the stream (electivity values = -1.0 and -0.8 respectively).

(Table 4-4).

In historic collections, the diet ofEtheostoma blenniodesconsisted of 82% chironomids followed by caddisflies of the genus Hydropsyche and other trichopterans that could only be identified to the taxonomic level of order (Table 4-9).

Etheostoma zonale

Etheostoma zonale collected in early summer at Woodcock Creek had 95% of their stomach contents identified as chironomids with an electivity value of 0.3. Simuliids comprised of 3.6% of the diet composition (electivity value = 0.6) and hydropsychid caddisflies were consumed at a much lower proportion compared to what was available in the stream (electivity value = -1.0) (Table 4-5).

Etheostoma zonale that were collected in late summer at Woodcock Creek again had chironomids as the most abundant stomach composition with an electivity of 0.4. While they had simuliids in their stomachs, they still consuming them at a lower proportion compared to what is available in the stream (electivity value = -0.2). Hydropsychid caddiflies were, again, consumed at a lower proportion than what was available in the stream (electivity value = -0.9)

(Table 4-6).

At French Creek, fish collected in early summer had a diet mainly comprised of chironomids (90%; electivity value = 0.5) and simuliids (7.5%; electivity value = 0.1). Results, again, indicate low consumption of heptageniid mayflies and hydropsychid caddisflies (electivity value: -0.7 and -0.9)(Table 4-7).

28 Stomach contents from historic collections indicated Etheostoma zonale primarily consumed chironomids (73%) followed by Simuliidae (23%) and Ephemeroptera (2.0%) (Table

4-9).

Etheostoma caeruleum

Etheostoma caeruleum collected in early summer at Woodcock Creek had stomach contents of 91% chironomids, with electivity of 0.3. One other genera of dipterans was found in the stomach contents of E. caeruleum. The crane fly,Antocha, had an electivity of 0.3. simuliid larvae constituted 4.3% with electivity of 0.7. This species also consumed mayflies of the family

Baetidae, with electivity of 0.7, and also had some Hydracaridae(Order: Trombidiformes)in the stomach contents, although none were found in the kicknet samples of macroinvertebrates in the stream. Again, this darter species consumed hydropsychid caddisflies in a small proportion compared to their availability in the stream (electivity value = -0.9) as well as Gammarus

(Gammaridae) with an electivity of -0.7 (Table 4-5).

In late summer at Woodcock Creek, E. caeruleum collected ate very few elmids with an electivity value of -1.0. Few simuliids larvae were eaten (electivity value = -0.8) while chironomids and baetid mayflies were consumed at higher proportions (electivity value =0.4). In the order Trichoptera, they were eating very few hyrdopsychids (electivity value = -0.8 ), but did have hyptropilids in their stomach with an electivity of 0.8.Polycentropodids with an electivity of

0.4 and psychomyiids with an electivity of 0.7 were also present (Table 4-6).

Etheostoma caeruleum were not collected in early summer at French Creek, but those collected in late summer (n = 3) consumed low proportions of elmids (riffle beetles) (electivity value = -0.4). They were eating chironomids, which constituted 18% of their diet (electivity value = 0.5), and they did eat other dipterans that were not identified as simuliids or

29 chironomids. They also consumed hydropsychid caddisflies (31.8%) with electivity of -

0.2. Unfortunately, 38% of individuals in the stomach contents were too digested for useful identification (Table 4-8).

Etheostoma caeruleumcollected in the main stem of French Creekfrom museum specimens mostly ate chironomids (83%) followed by caddisflies from the genus

Cheumatospycheand individuals from the order Ephemeroptera (mayflies) (Table 4-9).

Etheostoma flabellare

The stomach contents of Etheostoma flabellare collected in early summer at Woodcock

Creek, consisted of 78% chironomids with electivity of 0.2 and 17.5% simuliids with electivity of

0.9. Ephemeropterans were consumed at lower proportions compared to what was available in the stream with an electivity value of -0.9. They also consumed trichopterans, namely the hydropsychids (electivity value = -0.9) and some plecopterans (-0.6) (Table 4-5).

From E. flabellare collected in late summer at Woodcock Creek, the majority of the diet consisted of chironomids at 80% with an electivity value of 0.4. Results indicated that simuliids were consumed less than what was available in the stream (electivity value = -0.2) and heptageniidmayflies (-0.2). During this time, hydropsychids (electivity value = -0.9) were consumed at lower proportions (Table 4-6).

In the two years of sampling, Etheostoma flabellare were never collected at the site in

French Creek, Venango. The sample size of E. flabellare from historic collections was low (n =

2), but each fish’s diet consisted entirely of ephemeropterans followed by hydropsychidcaddisflies (Table 4-9).

30 Etheostoma nigrum

From E. nigrum collected in early summer at Woodcock Creek, only 1 individual was found during both field seasons. In this individual, 78.% of stomach contents was chironomids with an electivity value of 0.2. This species also consumed simuliid larvae (17.5%) with electivity of 0.9. Only a small percentage of ephemeropterans were found in the stomach contents with electivity value of -0.9. This individual also consumed hydropsychid caddisflies

(electivity value = -0.9) and plecopterans (electivity value = -0.6) at low proportions compared to what was available in the stream (Table 4-6).

This species was not collected in the site at French Creek.

Etheostoma variatum

Etheostoma variatum collected in French Creek during early summer indicated chironomids were a major portion of their diet at 43% (electivity value = 0.1), simuliids made up

50% of the diet (electivity = 0.7). They also ate heptageniids (electivity value = 0) and other families of ephemeropterans including Leptohyphidae and Siphlonuridae, while avoiding hydropsychid caddisflies (electivity value = -0.8) (Table 4-7).

In late summer at French Creek, chironomids were a substantial part of diet (27%; electivity value = 0.7) while the majority was hydropsychids at 52% with an electivity value of

0.0. This has been the only species to consume hydropsychids at proportions higher than found in the stream, even though they consumed less of them in earlier months (Table 4-8).

Historic data indicated E. variatum mainly consumed chironomids (83%) followed by simuliids (7%) and individuals from the order Plecoptera (5%) (Table 4-9).

31 Percina macrocephala

Two individuals of P. macrocephala were collected in early summer at Woodcock Creek.

Their diet consisted of chironomids and simuliids (electivity value = 0.2 and 1.0 respectively)

(Table 4-5).

In French Creek during early summer, P. macrocephala was collected, however,the sample size was low (n = 2). These individuals consumed freshwater shrimp in the genus

Gammarus at 41.7% (electivity value = 0.9), followed by chironomids (electivity value =-0.3 ), simuliids (electivity value = 0.0) and ephemerid mayflies (electivity value = 0.9) (Table 4-7).

From historic collections, P. macrocpehala had a diet that consisted of mainly chironomids at 67% followed by heptageniids and other mayflies that could only be identified to the taxonomic level oforder (Table 4-9).

Percina caprodes

No individuals were collected in early summer at either Woodcock Creek or French

Creek. In Woodcock Creek during late summer diet consisted of three main taxa, chironomids at

42.6% (electivity value = 0.1), simuliids at 13.8% (electivity value = -0.3) and hydrosychids

(electivity value = -0.2) (Table 4-6).

At French Creek in late summer a wide variety of food items including freshwater shrimp

(Gammarus)(electivity value = 0.1), chironomids (17.1%; electivity value = 0.6) and simuliids

(11.4%; electivity value = 0.3). They had a large proportion of caenidmayflies(electivity of 0.9).

They also ate heptageniids (electivity value = 0.1) and some plecopterans (2.1%; electivity value

= 1.0). Lastly, they consumed hydropsychids at 24.3% and with an electivity of -0.4. Of all the

32 fish species collected, P. caprodes are the only individuals that consumed individuals in the order

Megaloptera (Table 4-8).

Historic collections of Percina caprodes ate a variety of food items, including chironomids (18%) and hydropsychids (18%). Unfortunately, many of the stomach contents were unidentifiable due to high degree of digestion (44%) (Table 4-9).

Percina maculata, Etheostoma camurum and Etheostoma tippecanoe

The last three species are currently listed as threatened or endangered federally or in the state of Pennsylvania and include Percina maculata, Etheostoma camurum and Etheostoma tippecanoe which are found throughout the mainstem of French Creek and also at the study location at Venango in Crawford County, PA. The results from these diet compositions were from historic collections of these species, before they were listed as threatened or endangered.

The diet of Percina maculata consisted of chironomids (71%) followed by heptageniidmayflies(11%) and other specimens that could only be identified as ephemeropterans

(5%). Etheostoma camurum ate 85% chironomids, followed by trichopterans (5%). Lastly,

Etheostoma tippencanoe ate 94% chironomids followed by hydropsychid caddisflies (3.5%)

(Table 4-9).

E. E. E. E. E. E. A Ind. B Ind. C Ind. D Ind. E Ind. F Ind.

Taxon n = 24 n = 85 n = 20 n = 58 n = 1 n = 1 Order Family Coleoptera Chrysomelidae 0.0% 0.2% 1.0 ------Diptera Chironomidae 47.3% 94.7% 0.3 95.7% 0.3 91.1% 0.3 78.5% 0.2 100.0% 0.4 50.0% 0.2 Diptera Simuliidae 0.8% 1.4% 0.3 3.6% -- 4.3% 0.7 17.5% 0.9 -- -- 50.0% 1.0 Diptera Tipulidae 0.4% ------0.2% -0.3 ------Ephemeroptera Baetidae 0.1% ------0.4% 0.7 ------Ephemeroptera Baetidae Ephemeroptera Caenidae 4.2% -- -- 0.0% -1.0 ------Ephemeroptera Heptageniidae 1.1% ------0.1% -0.9 ------Ephemeroptera -- 1.6% 0.1% -0.9 0.2% -5.6 -- -- 0.3% -0.8 ------Oligochaeta -- 0.3% -- -- 0.0% -0.8 ------Plecoptera Chloroperlidae 0.2% ------0.1% -0.6 ------Plecoptera -- 0.1% ------0.3% 0.7 ------Plecoptera Pteronarcyidae 0.3% -- -- 0.0% -0.8 ------Trichoptera Hydropsychidae 13.2% 0.1% -1.0 0.3% -1.0 1.1% -0.9 1.0% -0.9 ------Trichoptera -- 0.3% -- -- 0.1% -0.3 0.1% -0.4 0.4% 0.0 ------Unidentified -- 0.0% 2.2% ------1.4% ------Hydracarina -- 0.0% ------2.3% ------Molluska -- 0.0% 0.1% -- 0.0% ------Terrestrial -- 0.0% 0.5% ------

Coleoptera Elmidae 5.4% ------0.1% -1.0 ------

Diptera Chironomidae 35.4% 73.1% 0.3 80.5% 0.4 92.0% 0.4 80.5% 0.4 42.6% 0.1 Diptera Empididae 0.5% ------0.4% -0.1 ------Diptera Simuliidae 23.4% 11.9% -0.3 15.2% -0.2 2.1% -0.8 15.2% -0.2 13.8% -0.3 Diptera -- -- 0.2% 0.0 ------0.2% ------Ephemeroptera Baetidae 0.1% ------0.3% 0.4 ------Ephemeroptera Baetidae Ephemeroptera Heptageniidae 1.0% ------0.2% -0.6 -- -- Ephemeroptera Heptageniidae Ephemeroptera -- 0.3% 0.2% ------0.7% -- Trichoptera Hydropsychidae 27.4% 12.8% -0.4 1.5% -1.0 3.3% -0.8 2.2% -0.9 19.3% -0.2 Trichoptera Hydroptilidae 0.0% ------0.3% 0.8 ------Trichoptera Polycentropodidae 0.0% ------0.1% 0.4 ------Trichoptera Polycentropodidae Trichoptera Psychomyiidae 0.1% ------0.3% 0.7 ------Trichoptera -- -- 1.5% -- 0.5% ------0.5% ------Turbellaria -- 0.6% ------Unidentified -- -- 0.2% -- 1.9% ------1.9% -- 19.1% -- Hydracarina ------0.9% ------Terrestrial ------4.5% --

Table 3-7: Electivity Index for darters collected at French Creek in early summer. Letters denote fish species while E. Ind. represents the electivity index value, ranging from -1 to +1. A = Etheostoma blenniodes; B = Etheostoma zonale; F = Percina macrocephala; H = Etheostoma variatum. Only benthic macroinvertebrates found in the stomach contents are shown in this table and used in the index. The index was calculated at the family level for benthic macroinvertebrates and could not be calculated if contents in the stomach were not collected in the stream. E. E. E. E. A Ind. B Ind. F Ind. H Ind. n = n = Taxon n = 59 63 n = 2 57 Order Family Amphipoda Gammaridae 2.3% -- -- 0.1% -0.9 41.7% 0.9 0.3% -0.8 Coleoptera Psephenidae 1.3% ------0.1% -0.9 Diptera Chironomidae 33.4% 95.2% 0.5 90.2% 0.5 16.7% -0.3 43.1% 0.1 Diptera Simuliidae 8.8% 4.4% -0.3 7.5% -0.1 8.0% 0.0 49.8% 0.7 Ephemeroptera Ephemeridae 0.3% ------8.0% 0.9 -- -- Ephemeroptera Heptageniidae 1.1% 0.1% -0.9 0.4% -0.5 -- -- 1.2% 0.0 Ephemeroptera Tricorythodidae 0.1% ------0.1% -0.1 Ephemeroptera Siphlonuridae 0.0% ------0.1% 1.0 Ephemeroptera -- -- 0.0% -- 0.1% ------1.2% -- Oligochaeta -- 7.7% -- -- 0.1% -1.0 ------Plecoptera Perlodidae 0.1% -- -- 0.1% 0.0 ------Plecoptera Pteronarcyidae 0.2% ------8.0% 1.0 -- -- Plecoptera ------16.7% -- -- Trichoptera Hydropsychidae 24.3% -- -- 0.4% -1.0 -- -- 2.5% -0.8 Trichoptera -- 0.0% 0.0% ------1.0% -- Unidentified -- -- 0.1% -- 1.2% ------Fish Eggs ------0.8% --

E. E. E. E. E. A Ind. B Ind. C Ind. G Ind. H Ind. Taxon n = 15 n = 86 n = 3 n = 5 n = 9 Order Family Amphipoda Gammaridae 1.6% ------2.1% 0.1 -- -- Coleoptera Elmidae 3.0% ------4.5% 0.2 ------Diptera Chironomidae 4.8% 82.4% 0.9 79.1% -0.1 13.6% 0.5 17.1% 0.6 27.8% 0.7 Diptera Simuliidae 6.2% 7.5% 0.1 5.7% 0.0 -- -- 11.4% 0.3 -- -- Diptera -- 0.1% ------18.2% ------Ephemeroptera Caenidae 0.5% ------14.2% 0.9 -- -- Ephemeroptera Heptageniidae 8.3% 0.2% -1.0 1.6% -0.7 -- -- 9.3% 0.1 -- -- Ephemeroptera -- 0.4% 0.3% -- 4.3% ------1.4% -- 2.8% -- Megaloptera Corydalidae 0.1% ------0.7% 0.8 -- -- Plecoptera Perlidae 0.0% ------2.1% 1.0 -- -- Trichoptera Hydropsychidae 50.6% 4.2% -0.8 3.3% -0.9 31.8% -0.2 23.6% -0.4 -- -- Trichoptera -- 0.0% 3.2% -- 1.2% ------Turbellaria -- 0.0% ------Unidentified -- -- 0.1% -- 9.3% -- 38.1% -- 30.0% -- 16.7% Terrestrial -- -- 2.0% 0.0 ------

Table 3-9: Stomach contents represented as percentages from darters collected between 1985 and 1992 in French Creek. Fish were selected for dissection from Penn State museum based on location of collection and species.Letters denote fish species:A = Etheostoma blenniodes; B = Etheostoma zonale; C = Etheostoma caeruleum; D = Etheostoma flabellare; F = Percina macrocephala; G = Percina caprodes; H = Etheostoma variatum; I = Percina maculata; J = Etheostoma camurum; K = Etheostoma tippecanoe. Electivity indices could not be calculated for these specimens because stream benthic macroinvertebrate collections were not made at the time of fish collections. Stomach Contents A B C D F G H I J K Taxon n = 18 n = 30 n = 30 n = 2 n = 16 n = 12 n = 27 n = 23 n = 9 n = 21 Order Family Genus Coleoptera Elmidae Stenelmis ------1.0% ------Diptera Chironomidae -- 86.5% 73.0% 83.0% -- 68.0% 18.0% 83.0% 71.0% 85.0% 94.0% Diptera Simuliidae -- -- 23.0% 2.0% -- 3.0% 0.3% 7.0% -- 1.0% -- Diptera ------2.0% ------Ephemeroptera Baetidae ------1.0% ------Ephemeroptera Caenidae Caenis ------1.0% ------Ephemeroptera Heptageniidae ------0.7% -- 13.0% 2.0% 0.1% 11.0% -- 0.5% Ephemeroptera Potamanthidae Anthopotamus ------1.0% -- -- Ephemeroptera Siphlonuridae Ameletus ------1.0% ------Ephemeroptera -- Macrostemum ------2.0% ------Ephemeroptera Polymitarcydae Ephoron ------1.0% ------Ephemeroptera -- -- 2.4% 2.0% 3.3% 7.0% 7.9% 1.0% -- 5.0% 2.0% 0.5% Molluska ------1.0% -- -- Plecoptera Taeniopterygidae ------1.0% ------Plecoptera ------0.7% -- 1.0% 0.3% 5.0% 1.0% -- -- Trichoptera Hydropsychidae Cheumatopsyche 0.6% 0.4% 4.0% -- -- 9.0% -- 3.0% 1.0% 3.0% Trichoptera Hydropsychidae Hydropsyche 4.3% 1.0% ------9.0% 0.1% 2.0% 2.0% 0.5% Trichoptera Hydropsychidae -- -- 1.0% 0.4% 33.0% ------Trichoptera -- Chimarra ------2.0% ------Trichoptera Polycentropodidae -- 0.6% ------Trichoptera -- -- 4.3% 0.9% 4.0% ------5.0% 1.0% Terrestrial ------1.0% 6.0% ------Unidentified -- -- 1.2% 0.2% 1.0% -- 6.0% 44.0% 2.0% 6.0% 4.0% 0.5% Fish Eggs ------0.1% ------

Diet Comparison Using Principle Component Analysis and ANOVA

There were differences in diet among species at Woodcock Creek in early summer (p =

0.001 at alpha = 0. 0125). While there are no statistical differences between Etheostoma blenniodes and E. zonale collected at Woodcock Creek in the early summer (Figure 3-1A), based on PCA results Etheostoma zonale has the more specialized diet, while the diet of E. blenniodes and E. zonale overlaps with E. blenniodes, which are eating more of a variety of food items compared to E. zonale. There is a difference in diet among E. caeruleum and E. flabellare (p =

0.01) and between E. caeruleum and E. zonale (p = 0.0001). Woodcock Creek in early summer compared to late summer shows very little different in diet within a species (Figure 3-1A; Figure

3-2A).

Results show a significant difference among dietary items in fishes collected (p = <0.001 at alpha = 0.01) at Woodcock Creek in late summer based on principle component 1 (PC1). PC1

Tukey test results show that that P. caprodes had a different diet than E. caeruleum(p <0.001 ), E. blenniodes (p <0.001 ), E. zonale (p <0.001 ) and E. flabellare (p <0.001 ). While E. caeruleumwere eating different prey items based on PCA results alone, there was no significant differences. Figure 3A also illustrates considerable diet overlap among E. blenniodes, E. zonale and E. flabellare. Principle components 2 through 5 showed no significant differences among diet items consumed (p values >0.01).

At French Creek in early summer, only three species could be collected for analysis due to conservation statuses (Figure 3-1B) and there were differences among diet in fishes (p <0.001 and alpha = 0.0125). Principle component 1 show differences between E. variatum and E. blenniodes( p< 0.001), E. zonale and E. blenniodes (p = 0.007) and E. zonale to E. variatum (p

39 <0.001). Principle component 2 also showed a statistical difference among prey items (p <

0.001), specifically between E. zonale and E. blenniodes (p < 0.001) and E. zonale to E. variatum

(p < 0.001).Etheostoma blenniodeswas consuming mainly dipteran larvae, compared to E. variatum, while E. zonale shows a generalized diet that overlaps with prey items from the other two species. Results from French Creek in late summer indicatediet partitioning was occurring among P. caprodesandE. zonale (p = 0.002). Etheostoma variatum and E. zonale have more specialized diets (Figure 3-2B). In terms of diet shifts in different seasons, the diet of E. variatumchanges from eating a variety of prey items in early summer, to becoming specialized in late summer. Etheostoma blenniodes shifts from a more specialized diet in early summer, to generalized diet in late summer. The diet of Etheostoma zonale remains similar during both seasons. To understand a clearer picture of how each species diet is changing by site and by time,

PCA results are also broken down by individual fish species.

Etheostoma blenniodes diet changed (p < 0.001 at alpha = 0.0125) from French Creek in early summer as a specialized diet, moving towards a extremely diversified diet in late summer (p

= 0.007). At Woodcock Creek, their diet also differedconsiderably from early summer to late summer (p < 0.001). It is also important to note there is a different in diet between sites (WC late summer to FC early summer p < 0.001), where again, French Creek individuals tend to have a more generalized diet (Figure 3-3). Historically, from the individuals stomach contents used in this study, they had a low diversity in prey items identified in the stomach contents (Figure 3-4).

There was a difference between historic collections to French Creek late summer for PC3 (p =

0.007), and PC4 showed a difference between Woodcock Creek late summer to historic collections (p < 0.001).

The diet of E. zonale changes considerably between French Creek early summer and

French Creek late summer, moving from a specialized diet to more generalized. French Creek early summer diet is also different than prey items that are eaten at Woodcock Creek during either

40 early or late summer. Lastly, the diet of E. zonalein late summer is generalized, compared to diets at Woodcock Creek (Figure 3-5).Figure 3-6 includes the diet of E. zonale from each season at both locations and also includes historic collections. French Creek late summer shows a very different diet compared to early summer and both seasons at Woodcock Creek. In late summer, their diet is less specialized and includes different prey items compared to early summer and

Woodcock Creek. Historically, there are statistical differences ( p< 0.0001 at alpha - 0.125) among PC1 with differences specifically between French Creek early summer to historic collections (p < 0.0001), Woodcock Creek early summer to historic ( p< 0.0001) and Woodcock

Creek late summer to historic (p < 0.0001).

Etheostoma caeruleumhad a visual difference in PCA results between Woodcock Creek in early and late summer, where diet shifted from a narrow range of prey items in early summer to a more generalized diet in late summer (Figure 3-7). Specimens collected historically had a difference in diet items compared to Woodcock Creek late summer (Figure 3-8; p = 0.003).

French Creek was not included in this analysis because the sample size was too low to give a proper representation of the species diet.

Percina caprodes was only located at each site in late summer. There is a distinct different in prey items from Woodcock Creek to French Creek (Figure 3-9; p = 0.008 at alpha =

0.025). Historically, this species diet to that of French Creek but tended to have a narrower range of food items. Historic diet was considerably different than that of Woodcock Creek specimens

(Figure 3-10).

Figure 3-5:PCA scores plot showing the relationship between diet contents in Etheostoma zonale in terms of PC 1 (x axis) versus PC 2 (y axis) from both Woodcock and French Creek and early to late summer. Seasons and sites were plotted and color coded, diet was broken into the taxonomic level of Order.

Chapter 4

Discussion

Observations of competition among syntopic species are necessary to understand resource utilization and partitioning (Grossman et al., 1998; Smart and Gee, 1979). The comparison of a location where as many as 13 species co-existed in a single riffle with one of low diversity (i.e. 5 spp) in the same sub drainage (French Creek, Allegheny River) was unique. Many of these fishes had to be immediately released because they were listed as either threatened or endangered. Historic collections of darters made in French Creek gaveus the opportunity to study diets from otherwise restricted fishes to current fishes that are not under protection and both were utilized in this study.

Rather than competing for resources when living in closely related habitats, such as a single riffle, Gause's principle (1934) states that closely related coexisting species with similar niche requirements partition their resources rather than competing. Speciation in darters has also been extensive (Page, 1972), and the French Creek watershed holds 16 species that live in very similar habitats and have very similar energy requirements. For darters living in the French Creek watershed, Gause's principlewas supported with regard to dietary shifts to partition certain food items in areas with high interspecific competition with low.

Competitive interactions among darter species are of great interest in French Creek not only because there are so many species living together, but because darters are the primary consumers of benthic macroinvertebrates in mid-order streams (Small, 1975; Martin, 1984). In

Small's (1975) study, he noted that the only groups of fishes feeding on aquatic, benthic macroinvertebrates weredarters, sculpin and minnows of the genus Rhinichthys. During his collections, the latter two groups were not present indicating a probable large prey base on which

52 darters were able to feed. My collections also resulted in few sculpin and Rhinichthys species, supporting Small’s (1975) study and indicating darters collected probably had a large resource base from which to choose prey. It is also worth noting, that smaller fish maybe limited on food types while larger individuals have greater possibility of prey items/choice (Turner, 1921).

VanSnik Gray et al. (1997) also support that juvenile darters had fed on a narrow range of taxa than adult darters in the Allegheny River system. For example, this study demonstrated that E. flabellare selected prey size of 3 -4 mm while other darter species consumed items that were more abundant, while prey greater than 5 mm was generally avoided by Etheostomaspecies.

During this same study, in July, most darters consumed diet items that were 1-2mm in length with a few exceptions. In addition, prey items 3-4mm wereavoided by all species except E. variatum, which fed opportunistically on this size class (van Snik Gray et al., 1997).

Literature supports that species, in general, tend to decrease interspecific competition by minimizing overlap on limited resources (Grossman et al., 1998; Grossman et al., 1982). In addition, coexistence with high resource overlap, including prey, is possible within fish communities because of environmental variation which can reduce species abundance to levels below resource limitations, or shifts in resource use can occur to increase competitive advantages among species (Grossman et al., 1982; Grossman et al., 1998; van Snik Gray et al., 1997). Diets of trophic competitors, such as darters, which occupy benthic habitats, are expected to converge when prey is abundant and to diverge in times of low food availability (Zaret and Rand,1971;

Fisher and Pearson, 1987).Grossman et al. (1998) also suggested that habitat is not a limiting resource within a study site because of the general variability within the area as well as behavior, morphological, and physiological capabilities of assemblage constituents. Although resource overlap does not directly indicate competition (Colwell and Futuyma, 1971), it is a good estimate of a shared resource use (Adams, 1980). Even if species are utilizing the same habitat, mouth morphology, gill rakers, as well as pecking capabilities can be responsible for the consumption of

53 specific prey items (Smart and Gee, 1979; Antonelli et al., 1972; Schoener, 1974; Findley, 1976).

Even morphological differences beyond mouth shape and size can play a role in foraging behavior. As previously mentioned, darters are visual predators and position of the eyes on the head and retinal specializations can also be correlated with feeding habits (Tamura, 1957;Wynes and Wissing, 1982; Daugherty et al., 1976; Adamson and Wissing, 1977).). Another physical barrier in terms of foraging is the presence or absence of a swim bladder, which can alter the capability of darters to find food in certain habitats (Smart and Gee, 1979). Lack of a swim bladder in the genera Etheostomamay restrict fish to benthic habitats and therefore food items that are located on the bottom of the stream.

There were no major dietary shifts between species collected historically to currently collections made during this study. Currently, E. caeruleum are still consuming a variety of food items, including a high percentage of chironomid larvae. Historic collections of E. caeruleum were taken in April, during the spawning season, when opportunistic feeding intensity increases for females (van Snik Gray et al., 1997). Historically, they are also feeding on high percentage of chironomids, and also low percentages of other taxa. Because historic macroinvertebrate assemblages were not collected, an electivity index could not be calculated which may have shown that E. caeruleum was actually eating less chironomids that was available in the environment, indicating very little change in diet over the years.

Etheostoma blenniodes, E. variatum and E. zonale,also had a diet that was relatively unchanged from historic collections. Etheostoma blenniodeshad a diet that was relatively narrow historically to eating a wider variety of taxa, especially in French Creek. Etheostoma blenniodes was historically collected in June, and based on percentages of macroinvertebrates consumed, their diet has remained relatively unchanged with major consumption still of chironomids and

Hydropyschidae caddisflies. Etheostoma variatum was collected in January historically, which does not correspond to any of the collections made in this study. Based on percentages of taxa

54 consumed, their major food items have not changed throughout the years. Historically, E. zonale was collected in July which had a very similar diet to late summer in French Creek presently.

Percina caprodes was historically collected in June and July from French Creek. When diets are compared between late summer in French Creek to historic collections, the percentages of food items consumed from each taxa are similar. There was no available macroinvertebrate data from these sites to perform an electivity index on diet for each species to determine what proportion they are consuming compared to what was available. Based on percentages of items consumed, P. caprodes has retained a generally broad diet, and had more terrestrial insects which indicate they may have been utilizing their retained gas bladder and eating prey items from the water column during that time rather than from the bottom of the stream.

Differences in diet historically to present day could have been due to a number of factors including collection season and time of day as well as location of collections. Collections made historically were taken from various times of day, including morning and afternoon hours.Also, historic collections were made from the mainstem of French Creek, current specimens of some species, including E. caeruleumand E. flabellare, were only collected from Woodcock Creek which could have had changes in substrate and hydrologic features throughout the years, leading to alternative communities of benthic macroinvertebrates and therefore fishes. On the other hand, the main stem of French Creek's high species diversity may be due to its relatively unaltered watershed through history. While other parts of the Allegheny River drainage were under intense timber harvest as well as drilling for oil and gas, French Creek has remained comparatively intact and undisturbed.One change that did occur at the Venango site on French Creek is the removal of the historic Veterans Bridge. This bridge was constructed in 1839, closed to traffic in 2002 and removed completely in 2010 by PennDOT. After the removal of the bridge, substrate presumably changed downstream of the rock pillars that formed the foundation for the bridge which would ultimately change macroinvertebrate communities and microhabitat for darters.

55 As previously mentioned, the majority of diet items from fish specimens taken historically to current collections has remained relatively unchanged and assumptions can be made for species under conservation protection in French Creek that their diets have also stayed similar. Many of the currently listed species found in the mainstem of French Creek, and specifically at the Venango site, are proposed for delisting (Table 2-1) in 2015 which means populations are doing well in the state of Pennsylvania. Delisting also means that these species have less conservation protection and as a result there will be less policies in place for habitat preservation in the areas these species are found. Another opportunity would also arise when delisting does occur such as a diet analysis of historic to current collections of those species which were unable to be collected in this study. If diets have indeed remained the same for those species, this could be valuable tool for other listed species not only in French Creek, but other aquatic systems throughout the world to help understand community structure and diet partitioning.

Results of the diet analysis among all species of darters in both French Creek and

Woodcock Creek generally are congruent with other findings in the literature regarding major prey items (Forbes 1880; Turner, 1921; Lotrich, 1973; Hlohowskyj and White 1983; van Snik

Gray et al., 1997). Overall, species want to minimize competition and maximize resource utilization.Chironomids were a major food item that was consistent in diets of all darters collected, and were also abundant in the environment at both locations. Because of the high overlap in all species collected from both locations, chironomids are probably not a limited resource and therefore were available in excess supply for consumption. These findings are consistent with other studies, which indicated chironomids dominated lotic systems. Many times they account for half of the total number of macroinvertebrates present (Pekarsky et al., 1990;

Hlohowskyj and White, 1983). They inhabit many habitat types even including marine springs and tree holes (Merritt et al., 2008, Peckarsky et al., 1990). Chironomids have a worldwide

56 distribution, are the most widespread of insect families(Merritt et al., 2008) and are considered ecologically important group of insects often found in high numbers. They can tolerate a wide range of temperatures, pH, salinity, oxygen concentrations, water velocity, depth, altitude, latitude and other abiotic parameters (Merritt et al., 2008). Chironomids are also small, mature larvae ranging from 2 to 30mm (Merritt et al., 2008), normally benthic sprawlers or crawlers

(Coffman, 1978) that also drift for dispersal (William and Hynes, 1976)making them easy prey for many insectivorous fishes such as darters. This is true even when fishes exhibit small body sizes as adults or only eat chironomids in youth. Another Family of Dipterans, Simuliidae, or black flies, are widespread in lotic systems and were also abundant in both French Creek and

Woodcock Creek. Larvae are found exclusively in flowing freshwater and are often intolerant of pollution (Merritt et al., 2008).Additionally, van Snik Gray et al. (1997) also found simuliids were important prey items at various times, for instance, primary food item in April, but not in

July.A study conducted in the Allegheny River drainage by van Snik Gray et al. (1997) also had similar findings, reporting that aquatic macroinvertebrate larvae were primary prey items for nine species of darter which is representative to the diets of fishes in this study. van Snik Gray et al.,

1997 also noted that the common taxa ingested in 9 species of darters in the Allegheny River system were Chironomidae, Simuliidae, Hydropsychidae(Cheumatospyche, Hydropsyche, and

Macronema), Ephermeliidae, Heptageniidae, Elmidae larvae, and Hydracarina species. All of the taxon listed above were also found within stomach contents of the some or all of the darters in this study.

Darters are visual predators and therefore rely on movement to locate prey (Roberts and

Winn, 1962; Mathur, 1973; Daugherty et al., 1976), so a diet consisting entirely of aquatic insect larvae is not unusual. Also, with few exceptions, they feed exclusively on benthic organisms

(Small, 1975; Wynes and Wissing, 1982; Martin, 1984), which supports the findings in this study.Stomach content analysis from Wynes and Wissing (1982) study discovered that these

57 species were not feeding primarily on drift, probably because peak macroinvertebrate drift occurs at night (Walters, 1965) when darters are not actively hunting. This indicates overlap in diet items for the seasons sampled (May to September) and suggested that there was shared resource used (Hlohowskyj and White, 1983).

Etheostomazonale and E. blenniodeswere collected at both locations and had a relatively narrow diet compared to other species, consisting primarily of chironomids during all seasons and both locations. When comparing only E. blenniodesto E. zonale, the diet of E. zonale was narrower and contained almost entirely dipteran larvae with very few other taxa. Cordes and Page

(1980) found similar results with regard to food items in E. zonale. This study found that E. zonale fed mostly on chironomids, and during a 24 hour sampling period the lowest proportion of chironomids was found at 0200, when their diet consisted of higher proportions of ephemeropterans and trichopterans. Adamson and Wissing (1977) also found that the diet of E. zonale mostly consisted of chironomids (63%) and simuliids (26%) and, again, related it to the diel activities of prey (Keast and Welsh, 1968). The fishes in this study were collected mainly in the morning hours, which would constitute why chironomids, and not nocturnal aquatic insect larvae, comprised such a large portion of the diet. Also, as previously mentioned, both chironomids and simuliids were abundant in the kicknet stream samples which could indicate this particular food item is not a limited resource, easy to prey upon and found in many microhabitats.

Hlohowskyj and White (1983) supported diet composition from findings in E. blenniodes and noted that the subfamily Orthocladiinae (Family: Chironomidae) was the most important food item for E. blenniodes during all seasons, even when they occurred at low proportions in the stream. In this same study, E. blenniodes also consumed Simulium larvae, which were not abundant in the environment. This study also mentioned that other food items were important, but never appeared at such high proportions as orthoclads(Hlohowskyj and White, 1983). Wynes and Wissing (1982) also found that E. blenniodesfed heavily on chironomids during May and

58 June (in 1978) and that this species consumed more individual food items than either E. caeruleumor E. zonale. Other studies have reported that in early stages of life, this species eat mayflies and midge larvae in both small and large fish specimens (Turner, 1921). Etheostoma blenniodespasses over stage of fry very quickly and begins eating food characteristic of juveniles by 20mm and reaches maturity by 25mm (Turner, 1921). There are noticeable changes in diet during seasons at Woodcock Creek, where diet in the Spring was narrow and moves towards a diet consisting of Trichopterans, primarily from the Family Hydropsychidae. Hydropsychids, in general, are retreat-making caddisflies that construct nets and cling to rocks in fast moving water

(Grenier, 1949).They are different from case-makers because they are found in stream currents, which bring food to their fixed refuge (Merritt et al., 2008).The behavior of these caddisflies and habitat and energy requirements explains why they played such a large role in the kicknet samples from both locations as well as stomach contents in many of the darters collected.French

Creek in late summer again produced a diet of E. blennioidesthat was broad and includes more variety in taxa consumed.

Habitat studies have shown that E. blenniodes are often found in microhabitats with large substrate filled with aquatic plants which are used as foraging sites for preying upon aquatic insect larvae and for spawning habitat (Fahy, 1954; Winn, 1958b). Physical body characteristics may aid this species in foraging in large substrate types covered in algae such as a subterminal mouth, which allows for foraging on surface of substrates (Hlohowskyj and Wissing, 1986).

Etheostoma blenniodes are also one of the largest darter species in French Creek, and body size alone can restrict foraging to only very large crevices and surfaces of substrate (Hlohowskyj and

Wissing, 1986) (Figure 5-1). Although this species is large for the genera of Etheostoma, it’s small, blunt, subterminal mouth may prevent it from consuming larger prey items relatively to body size (Page, 1983).

Figure 4-1. Photograph illustrating terminal mouth of Etheostoma flabellare collected from Woodcock Creek.

Etheostoma flabellare was not collected in the main stem of French Creek. Studies have indicated that this species inhabits cobble substrate (Schlosser and Toth, 1984) which corresponds perfectly with its body shape, flexible and narrow, well adapted to crevice microhabitats (Chipps et al., 1994). The mainstem of French Creek not only had more species of darters, but the substrate was also mostly small gravel substrate, particularly in the swift riffles, and could account for the lack of E. flabellarecollected. A unique feature of E. flabellare is the position of a terminal mouth where the lower jaw is projecting outwards, perfectly designed to peck organisms from the sides and undersides of stones (Page and Swofford, 1984) (Figure 5-2).

Other studies have suggested that this morphology seems correlated with habit of taking large and active prey (Turner, 1921).

Etheostomaflabellare overlapped in diet with other species in Woodcock Creek mainly due to their consumption of chironomids. Other food items played a small role in the overall

60 composition of the diet, although some large food items (such as plecopterans and ephemeropterans) were eaten at low proportions. Turner (1921) also noted that E. flabellare consumed prey items that were large compared to fish body size and when compared with E. variatumand E. caeruleum, they have the highest degree of specialization in diet. Literature indicates that Etheostoma variatum is typically found under rocks (Welsh and Perry, 1998) and consumes a variety of taxa (typically 6-10 taxa) (van Snik Gray et al., 1997) but commonly have high proportions of chironomid and ephemeropteran larvae (Turner, 1921).

Hlohowskyj and White (1983) discovered changes inE.flabellare diet compared to earlier counts. Where Adamson and Wissing (1977) found caddisfly larvae to be particularly important,

Hlohowskyj and White's (1983) study found that caddisfly larvae were seasonally important, chironomid larvae and stonefly larvae in winter were consumed more in northeastern Ohio. This study also found strong seasonal trends and that in winter, for instance, Plecoptera became more important prey. Zaret and Rand (1971) observed a dietary shift to larger prey by E. flabellare in the presence of the redline darter E. rufilineatum. In this study, we were unable to compare diet overlap in areas with high competition (French Creek) versus low competition (Woodcock Creek) because this species was not collected in French Creek and historic collections had a low sample size of individuals with identifiable stomach contents.

Figure 4-2.Photograph illustrating terminal mouth of Etheostoma flabellare collected from Woodcock Creek.

Etheostomacaeruleumwere often found in similar habitats as E. flabellare, namely in fast moving riffles. Although the mainstem of French Creek has swift and under normal flow conditions, shallow riffles, very few were actually collected; however, E. caeruleum in

Woodcock Creek was always collected in similar habitats as E. flabellare. Studies have shown that both species food is similar after adulthood, primarily mayflies and midge larvae in the stomachs after fish reach 15mm in length (Turner, 1921). In this study, E. caeruleum had a variable diet, even consuming water mites and riffle beetles (Order Coleoptera) at times. This was the only species that consumed beetles in any of the collections during any season. Riffle beetles, both larvae and adults, were common in the kicknet samples yet not consumed in high proportion, or any proportion, of the darters collected. Tzilkowski and Stauffer (2004) also noticed this when analyzing diets of madtoms, another small benthic fish, and noted that riffle beetles may be abundant, yet unpalatable for madtoms or unavailable at the microhabitat scale.

Like madtoms, darters may also have trouble successfully consuming riffle beetles, whether

62 larvae or adult, based on physical characteristics or defensive mechanisms such as cryptic coloration, speed, hardened bodies, spines and claws and even distasteful chemicals (Merritt et al., 2008). In addition, many remain buried in the sediment or rock crevices during daylight hours, when darters are primarily feeding.

Other literature has shown chironomids to be an important food items in all seasons for E. caeruleum and little differences in diet when looking at non-chironomid prey items, in particular regard to ephemeropterans and plecopterans (Hlohowskyj and White, 1983). Wynes and Wissing

(1982) found that E. caeruleumfed entirely on benthic organisms including chironomids and hyrdopsychid caddisflies (Hydropsyche and Cheumatospyche), the Baetis mayflies and Simulium larvae. There is great variety among literature regarding chironomids as important prey items in

E. caeruleum, some regarding midge larvae important and representing large portions of the diet

(Hlowhowskyj and White, 1983) while other studies found them to be less important (Adamson and Wissing, 1977; Nemecek, 1980; Stewart, 1988). Wynes and Wissing (1982) did notice a variation in diet from April to August and were not closely linked to abundance variation in prey organisms. This study showed that in July and August, increased numbers of caddisflies and mayflies were consumed, even though they comprised small portion of the total diet (Wynes and

Wissing, 1982). They have an ability to take prey of various sizes and therefore, larger biomass, may contribute to why E. caeruleum does so well in riffles, whereas E. zonale and E. blenniodes consume smaller prey (E. zonale consuming smallest prey) (Wynes and Wissing, 1982).

Foraging strategies for E. caeruleum include consumption of prey items on the surface of substrates, unlike E. flabellare (Hlohowskyj and Wissing, 1986; Schlosser and Toth, 1984). This species has a subterminal, horizontal mouth that is specially designed for foraging on the surface of substrate (Page, 1983), and it's larger scales, deeper body design and rigidity when compared to E. flabellare inhibits individuals to forage between crevices in large substrate (Schlosser and

Toth, 1984; Hlohowskyj and Wissing, 1986). Hlohowskyj and Wissing (1986) have also

63 supported this theory, finding E. caeruleum occupying the surface of large substrate, while E. zonale were concealed in crevices.

It is not surprising that my results indicated P. caprodeshad a broader diet than species from the genera Etheostoma. Their habitat is more diverse, therefore they can consume prey items that are more diverse behaviorally and taxonomically (Smart and Gee, 1979). Diets also varied considerably between sites and historically. Fish collected from French Creek, again, had the broadest diet compared to Woodcock Creek and historic collections. Other studies reported these differences (Adamson and Wissing, 1977; Smart and Gee, 1979; Turner, 1921).One notable difference between the genera Percinaand Etheostomawas that Percinacaprodes, specifically, had a unique feeding strategy that involved stone-rolling and consuming macros beneath rocks

(Welsh and Perry, 1998; J. Stauffer personal communication, 2013).Turner (1921) noted that P. caprodes ate crustaceans in early stages of life and their diet became more complicated in terms of food items as they got bigger. He also noted that this species most nearly meets the specifications of being a generalist in terms of diet and that the period of maturity is marked by omnivorous habit. Turner (1921) documented the stage of youth at 25mm and that as fry, P. caprodeswere surface feeders, then used a benthic feeding mode where and large debris was accidentally ingested. Because P. caprodeswere capable of adapting to a variety of habitats they developed a more generalized diet, which was again, represented in this study.

In French Creek, especially in late summer, it appears that all species were feeding opportunistically, which is supported by Adamson and Wissing (1977), van Snik Gray et al.

(1997)and Martin (1984). French Creek at late summer also had the highest abundance of benthic macroinvertebrates collected from kicknet samples as well as family level richness. The mainstem of French Creek, at Venango, was home to 13 species of darters, each acting as benthic insectivores in the overall fish community.Wynes and Wissing (1982) support the findings in this study and found high overlap values between E. caeruleum and E. zonale; between E. zonaleand

64 E. blenniodes; and E. caeruleumand E. blenniodes. The more generalized diet and lower overlap in food items may be a result of the high abundance of aquatic macroinvertebrate assemblages, which would reduce diet partitioning among darters (Martin, 1984; Schlosser and Toth, 1984).

Literature supports that darters diet changed seasonally as benthic macroinvertebrate abundances changed (Brassche and Smith, 1967; Page and Burr, 1976; Wynes and Wissing, 1982).During certain times of the year, as macroinvertebrate communities change, both interspecific and intraspecific competition may increase.

Darters are dependent on the amount and variety of food with the seasons in which prey items grow and hatch (Turner, 1921). Overall, this study suggested that diet changed over time for the species that were collected both historically and presently.

Cost and benefit analysis of prey items is lacking in the literature with regard to prey selection of darters. Gillette (2012) suggested that darter prey selection was taxon-specific and not based on prey size. Other authors have suggested that predator diet breadth is a function of the abundance of profitable prey and that when profitable prey items are abundant, diet breadth was low; diet breath was higher when low profit prey items were abundant and consumed

(Charnov, 1976; Turner, 1982; Gillette, 2012).Chironomids may be both abundant, small for easy consumption, and energetically beneficial which explains why all species are consuming them in higher proportions than found in the stream kicknet samples. Etheostomacaeruleum and P. caprodes, whose diet varies, may be pushed towards eating macroinvertebrate taxon that are unpalatable toother species or living within microhabitats that are inaccessible to these species.

Gillette (2012) also commented that the assumption that larger prey items had higher energetic gains is a simplification, and that bioenergetics of small benthic fishes, such as darters, is poorly understood (Gatz, 1983). Diets among sympatric darter species may also be related to habitat segregation which has been observed in other fish species (Zaret and Rand, 1971; van Snik Gray et al., 1997).

65 Most of the darters in the French Creek watershed spawn between April and June, which can also account for differences in diets between species. Although sex was not analyzed in specimens from this study, female and male darters tend to occupy different habitats during the spawning seasons and therefore may have access to different food items in terms of species richness and abundance (Orth and Maughan, 1983). In addition, males from many species (i.e. E. blenniodes, E. variatum, E. zonale) defend territories in riffles while females inhabit runs or pools before mate selection begins (Winn, 1958). Females may also be consuming more prey items before spawning (van Snik Gray et al., 1997).

As previously mentioned, mouth morphology may also play a large role in aiding to explain diet variation between sympatric species (Wood and Bain, 1995; van Snik Gray et al.,

1997; Matthews et al., 1982). What fishes eat is morphologically related to the structure of the jaw, dentition, as well as prey-body size relationships (Gosline, 1987; Ross, 2013).Foraging behavior based on mouth morphology can influence microhabitat selection. For instance,

E.caeruleumand E. zonaleoccurs synopticallybetween rocks. Etheostoma zonale have been found foraging along the sides of rocks and literature suggests that its subterminal mouth may contribute to this feeding strategy (Kessler et al., 1995; Welsh and Perry, 1998). Welsh and Perry (1998) also found that E. blenniodeswere typically found on top of rocks; that E. caeruleumused significantly larger substrate than E. zonale in riffles from the Birch River while

Percinacaprodesutilize a stone-rolling techniques and consuming macros beneath rocks (Welsh and Perry, 1998; J. Stauffer personal communication, 2013) and fed at the surface and in the water column at earlier life stages as well. Body size may also play a role, especially when adult body size varies between species. Darters with smaller body sizes (e.g. E. zonale, E. tippecanoe) tended to eat primarily larval insects while species with larger adult body size (i.e. P. caprodes,

P. macrocephala) tend to feed on larger prey items and crustaceans (Martin, 1984; Burr and Page,

1978; Cordes and Page, 1980).

66 Limitations did occur during this study due to weather. During the summer of 2014, there were many rain events that left both French Creek and Woodcock Creek at high flow and turbid conditions, which prevented collections at both locations. In addition, the site at

Woodcock Creek is below the dam and high rain events caused the dam to be released for extended periods of time even after rain. This may have changed the natural flow regime of the stream and caused changes in species richness and abundance, especially in aquatic insect larvae.Many studies have indicated that variability, particularly in water velocity, changed fish assemblages (Poff and Allan, 1995; Grossman et al., 1998; Facey and Grossman, 1992). Floods and droughts may play a strong role in the organization in stream biota (Poff and Allan, 1995;

Grossman et al., 1982). High flow, especially, can have negative effects on fishes such as increased energy expenditure and decreased foraging success (Facey and Grossman, 1992; Hill and Grossman, 1993). There was also a natural seasonal cycle in fauna and flora.Alternatively, low flow conditions will also change behavior, especially in fishes, when they will respond to low water by moving to alternative habitats (Hlohowskyj and Wissing, 1986). Below dams, habitat changes including siltation and sedimentation which causes embeddeness between substrate can take place and inhibit foraging (Poff et al., 1997).Literature supports that water depth and flow are not direct measures of darter habitat, but have been shown to affect foraging (Flore et al.,

2000; Gillette, 2012). This could also affect their ability or behavior to find food items if water remains turbid for periods of time (Daugherty et al., 1976).

While microhabitat partitioning was not examined in the present study, it is worth noting that these variables inevitably play a role in lessening competitive interactions between syntopic species of darter (Hlohowskyj and Wissing, 1986). One factor alone, but a combination of abiotic parameters including water velocity, depth and substrate type, most likely account for microhabitat differences between species (Pratt and Laurer, 2013) as well as morphological differences between fishes. Darters, especially those in the genus Etheostoma, are valuable

67 research specimens to help understand complex community interactions in lotic systems. French

Creek has historically remained relatively unaltered throughout history,although some non- point source pollution has inevitably occurred. With changes in habitat, darter communities can also change (Lau et al., 2006), especially when microhabitats change to eliminate cover objects, foraging and spawning sites (Pratt and Lauer, 2013). Deemed one of America's "last great places" by the Nature Conservancy, French Creek remains one of the few streams in the United States that holds such a high abundance and diversity of fish species, including many under conservation protection. It remains an important refuge for species in the upper Allegheny River, both terrestrial and aquatic. With undeniable threats to the biodiversity in French Creek, including invasive species (i.e. round gobies), global climate change, riparian zone alterations and non-point source pollution, it is important to investigate aquaticcommunity structures and ensure preservation of one of North America's last aquatic treasures.

Summary of Major Findings

 Dietary trends for historic to current darter collections have remained similar, indicating

species under current conservation protection have a diet that has also remained relatively

unchanged.

 There were obvious seasonal trends in dietary components for darters and differences in

diet between Woodcock Creek and French Creek.

 Darters found in French Creek, especially in late summer, had a broader diet than those

from earlier seasons and from Woodcock Creek, which indicates food is probably not a

limited resource and therefore both intraspecific and interspecific competition is low.

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VITA

Casey R. Bradshaw-Wilson

17897 State Highway 198 Allegheny College Saegertown PA, 16433 520 N. Main Street Meadville, PA 16335

Education______

•The Pennsylvania State University, University Park PA Expected Graduation Date: December 2015 Wildlife and Fisheries Science; Department of Ecosystem Science and Management

•Marshall University, Huntington WV Graduated: May 2010 M.S. in Biological Sciences; concentration in Herpetology

•The Pennsylvania State University, Behrend College, Erie PA Graduated: May 2007 B.S. in Biology, concentration in Ecology

Work Experience______• 2013 – Present: Assistant Professor of Environmental Science; Allegheny College, Meadville PA • 2010 – 2013: Research Technician, Regional Science Consortium, Erie PA • 2008 – 2010: Teaching Assistant, Marshall University, Huntington WV • 2008: Summer Field Technician, SSRAA, Ketchikan AK • 2007 – 2008: Agricultural Engineering Assistant, Penn State Extension, Meadville PA

Current Research Projects______

• Status of Round Gobies in French Creek Watershed and Their Influence on Native Benthic Fishes • Determining the ability of Round and Tubenose Gobies to move into Lotic Systems from Lake Erie

Professional Organizations and Current Committees______• Member: ASIH (American Society of Ichthyologists and Herpetologists) • Vice President: NWPWA (Northwestern PA Woodland Association) • Vice President: RSC (Regional Science Consortium)