Diet Analysis and Community Characteristics of Lepomis (Sunfish) Species Congregating Near Natural Spring Vents of the Rainbow R

Diet analysis and community characteristics of Lepomis (sunfish) species

congregating near natural spring vents of the Rainbow River, Florida

Michael S. Sears

A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science Department of Environmental Science, Policy, & Geography College of Arts and Sciences University of South Florida St. Petersburg

Major Professor: James Gore, Ph.D. Randy Edwards, Ph.D. Melanie Riedinger-Whitmore, Ph.D.

Date of Approval: November 1, 2010

Keywords: natural spring vents, Lepomis , stomach content, spring-fed river, diversity, electroshocking, stomach pump

Table of Contents

List of Tables ...... ii

List of Figures ...... iii

Abstract ...... iv

Introduction ...... 1

Materials & Methods ...... 12 Study Site ...... 12 Sampling Methods ...... 17 Electroshocking Sampling Methods ...... 18 Hook and Line Sampling Methods ...... 22 Plankton Net Sampling Methods ...... 24 Statistical Analysis ...... 27

Results ...... 29 Fish Populations ...... 29 Stomach Content Analysis ...... 32 Plankton Net Sampling Results ...... 36

Discussion ...... 37

List of References ...... 44

Appendix ...... 51

List of Tables

Table 1. Upstream and downstream Rainbow River pH data from 2004 and 2005 ...... 17

Table 2. Mean monthly discharge (cfs) from the Rainbow River during sampling events ...... 17

Table 3. Upstream and downstream Fish species composition percentages ...... 29

Table 4. Upstream fish species diversity measurements ...... 30

Table 5. Downstream fish species diversity measurements ...... 31

Table 6. Upstream and downstream Lepomis species length results ...... 31

Table 7. Upstream Lepomis species stomach contents ...... 33

Table 8. Downstream Lepomis species stomach contents ...... 34

Table 9. Upstream Lepomis species stomach content diversity ...... 36

Table 10. Downstream Lepomis species stomach content diversity ...... 36

List of Figures

Figure 1. Rainbow River Watershed Land Use Map of 1944 ...... 3

Figure 2. Rainbow River Watershed Land Use Map of 1999 ...... 4

Figure 3. Spring vent locations within the Rainbow River ...... 12

Figure 4. Rainbow River water quality sampling site ...... 15

Figure 5. Upstream and downstream Rainbow River temperature data between 2006 and 2009 ...... 16

Figure 6. Upstream and downstream Rainbow River dissolved oxygen data between 2006 and 2009 ...... 16

Figure 7. Upstream electroshocking sampling locations ...... 19

Figure 8. Downstream electroshocking sampling locations ...... 20

Figure 9. Hook and line sampling locations ...... 22

Figure 10. Upstream plankton net spring vent and tow sampling locations ...... 25

Figure 11. A Lepomis species at a sample site ...... 26

Figure 12. View of the plankton net setup over a spring vent ...... 26

Figure 13. Up-close view of a spring vent/boil ...... 28

iii

Abstract

Little is known about the ecology of natural springs and the influence spring vents have on fish populations. This study explores fish population characteristics, such as abundance, diversity, and length distributions near headwater spring vents and downstream away from the spring vents within the Rainbow River, located in central Florida. The population characteristics examined are useful to resource managers when evaluating the possible impacts on fish populations from increased groundwater withdrawals and subsequent lower discharge rates into large spring-fed rivers. Initial field observations led to the hypothesis that Lepomis species feed on organisms expelled from the spring vents. Electrofishing and hook and line sampling were utilized to collect fish specimens for analysis, and

Lepomis species were frozen for later stomach analysis, or stomach contents were pumped in the field. Spotted sunfish ( Lepomis punctatus ) were dominant upstream (28.8% composition), while bluegills ( Lepomis macrochirus) were most abundant downstream (38.0% composition). Upstream populations of Lepomis species had significantly larger individuals (P < 0.05) than Lepomis species sampled downstream. Lepomis species stomach analysis revealed Hyalella azteca as the most abundant food source upstream, and chironomids as the most abundant prey taxa downstream. Upstream Lepomis species stomach analysis yielded an average of 19.05 organisms identified per individual fish, while 4.01

organisms per fish were observed downstream. These differences in Lepomis species populations and their diets indicate that future groundwater withdrawals could impact these stable state spring environments, and have substantial impacts on the ecology of the Rainbow River.

Introduction

Natural freshwater springs are unique ecosystems that provide essential habitat for many species of flora and fauna (Odum 1957; Hubbs 1995; Walsh

2001). These habitats are vulnerable to flow alterations and habitat loss from increased demand for groundwater withdrawals by residential and agricultural land use. This demand continues to increase on aquifers that sustain natural springs, like the Rainbow River, in central Florida. Over the last several decades, much of the Rainbow River watershed and recharge area were converted from forested lands to agricultural and residential land uses (Figures 1 and 2). As of 2004, the population of the area has increased by 37.5 percent since ten years prior

(SWFWMD 2004).

The increasing development of the area and demand on water poses a threat to the relatively constant water flows, water temperatures, and water quality that makes the Rainbow River and other spring habitats unique natural resources

(Odum 1957; Hubbs 1995; Berndt et al . 1998; Barquin and Scarsbrook 2008).

Spring-fed rivers are unique for providing a annually stable habitat for many species, including resident endemic species (Hubbs 2001), and seasonal species seeking thermal refuge in extreme cold or warm conditions (Patterson 1996).

Future impacts from groundwater withdrawals on spring-fed rivers would have negative effects on these unique ecosystems by decreasing flows from springs and possibly altering fish and macroinvertebrate communities (Barquin and

Scarsbrook 2008).

Initial visual site observations from a boat, with confirmation by preliminary electrofishing, indicated that fishes of the family Centrarchidae are abundant and likely dominant in the Rainbow River system, including bluegill ( Lepomis macrochirus ), spotted sunfish ( Lepomis punctatus ), and largemouth bass

(Micropterus salmoides ). During these field visits, Lepomis species were observed congregating around the spring vents at high frequencies for reasons unknown. It is expected that decreased flow from the spring vents might alter this unique habitat and its fish populations.

Figure 1. Rainbow River watershed land use map of 1944 (SWFWMD 2004).

Figure 2. Rainbow River watershed land use map of 1999 (SWFWMD 2004). 4

Lepomis species, such as bluegill are characterized as generalist feeders

(Werner et al. 1981), and select prey according to size (Werner and Hall 1974;

O’Brien et al. 1976). As flow is concentrated directly adjacent to and downstream from individual spring vents, Lepomis may utilize the area of flow to maximize

predation on drift prey expelled directly from the spring vents or sediment, much

like salmonid feeding behavior on drifting macroinvertebrates in many temperate

riverine ecosystems (Elliot 1967; Flecker 1992; Giroux et al . 2000).

Several studies suggest that invertebrates from aquifers (i.e. hypogean

fauna) travel through interstitial space to surface waters through spring vents or

stream upwelling (Notenboom et al. 1996; Ward and Palmer 1994), and through

sediments of the hyporheic zone (Palmer and Swan 2000), but little analysis has

been conducted to determine if hypogean fauna contribute to the diets of fishes

that congregate near spring vents. Hypogean fauna prefer low oxygen

environments indicative of groundwater conditions. Amphipods, such as

Salentinella , have been identified as hypogean source indicators, while Gammarus

appears to be an epigean (i.e. surface water) source indicator (Ward and Palmer

1994). Ward and Palmer (1994) also observed benthic invertebrates originating

from a stream’s hyporheic zone, near the substrate surface where groundwater

upwells into the surface waters of the stream. In addition, invertebrates have been

found in karstic spring systems in high abundance (Dumnicka et al. 2007), and have been found at their greatest abundances in springs with the highest discharges (Mattson et al. 1995; Notenboom et al. 1996; Dumnicka et al. 2007), which is similar to the karstic high flow environment of the Rainbow River.

Fish assemblage and composition in spring-fed rivers provides important information about particular functions of these ecosystems (i.e. water quality, nutrient levels, productivity, etc.). Among the few spring studies in Florida, fish assemblages from Silver Springs, Singing Springs, Homosassa Springs, and

Weekiwachee Springs have been examined (Sloan 1956; Odum et al. 1957;

McKinsey and Chapman 1998). Other studies in Texas and Oklahoma were conducted in similar karstic springs (Hubbs 2001; Bergey et al . 2008).

Centrarchids are dominant in many large karstic spring-fed rivers of Florida

and the southeast United States (Sloan 1956; Odum et al. 1957; McKinsey and

Chapman 1998). Sloan (1956) studied Homosassa and Weekiwachee Springs in

Florida, examining the distribution of aquatic insects relative to dissolved oxygen

(DO), fish assemblages near the springs. Sloan found that benthic invertebrate diversity increased as DO increased downstream, and hypothesis that spring vents with constant and predictable environments create fewer niches, and harbor low species diversities. Sloan observed smaller Centrarchid species near the spring vents compared to downstream, and hypothesized that the higher abundances of small fish may contribute to the lower abundances of aquatic insects. These small fish may have a feeding advantage on small hypogean fauna, if in fact hypogean fauna are expelled from spring vents. Lepomis species have been observed feeding on microscopic prey (zooplankton species) when the energy gain from this behavior is advantageous (Mittlebach 1981; Mittlebach and Osenberg 1994).

Odum (1957) also questioned the possibility of hypogean fauna being dispersed from spring vents as a hypothesis for high abundances of fish near

spring vents of Silver Springs, Florida, but found no evidence supporting this hypothesis. Silver Springs has similar attribute to the Rainbow River, including a constant chemical environment and temperature of approximately 23°C, with the dominant submerged aquatic vegetation consisting of eelgrass ( Vallisneria americana ) (Odum 1957). Odum estimated that Centrarchid species composed

68% of the community, with spotted sunfish representing the dominant Lepomis species (Odum et al. 1957). Periphyton, which consisted of algae and invertebrates, was found in all fish stomachs, along with high abundances of amphipods, chironomids, and tricoptera. Overall, fish were found to have moderate to low growth rates, reproduction occurring among juvenile-sized fish, and year-round reproduction of spotted sunfish due to the nearly constant environmental conditions (Odum et al. 1957).

McKinsey and Chapman (1998) found that fish diversity increased as DO increased downstream, and that greater numbers of low oxygen tolerant species

(i.e., Gambusia and Centrarchids) were observed near the spring vents. This study on Singing Springs, Florida, by McKinsey and Chapman (1998), focused upon fish diversity relative to progressing increases of DO gradients downstream.

Mosquitofish ( Gambusia spp.) were the focus of this study as species of this genus, found commonly in Signing Springs, are spring specialists while other species are generalists. Centrarchids are also common here and were observed in the study.

Hubbs (2001) analyzed Gambusia species distributions in six Texas springs, and concluded that temperature, pH, and flow stability were the most

important factors influencing fish abundance. Decreased flows decrease amphipod abundance, which were an important food source in springs (Hubbs

2001). Centrarchids (including, but not limited to, spotted sunfish, bluegill, and largemouth bass) were also commonly found congregating in springs with higher discharges. The pH, temperature, DO, and species diversity of fishes increased downstream from spring vents in these springs (Hubbs 2001).

Bergey et al. (2008) analyzed 22 springs in Oklahoma that had altered flow regimes between 1981 and 2001 to compare changes in fish abundance. Fish populations were susceptible to flow alterations due to groundwater extraction, and groundwater pollution. Endemic freshwater spring species were negatively affected, like the protected Arkansas darter ( Etheostoma cragini ). Fish biodiversity

and abundance increased in springs that had higher flows in 2001 than in 1981.

Seasonality influences relative fish sizes and diversity in spring vent areas

(Patterson 1996). In winter months, stream dwelling fish move into spring areas

for thermal and feeding refuge, and larger fish become dominant over smaller fish

as a result of competition and predation (Patterson 1996). Seasonal shifts in fish

composition and abundance have direct influences on the composition and

abundance of macroinvertebrate communities. When fish abundances increase

and species compositions shift to fish that forage on macroinvertebrates,

macroinvertebrate populations decrease, and vice versa (Aday et al . 2005).

Macroinvertebrate populations also experience seasonal shifts dependent on life

stage cycles, and are more subject to predation during certain phases, such as the

emergent phase, when morphing adults rising to the water surface frequently fall prey to fish (Boulton et al . 1992).

Fish abundance in relation to physicochemical properties in Florida’s karstic springs (Sloan 1956; Odum et al. 1957; McKinsey and Chapman 1998). Benthic macroinvertebrate sampling in groundwater influenced ecosystems have been conducted, but with minimal fish predation analysis (Palmer 1990; Glazier 1991;

Ward and Palmer 1994), and no studies fully investigate the presence of hypogean fauna contribution to fish diet in natural springs. The lack of studies related to hypogean fauna contribution to fish diet and surface environments, in general, is due to the extreme difficultly in identifying these organisms (Palmer and

Hakenkamp 2000). Many hypogean species are also habitat generalists common in surface (epigean) waters (Reid and Strayer 1994), which increases the difficultly of knowing the contribution of hypogean species to epigean systems. A few known Floridian hypogean species, such as the Hobb’s cave isopod ( Crangonyx hobbsi ) or the Florida cave shrimp ( Palaemonetes cummingi ) are listed species associated with karst communities (Mattson et al . 2005).

Spring-fed rivers have unique species, habitats, and fish population characteristics that might be important to resource managers (Walsh 2001,

Barquin and Scarsbrook 2008). Comparison of fish assemblages of the Rainbow

River to similar karstic springs may provide valuable information for fisheries management of spring systems in Florida, when conserving habitat for rare or recreationally important species. In addition, state conservationists and environmental managers would be greatly concerned if protected hypogean

species are found in the Rainbow River. Successful resource management, including strict regulations on groundwater withdrawals and water quality monitoring, is necessary for sustaining these unique habitats and their fauna

(Mattson et al . 2005; Barquin and Scarsborough 2008). The anticipated results of this study may help river and fisheries managers to better understand the ecology of spring-fed rivers, and more accurately assess possible impacts of decreased flow to fish populations and their forage base, especially if hypogean fauna are observed as a food source for Lepomis species.

Little is known about ecological processes in freshwater spring systems, and even less about fish dependence upon the habitats created by spring vents.

Invertebrate passage through the hyporheic zone may give some indication that spring vents may create a habitat where hypogean organisms can integrate into the higher vertical hyporheic zones near the streambeds. “Popcorn” vents are comprised of sediments bubbling and mixing over the vents as groundwater is discharged, which may act as a vector for hypogean organisms to enter surface waters. The phenomenon could be possible from groundwater discharges through spring vents (especially popcorn vents) of the Rainbow River, leaving these organisms highly vulnerable to predation by Lepomis species, which are generalist feeders (Werner et al . 1981), and therefore identifying spring vents as preferred fish habitat in spring-fed rivers. In this study, I test the hypothesis that spring vents are essential fishery habitat in spring-fed rivers through analysis of Lepomis species congregating around spring vents, including stomach content analysis of

Lepomis species, fish abundance, and fish diversity of species captured around

these spring vents in the Rainbow River. A secondary hypothesis tested is the possibility of hypogean fauna providing a food source for Lepomis species in this natural spring habitat.

Materials and Methods

Study Site:

The Rainbow River is located in Marion County, Florida, and is the fourth largest, first magnitude natural spring system in Florida. The river flows approximately 9.2 kilometers to the Withlacoochee River, where an average of

1.87 billion liters (493 million gallons) of water flows from the Rainbow River daily

(FGS 2001; SWFWMD 2004). The flow is primarily of spring origin from several large spring vents at the headwaters, and other smaller vents along much of the spring run until the confluence with the Withlacoochee River (Figure 3) (FDEP

2007).

Figure 3. Spring vent locations within the Rainbow River (Jones et al. 1996). 12

Submerged aquatic vegetation dominates the river bottom, comprised of strap-leaf sagittaria ( Sagittaria kurziana ) and eelgrass ( Vallisneria americana ), with sparse patches of exposed limestone and sandy substrates primarily found surrounding spring vents (SWFWMD 2004). The water quality of this river is considered excellent, but nitrate concentrations have been increasing over the past several decades, and nuisance plant species, such as Lyngbya and Hydrilla are beginning to invade higher reaches of the river to take advantage of the increased nutrient levels (Jones et al. 1996; SWFWMD 2004).

Water quality parameters important to fish habitat, such as temperature,

DO, and pH remain relatively constant throughout the river (Figures 5 and 6; Table

1). However, subtle seasonal changes in temperatures and DO occur from upstream to downstream. Figure 4 shows SWFWMD’s continuous water quality sampling locations, from which upstream samples (sample locations RR1, RR2, and RR3) and downstream samples (sample locations RR7, RR8, and RR9) were used to construct Figures 5 and 6, as these areas represent the sampling locations used within this study (see Figures 7 and 8). Figures 5 and 6 display nearly constant temperature and DO levels near the upstream spring vents over four years, but more variable downstream data that correlates with the changes of the four seasons between 2006 and 2009.

Specifically, summer downstream temperatures were higher than upstream temperatures, but downstream temperatures were lower than upstream spring vents in winter months. Upstream temperatures remained relatively constant throughout each year, as fluctuations were within 0.5 C °. Constant DO levels were

also observed upstream, while the downstream levels were more variable, and predominately higher than upstream DO levels throughout each year.

Upstream and downstream, pH levels were relatively constant throughout each year (Table 1). Specific conductivity ranges between 150-300 uS/cm throughout the entire river, gradually increasing when descending downstream and away from the main headwater spring vents (SWFWMD 2010). Depths range between 2m and 5m throughout the river, but spring vents reach greater depths up to 12m (SWFWMD 2010). The mean discharge from 1975 to present is 15.99 cms

(cubic meters per second), ranging between 14.75 and 24.35 cms (USGS 2010).

Figure 4. Rainbow River water quality sampling sites (SWFWMD 2010).

Rainbow River - Up & Downstream Temperatures 2006-2010

24.50

24.00 US-2006 23.50 US-2007 US-2008 23.00 US-2009 DS-2006 22.50 DS-2007 DS-2008 22.00 DS-2009

21.50

21.00 Spring Summer Fall Winter Season

*US measurements from RR1, RR2, and RR2 and DS measurements from RR 7, RR8, and RR9 as seen in Figure 4.

Figure 5. Upstream (US) and downstream (DS) Rainbow River temperature data between 2006 and 2009.

Rainbow River - Up & Downstream Dissolved Oxygen 2006-2009

10.50 US-2006 9.50 US-2007 US-2008 8.50 US-2009 DS-2006 7.50 DS-2007 DS-2008 6.50 DS-2009

5.50 Spring Summer Fall Winter Season

*US measurements from RR1, RR2, and RR2 and DS measurements from RR 7, RR8, and RR9 as seen in Figure 4.

Figure 6. Upstream (US) and downstream (DS) Rainbow River dissolved oxygen data between 2006 and 2009.

Table 1. Upstream (US) and downstream (DS) Rainbow River pH data from 2004 and 2005. 2004 US DS

Spring 7.54 7.81

Summer 7.80 8.05

Fall 7.55 7.78

Winter 7.72 7.73

2005 US DS

Spring 8.09 8.30

Summer 7.95 8.11

Fall 8.06 7.96

Winter 7.87 7.54

*US measurements from RR1, RR2, and RR2 and DS measurements from RR 7, RR8, and RR9 as seen in Figure 4.

Table 2. Mean monthly discharge (cfs) from the Rainbow River during sampling events (USGS 2010). Sept Oct 2008 745.1 715.7 2009 582.7 -

Sampling Methods:

Electroshocking, hook-and-line sampling, plankton tows, and Lepomis species stomach analysis were utilized to reveal possible differences in fish population characteristics, including Lepomis diets near upstream spring vents and downstream near the confluence of the Withlacoochee River.

Electroshocking Sampling Methods:

Fish samples were collected by electroshocking (Cowx 1983) from a 12-foot aluminum Jon-boat. Electroshocking took place near the upstream spring vents in

September and October of 2008. Downstream electroshocking was conducted in

October of 2008. Lepomis seasonality and the associated seasonal sampling was not a concern in this study, as Lepomis are not migratory species (Scott and

Crossman 1973), and the relatively constant environment of the springs does not trigger a need for migration as adequate conditions for Lepomis foraging and spawning exist throughout the entire Rainbow River and throughout each year. All samples were collected during mid-day hours. Electroshocking was conducted in water depths of approximately one to three meters along the banks and around spring vents. The localized electrical field did not sufficiently affect fish at greater depths for successful capture. Fish temporarily stunned by the electrical currents were collected with nets from the Jon-boat.

Electroshocking sample sites were located in the upstream portions of the river around the headwater spring vents, and downstream near the confluence with the Withlacoochee River (Figures 7 and 8). Downstream sample sites where located far downstream and near the confluence of the Withlacoochee River, and served as control sample sets for comparison of fish population characteristics and

Lepomis species diet to the upstream population. Upstream sampling locations were conducted near springs at and near the headwaters. Four upstream, and four downstream electroshocking sampling trials were conducted (Figures 7 and

8). Upstream trials 2 and 4, and downstream trials 2, 3, and 4 included sampling

on both sides of the banks. All electrofishing samples were done in 30-minute intervals.

1 2

Figure 7 – Upstream electroshocking sampling locations.

1 2 3

Figure 8 – Downstream electroshocking sampling locations.

The number of samples was determined from data collected during

preliminary sampling events (September 2008) that indicated at least 34 fish were

needed to accurately represent the population with a 90 percent confidence level

where:

Population = µµµ(0.1) ±±± τττ (1- ααα/2) (s/ √√√n)

and µ = 6.5, τ (1- α/2) = 2, s = 1.89, and 0.1 represents 90% confidence

(n = 33.7 samples needed).

All Lepomis species captured by electrofishing were placed in sealed and labeled plastic bags, placed in a cooler with ice, and frozen within five hours. This approach is similar to another Lepomis diet study that successfully extracted identifiable macroinvertebrates from Lepomis stomach contents ( Lobinske et al.

2002) . For cost-savings and to save time in the field, fish were preserved by freezing rather with formalin or ethanol. Fish other than Lepomis species were

captured during electroshocking were identified, and length and body depth

measurements were recorded. Unidentified fish specimens were bagged and

frozen for later identification. Notes on substrate type, vegetation, water depths,

sampling conditions, and location were recorded in the field.

Frozen Lepomis species were thawed in the laboratory. Stomachs were

removed and placed within vials containing 70 percent ethanol after fish were

identified and measured. Stomachs were then placed within Petri dishes and the

contents were extracted. A compound and dissecting microscope were used to

tally macroinvertebrates found in each individual Lepomis stomach, and were

identified to the lowest practical taxonomic level (Epler 1995; Covich and Thorp

2001; Merritt and Cummins 2009). As there was a high probability of the

organisms being partially digested due to their small size (Hayes and Taylor 1991),

many body parts were found and several organisms were not identified.

Hook-and-Line Sampling Methods:

Fish specimens were also collected by hook-and-line sampling (Everhart et al . 1975) directly over and adjacent to spring vents (Figure 7). This alternative sampling method was utilized because electroshocking was not successful at capturing fish congregating near spring vents, due to a combination of deep water depths and the low conductivity levels. It was important to this study that fish were collected from directly over and adjacent to spring vents for stomach analysis to determine what the dominant prey species are and investigate the presence of hypogean fauna.

Figure 9 – Hook and line sampling locations.

Small pieces of worms purchased from a local bait store were attached to a size-16 fishing hook on five-pound test fishing line. Weight was attached to present the bait to fish congregating near the substrate around spring vents. Five hook and line sampling trials were conducted for 45-minutes in each location

(Figure 10). Hook-and-line samples were collected in September, October and

December of 2009. All fish caught by hook and line sampling were identified to species, measured, and released alive.

Stomach contents of all Lepomis species captured by hook-and-line sampling were collected with a stomach pump model commonly used by fly-fishing anglers interested in identifying stomach contents to mimic the organisms their target fish are eating. This type of device has also been widely used for extracting stomach content of fishes for research purposes (Seaburg and Moyle 1964;

Hakala and Johnson 2004). The specific stomach pump model used for this study has a manual suction device and a five-millimeter diameter shaft that fits down the mouth and into the stomach of captured Lepomis species. Stomach contents of

Lepomis species were preserved in the field, within small vials containing 70 percent ethanol.

Stomach pumping was used to collect stomach contents rather than

freezing the fish for laboratory processing, because this sampled was primarily

done from a small kayak. In addition, preliminary stomach pump testing revealed

that nearly the entire amount of contents within Lepomis stomachs were extracted,

and was therefore a comparable to the laboratory method extraction.

All macroinvertebrates within each Lepomis stomach were identified in the laboratory to the lowest practical taxonomic level (Epler 1995; Covich and Thorp

2001; Merritt and Cummins 2009) using dissecting and compound microscopes.

Specifically, macroinvertebrates were recorded by tallying identified species within the stomach of each Lepomis species for all sampling methods and labeling each fish in accordance with the date and location sampled. Individual macroinvertebrates were sorted into vials labeled with the individual fish number and macroinvertebrate species identified. This maintained a record of the macroinvertebrates identified in each Lepomis species individual stomach.

Plankton Net Sampling Methods:

Plankton tows were conducted to identify the prey items available for

Lepomis species near spring vents and in the surrounding water column. A total of four plankton net samples were collected in March of 2010. Plankton tow net samples were collected directly over two spring vents, in the water column near the upstream headwater spring vents, and in the water column, downstream, near the confluence with the Withlacoochee River (Figure 8). These data were collected to derive qualitative information to determine if organisms were being expelled from vents and into the water column, becoming subject to fish predation.

In addition, the composition of species captured within the plankton nets were compared to species observed in Lepomis stomach contents.

Figure 10 – Upstream plankton net spring vent and tow sampling locations.

A 90-µm mesh plankton net was used for all plankton tows and net sets over the spring vents. Water column samples were collected at approximately 1.5 meters under the water surface by slowly towing the plankton net upstream for fifteen minutes. The nets placed over the spring vents were set in place for fifteen minute. Rose bengal was added to the samples in the lab to stain mieofauna

(organisms < 1000 µm in size) in the samples for easier observation under microscopes. A compound and dissecting microscope were used to identify macroinvertebrates to the lowest practical taxonomic level in the laboratory (Epler

1995; Covich and Thorp 2001; Merritt and Cummins 2009).

Figure 11 – A Lepomis species at a sample site.

Figure 12 – View of the plankton net setup over a spring vent.

Statistical Analysis:

Fish length measurements were recorded to document size variations of fish in downstream and upstream (near the spring vents) habitats. Body depth measurements were collected to verify that species captured around spring vents were less than the 152-millimeter (0.6 feet) to meet fish passage criteria mandated by the SWFWMD. According to SWFWMD, fish observed utilizing the springs that have heights greater than 152-millimeter would not utilize the habitat if future groundwater withdrawals resulted in drastic decreases of water depths (SWFWMD

2004).

Species compositions of sampled fish were measured for upstream and downstream population segments to identify dominant species in each zone.

Species diversity was measured by using the Shannon-Weaver Index (Shannon and Weaver 1949), the Simpson Diversity Index, species richness, and species evenness (Simpson 1949) for upstream and downstream populations of fishes, and macroinvertebrate populations found in Lepomis species stomach contents.

Catch-per-unit-effort (CPUE) was calculated for electroshocking and hook- and-line sampling to standardize the catch rates between the two different methods for comparison (Everhart et al. 1975). Parametric t-tests (Press et al .

1992) were used to compare length and body depth data, and to determine if significant differences existed between the fish populations observed upstream near the spring vents to downstream. Specifically, t-tests were performed to compare all Lepomis species lengths, and compare bluegill and spotted sunfish

lengths between upstream and downstream populations with 95% confidence limits.

Figure 13 – Up-close view of a spring vent/boil, or “pop-corn” spring with fine sandy substrate bubbling over the vent and limestone surrounding the vent opening.

Results

Fish Populations:

Electroshocking and hook-and-line sampling captured a total of 163 fishes, and 120 (73.6%) were Lepomis species. Variations in species compositions were

observed between upstream and downstream fish populations (Table 1). Fishes in

the Centrarchidae family were dominant throughout the entire river, and Lepomis

species were dominant in both sampling regions. Bluegill (Lepomis macrochirus)

were the dominant species found downstream (48.1% composition), while spotted

sunfish (Lepomis punctatus) were the dominant species collected upstream near

the headwater spring vents (52.7% composition). Electroshocking yielded higher

CPUE values than hook and line sampling upstream near the spring vents

(Appendix A – Table A1), but this method did not capture fish congregating directly

over the spring vents.

Table 3. Upstream and downstream Fish species composition percentages. Species Compositions (%) Species Upstream Downstream Spotted sunfish ( Lepomis punctatus ) 52.7 16.7 Bluegill ( Lepomis macrochirus ) 18.2 48.1 Longear sunfish ( Lepomis megalotis ) 7.3 0.9 Redbreasted sunfish ( Lepomis auritus ) 1.8 0.9 Warmouth ( Lepomis gulosus ) 0.0 3.7 Largemouh bass ( Micropterus salmoides ) 14.5 24.1 Golden shiner ( Notemigonus crysoleucas ) 0.0 2.8 Bowfin ( Amia calva ) 0.0 2.8 Common carp ( Cyprinus carpio ) 1.8 0.0 Seminole killifish ( Fundulus seminolis ) 1.8 0.0 Gizzard shad ( Dorosoma cepedianum ) 1.8 0.0 *Full data set available in Appendix A – Tables A1 and A2.

Florida gar ( Lepisosteus platyrhincus ) were visually observed from the sampling boat in the upstream areas but were not captured by any of the sampling methods described in this study. Gizzard shad ( Dorosoma petenense ) and

Seminole killifish ( Fundulus seminolis ) were also visually observed upstream in greater numbers than revealed in the sampling data, as only one individual of each species were captured. Both of these species were observed near the spring vents, and gizzard shad were only observed during sampling events that took place in September 2008.

Fish diversity indices between the upstream spring vents and downstream showed slightly different results (Tables 3 and 4). The Shannon-Weaver Diversity

Index was slightly higher for the upstream fish populations than downstream.

Species richness was identical for both habitats sampled, but different species were present within each habitat (Tables 3 and 4). Species evenness for both zones was also nearly identical, with a value of 6.78 upstream and 6.74 downstream. The Simpson Diversity Index indicated that there is a 33.9 (D x 100) percent chance that two randomly selected individuals are of the same species in the upstream zone, while there is a 32.1 (D x 100) percent chance in the downstream zone. These results show a fair diversity for each habitat, but slightly more diversity downstream (Tables 3 and 4).

Table 4. Upstream fish species diversity measurements. Shannon-Weaver Species Simpson Total Diversity Index Richness Diversity Index Evenness Abundance 1.410 8 D: 0.339 0.678 55

Table 5. Downstream fish species diversity measurements. Shannon-Weaver Species Simpson Total Diversity Index Richness Diversity Index Evenness Abundance 1.401 8 D: 0.321 0.674 108

Paired T-tests were used to analyze differences in the two populations of

Lepomis species upstream and downstream (Table 5). The length data showed a

statistically significant difference (P < 0.05) between the upstream and

downstream Lepomis species populations, as the upstream population individuals

were significantly larger in size than the Lepomis individuals in the downstream

population (Table 5).

Table 6. Upstream and downstream Lepomis species length results. Mean Standard Unpaired T- (mm) Deviation (mm) test (P value) Upstream 129.74 24.56 Lepomis Lengths 0.048 Downstream 112.61 27.89 Spotted Sunfish Upstream 115.33 20.46 0.298 Lengths Downstream 107.63 26.27 Upstream 133.40 28.66 Bluegill Lengths 0.053 Downstream 112.41 1.049 *Full data available in Appendix A – Tables A1 and A2

Bluegill populations and spotted sunfish populations were compared fto

analyze differences between species, as they were the most dominant Lepomis species observed. Unpaired t-tests indicated that lengths of spotted sunfish and bluegill were not significantly different (P > 0.05) (Table 5). However, upstream bluegill populations were significantly larger in size than downstream bluegill at a

90 percent confidence level (P < 0.10).

Lepomis species depth measurements were all below the 152-millimeter

SWFWMD mandated fish passage criteria. However several largemouth bass exceeded 152-millimeter in body depth. Only the two largest bluegill captured exceeded 76-millimeters (or 3 inches) of body depth.

Stomach Content Analysis:

Although there was a larger sample of fish taken from downstream, a greater number (> 4:1) of macroinvertebrates were identified in the stomach contents from the smaller upstream fish sample set (Tables 6 and 7). Only 44

Lepomis species stomachs were analyzed upstream, compared to the 72 Lepomis species stomachs analyzed downstream. A total of 724 organisms were identified in upstream Lepomis , while 305 organisms were identified in downstream Lepomis stomachs. An average of 19.05 organisms per fish upstream and 4.01 per fish downstream were found within stomach contents. As there was a high probability of the organisms being partially digested due to their small size (Hayes and Taylor

1991), many body parts were found and several organisms were not identified.

Much of the downstream stomach content consisted of periphyton, which is a complex of plant and animal material masses that are found attached to submerged plants and various substrates (EPA 2010). Most of the animal material within the periphyton was not identifiable, and the amounts of periphyton within each stomach were not quantified. Although periphyton was not quantified in the upstream or downstream samples, very little periphyton contributed to upstream stomach samples. Warmouth ( Lepomis gulosus ) stomach contents were not

analyzed because warmouth were not captured upstream during the sampling events.

Table 7. Upstream Lepomis species stomach contents. Species Order Family Genus Species Totals Composition Ephemeroptera Leptohyphidae Tricorythodes albilineatus 204 28.2 Baetidae Acentrella parvula 1 0.14 Trichoptera Hydroptilidae Orthotrichia spp. 15 2.07 Leptoceridae - 1 0.14 Hydropsychidae Cheumatopsyche spp. 1 0.1 Phryganeidae - 1 0.1 Amphipoda Hyalellidae Hyalella azteca 246 34.0 Lepidoptera Pyralidae Petrophila spp. 1 0.1 Nymphalidae spp. 1 0.1 Diptera Chironomidae Subfamily Tanypodinae Ablabesmyia mallochi 1 0.1 Larsia spp. 3 0.4 Procladius spp. 1 0.1 Tanypus spp. 7 1.0 Hudsonimyia spp. 4 0.6 Chironominae Pseudochironomus spp. 50 6.9 Paratanytarsus sp. B 16 2.2 Paratanytarsus sp. D 24 3.3 Paratanytarsus dissimilis 1 0.1 Tanytarsus sp. C 7 1.0 Rheotanytarsus exiguus group 2 0.3 Rheotanytarsus sp. A 23 3.2 Dicrontondipes neomodestus 58 8.0 Cryptochironomus spp. 1 0.1 Beardius truncatus 1 0.1 Manoa spp. 8 1.1 Orthocladiinae Orthocladius dorenus 2 0.3 Orthocladius robacki 13 1.8 Orthocladius annectens 6 0.8 Thienemanniella similis 2 0.3 Stratiomyidae - - 1 0.1 Thaumaleidae - - 1 0.1 Ceratopogonidae Bezzia spp. 7 1.0 Gastropoda Amicolidae - - 4 0.6 Acariformes Hydrachnidae - - 10 1.4 Total 724 100.0 Note: Macroinvertebrates were collected from 29 Lepomis puntatus , 10 Lepomis macrochirus , 4 Lepomis megalotis , and 1 Lepomis humilis directly over and adjacent to spring vents. Full data set available in Appendix A – Table A1.

Table 8. Downstream Lepomis species stomach contents. Species Order Family Genus Species Totals Composition Ephemeroptera Leptohyphidae Tricorythodes albilineatus 50 16.4 % Baetidae Acentrella parvula 1 0.3 % Trichoptera Hydroptilidae Orthotrichia spp. 15 4.9 % Amphipoda Hyalellidae Hyalella azteca 30 9.8 % Diptera Chironomidae Subfamily Tanypodinae Hudsonimyia spp. 1 0.3 % Larsia spp. 2 0.7 % Chironominae Pseudochironomus spp. 63 20.7 % Polypedilum illinoense group 6 2.0 % Paratanytarsus sp. D 13 4.3 % Tanytarsus L 1 0.3 % Tanytarsus sp. C 1 0.3 % Rheotanytarsus sp. A 2 0.7 % Dicrontondipes neomodestus 15 4.9 % Orthocladiinae Cricotopus tricinctus 1 0.3 % Orthocladius robacki 80 26.2 % Orthocladius dubitatus 1 0.3 % Nanocladius alternanthorae 1 0.3 % Ceratopogonidae Bezzia spp. 7 2.3 % Gastropoda Amicolidae - - 4 1.3 % - - 6 2.0 % Lumbriculidae 5 1.6 % Lumbriculida (Oligochaeta) Total 305 100.0 % Note: Macroinvertebrates were collected from 18 Lepomis puntatus , 52 Lepomis macrochirus , 1 Lepomis megalot is , and 1 Lepomis humilis directly over and adjacent to spring vents. Full data set available in Appendix A – Table A2.

Macroinvertebrate species composition within Lepomis stomachs varied between upstream and downstream samples. Hyalella azteca was the dominant species observed in upstream samples (34 percent), while the mayfly species

Tricorythodes albilineatus was the next dominant at 28.2 percent (Table 6). The contribution of the remaining 32 macroinvertebrate species to the total species composition within Lepomis stomach content was much lower. However, eleven

species within the Chironominae subfamily comprised 26.3 percent of upstream fish stomach contents, making them the second most dominant taxon in upstream stomach contents.

Downstream species composition within Lepomis species stomach contents yielded two Chironomidae species as dominant. Orthocladius robacki comprised

26.2 percent and Pseudochironomus spp. comprised 20.7 percent of the downstream sample (Table 7). The next most dominant species was

Tricorythodes albilineatus at 16.4 %, and then Hyalella azteca at only 9.8 percent, which was much lower than this species’ presence upstream near the spring vents

(34 percent). The Chironomidae subfamily contributed 13 species and had the highest total downstream abundance, at 61.3 percent. No confirmed hypogean species were identified in any of the Lepomis species stomachs sampled.

Species richness of Lepomis species stomach contents was higher for the

upstream samples than downstream. However, species evenness indicated that

the downstream species found within stomach contents were more diverse than

the upstream species (Table 8 and 9).

Other diversity measures also revealed a greater diversity of macroinvertebrates within Lepomis species stomachs in downstream samples.

The Shannon-Weaver Index (upstream = 2.100; downstream = 2.195) and the

Simpson’s Diversity Index (upstream D = 0.210; downstream D = 1.57) indicate that the downstream macroinvertebrate population was slightly more diverse than upstream near the spring vents (Table 8 and 9).

Table 9. Upstream Lepomis species stomach content diversity. Shannon - Weaver Diversity Species Simpson Total Index Richness Diversity Index Evenness Abundance 2.100 34 D: 0.210 0.596 724

Table 10. Downstream Lepomis species stomach content diversity. Shannon - Weaver Species Simpson Total Diversity Index Richness Diversity Index Evenness Abundance 2.195 21 D: 0.157 0.721 305

Plankton Net Sampling Results:

The downstream plankton net tow yielded some macroinvertebrate species,

while the plankton net tow near the headwaters yielded less. One of the two

samples taken from setting a plankton net directly over a spring vent produced no

organisms, while the other captured a total of two Ostracods. Ostracods were also

found in the upstream and downstream plankton net tows, but these organisms

were not identified during stomach content analysis. A few Hyalella azteca were

only observed in the downstream plankton net tow. Several Chironominae were

captured in the plankton net tows that were not observed in the stomach content

analysis including Thienemanniella sp. C, Thienemanniella xena, Tanytarsus sp.

T, Orthocladius jacobsen, and an unidentified species of the genus Hydrabaenus .

A total of only 26 organisms were collected through plankton net sampling, most

from the downstream net tow.

Discussion

The fish composition and diversity data show differences in fish

assemblages between the upstream spring vent habitats and downstream,

especially a shift in dominant species and species sizes (lengths). This study

demonstrates that the upstream spring vent habitats are dominated by spotted

sunfish, which were found in a similar river (Silver Springs) in Florida (Odum

1957), and that upstream Lepomis species as a whole are significantly larger than

the downstream Lepomis population. Sloan (1956) found that Lepomis species

near spring were actually larger than downstream fish, but he did not observe

amphipods as abundant near the springs.

When analyzing the length data for bluegill and spotted sunfish alone in

each sampling zone, the size differences were not significant (P > 0.05). The

bluegill size comparison does have a P value of 0.053, but the upstream sample

size is only ten, compared to 52 bluegill captured downstream. It appears that

spotted sunfish prefer the upstream spring vent and sandy substrate habitat while

bluegill might prefer downstream habitat consisting of more vegetative cover

growing from the substrate and along the riverbanks. Increased vegetative cover

may also be correlated to the smaller sized Lepomis species observed

downstream, since this habitat type serves as preferred nursery habitat for bluegill

and spotted sunfish (Scott and Crossman 1973). Bluegill are generalist feeders,

and prefer larger sized prey as it becomes available (Werner et al. 1981; Ehlinger

1990), and will move from the vegetative habitat as they become larger to feed on

larger prey items (Ehlinger 1988), like the high concentrations of amphipods found near spring vents.

The differences in Lepomis species sizes might also be connected to the dominance of Hyalella azteca in the stomach contents of Lepomis species in the upstream spring vents habitat. This amphipod species might also be a high quality or preferred food source, resulting in higher growth rates and larger fish on average, as opposed to Lepomis species downstream that mainly foraged on chironomids. Another explanation for Lepomis species size being greater upstream than downstream is simply that prey is far more abundant upstream than downstream, since stomach analysis found an average of 19.05 organisms identified per fish, compared to only 4.01 organisms per fish downstream, suggesting that there are higher growth rates of fish upstream than downstream.

Sediment macroinvertebrate grab sampling has been done on the Rainbow River that found great abundances of the same species found within Lepomis stomach content analysis (James Banning, University of Tampa; Personal Communication;

2010), however upstream and downstream abundances were not derived.

Previous studies have shown bluegill to be generalists in habitat and food choice (Webb 1984; Werner and Hall 1988), but they can specialize on certain habitats where foraging is most beneficial to them (Werner et al. 1981). The results of the Rainbow River predict that bluegill prefer the more vegetated downstream habitat dominated by chironomid species, and spotted sunfish prefer the sandy bottom open water spring vent habitats dominated by amphipods.

My results support a recent study conducted on spotted sunfish that concluded this species is highly sensitive to flow reductions in Florida’s rivers.

Dutterer and Allen (2008) found that 20 to 70 percent population reductions could result from a 0.3-meter reduction in daily average water staging. The Rainbow

River and other similar spring fed systems could experience drastic impacts in spotted sunfish populations from flow reductions, which may also impair their main food source near the springs, Hyalella azteca . Spring flow losses might decrease the likelihood of amphipods and other macroinvertebrates being expelled into the water column and made available to Lepomis species and other fish.

The differences in Lepomis population characteristics between those found upstream near the spring vents and those found downstream in the main stem of the river indicates that the initial hypothesis of this study is true, since spring vent habitat is essential to Lepomis species in the Rainbow River, especially spotted sunfish. The secondary hypothesis that hypogean fauna exist in the surface waters near spring vents was not confirmed by the stomach content analysis results. However, further studies are needed to verify this conclusion.

Although I did not observe endemic hypogean species in the stomach contents analysis, the plankton net sets did capture ostracods. Ostracods have been observed in groundwater and interstitial habitats in great abundances and diversity (Reeves et al . 2007). Since two ostracods were captured in a plankton net set directly over a spring vent with considerable flow and others in the water column tows, ostracods from the groundwater or interstitial zone may be dispersed into the water column and made available to the surface water food web and fish

predators in the Rainbow River, however no ostracods were found in Lepomis stomach analysis.

It has also been found that tropical cave or karsts communities can have very few endemic hypogean species, as compared to temperate climates (Mitchell

1969). Mitchell (1969) hypothesized that this is due to the constant gene mixing between epigean and hypogean environments in tropical areas, as opposed to more genetically isolated temperate epigean and hypogean environments, limited by extreme temperature changes between zones. The tropical/subtropical environment of the Rainbow River may support this idea, indicating species like

Hyalella azteca or ostracod species freely move between the groundwater, hyporheic zone, and water column, resulting in the unlikely presence of hypogean endemic species, but indicate a dependence on spring flows to disperse these organisms from vents to fish for food.

Furthermore, spring vents and especially “pop corn” springs or boils common within the Rainbow River could aid the mixing of the sediments and organisms of the groundwater environment into the hyporheic zone, and finally into the water column to be foraged by fish. This has been observed in river systems with laterally shallow hyporheic zones and fine sediments, where flows between the zones have increased during flood events and dispersed greater numbers of organisms into the surface waters (Palmer 1990). This observation is similar to the

Rainbow River springs since the vents consist of fine sands within high flow spring vents discharging from the aquifer and hyporheic zones.

Several future studies on the Rainbow River or other similar habitats would

be useful to further support the findings of this study, and the need for additional

protections of natural spring habitats. Macroinvertebrate sampling within the

hyporheic zone of spring vents and deeper within the supporting aquifer would

show whether species are indeed moving through these zones and becoming prey

to fish congregating around spring vents. Stable isotope analysis of fish captured

around the springs and species within the subterranean zones (especially

ostracods) could also indicate whether fish are feeding on subterranean species.

Stable isotope analysis could also identify the possibility hypogean organisms

contributing to the surface water food web through epigean macroinvertebrates

feeding on small hypogean meiofauna, as previous analyses have confirmed

(Palmer and Hakenkamp 2000), but financial and logistical constraints restricted

the possibility of stable isotope analysis tests in this study.

Modifications of this study would produce additional supporting data.

Overnight plankton net sets may have been beneficial in collecting more species

dispersed from the spring vents, as specific subterranean species may move from

the springs more during twilight. Fish weight measurements may have given

useful condition factor data for more comprehensive analysis between upstream

and downstream fish populations. In addition, stomach content analysis has been

done by freezing Lepomis species before thawing them for lab processing

(Lobinske et al. 2002), but stomach content extraction in the field may have yielded better samples for identification in the lab, which effects the results of this study.

Much of the upstream stomach data were collected by stomach pumps and

preserved in the field with ethanol, while the downstream samples were put on ice in the field and frozen later the same day.

Although the preservation methods were not consistent throughout the study, both methods are useful and thawed stomach specimens were intact during lab analysis. Field preservation was a more time efficient method that enabled fish to be released alive. Studies comparing each method would be useful for future fisheries studies, especially since preserving the life of fish samples for stomach analysis studies is becoming preferred in today’s fishery sciences, especially for listed fish species (Gray et al. 2002).

The protections enacted in current public policies (i.e., SWFWMD fish

passage depth criteria) do not address impacts on the habitat requirements of the

forage base of spotted sunfish and bluegill associated with spring vents. Further

investigation and policy revisions are necessary for preserving the flows into the

Rainbow River that support abundant macroinvertebrate populations, whether they

existing within the vents or directly adjacent to them. Other studies have shown

detrimental impacts on Lepomis species (Dutterer and Allen 2008) and amphipods

(Hubbs 2001) resulting from decreased flows, indicating that the 152-millimeter

minimum flow level is not sufficient in sustaining fish populations in the Rainbow

River.

The results of the study demonstrated differences among Lepomis species

populations and their diets of macroinvertebrates near the Rainbow River

headwater spring vents and downstream near the confluence with the

Withlacoochee River. The population characteristics of these organisms near the

spring vents are important observations for resource managers to use when developing policies that impact water discharge rates from spring vents. Impacts to organisms at the base of the food chain, such as Hyalella azteca may have great impacts on spotted sunfish and bluegill populations, which also contribute to the forage base of largemouth bass, Florida gar, and other large piscivorous species that provide substantial ecological and recreational values.

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Appendix

Appendix (continued) Upstream Data Length Depth Date Method Location Fish # Species CPUE LTa BAp THo TL TC TP AHa Pp Pn S A H T CB Tam TL- TP TT TH Cp CP-B CP-D CC CPd Ct-C CReg CR-A CDn CBt CM OOd OOr OOa OTs (mm) (mm) Spotted 10/17/2009 Hook/Line Up (Area 1) 1 7 2 1 1 1 sunfish 108.0 54.1 Lgmouth 2 4.0 bass 209.8 53.3 Spotted 3 4 1 1 4 2 1 2 sunfish 116.6 44.7 Spotted Up (Area 2) 4 sunfish 114.3 39.6 Spotted 5 17 1 7 2 1 4 1 2 sunfish 134.9 44.5 Spotted 6 94.0 28.7 58 1 17 1 1 4 4 6 1 1 12 sunfish 8.0 Spotted 7 8 2 2 1 1 2 1 sunfish 114.3 38.9 Spotted 8 34 1 1 4 1 1 4 sunfish 133.4 55.9 Spotted 9 sunfish 120.9 38.9 Up (Area 3) 10 Bluegill 140.0 53.9 Spotted 11 sunfish 132.8 49.5 Spotted 12 66.0 16.0 1 sunfish 8.0 Spotted 13 sunfish 88.9 29.2 14 Bluegill 159.8 63.3 3 1 1 6 1 1 Spotted 15 sunfish 119.6 38.1 Spotted 12/4/2009 Up (Area 4) 19 30 2 1 1 1 3 2 9 1 3 1 sunfish 120.7 45.7 Spotted 20 6 1 19 1 3 2 1 sunfish 132.1 49.0 Spotted 21 4 129 sunfish 136.1 55.9 Spotted 22 101.6 43.2 sunfish 10.7 Spotted 23 sunfish 129.5 52.8 Spotted 24 16 7 1 3 1 5 2 17 7 5 3 1 sunfish 109.7 44.2 Spotted 25 2 sunfish 86.6 26.2 Spotted 26 8 6 1 1 4 sunfish 114.3 44.5 Spotted 9/6/2008 Electrofish Up (Area 1) 1 sunfish 130.3 47.8 3 Bluegill 146.3 55.9 4 Bluegill 140.7 55.1 14.0 5 Bluegill 108.0 35.3 9 1 2 1 6 Bluegill 78.7 23.4 1 Spotted 7 sunfish 95.5 35.1 Lgmouth Up (Area - 2) 9 419.1 156.2 bass 4.0 Spotted 10 sunfish 141.0 54.1 Spotted Up (Area - 3) 12 sunfish 76.2 25.4 Spotted 13 6.0 sunfish 92.2 35.1 Gizzard 14 shad 416.1 135.9 Lgmouth 10/19/2008 Electrofish Up (Area - 4) 20 bass 574.8 148.8 Lgmouth 21 bass 494.0 128.3 Lgmouth 22 bass 349.3 98.6 Lgmouth 23 bass 213.6 93.7 Lgmouth 24 bass 142.2 33.8 Lgmouth 25 bass 133.4 25.9 common 26 28.0 carp 311.2 99.1 30 Bluegill 147.6 52.8 1 6 3 1 Spotted 31 3 sunfish 138.9 50.8 32 Bluegill 110.5 43.2 13 1 2 1 6 5 Longear 33 1 2 6 1 3 2 sunfish 134.9 46.2 34 Bluegill 124.5 44.5 1 5 1 1 Spotted 35 34 10 1 sunfish 121.2 47.5 Longear 36 8 6 17 3 1 2 7 1 sunfish 123.2 38.1 Hook/Line Spotted 9/27/2009 (Area - 5) 1 (no guts) sunfish 129.8 50.8 Seminole 2 Killifish 170.4 33.0 Longear 3 sunfish 146.1 53.6 4 Bluegill 178.1 74.2 9.3 Spotted 5 sunfish 145.0 57.2 Red 6 breasted sunfish 178.6 74.4 Longear 7 sunfish 139.5 44.5 TOTALS 204 1 15 1 1 1 246 1 1 1 4 10 1 7 1 3 1 7 4 50 16 24 1 1 7 2 23 58 1 8 2 13 6 2 All Fish mean 160.62 54.69 TOTAL 724 stdv 102.60 29.92 n 55 Lepomi mean 129.74 45.21 s stdv 24.56 12.08 Percent Abundance TOTAL 100 n 44 28 0.1 2.1 0.1 0.1 0.1 34 0.1 0.1 0.1 0.6 1.4 0.1 1 0.1 0.4 0.1 1 0.6 6.9 2.2 3.3 0.1 0.1 1 0.3 3.2 8 0.1 1.1 0.3 1.8 0.8 0.3 Leptohyphidae Tricorythodes albolineatus (LTa), Baetidae Acentrella parvula (BAp), Trichoptera Hydroptilidae orthotricia (THo), Trichoptera Leptoceridae spp. (TL), Trichoptera Cheumatopsyche spp. (TC), Trichoptera Phryganeidae spp. (TP), Amphipoda Hyalella azteca (AHa), Pyralidae petrophila (Pp), Pyralidae nymphalidae (Pn), Stratiomyidae (S), Amicolidae (A), Hydrachnidinae (H), Thaumaleidae (T), Ceratopogodinae Bezzia (CB), Tanypodinae Ablabesmyia mallochi (TAm), Tanypodinae Larsia (TL-), Tanypodinae Procladius (TP), Tanypodinea Tanypus (TT), Tanypodinae Hudsonimyia (TH), Chironominae pseudochironomus spp. (Cp), Chironominae paratanytarsus sp. B (Cp-B), Chironominae Paratanytarsus sp. D (CP-D), Chironominae Cryptochironomus spp. (CC), Chironominae Paratanytarsus dissimilis (CPd), Chironominae Tanytarsus sp. C (CT-C), Appendix (continued) Downstream Data Date Method Location Fish # Species Length (mm) Depth (mm) CPUE LTa BAp THo AHa A H CB TL- TH Cp CP-D CT-L CT-C Cpig CR-A CDn OOr OOd ONa OCt O 12//200 Hook/Line Downstream 1 Spotted sunfish 126.5 45.7 1.33 10/18/2008 Electrofish Area 1 1 Bowfin 558.8 133.4 2 Bowfin 489 95.3 3 Lgmouth bass 180.3 44.2 4 Lgmouth bass 196.1 52.1 5 Lgmouth bass 189.2 39.1 6 Lgmouth bass 191.8 43.4 fish 1-1 Bluegill 109.5 38.1 fish 1-2 Bluegill 122.2 44.5 3 5 1 1 1 1 fish 1-3 Bluegill 97.8 37.6 1 2 fish 1-4 Bluegill 179.1 78.538.00 3 1 1 1 1 1 fish 1-5 Bluegill 96.5 32.3 2 1 1 30 fish 1-6 Bluegill 112.8 38.1 6 2 1 fish 1-7 Bluegill 79.5 25.4 fish 1-8 Bluegill 107.2 34.3 7 2 1 4 2 1 11 2 fish 1-9 Bluegill 76.7 27.4 1 2 fish 1-10 Bluegill 97 38.4 3 fish 1-11 Bluegill 86.4 30.7 1 fish 1-12 Bluegill 84.8 30.5 fish 1-13 Bluegill 70.4 21.6 Electrofish Area 2 8 Lgmouth bass 352.8 96.8 9 Bowfin 511 121.7 10 Lgmouth bass 230.6 51.3 11 Golden shiner 183.4 44.2 12 Lgmouth bass 231.9 50.8 13 Lgmouth bass 117.6 22.6 14 Golden shiner 112.8 24.9 15 Golden shiner 93.5 19.1 16 Lgmouth bass 133.4 25.7 17 Lgmouth bass 139.2 29 18 Lgmouth bass 107.7 23.1 19 Lgmouth bass 98.3 20.6 fish 2-14 Spotted sunfish 104.9 40.6 1 1 fish 2-15 Bluegill 92.7 38.4 2 1 1 fish 2-16 Bluegill 129.8 51.6 2 3 3 60.00 fish 2-17 Bluegill 127 45.7 fish 2-18 Bluegill 95.3 33.3 1 4 5 2 7 fish 2-19 Bluegill 117.3 29 1 fish 2-20 Spotted sunfish 104.1 38.4 6 4 2 1 1 1 1 fish 2-21 Bluegill 114.3 39.4 1 1 1 1 1 fish 2-22 Bluegill 127.5 45.2 4 1 1 1 1 fish 2-23 Bluegill 97.5 37.8 1 30 3 1 1 fish 2-24 Bluegill 80 37.3 1 3 fish 2-25 Bluegill 142.7 57.2 12 8 2 1 1 1 2 9 fish 2-26 Spotted sunfish 121.9 50.8 3 fish 2-27 Bluegill 88.1 29.2 fish 2-28 Bluegill 105.2 38.1 1 fish 2-29 Bluegill 114.6 44.5 fish 2-30 Bluegill 100.3 37.3 fish 2-31 Bluegill 81.3 25.4 4 1 3 1 1 3 Electrofish Area 3 23 Lgmouth bass 107.4 22.6 fish 3-7 Warmouth 166.1 57.2 fish 3-8 Bluegill 209.3 96.5 fish 3-9 Bluegill 120.4 48 1 2 4 2 2 fish 3-10 Bluegill 177.3 72.1 fish 3-11 Bluegill 140.7 50 fish 3-12 Bluegill 96.3 37.1 fish 3-13 Bluegill 109.2 43.4 fish 3-14 Bluegill 100.6 38.4 fish 3-15 Bluegill 102.1 36.8 fish 3-16 Bluegill 114.8 42.9 1 44.00 fish 3-17 Bluegill 114.3 38.9 4 1 fish 3-18 Bluegill 87.9 28.2 fish 3-19 Redbreasted sunfish 97 38.1 fish 3-20 Bluegill 120.7 48.3 fish 3-21 Bluegill 114.3 44.7 1 1 fish 3-22 Bluegill 109 38.9 fish 3-23 Longear sunfish 90.7 31.8 fish 3-28 Spotted sunfish 70.9 26.9 fish 3-29 Spotted sunfish 57.9 20.1 fish 3-30 Spotted sunfish 120.4 43.9 fish 3-31 Spotted sunfish 100.3 37.8 Electrofish Area 4 24 Lgmouth bass 340.9 76.2 25 Lgmouth bass 283 95.5 26 Lgmouth bass 291.1 77.5 27 Lgmouth bass 179.6 40.4 28 Lgmouth bass 225.6 52.3 29 Lgmouth bass 238 54.4 30 Lgmouth bass 267 64 31 Lgmouth bass 205.5 44.7 32 Lgmouth bass 149.1 35.3 33 Lgmouth bass 164.6 31.5 34 Lgmouth bass 104.4 23.1 35 Lgmouth bass 108 25.7 36 Lgmouth bass 154.9 35.3 fish 4-1 Bluegill 119.1 43.9 fish 4-2 Bluegill 111.5 43.2 fish 4-3 Spotted sunfish 141.5 57.2 fish 4-4 Spotted sunfish 114.3 43.7 fish 4-5 Spotted sunfish 108 40.1 72.00 fish 4-6 Spotted sunfish 94.2 33.3 fish 4-32 Bluegill 113.3 43.9 fish 4-33 Bluegill 106.9 35.6 fish 4-34 Spotted sunfish 75.7 28.4 fish 4-35 Bluegill 114.6 41.9 2 1 7 1 fish 4-36 Spotted sunfish 101.6 38.9 fish 4-37 Spotted sunfish 95.3 35.3 1 1 2 1 fish 4-38 Bluegill 134.1 51.8 fish 4-39 Spotted sunfish 91.9 32.5 fish 4-40 Bluegill 108.5 40.9 fish 4-41 Bluegill 86.4 31.2 2 1 3 2 1 1 fish 4-42 Bluegill 158.8 63.8 fish 4-43 Spotted sunfish 155.2 57.2 1 fish 4-44 Spotted sunfish 152.7 58.2 fish 4-45 Warmouth 126.2 45 fish 4-46 Warmouth 104.9 32.3 fish 4-47 Warmouth 191 71.1 fish 4-48 Bluegill 142 52.8

TOTALS 50 1 15 30 4 6 7 2 1 63 13 1 1 6 2 15 80 1 1 1 5 All Fish mean 143.48 44.45 TOTAL 305 stdv 84.04 19.85 n 108 Lepomis mean 112.61 41.90 stdv 27.89 12.80 Percent Abundance TOTAL 100 n 76 0.164 0.003 0.049 0.098 0.013 0.020 0.023 0.007 0.003 0.207 0.043 0.003 0.003 0.020 0.007 0.049 0.262 0.003 0.003 0.003 0.016 Leptohyphidae Tricorythodes albolineatus (LTa), Baetidae Acentrella parvula (BAp), Trichoptera Hydroptilidae orthotricia (THo), Trichoptera Cheumatopsyche spp. (TC), Trichoptera Phryganeidae spp. (TP), Amphipoda Hyalella azteca (AHa), Amicolidae (A), Hydrachnidinae (H), Ceratopogodinae Bezzia (CB), Tanypodinae Larsia (TL-), Tanypodinae Hudsonimyia (TH), Chironominae pseudochironomus spp. (Cp), Chironominae Paratanytarsus sp. D (CP-D), Chironominae Tanytarsus sp. L (CT-L), Chironominae Tanytarsus sp. C (CT-C), Chironominae polypedilum illinoese group (Cpig).CHironominae Rheotanytarsus sp. A (CR-A), Chironominae Dicrontondipes neomodestus (CDn), Orthocladius robacki (OOr), Orthocladiinae Orthocladius dorenus (OOd), Orthocladiinae Nanocladius alternanthorae (ONa), Orthocladiinae Cricotopus trincinctus (OCt), Oligochaeta (O).