ECOLOGICAL INDICATORS OF RESTORATION SUCCESS:

FISH COMMUNITY DISTRIBUTION, COMPOSITION, AND SAMPLING STRATEGIES

WITHIN THE PICAYUNE STRAND RESTORATION PROJECT

A Thesis Presented to

The Faculty of the College of Arts and Sciences

Florida Gulf Coast University

In Partial Fulfillment

Of the Requirement for the Degree of

Master of Science in Environmental Science

By

Ryan C. Young

May 2013

ii

APPROVAL SHEET

This thesis is submitted in partial fulfillment of

the requirements for the degree of

Masters of Science

__________________________________________

Ryan C. Young

Approved: May 2013

___________________________________________

Edwin M. Everham, III, Ph.D.

Committee Chair

___________________________________________

David W. Ceilley, M.S.

Committee Co-chair

__________________________________________

Michael Duever, Ph.D.

Committee Member

The final copy of this thesis has been examined by the signatories, and we find that both the content and the form meet acceptable

presentation standards of scholarly work in the above mentioned discipline.

iii

ACKNOWLEDGEMENTS

I would like to genuinely thank the Florida Fish and Wildlife Service for granting me access and

the necessary permits to conduct this research; the Florida Gulf Coast University and the Office

of Research and Sponsored Programs for providing funding to conduct this study; the Inland

Ecology Research Group for providing sampling equipment and guidance; my professors who

provided me with the core knowledge necessary to understand complex concepts and give me the

background knowledge necessary to have a fulfilling graduate experience; Jennifer Nelson for

her encouragement; my faithful volunteers including Geoffrey Rosenaw and Garrett Coe who

gave their time and energy to help make my field sampling possible; and my loving family

Shane, Donna, and Steve Young who came to my rescue whenever I needed assistance, provided

endless support and encouragement, and stood behind me every step of the way. This project

would not have been possible without these generous aids, tremendous support, and continuous

encouragement.

I would also like to express my sincere appreciation and gratitude to my committee chair Dr.

Edwin Everham with Florida Gulf Coast University for always giving me the push I needed, the

wise words of advice that helped to organize and bring together my thoughts and focus, teaching

me to think like a scientist, and sharing with me his passion and love for making a difference in

the world; my co-chair David Ceilley with Florida Gulf Coast University for teaching me about

experimental design and methodology, helping me to identify my fish samples, teaching me

about the Picayune, helping me understand the most current scientific data on inland freshwater

fish research in Florida, showing me how to analyze the data I collected, and how fun data

analysis can be by letting the information tell a story about the natural world; and Dr. Michael

Duever with the South Florida Water Management District for assisting in project design and

analysis, critically reviewing my text, sharing his expertise about the area of the Picayune and

the great stories about his time in the beautiful area, and providing guidance and encouragement

through the project. Their edifying assistance and research experience was the backbone of this

study

My heartfelt thanks and endless appreciation goes out to everyone who helped me to complete

this project because I could not have done it alone!!

iv

ABSTRACT

Increasing awareness of the damage inflicted upon natural systems by human beings has

brought environmental and ecological restoration to the forefront of environmental research and

monitoring efforts of the 21st century. Florida leads the country with some of the largest

restoration and monitoring projects in our nation’s history. This study was designed to evaluate

the success of restoration activities within the Picayune Strand Restoration Project, part of one of

the world’s largest restoration efforts the Comprehensive Everglades Restoration Plan. This

study was conducted by collecting and analyzing data on fish community structure, species

diversity, and species abundance in relation to various restoration phases. These restoration

phases included two treatments (impacted unrestored areas, transitional recently restored areas),

and reference wetlands (non-impacted natural wetland). Fish community data was collected

monthly through the period of inundation with passive sampling using Breder traps as well as

active dip net sampling. Based on the abundance and diversity data, results indicated that

species richness, abundance, and diversity was lowest in impacted areas, increased in transitional

recently restored areas, and was highest in both abundance and diversity in natural reference

areas. Fish community data also indicated distinct groupings and similarities within each

restoration phase and indicated varied species distribution among sites of different restoration

phases. This analysis confirmed that fish community assemblages differed significantly among

all three restoration treatments. Several indicator species were identified including Gambusia

holbrooki, Jordonella floridae, and Fundulus confluentus which helped to drive the dissimilarity

between different phases of restoration. In addition, the majority of species captured were only

found in reference wetlands. These findings serve as an indicator that the restoration activities in

the Picayune Stand are effective, and that several fish species may be used as indicators of

hydrologic restoration success in ephemeral wetlands of Southwest Florida. Further analysis

v

was conducted to observe patterns in sampling effort and temporal changes in community

structure in order to determine the sampling frequency required to obtain a robust signal, the time

of year most appropriate for collecting samples of a mature fish community, and patterns of

dispersion over multivariate space through the period of inundation. Based on this one-year

study, community data suggested that the months of October and November provided the best

examples of a mature fish community and that sampling at a frequency of every third month

(September, December, and March) provided sufficient community data to obtain a robust

signal. These findings serve as indication that a sampling frequency of every second month is

required to obtain the information necessary to make informed decisions about restoration

activities, and that the optimal time period for sampling a mature fish community occurs during

the months of October and November.

vi

TABLE OF CONTENTS

Page

Approval Sheet……………………………………………………………………………………ii

Acknowledgements………………………………………………………………………………iii

Abstract…………………………………………………………………………………………...iv

Table of Contents…………………………………………………………………………………vi

List of Figures ............................................................................................................................... vii

List of Tables ................................................................................................................................. xi

Introduction………………………………………………………………………………………..1

The Florida Everglades……………………………………………………………………1

Study Site – The Picayune Strand Restoration Project…………………………………...3

Ecological Indicators…………………………………………………………………….10

Fish as Indicators………………………………………………………………………...13

Fish Sampling Methods………………………………………………………………….16

Objectives………………………………………………………………………………………..18

Hypothesis……………………………………………………………………………………….19

Methods………………………………………………………………………………………….20

Experimental Design…………………………………………………………………….20

Sample Regions………………………………………………………………………….20

Deep Cypress…………………………………………………………………….21

Cypress Gramminoid…………………………………………………………….21

Gramminoid……………………………………………………………………...22

Restoration Phases……………………………………………………………………….22

Miller Canal……………………………………………………………………...22

Prairie Canal……………………………………………………………………...22

Fakahatchee Strand………………………………………………………………23

Survey Techniques……………………………………………………………………….23

Breder Traps……………………………………………………………………..24

Dip-Netting………………………………………………………………………24

Data Analysis……………………………………………………………………………25

Results……………………………………………………………………………………………28

Fish Community Data…...……………………………………………………………….29

Species Richness…………………………………………………………………30

Relative Abundance……………………………………………………………...33

Potential Indicator Species…………………………………………..…………...38

vii

TABLE OF CONTENTS (Continued) Page

Sampling Frequencies……………………………………………………………………42

Temporal Community Structure…………………………………………………44

Fish Community Structure………………………………………………………….……46

Temporal Dispersion ..............................................................................................49

Restoration Success .......................................................................................................................51

Discussion ......................................................................................................................................53

Potential Indicator Species .................................................................................................59

Sampling Frequency ..........................................................................................................60

Temporal Sampling ............................................................................................................61

Fish Community Structure .................................................................................................61

Temporal Dispersion ..........................................................................................................62

Restoration Success ...........................................................................................................63

Future Research Opportunities ..........................................................................................65

Conclusions ....................................................................................................................................68

References Cited ............................................................................................................................69

Appendix A – Fish Sampling Data Form ................................................................................... A-1

Appendix B – Fish Sampling Data ..............................................................................................B-1

Appendix C – Total Fish Abundance...........................................................................................C-1

Appendix D – Restoration Phases: Cluster Analysis and MDS Ordination ............................... D-1

viii

LIST OF FIGURES

Page

Figure 1. Picayune Strand Restoration Project location map………………………………………..3

Figure 2. Picayune Strand Restoration Project with adjacent restoration lands……………………..3

Figure 3. Aerial photograph of the Picayune Strand Restoration Project boundary………...............4

Figure 4. Originally constructed roadway network……………………………………….................5

Figure 5. Plans for the rehydration of the Picayune Strand Restoration Project…………................6

Figure 6. Study area with fish sampling locations…………………………………………………20

Figure 7. Average species richness and relative abundance of fishes within each samplable

site………………………………………………………………………………………..32

Figure 8. Average diversity metrics including Margalef’s (d), Pielou’s indices (J’), Shannon

(H’(loge)), and Simpson (1-Lambda’) for each of the represented sampling sites….......33

Figure 9. Percent composition of species documented over the entire study………………………35

Figure 10. Cluster analysis with sample sites representing habitat type and restoration phase

using Bray-Curtis similarity from the entire sampling period………………………..….37

Figure 11. MDS ordination with sample sites represented by habitat type and restoration

phase using Bray-Curtis similarity from the entire sampling period…….........................38

Figure 12. MDS ordination with superimposed raw abundance data for Gambusia

holbrooki…………………………………………………………………………………40

Figure 13. MDS ordination with superimposed raw abundance data for Fundulus confluentus……40

Figure 14. MDS ordination with superimposed raw abundance data for Jordonella floridae……....41

Figure 15. MDS ordination with superimposed raw abundance data for Lepomis sp…………………41

Figure 16. MDS ordination with superimposed raw abundance data for Poecilia latipinna………..42

Figure 17. MDS ordination including composite fish community data from the entire sampling

period including the months of September, October, November, December, January,

February, and March…………………………………………………………………..…43

Figure 18. MDS ordination including composite fish community data from every other

month of sampling including the months of September, November, January, and

March…………………………………………………………………………….……...43

Figure 19. MDS ordination including composite fish community data from every third

month of sampling including the months of September, December, and March……….44

ix

LIST OF FIGURES (Continued)

Page

Figure 20. MDS ordination including fish community data from the month of October at all

samplable sites…………………………………………………………………………...45

Figure 21. MDS ordination including fish community data from the month of November at all

samplable sites…………………………………………………………………………...45

Figure 22. MDS ordination representing reference sites of various habitat types sampled

each month during the entire sampling period with superimposed relative

abundance data for Gambusia holbrooki for each represented month when

sampling occurred……………………………………………………………………….47

Figure 23. MDS ordination representing reference sites of various habitat types sampled

each month during the entire sampling period with superimposed relative

abundance data for Jordonella floridae for each represented month when

sampling occurred………………………………………………………………………..48

Figure 24. MDS ordination representing reference sites of various habitat types sampled

each month during the entire sampling period with superimposed relative

abundance data for Fudulus confluentus for each represented month when

sampling occurred……………………………………………………………………….48

Figure 25. MDS ordination of monthly composite fish community data in transitional

sites with superimposed trajectory…………………………………………………….....49

Figure 26. MDS ordination of monthly composite fish community data in reference

sites with superimposed trajectory……………………………………………………...50

Figure 27. Cluster analysis of Bray-Curtis similarity representing composite fish

community data for sampling sites of various restoration phases and

habitat types through the entire sampling period……………………………………….51

Figure 28. MDS ordination of composite fish community data for each sampling

site representing various restoration phases and habitat types through the entire

sampling period using Bray-Curtis similarity from all sampling events……………….52

Figure 29. MDS ordination of composite fish community including superimposed

similarity groupings of 40 percent, 50 percent, and 60 percent similarity………….…52

x

LIST OF FIGURES (Continued)

Page

Figure 30. Habitat maps of the Picayune Strand from 1940 and 1995……………………………...57

Figure 31. Principal component analysis including an MDS ordination of all

sampling events with superimposed environmental factors including

dissolved oxygen, average depth, average conductivity, salinity,

and temperature…………………………………………………………………………..58

xi

LIST OF TABLES

Page

Table 1. Site descriptions with locations and adjacent wells……………………………………...28

Table 2. Presence / absence of water in each sampling site per month…………………………...29

Table 3. Fish species documented via Breder traps and dip-net sampling

by scientific name, common name, and species code abbreviations………………….....29

Table 4. Fish species documented in each restoration phase listed by scientific name………..….30

Table 5. Univariate diversity indices including species richness (R) and relative

abundance (A), Margalef’s (d), Pelou’s indices (J’), Shannon-Weaver index

(H’(loge), and Simpson’s diversity (1-Lambda’) for fish species documented…….…....31

Table 6. Total relative abundance values per restoration phase, total abundance

for all species, and total percent composition of fish species

documented during the sampling period………………………………………………....34

Table 7. SIMPER analysis of fish species and restoration phases………………………………..39

Table 8 Relative abundance data for Gambusia holbrooki, Jordonella floridae,

and Fundulus confluentus over the entire sampling

period…………………………………………………………………………………….46

Table 9. Dissimilarity values between consecutive months within various restoration

phases and average dissimilarity for each restoration phase through

the entire sampling period…………………………………………………………….….50

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INTRODUCTION

The Florida Everglades

The Everglades is a vast wetland comprised of a variety of habitat types including saw

grass marshes, wet prairies, sloughs, ponds, tree islands, and mangrove estuaries that dominate

Southern Florida and are characterized by habitat heterogeneity, large spatial extent, and a

distinctive hydrologic regime (Dixon 2009; Chimney & Goforth 2006; Davis 1943a,b; Loveless

1959, McCally 1999, Whitney 2004).

Attempts to drain the Everglades for human uses began in the 1850s with the Swamp and

Overflowed Lands Act which allowed for large scale dredging and channelization operations,

destroying five million acres of wetlands and creating over 400 miles of drainage canals (Florida

Conservation Foundation (FCF) 1993). Shifting attitudes led to the protection of these wetland

systems with the Wetland Protection Act of 1984, which helped to preserve these wetland

swamp systems that still cover about 10% of Florida’s land area today (Myers & Ewel 1990).

Nonetheless, this area has also been severely impacted by anthropogenic activities and is

severely threatened if it is not protected and restored (COE & SFWMD 1999).

In an effort to protect and restore this landscape, the SFWMD and COE developed a plan

titled “Central and Southern Florida Project Comprehensive Review Study Final Integrated

Feasibility Report and Programmatic Environmental Impact Statement” in 1999. The plan, also

referred to as the Comprehensive Everglades Restoration Plan (CERP), was designed to create

and restore wetlands and reservoirs to increase water storage and water supply (COE & SFWMD

2001). CERP is a multi-billion dollar federal-state partnership that seeks to restore historical

hydrologic conditions of the Everglades and encompasses over 200 components addressing

water management and ecosystem restoration needs over the southern third of peninsular Florida

(Doren et al. 2009). It was created as, “a framework for modifications and operational changes to

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the Central and Southern Florida Project that are needed to restore, preserve, and protect the

South Florida ecosystem while providing for other water-related needs of the region, including

water supply and flood protection” (COE & SFWMD 2001 p. 1-2). The plan was reviewed and

finally approved through the Water Resource Development Act on December 11, 2000. The new

plan gave the opportunity to reverse the course of declining health of the Everglades system and

leave the irreplaceable system as a legacy for generations to come (COE & SFWMD 1999 p. i).

This document provided a tool to help sustain the environment, economy, and social well-being

of South Florida (COE & SFWMD 2004).

Increasing awareness of the damage inflicted upon natural systems by human beings has

brought environmental and ecological restoration to the forefront of environmental research and

monitoring efforts of the 21st century. The Society for Ecological Restoration International

defines ecological restoration as, “the process of assisting the recovery of an ecosystem that has

been degraded, damaged, or destroyed.” They also describe the process as an, “activity that

initiates or accelerates the recovery of an ecosystem with respect to its health, integrity and

sustainability.” (Society for Ecological Restoration International Science & Policy Working

Group 2004.) Restoration projects often require long term commitments by participating parties.

Monitoring is a vital facet in the restoration process as it allows us to assess the progress and

effectiveness of restoration efforts, and helps inform the discipline on the relative merits of

different approaches to restoration.

Acting as the vanguard for this movement of ecological restoration efforts, Florida leads

the country with some of our nation’s largest restoration and monitoring projects. Often noted as

the world’s largest restoration effort, the Comprehensive Everglades Restoration Plan (CERP)

sets many complex guidelines for evaluating the success of the restoration efforts involving

hydrology, water quality analysis, and a variety of other assessment models. The overall CERP

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Figure 2. This map shows the location of the Picayune Strand Restoration Project in the state of Florida, Collier County, and the immediate areas surrounding the project (Dixon 2009).

Figure 1. This map shows the Picayune Strand Restoration Project with adjacent conservation lands including Belle Meade, the Florida Panther National Wildlife Refuge, Fakahatchee Strand Preserve State Park, the Ten Thousand Islands National Wildlife Refuge, Collier-Seminole State Park, Deltona Lands, and the Rookery Bay National Estuarine Research Reserve (Dixon 2009).

project implementation will take over 20 years but, monitoring and evaluation efforts will extend

long beyond the project’s completion, well into the future.

Study Site - The Picayune Strand Restoration Project

The Picayune Strand Restoration Project is one of the largest individual CERP projects and as

part of the Governor’s ACCELER-8 program in 2004, the first CERP wetland restoration

project. Formerly intended for development as a large-scale residential area called the Southern

Golden Gate Estates, Picayune encompasses approximately 85 square miles and is located

immediately south of Interstate 75 and extends to US 41 in western Collier County (COE &

SFWMD 2004). Picayune Strand was historically characterized by seasonal flooding and slow-

moving sheet flow prior to efforts to develop the area.

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These characteristics supported a

typical Southwest Florida wetland

landscape which consisted of cypress

swamps and sloughs, wet prairies,

hardwood hammocks, marshes, pine

and cabbage palm flatwoods, and tidal

areas (Duever & SFWMD, pre-

development vegetation map). The

area is open to the public for various

recreational activities including

hunting, fishing, camping, horseback

riding, hiking, photography, and

wildlife observations and sits in an area

surrounded by additional conservation

lands including the Fakahatchee Strand

Preserve State Park on the east, Belle

Meade to the west. As a result, sheet flow was eliminated, the water table dropped more than 1

meter in some areas, hydroperiod was reduced by 2 to 4 months, drainage rate increased by 16

times, the frequency and intensity of forest fires increased, and the area was invaded by exotic

vegetation and other invasive non-native species (SFWMD 2008a). As the water was

channelized in the late 1960s by an extensive 48 mile canal system, the overall hydrology of

watershed was altered by the discharge of approximately 18 inches of run off annually and the

interception of shallow groundwater outflow. This canal system has continually lowered the

water table and can still be seen today in the areal image provided in Figure 3. This over-

Figure 3. This map shows an arial photograph with the Picayune Strand Restoration Project boundary. Also visible in the map are the system of 290 miles of road and canal system built during the lands intended development (Dixon 2009).

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drainage of the area also resulted in large point source

freshwater discharge into the estuarine system of the 10,000

islands. The decline of ecological function including the

degradation of upland, wetland, and estuarine plant

communities, reduced abundance of native fish, wildlife,

and estuarine shellfish populations, and reduced aquifer

recharge showed even more evidence of the severe negative

impacts that this project had on the natural system. In

addition to the over-drainage by the canal system, 290

miles of roads were constructed in a grid system throughout

the property (Figure 4). The roads were graded and sat approximately 15 centimeters above the

surrounding ground elevation, acting as barricades to sheet flow and redirecting any standing

water into the nearby canals. Although these modifications severely lowered water levels, they

failed to prevent the property from holding water during the wet season, causing the

development project to fail (COE & SFWMD 2004, U.S. Department of the Interior (DOI)

2005).

After the company that owned the Southern Golden Gate Estates, the Gulf American

Corporation, went bankrupt concerns over the degradation of environmental quality and water

supply potential of the region began to arise. This prompted the State of Florida to include

Picayune Strand in the Save Our Everglades program in 1985 and direct the South Florida Water

Management District (SFWMD) to develop a hydrologic restoration plan to reduce over-

drainage, restore historic sheetflow patterns while maintaining the existing levels of flood

protection for areas in the north, and enhance the areas environmental value and water resources.

Re-attaining pre-development conditions was the main goal of the proposed restoration project

Figure 4. This map shows the originally constructed roadway network including 290 miles of paved roads (Dixon 2009).

-6-

(COE & SFWMD 2004, Duever 2005) and restoration of the native fish communities was

determined to be an important component of the restoration project (Ceilley 2008).

Prior to the onset of CERP, the need for restoration of this area was recognized. A jointly

funded plan by the SFWMD and the COE was developed and land acquisition of more than

20,000 parcels began with funding assistance from the Department of the Interior in 1984. The

effort extended for over 20 years, continuing through 2005. Restoration efforts began in 2003

after the implementation of CERP,

beginning with the installation of

culverts under US-41 and the

plugging of the northern portion of

Prairie Canal, which lies on the

northeastern most side of the

property. Restoration efforts

continued for four years as the

SFWMD finished the plugging of

Prairie Canal, removed 65 miles of

roads east of Merritt Canal,

demolished 160 structures, and

conducted soil remediation on

approximately 28 acres of

contaminated soil. Activities halted in 2008 due to lack of funds as a result of the economic

downturn, but have since resumed. The remainder of the project includes continued road

removal to the west of Merritt Canal, plugging of the remaining three canals, and completing the

installation of three pumping stations as well as spreader canals (COE & SFWMD 2004, Duever

Figure 5. This map shows the plans for the intended areas of rehydration in the Picayune Strand Restoration Project including canal plugs, road removal, pump station installations, and existing weirs (COE & SFWMD 2004).

-7-

2005). Land management activities are being conducted by the Florida Division of Forestry

(DOF), and other cooperating agencies include the U.S. Fish and Wildlife Service, U.S.

Environmental Protection Agency, the National Park Service, and the Florida Department of

Environmental Protection (Dixon 2010, COE & SFWMD 2004).

One of the key restoration components of this project is the plugging of the 48 ± mile

canal system. In an effort to restore a more natural hydrologic regime in the area, spoil from the

removed roads are being used to plug the canal system. This backfilling technique has been used

to return areas to a more natural hydrologic regime and has great potential to improve aquatic

wildlife habitat and the area’s hydrologic connectivity. Backfilling is a reasonably simple and

short term management strategy which requires simple equipment and no on-site maintenance

that has been proven to demonstrate stability over decades (Turner et al 1994). Despite the fact

that Prairie Canal was not entirely backfilled, leaving pools of surface water between plugs, the

plugs help to prevent water from being released directly into downstream estuaries. The

SFWMD stated that once the water is no longer being conveyed that the canals would no longer

over-drain the landscape and surface aquifer resulting in a longer period of inundation for the

surrounding area (COE & SFWMD 2004 p. vi). Due to monitoring efforts, SFWMD has already

began to document higher water levels near the filled Prairie Canal compared to areas near the

unrestored Merritt Canal during the winter of 2006-2007 (Dixon 2010, Ceilley 2008, Chuirazzi

& Duever 2008).

Plugging of the canals in the Picayune Strand, along with the grading of the road system,

removal of existing structures, and soil remediation are expected to have significant benefits on

the area, as well as adjacent lands, helping to achieve the overall Everglades restoration goal.

“Expected project benefits include restoration of historic wetland communities, restoration of

sheetflow towards the coastal estuaries, reduction of harmful surge flows through the Faka Union

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Canal into Faka Union Bay, improved freshwater overland flow and seepage into other bays of

the Ten Thousand Islands region, improved aquifer recharge, decreased frequency of forest fires,

improved habitat for fish and wildlife and threatened and endangered species, reduced invasion

of exotic species, and increased spatial extent of wetlands,” (Dixon 2009; Ceilley 2008; COE &

SFWMD 2004; Chuiarazzi & Duever 2008 p.3). These hydrologic improvements are is

expected show very positive responses from water dependent animals by increasing their

numbers and returning natural distribution patterns once the project is complete (Dixon 2009;

COE & SFWMD 1999).

Although the outcomes of the canal backfilling are expected to be positive, it should be

noted that negative impacts of canal backfilling have been documented in the past. A study in

the Upper St. John’s River Basin, Florida in 1986 stated that while the plugs slowed canal flow

and re-hydrated the marsh, it caused shorter intensity drying events in upstream locations,

causing undesirable shifts to flood-tolerant vegetation, and converted areas of marsh to open-

water (Chandy, Miller, & Morris 2001). Although some negative impacts of canal plugs have

been found in the past, the main objective of the Picayune Strand Restoration Project is to restore

the historical hydrology and ecology of the area and surrounding areas. These positive impacts

were documented by Baustian and Turner (2006), stating that backfilling restores local

hydrologic conditions, creates marsh, and improved ecological processes over a period of 20

years (Baustian & Turner 2006). There may be some negative impacts associated with the canal

plugs in the Picayune Strand, but the resulting hydrologic restoration is expected to have a

significantly positive impact on the ecological and hydrologic regimes that are currently found

there and in the surrounding areas.

Prior to the onset of these restoration efforts, a baseline study was conducted to evaluate

the status of Picayune’s habitats and eventually evaluate environmental changes after the PSRP

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has been completed. The baseline assessment study established reference sites in cypress, wet

prairie, hydric pine, and brackish marsh habitats in the Florida Panther and Ten Thousand Islands

National Wildlife Refuges and in Fakahatchee Strand State Preserve and compared them to

similar, but impacted wetlands of PSRP (Ceilley 2008, Duever 2005). Aquatic faunal samples

were collected from 42 locations six times from August 2005 to February 2007.

For the baseline assessment, fish communities were surveyed at a frequency of three

times per year at each location. Wetland ecologists at Florida Gulf Coast University (FGCU)

identified a total of 6,381 individual fish collected using Breder traps which consisted of 25

species including nine families, two non-native families, and two non-native species. Eastern

mosquito fish was the most abundant species present being found at every sample site. The

reference sites had a 58% similarity of fish community structure with seven species contributing

to >93% of the similarity, while impacted sites showed an average of 45% similarity with only

five species contributing to >93% of the similarity. Reference wetlands showed a higher

diversity in fish communities than that of impacted wetlands with a more even distribution of

species abundances within and between sites (Ceilley 2008). Abundances of specific fish species

will also provide a good indicator of ecosystem health. Deep-water refugia allow certain fish

species and higher trophic level fishes to persist in areas where they would not survive during

period of frequent dry-downs. Frequent dry-downs may alter the dynamics of native fish

communities and may diminish the value of these habitats by not providing access to dry season

refugium for aquatic fauna during the dry-down throughout the Picayune. Assessing the change

in abundance of specific fishes after the plugging of Prairie and Merritt canal can provide us with

another indicator of restoration success.

The baseline study was conducted to eventually evaluate habitat quality and restoration

success at the completion of the project. As restoration continues it is expected that fish

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communities will recover at the impacted wetlands and become more similar to those of

reference wetlands. At this point in the restoration process, Phase 1 has been completed, and the

continued plugging of additional canals, removal of roads, and the installation of pump stations

is yet to occur. We have a unique opportunity, at this time, to measure the improvement of the

ecosystems after the completion of Phase 1 of the project, and to examine possible methods for

quantifying success.

ECOLOGICAL INDICATORS

Monitoring is required to determine whether the benefits of the project are being

achieved (COE & SFWMD 2004). In 2005, the Department of the Interior produced a Science

Plan outlining the importance of monitoring within South Florida to develop and effectively

synthesize and communicate the best available science in order to apply it to the adaptive

management program for the large-scale restoration project (DIO 2005). The complexity of

ecological systems makes it extremely difficult to measure and communicate the effects of such

large restoration projects. Despite the difficulty, these monitoring efforts are essential for

complex regional restoration programs as stated by Dale & Beyeler et al. (2001), “Ecological

systems are complex, and developing effective strategies for measuring and communicating

restoration success (or failure) is an extremely difficult, but essential task in any large, complex

regional restoration program” (Schiller et al. 2001; Lausch & Herzog 2002; Niemi & McDonald

2004; Ruiz-Jean & Aide 2005; Thomas 2006; Doren et al. 2009 p. 2).

The task of monitoring the entirety of ecological processes within a system would be

unmanageable, extremely expensive and time consuming. Ecologists have extensively sought

concise and cost-effective measures that can characterize ecosystem conditions (Schiller 2001).

In a large and complex region such as South Florida, there must be means to determine how

restoration goals are being met (Doren et al. 2009).

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Environmental, biological, and ecological indicators may provide an effective and

efficient method of characterizing and assessing ecosystem conditions. The first reference to

environmental indicators is attributed to Plato, who cited the impacts of humans on fruit tree

harvest (Niemi 2004). The concept of indicators for plant and animal communities can be traced

as far back as the 1600s. Examples of early indicators include using migratory bird populations

to provide insight into changing environmental conditions, or the use of canaries in coal mines to

detect the presence of noxious gasses in the 1920s. One of the more elaborate examples of early

biological indicators was the saprobian system developed by Kolkwitz & Marsson in 1908,

which used benthic and planktonic plants as indicator species for classifying stream

decomposition zones (Niemi 2004). Variations of this system are still used in ecological

assessments today in the Florida Department of Environmental Protection’s stream condition

index (FL DEP).

Ecological indicators are defined by Niemi (2004) as, “measurable characteristics of the

structure (e.g. genetic, population, habitat, and landscape pattern), composition (e.g. genes,

species, populations, communities, and landscape types), or function (e.g. genetic,

demographic/life history, ecosystem, and landscape disturbance processes) of ecological

systems.” Ecological indicators serve several purposes including assessing the condition of the

environment, or monitoring trends in condition over time. In addition, these indicators can be

used to provide an early warning signal for changes in the environment, or diagnose the cause of

an environmental problem (Dale 2001).

The selection of specific indicators for large scale restoration projects comes with many

challenges. The selected indicator must be representative of the system as a whole by capturing

key pieces of ecosystem function that are tied to the underlying values of the restoration

program. The goals and objectives of the monitoring program will help to determine which

-12-

ecological indicators are used (Doren 2009; Niemi 2004). In addition, the selected indicator

should be easily and routinely measurable, be sensitive to stresses on the system, have a known

response to disturbances, and can be integrated into management. Indicators can be selected at

different hierarchal levels including organism, species, population, ecosystem, and landscape in

order to best provide a simple and efficient method to examine the ecological composition,

structure, and function of complex ecological systems. This ecological hierarchy includes

functional, compositional, and structural elements that can be combined to define ecological

systems and provide a means to select indicators representative of the key characteristics of the

system (Dale 2001).

Once an indicator is selected, it is necessary to determine the environmental metrics that

will be used to most effectively represent the indicator’s response. Indicators previously used for

Florida’s restoration projects have embodied components of the South Florida ecosystem by

using the organisms including characteristics distinctive of the Everglades landscape, trophic

constituents, biodiversity, physical properties, and associated ecological structure and function

(Doren 2009). These indicators can then be used to reflect the biotic and abiotic state of the

environment, reveal evidence for impacts of environmental change, and/or indicate the diversity

of other species, taxa, or communities within an area (Niemi 2004).

Although few scientists deny the benefits that indicators provide to research, monitoring,

and management efforts, it should be noted that there are three concerns that hinder the use of

ecological indicators as a resource management tool. Those concerns include: the use of

indicators that may not be able to convey the full complexity of the ecosystem; choosing

indicators without defined project goals and objectives, or goals and objectives associated with

short-term response rather than long-term maintenance of healthy ecosystems; and failure to

establish standard procedures for the selection of indicators to ensure repeatability, avoid bias,

-13-

impose discipline upon the selection process, and encompass management concerns to ensure

scientific rigor (Dale 2001). If these concerns are addressed in the planning process, indicators

provide a valuable tool to research, monitoring, and land management activities to conduct

concise and cost-effective evaluations of ecosystems (Schiller 2001).

The goals of the backfilling efforts in the Picayune Strand encompass the goals of the

greater Everglades restoration. Some of the expected benefits surrounding these goals are: the

restoration of historic wetland communities; restoration of sheetflow towards coastal estuaries;

reduction of harmful surge flows through the Faka Union Canal into Faka Union Bay, improved

freshwater overland flow and seepage in to other bays of the Ten Thousand Islands region;

improved aquifer recharge; decreased frequency and intensity of forest fires; improved habitat

for fish and wildlife and threatened and endangered species; reduced invasion of exotic species;

and increased spatial extent of wetland (COE & SFWMD 2004; Chuirazzi & Duever 2008 p. 3).

With these goals and intended benefits in mind, it is imperative to monitor and track changes in

environmental and ecological conditions, and the progress and extent of expected benefits as

hydrologic restoration advances.

FISH AS INDICATORS

Fish have been identified as key indicators by which to measure restoration success in

South Florida. As essential components to the successful functioning of wetland food webs,

changes in fish population sizes and community composition can provide essential information

about the structure, composition, and function of an ecosystem (Loftus et al. 2000). Fish are also

greatly affected by large scale ecosystem processes, especially in highly variable systems such as

the seasonal wetlands of the Picayune Strand. Colonization by fish in these systems depends on

the frequency and duration of hydrologic connections, and species specific dispersal abilities.

“The longer and more frequent the connections to other water bodies, the greater the opportunity

fish species have to colonize a wetland,” (Barber et al. 2002). Fish assemblages are known to be

-14-

greatly influenced by these parameters, and it has been noted that higher hydrologic connectivity

results in greater richness and diversity. These fundamental effects on fish community structure

provide a means for evaluating impacts of changing hydrologic conditions on the health of

seasonal wetlands. Monitoring fish assemblages may provide meaningful information on the

ecological health of these systems (Main et al. 2007). Fish assemblages have also been noted

(Niemi 2004) as excellent indicators because:

1. They are relatively easy to identify;

2. They are of interest to the public;

3. They are relatively easy to measure;

4. They often contain various locally abundant species with known responses to

disturbance; and

5. They can be monitored at a relatively low cost.

Using fish assemblages as indicators is particularly successful in South Florida due to the

topographical characteristics of the area. The lack of topographical relief allows fish species to

move throughout the landscape as a function of hydrologic connectivity (Barber et al. 2002). In

addition, wetland fish species in south Florida are adapted to deal with historic environmental

conditions, which decrease the influence of local abiotic factors on community composition

(Ruetz et al. 2005). Anthropogenic hydrologic alterations such as channelization and

compartmentalization of the previously sheetflow-driven system of the Picayune Strand resulted

in major changes in the physical and hydrologic processes which has had far reaching effects on

the ecological processes and habitat in the area. The physical alterations brought about by

canals, impoundments, and roads have resulted in loss of connectivity necessary for sheetflow in

this area (Ogden et al. 2005).

-15-

The monitoring of ecological responses of fish to hydrologic change within the Picayune

Strand was identified as a necessary science for the south Florida adaptive management process

(DOI 2005). A unique aspect of the current Picayune Strand is that it is in the process of being

restored, which allows for immediate biological comparisons to be made through the various

stages of hydrologic restoration (Dixon 2009). Monitoring of fish assemblages within various

restoration phases would provide excellent biological indication of the hydrologic conditions of

these different areas, and the progress of hydrologic restoration over the landscape as a function

of connectivity.

Fish community investigations have been conducted in the Picayune Strand since 2001

including pre-restoration wildlife surveys from 2001-2004 (Addison et al. 2006) and continued

biological surveys from 2005-2007 (Ceilley 2008). Fish were sampled using Breder traps

(Breder 1960) to develop a baseline for fish communities to be assessed in the future after

restoration occurred. Sampling took place over a broad area of the Picayune Strand including the

tidally influenced southern portions. During early pre-construction sampling, a total of seven

families and 23 species were collected from a variety of habitats (Addison et al. 2006). In the

2005-2007 study, 25 fish species from nine families were collected from a variety of habitat

types across Picayune Strand. The study showed that reference wetlands showed a mean species

richness of 8.1 species per site, while impacted wetlands showed a mean species richness of 4.5

species per site (Ceilley 2008). This data compared impacted wetlands to reference wetlands,

but did not focus on the change in the transitional, recently restored areas of the Picayune Strand.

-16-

FISH SAMPLING METHODS

Many techniques, including active and passive sampling methods, have been used to

sample fish communities in wetland habitats. In sampling fish communities, it is important to

provide information about the spatial and temporal resolution of fish movement at a low cost and

low catch per unit effort, while providing statistical validity through replicable methods (Sargent

& Carlson 1987). Some of the more most commonly used methods are: Breder traps; throw

traps; seines; dip-netting; and drop traps. A brief summary of each of these methods advantages

and disadvantages are provided below:

1) Breder Traps – This passive sampling method is suitable for use in all types of

vegetation and water levels, replicable with low labor, and inexpensive and

simple to apply. A limitation to this method is the behavior sampling bias against

transient and predatory species, and for active forage fish species (Ceilley et al. in

press, Main et al. 2007, Sargent and Carlson 1987).

2) Throw Traps – This active sampling technique works in herbaceous vegetation,

but can be very destructive to sampling areas and are both labor and time

intensive.

3) Seine Nets – This active sampling method collects large numbers of individuals

and species in the sampled area and provides a largely comprehensive inventory

of fish species in the area. A limitation to this method is that it can only be used

in open water with flat bottoms (Sargent & Carlson 1987).

4) Dip-Netting - This active sampling technique is effective in areas that are difficult

to sample using other active sampling techniques, and are inexpensive and easy to

use. Limitations to this method include disturbance to the sampling area, and

-17-

difficulty in standardizing sampling effort and efficiency among individuals and

locations (Main et al. 2007).

5) Drop Traps – This active sampling technique is effective to conduct quantitative

sampling. Some limitations of this method are that it is a destructive method of

sampling, capture is selective due to avoidance behaviors, they are expensive, and

they are complicated to build, set up, and transport (Sargent & Carlson 1987).

There are many fish sampling methods that have been proven effective. “Selection of the

appropriate technique or techniques depends on the question being asked, the information

required, the nature of the organism(s) or habitat being studied, and the resources available for

the project,” (Heyer et al. 1994; Dixon 2009). Since previous research within the Picayune

Strand and Babcock Ranch in Charlotte and Lee Counties has been successfully conducted using

a combination of Breder traps and D-frame dip nets (Ceilley 2008 and Ceilley et al. 2013), and

because results can be compared to new data collected, these methods were employed during this

research project. Breder traps have been identified as one of the most efficient and cost effective

techniques for attaining a statistical validity while measuring communities of wetland fish

species (Ceilley et al. 2013; Sargent & Carlson 1987). Based on survey methods that have been

proven successful within the Picayune Strand, and the identified need for fish community

sampling (DOI 2005) in the area, Breder traps in combination with dip-netting appear to be the

most effective sampling techniques. By employing these survey methods in the appropriate

areas, adequate and appropriate fish community information was expected to be obtained to help

determine whether the benefits of the restoration project are being achieved.

-18-

OBJECTIVES

Previous research conducted in the Picayune Strand was intended to eventually evaluate

habitat quality and restoration success at the completion of the project. As restoration continues

it is expected that fish communities will recover at the impacted wetlands and become more

similar to those of reference wetlands. At this point in the restoration process, Phase 1 has been

completed, and the continued plugging of additional canals, removal of roads, and the

installation of pump stations is in progress. We have a unique opportunity, at this time, to

measure the improvement of the ecosystems after the completion of Phase 1 of the project, and

to examine possible methods for quantifying success. It is also important to consider sampling

cost and efficiency as financial restraints have been a factor in the progress of restoration and

monitoring in the Picayune.

Therefore, the objective of this research included the following:

1. Identify specific species to use as performance indicators;

2. Examine the implications of various sampling frequencies to determine the

minimum required to adequately track changes in community structure;

3. Determine the time of year most appropriate to collect samples from a mature fish

community;

4. Determine how fish communities change from the beginning of the wet season

until the dry down;

5. Examine cyclical patterns of temporal dispersion over multivariate space caused

by seasonal hydrologic changes; and

6. Determine whether fish communities at Transitional sites undergoing restoration

are moving from impacted conditions towards minimally impacted Reference

conditions.

-19-

HYPOTHESIS

Based on the objectives described above, the hypotheses of this research include the following:

1. There will be significant differences in the distribution of various fish species

relative to the different restoration phases which will lead to the identification

of “indicator species” of restoration success.

2. Monthly sampling of fish communities will provide a similar signal to that of

an abbreviated sampling frequency, identifying the minimum sampling

frequency required to acquire an accurate depiction of fish community

structure among restoration phases.

3. Sampling will be most effective during the months after the start of the dry-

down while the majority of sites are still inundated to allow sampling of fish

communities.

4. Richness, relative abundance, and diversity of fish species will increase

through the dry down as fish communities are concentrated and isolated in

smaller areas.

5. A pattern will emerge from multivariate analysis that can be compared to

additional data in an effort to examine temporal fish community structure

changes, and identify a dispersion pattern through multivariate space

representing fish communities in different phases of restoration.

6. Fish communities in Transitional sites undergoing restoration with show

greater similarity to Reference sites than Impacted sites as hydrologic

restoration will allow movement of fishes across an improved hydrologic

landscape.

-20-

Figure 6. This map shows the sites selected for this study including three restoration phases with representative habitat types. Sites include two impacted sites (cypress gramminoid and gramminoid habitats), four transitional sites (two deep cypress, and gramminoid habitats), and three reference sites (deep cypress, cypress gramminoid, and gramminoid habitats). An impacted deep cypress site and a transitional gramminoid site were eliminated due to lack of water and samplable fish communities. An additional transitional deep cypress site was added due to its position between recently restored prairie canal and the reference wetlands of the Fakahatchee Strand.

METHODS

Experimental Design

There are three general phases specifically related to this study; 1) natural wetland habitats

including deep cypress, cypress gramminoid, and gramminoid marshes (reference); 2) restored canal –

former Prairie Canal including all habitat types (transitional treatment); and 3) un-restored Miller Canal

including all habitat types(impacted treatment). The study area occurred across the center portion of the

Picayune Strand representing the progression of the restoration and hydrologic regimes of the area on a

latitudinal basis.

The study area was divided in to three regions from east to west containing 1) three sample

locations within impacted un-restored sites around Miller Canal; 2) four sample locations within restored

locations around former Prairie Canal; and 3) three sample locations within the natural wetland habitats of

the Fakahatchee Strand. This study looked at these different restoration phases in an attempt to identify

patterns among fish community use and composition in

relation to restoration progress. Survey methods

included using Breder Traps and dip-netting to

incorporate both active and passive sampling techniques.

These surveys were conducted monthly during the end of

the wet season through the dry-down of 2011-2012 (i.e.

September through March).

Sample Regions

The study was divided into three regions with

three habitat types per region to represent major wetland

habitat types across different phases of the overall

restoration project. Ten sample sites were distributed

along an East to West transect of the Picayune and

-21- Fakahatchee Strands. Each restoration phase contained 3 to 4 sites representing 3 habitat types including

deep cypress, cypress gramminoid, and gramminoid marsh. Therefore, each habitat type could be

analyzed independently and/or together by restoration phase.

Deep Cypress

Deep cypress habitat is characterized by channelized

water flow through a “strand” dominated by cypress tree,

epiphytes, and a shaded understory. During the dry season,

these areas retain water for longer time periods than

surrounding wetland areas giving it the longest period of

inundation of all the represented habitats in this study

(Whitney, Means, & Rudloe 2004). These habitats may

provide dry season refuge for fish species in the area, and

contain larger more predatory species in addition to

omnivorous forage fish that dominate South Florida

wetlands (Odgen 2005).

Cypress Gramminoid

Cypress gramminoid habitat is

characterized by a shallower flow way with a

less shaded understory, allowing for the growth

of herbaceous wetland place such as Jamaica

swamp saw grass, ferns, and shrubs (Whitney,

Means, & Rudloe 2004). These habitats are

usually ephemeral and provide excellent

habitat for colonizer fish species such as Gambusia holbrooki and Jordonella Floridae (Addison et al.

2006). It also provides an advantage to smaller forage fish species that use submerged and emergent

plants as critical habitat and allow protection from predation (Main et al. 2007).

-22- Gramminoid

Gamminoid marshes are

ephemeral, broad level plains containing a

variety of herbaceous wetland plants and

grasses. These habitats become inundated

for part of the year, but can remain dry for

extended periods of time (Whitney, Means,

& Rudloe 2004). These habitats are also

beneficial to colonizer and small forage

fish species who take advantage of newly inundated areas for protection from predation and new food

sources (Barber 2002).

Restoration Phases

Miller Canal

The Miller Canal is the furthest major unrestored drainage canal from the recently restored Prairie

Canal and the natural reference site of the Fakahatchee Strand. This canal runs parallel with the former

Prairie Canal and is located on the far west side of the Picayune Strand. Three sampling sites were

selected in the region representing the three habitats of focus in this study. Each of the three habitat sites

contained vegetative communities that are characteristic of those habitats. The sites around this canal

provided treatment stations for this study which demonstrated pre-restoration characteristics around an

existing un-restored canal that were compared against sites around restored sites which should be

demonstrating ecological recovery, and un-impacted reference sites located in the Fakahatchee Strand

(Ceilley 2008). All three sites were located around Stewart Boulevard, which is located in the central

portion of the Picayune Strand and runs east to west.

Prairie Canal

Restoration on the former Prairie canal was completed in July 2007 which included the

installation of 7 miles of canal plugs (Dixon 2009). This former canal is located on the easternmost side

-23- of the Picayune Strand running north to south. Three sampling sites were selected in this region

representing the three habitats of focus in this study. Each of these habitat sites also contained vegetative

communities that are characteristic of those habitats. The sites around this former canal provided

treatment stations for this study which demonstrated post-restoration characteristics around a canal that

has undergone complete restoration. These sites were compared against sites around the un-restored

Miller canal, and un-impacted reference sites located in the Fakahatchee Strand. All three sites were also

located around the east-west transect of Stewart Boulevard.

Fakahatchee Strand

The Fakahathee Strand is a State Park located to the east of the Picayune Strand. The majority of

the Picayune Strand has not been negatively impacted by the Southern Golden Gate Estates (SGGE)

project, and retains a natural habitats and hydrologic regime. Four sampling sites were selected in this

region representing the three habitats of focus in this study, and an additional deep cypress habitat site at a

location which had been previously noted as being impacted by the altered hydrology of the Picayune

Strand due to its close proximity to the previously impacted areas (US Army Corps of Engineers 2004).

Each of the selected sites contained a vegetative community that was characteristic of those habitats. The

sites within the Fakahatchee Strand provided reference stations for this study which demonstrated natural

habitats and hydrology unaffected by the hydrologic alterations in the Picayune Strand, with exception of

the additional deep cypress site. These sites were compared against the restored and unrestored sites

located in the Picayune Strand. All sites in the Fakahatchee Strand were located along Jane’s Scenic

Drive, which is an extension of Stewart Boulevard bisecting both the Picayune and Fakahatchee Strands

on an east to west transect.

Survey Techniques

Fish were sampled using both active and passive capture methods to inventory present fish

species and determine species richness and relative abundance. Each sample location included Breder

trap sampling, and dip-netting. The survey data was recorded on a pre-prepared observation form

(Appendix A). Environmental data was obtained for each sampling event using a YSI Model 85 handheld

oxygen, conductivity, salinity, and temperature system (YSI 1998).

-24- Breder Traps

Many fish sampling techniques

can be both destructive to habitat and

difficult to conduct in areas with thick

wetland vegetation (Main et al. 2007;

Sargent and Carlson 1987). As Breder

traps have been shown to be a successful

technique in measuring absolute or

relative densities of wetland fish species (Ceilley et al. in press, Sargent and Carlson 1987), and they were

used in previous studies in the Picayune Strand (Ceilley et al. 2007, Ceilley 2008), this method was also

selected for use in this study. For sampling, ten of these plastic fish traps (Breder 1960) were deployed at

each sampling location when depths were sufficient to permit effective sampling (>2cm). Breder traps

were placed in a stratified pattern throughout the aquatic habitat to sample available fish habitat at each

location and to maximize capture efficiency (Sargent and Carlson 1987; Main et al. 1997; Ceilley et al.

1999). Following the protocol employed for the baseline assessment (Ceilley 2008), traps were

submerged for a period of one hour and then retrieved. The fish collected were field identified,

enumerated, and recorded on field sheets before being released live back into the water. Voucher

specimens for each fish species, along with any unidentified species in the field were anesthetized using a

solution of benzocaine and then preserved in 10% formalin and labeled by site and date to be brought

back to the FGCU lab for positive identification.

Dip Netting

Concurrent active dip net sampling was conducted by 2 persons with standard D-frame, 500

micron mesh dip nets in all of the major and minor micro-habitats (e.g. emergent and submerge

vegetation, snags, roots, algal mats, benthos, rocks, etc.) available at each site (Ross 1990; FDEP 1993).

Dip nets were selected for they were used in previous studies conducted in the Picayune Strand (Main et

al. 2007) and their relative ease of use and versatility in diverse habitats. “There are no definitive rules

about the number of sweeps needed to sample a habitat adequately…a reasonable procedure is to survey

-25- each aquatic habitat for an equal period of with an equal number of sweeps,” (Heyer et al. 1994). Dip

netting was standardized by sampling each represented habitat with three sweeps within each sampling

location. The net was vigorously worked through vegetation, open water, and/or surficial bottom

sediments. The net contents were then placed in a white pan and sorted with forceps. Fish were

identified, enumerated, and

recorded on field sheets before

being released live back into the

water. Any unidentified species in

the field were anesthetized using a

solution of benzocaine and then

preserved in 10% formalin and

labeled by site and date to be

brought back to the FGCU lab for

positive identification.

Data Analysis

Plymouth Routines in Multivariate Ecological Research, Volume 6 (PRIMER v6) was utilized to

perform analyses for the fish community data collected in this study (Clarke & Gorley 2006). Univariate

analysis was used for a comparison of treatments (reference, transitional, natural), for mean diversity

using several diversity indices, richness, and abundance. Non-parametric multivariate techniques were

also used to analyze the community data. All fish abundance data for this analysis were forth-root

transformed prior to analysis to down-weight the influence of extremely abundant species, (Clarke and

Warwick 2001; Ceilley 2008) which was consistent with previous studies. “Standardization is

appropriate for comparing data sets with varying levels of effort to obtain percent composition values for

individual taxa within a community (by samples) or percent composition of individual taxa between

communities (by variable) to identify relative distribution among habitat types and endemism where an

individual species or taxa is found only at one site or habitat type,” (Ceilley 2008). Differences in water

levels between reference, transitional, and impacted sites that allow for, or preclude the development of

-26- aquatic communities was considered as a primary variable to be monitored. Therefore, by weighting the

samples by averaging abundance and/or emphasizing percent composition between sites may mask

differences that may be ecologically important for measuring restoration of hydrology and sheetflow.

During scheduled wet season sampling events when dry conditions were observed (and therefore no

fishes were present) data were recorded as zero (0) fishes collected. Due to the small number of samples

collected at each site, data were pooled for each site for all sampling events for the overall analysis of site

conditions (Ceilley 2008). Data were also analyzed by sampling events to determine temporal trends, and

indicate adequate sampling frequencies over the sampling period.

Hierarchical agglomerative cluster analysis (using group average linking) based on Bray-Curtis

similarities was employed to examine natural groupings among samples and sampling locations. The

groupings produced in the cluster diagram were further evaluated for statistically significant evidence

(p<0.05) of genuine clusters in sample assemblages using a series of similarity profile (SIMPROF)

random permutation tests, a feature incorporated in PRIMER v6 (Clarke & Gorley 2006). Because

cluster analysis is best applied to communities responding to distinct environmental, non-metric

multidimensional scaling (MDS) was also employed. This statistical tool can also be based on Bray-

Curtis similarity matrices and has been recommended for use in examining communities structured by

environmental gradients (Clarke & Warwick 2001; Clarke & Gorley 2006). In the case of MDS, plots

may be considered useful for interpretation if associated stress values are >0.2 (Kruskal 1964).

Analysis of similarity (ANOSIM), a non-parametric analog of analysis of variance was used to

test for a priori groupings of samples. When using ANOSIM, global tests were considered significant at

p<0.05 and pair-wise tests were interpreted using the absolute value of the associated R statistics (Clarke

& Warwick 2001). Relative contributions, in terms of relative abundance were graphically displayed as

overlays on MDS plots to illustrate relative importance of ordination.

Additional statistical analysis was conducted on the data set using trajectories to observe temporal

fluctuations in species assemblages within and between sites through multivariate space. Given the

cyclical nature of inundation in south Florida landscape, cyclic season patterns in fish community were

observed in sites that provided a period of inundation long enough for analysis. The sampling frequency

-27- of this study allowed for the detection of changes in community structure through the period of

inundation as well as the presence/absence of individual taxa known to be indicators of wetland condition.

This information allowed for the observations of patterns to inform wetland restoration science of what

time of year was the most appropriate for collecting a mature wetland fish community, how wetland fish

communities change from the beginning of the wet season through the dry-down, and what sampling

frequency was necessary for obtaining an accurate representation of the temporal changes in community

structure and diversity metric s.

Baseline studies show high dissimilarity in fish communities at impacted sites and high similarity

between reference sites, suggesting that disturbance of sheetflow causes dispersion, or high dissimilarity

of fish communities even from like habitats. This dispersion observed in the baseline study at most of the

impacted wetland areas should show a decrease over time in transitional areas as wetlands are restored.

The analysis in this study allows for the observation of this trend at a critical time in the Picayune Strand

Restoration Project process that is currently not being evaluated.

-28-

RESULTS

A total of 8 sites were successfully sampled for fish including two impacted sites, three

transitional sites, and three reference sites (Table 1). Two sites, impacted cypress and transitional

gramminoid, did not have a long enough period of inundation, or enough hydrologic connectivity to

support a fish community, and therefore did not provide fish community data. These sites were

eliminated from analysis as they did not provide sufficient data for this study. A total of 18 sampling

events were conducted from September 2011 through March 2012 where adequate standing water was

available. All 8 sites were successfully sampled a minimum of 1 time during the sampling period as

water levels allowed (Table 2). For potential use in future research projects, a raw data set for all

sampling events are provided in Appendix B. A total of 15 species were documented within the study

area by use of the sampling methods described.

Table 1. Site descriptions with locations and adjacent wells. Sites highlighted in red have been eliminated from the study

due to lack of water and a samplable fish community.

Adjacent Well Restoration Phase Habitat Type Location

T3W1 Impacted Gramminoid Picayune

T3W2 Impacted Cypress Gramminoid Picayune

T3W2 Impacted Deep Cypress Picayune

T3W5 Transitional Gramminoid Picayune

T3W5 Transitional Cypress Gramminoid Picayune

T3W5 Transitional Deep Cypress Picayune

T3W7 Transitional Deep Cypress Fakahatchee

- Reference Gramminoid Fakahatchee

- Reference Cypress Gramminoid Fakahatchee

- Reference Deep Cypress Fakahatchee

-29- Table 2. This table shows the presence/absence of water in each sampling site per month. Cells highlighted in blue

represent inundated sites that held standing water and a samplable fish community during the represented month. Blank

cells represent dry sites without a samplable fish community.

Site September October November December January February March

Impacted Cg X X

Impacted C

X

Impacted G X X

Transitional C X X X

Transitional Cg X X

Transitional G X X

Transitional C2 X X X X X X

Reference C X X X X X X X

Reference Cg X X X X

X

Reference X X X X

Fish Community Data

Table 3. This table shows fish species documented via Breder traps and dip-net sampling by scientific name, common

name, and species code abbreviations.

Scientific Name Common Name Species

Code

Capture

Method

Belonesox belizanus Pike killifish PKKF Breder

Cichlasoma urophthalmus Mayan cichlid CIUR Breder

Elassoma evergladei Everglades pygmy sunfish ELEV Dip Net

Fundulus chrysotus Golden topminnow FUCR Breder

Fundulus confluentus Marsh killifish FUCO Both

Gambusia holbrooki Mosquito fish GAHO Both

Hemichromis letourneuxi Jewelfish cichlid HELI Breder

Heterandria formosa Least killifish HEFO Breder

Jordonella floridae Flagfish JOFL Both

Lepomis macrochirus Bluegill LEMAC Breder

Lepomis marginatus Dollar sunfish LEMAR Breder

Lepomis sp. (juv) Sunfish juvenile LESP Both

Lucania goodei Bluefin killifish LUGO Breder

Notropis petersoni Coastal shiner NOPE Breder

Poecilia latipinna Sailfin molly POLA Breder

A total of 15 species were documented through this study using Breder traps (Breder 1960) and

dip-netting. Each species was identified in the field and released alive unless positive identification was

required, upon which they would be preserved and brought to the FGCU laboratory for further

investigation. The fish species abbreviations for each fish species were based on the first two letters of

-30- the Genus and species in each scientific name. These species abbreviation are used in various graphs and

figures throughout the remainder of this report (Table 3).

Species Richness

To begin evaluating status and trends of fish communities, species richness was determined.

Table 4 summarizes fish species that were documented at least once in each of the represented restoration

phases. Impacted study sites contained a total of two species, the Transitional study sites contained a total

of 6 species, and reference sites contained a total of 15 species. A total of 15 fish species were collected

representing 7 families including 1 non-native family and 3 non-native species. The most abundant

species by far was the eastern mosquito fish (Gambusia holbrooki) (n = 2965) which was found at all

locations where fish were collected. Also abundant in some locations was the marsh killifish (Fundulus

confluentus) (n = 208), Florida flagfish (Jordonella floridae) (n = 107), and juvenile sunfish (Lepomis sp.

(juv)) (n = 30).

Table 4. This table shows fish species documented in each restoration phase listed by scientific name. An ‘X’ shows that a

fish species was present at a site at some time within the period of this study.

Scientific Name Impacted Transitional Reference

Belonesox belizanus X X

Cichlasoma urophthalmus X

Elassoma evergladei X

Fundulus chrysotus X

Fundulus confluentus X X X

Gambusia holbrooki X X X

Hemichromis letourneuxi X

Heterandria formosa X

Jordanella floridae X X

Lepomis macrochirus X

Lepomis marginatus X

Lepomis sp. (juv) X X

Lucania goodei X X

Notropis petersoni X

Poecilia latipinna X

Total Documented Species 2 6 15

Univariate diversity metrics were conducted for each of the sampling locations in all restoration

phases. The metrics applied to this analysis included six different indices: richness (species richness (R)

-31- and relative abundance (A)), evenness (Margalef’s (d) and Pielou’s indices (J’)), and heterogeneous

measures (Shannon-Weaver index (H’(loge)) and Simpson’s diversity index (1-Lambda’). Total

documented species for each site ranged from a low of 1 species at the impacted gramminoid site, to a

high of 13 species at the reference cypress gramminoid site (Table 5). The mean species richness was 1.5

for impacted sites, 3.3 for transitional sites, and 8.0 for reference sites. In general, the greatest diversity

values were reported in reference sites. Figure 7 illustrates the average species richness and relative

abundance of fishes within each restoration phase calculated from the values described in Table 5. The

lowest species richness and relative abundance values were documented in impacted sites. Relative

abundance was the greatest within transitional sites while diversity was greatest within reference sites

Table 5. : Univariate diversity indices including species richness (R) and relative abundance (A), Margalef’s (d), Pielou’s

indices (J’), Shannon-Weaver index (H’(loge), and Simpson’s diversity (1-Lambda’) for fish documented with highest

values for each region in bold and lowest value underlined are represented in this table. The highest diversity values were

found in reference sites and the highest abundance values were found in the transitional cypress site ‘C2’. The lowest

abundance and diversity values were found in impacted sites.

Diversity Metrics

Sample Species Abundance Margelef Pielou Shannon Simpson

S N d J' H'(loge)

1-

Lambda'

Impacted Cg 2.0 28.0 0.3 0.4 0.3 0.1

Impacted G 1.0 14.0 0.0

**** 0.0 0.0

Transitional C 3.0 376.0 0.3 0.0 0.0 0.0

Transitional Cg 2.0 108.0 0.2 0.1 0.1 0.0

Transitional C2 5.0 1574.0 0.5 0.1 0.1 0.0

Reference C 7.0 962.0 0.9 0.3 0.5 0.2

Reference Cg 13.0 442.0 2.0 0.5 1.4 0.7

Reference G 4.0 183.0 0.6 0.5 0.7 0.4

-32-

Figure 7. This is a graphic representation of average species richness and relative abundance of fishes within each restoration phase

documented during sampling. Species richness was highest in Reference sites, and relative abundance was highest at transitional site

‘C2’, followed by reference sites and transitional site ‘C’. Lowest values were observed in impacted sites.

Figure 8 illustrates the average diversity metric values (Margalef’s (d), Pielou’s indices (J’),

Shannon-Weiver (H’(loge)), and Simpson’s diversity (1-Lambda’)) of fishes within each restoration

phase calculated from the values described in Table 5. Diversity metrics were generally highest in

reference sites with the exception of the impacted cypress gramminoid site. Transitional sites showed a

general increase in diversity metric values from transitional sites, and reference sites showed a general

increase from transitional sites. Elevated diversity metrics in the impacted gramminoid site may have

been driven by the low relative abundance of species.

0.0

2.0

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10.0

12.0

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600.0

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1600.0

Spe

cie

s R

ich

ne

ss

Re

lati

ve A

bu

nd

ance

Total Abundance (N) Species Richness (S)

-33-

Figure 8. Average diversity metrics including Margalef’s (d), Pielou’s indices (J’), Shannon (H’(loge)), and Simpson (1-

Lambda’) for each of the represented sampling sites are shown in this graph. The highest values were found in reference

sites.

Relative Abundance

Fish species documented in this study are classified as small omnivorous fishes that move

through the hydrologic landscape via active dispersal (Trexler 2010; Main et al. 2007). Hydrologic

connectivity and distance shape the patterns of fish dispersal over the south Florida landscape which

impacts the relative abundance of species at locations of varying distance from source populations (Ruetz

et al. 2005). The relative abundance of fish species documented via sampling in this study was analyzed

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

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ive

rsit

y M

etr

ic V

alu

es

Sample Sites Margelef (d) Pielou (J') Shannon (H'(loge)) Simpson (1-Lambda')

-34-

Table6. The above table shows the total relative abundance values per restoration phase, total abundance for all species,

and total percent composition of fish species documented during the sampling period. The most abundant species

included Gambusia holbrooki and Fundulus confluentus, and the least abundant species included Cichlasoma

urophthalmus and Notropis petersoni. Gambusia holbrooki composed the majority of all species observed.

Relative Abundance

Scientific Name Species Impacted Transitional Reference Total

Belonesox belizanus PKFF 0 3 2 5

Cichlasoma urophthalmus CIUR 0 0 1 1

Elassoma evergladei ELEV 0 0 2 2

Fundulus chrysotus FUCR 0 0 2 2

Fundulus confluentus FUCO 2 20 186 208

Gambusia holbrooki GAHO 31 2027 907 2965

Hemichromis letourneuxi HELI 0 0 15 15

Heterandria formosa HEFO 0 0 6 6

Jordanella floridae JOFL 0 6 101 107

Lepomis macrochirus LEMAC 0 0 2 2

Lepomis marginatus LEMAR 0 0 4 4

Lepomis sp. (juv) LESP 0 1 29 30

Lucania goodei LUGO 0 1 1 2

Notropis petersoni NOPE 0 0 1 1

Poecilia latipinna POLA 0 0 13 13

Total Documented Species 33 2058 1272 3363

Percent Composition

Scientific Name Species Impacted Transitional Reference

Belonesox belizanus PKFF 0.00% 0.15% 0.16%

Cichlasoma urophthalmus CIUR 0.00% 0.00% 0.08%

Elassoma evergladei ELEV 0.00% 0.00% 0.16%

Fundulus chrysotus FUCR 0.00% 0.00% 0.16%

Fundulus confluentus FUCO 6.06% 0.97% 14.62%

Gambusia holbrooki GAHO 93.94% 98.49% 71.31%

Hemichromis letourneuxi HELI 0.00% 0.00% 1.18%

Heterandria formosa HEFO 0.00% 0.00% 0.47%

Jordonella floridae JOFL 0.00% 0.29% 7.94%

Lepomis macrochirus LEMAC 0.00% 0.00% 0.16%

Lepomis marginatus LEMAR 0.00% 0.00% 0.31%

Lepomis sp. (juv) LESP 0.00% 0.05% 2.28%

Lucania goodei LUGO 0.00% 0.05% 0.08%

Notropis petersoni NOPE 0.00% 0.00% 0.08%

Poecilia latipinna POLA 0.00% 0.00% 1.02%

-35- in an attempt to identify dominant fish species, species composition similarities among restoration phases,

and potential indicator species. Table 6 shows the total abundance values for each individual fish species

documented during this study. This table shows the total relative abundance for each species per

restoration phase, as well a total relative abundance for all sampling events and total percent composition

of each species per restoration phase.

Figure 9 illustrates the total percent composition for each fish species over the entire sampling

period. This was determined by totaling the relative abundance of each fish species over the entire

sampling period and calculating the overall percentage.

The fish species abbreviations in Figure 8 are described in Table 2 and Table 6. Gambusia

holbrooki (88 percent) comprised the overwhelming majority of species collected during this study and

was the most abundant of documented species. Other abundant species observed included Fundulus

confluentus (6 percent) and Jordonella floridae (3 percent). All other documented species comprised less

than 1 percent of the total collected specimen.

Figure 9. The above figure shows percent composition of species documented via sampling efforts based on overall relative abundance in

the form of a pie chart. Gambusia holbrooki makes up the majority of all fish collected during the sampling period.

0.03%

0.45% 0.06% 6.18% 0.06%

0.15%

88.17% 0.18%

0.39% 3.18% 0.89%

0.06%

0.12% 0.03%

0.06%

11.83%

CIUR

HELI

FUCR

FUCO

LUGO

PKKF

GAHO

HEFO

POLA

JOFL

LESP

LEMAC

LEMAR

88

-36- Abundance data for fish species was used to observe similarities and difference between and

among sites in the various restoration phases. Similarity percentage tests (SIMPER) for reference,

transitional, and impacted sites are included in Table 7. Reference sites averaged 58% similarity (32%

dissimilarity) of fish community structure, with 4 species contributing >91% of the similarity between

reference sites. Transitional sites averaged 63% similarity (37% dissimilarity) of fish community

structure, with 2 species contributing to 100% of the similarity between transitional sites. Of the two

sampled impacted sites, there was an average of 72% similarity (28% dissimilarity) of fish community

structure, with 1 species contributing to 100% of the similarity between impacted sites. Of the 12 species

that contributed to the >90% dissimilarity between sites of different restoration phases, the average

abundances of 10 species were higher in reference wetlands than in transitional wetlands including many

considered as indicators of wetland hydrology (Main et al. 2008), and the lowest average abundance of

various species was found in impacted wetland. These indicators include the Florida flagfish, marsh

killifish, mosquito fish, and sailfin molly which made the largest contribution to dissimilarity between

sites of varying restoration phases.

Using this data, a cluster analysis dendrogram was prepared of all sample locations with data

from all sampling events to illustrate natural groupings between fish communities based on Bray-Curtis

Similarity of fish abundance data (Figure 10). The restoration phase is provided by the symbol used to

represent each site and each habitat type is represented by the letters beneath. The cluster analysis

identified two main clusters. Reference and transitional sites grouped together in clusters that were

greater than 50 percent similar and impacted sites grouped together in a cluster that was greater than 70

percent similar. The less distinct groupings of transitional sites and reference sites may be a function of

other influences including the advanced restoration and geographic location of transitional site C2. The

measure of relative abundance were also assessed for each of the sampling sites using composite data

from all sampling events by compiling non-metric MDS ordination to illustrate the relative similarity

between fish communities based on restoration phase and habitat type (Figure 11).

The restoration phase is provided by the symbols used to represent each site, and the habitat type

is represented by the habitat letter codes assigned to each symbol. The results of the MDS ordination

-37- show distinct groupings of the sample locations among like restoration phases where impacted sites are in

one group and transitional and reference sites group together in another distinct group.

Figure 11 is a 2-dimensional (2D) ordination that was reduced from a 3-dimensional (3D)

ordination for incorporation into this document. The 2D stress value was 0.03 which provides a useful

image (Clarke & Warwich 2001). The 3D stress value was 0.01, which provides an even more support

for the flattened 2D image.

Figure 10. The above figure is a cluster analysis showing restoration phase and habitat type using Bray-Curtis similarity

from all samples. The cluster analysis shows two distinct groupings of sites at 95% confidence (p<0.05) with three

additional sub-groupings. Major groupings include a cluster of impacted sites, transitional sites ‘C’ and ‘Cg’, and

reference site ‘G’ at about 50% similarity. The second major cluster shows reference sites ‘C’ and ‘Cg’ as well as

transitional site ‘C2’ at >50% similarity. Sub groupings include impacted sites at >70% similarity in one group,

transitional sites ‘C’ and ‘Cg’ with reference site ‘G’ at >60% similarity in the second group, and transitional site ‘C2’

with reference sites ‘C’ and ‘Cg’ in a third sub grouping.

-38-

Figure 11. The above figure is an MDS ordination with restoration phase and habitat type for each sampling site using

Bray-Curtis similarity from all sampling events. The ordination through multivariate space is based on similarity values

and places points in a three-dimensional multivariate space. The three dimensional image has been compressed into a 2D

representation in order to represent the multivariate ordination effectively.

Potential Indicator Species

The MDS ordination was overlaid with superimposed total abundance data for each species that

had significant impact on the on the similarities and differences among and between sampling sites in an

attempt to identify “indicator species” of restoration success (Table 7). The scales for the MDS

ordinations with superimposed fourth root abundance figures represent 5 species including Gambusia

holbrooki, Jordonella floridae, Fundulus confluentus, Lepomis sp., and Poecilia latipinna. These species

made up most of the dissimilarity among various restoration phases in the SIMPER test due to their high

occurrence in reference sites, moderate occurrence in transitional sites, and low occurrence and/or

absence in impacted sites. Figures 12-16 are MDS ordination figures with superimposed fourth root

abundances for the 5 species attributing the most dissimilarity between sites of different restoration

phases. The highest overall abundances for all species except Gambusia holbrooki were documented in

reference sites, while G. holbrooki was most abundant in transitional sites.

-39- Table 7. SIMPER analysis showing the species contributing to similarity within groups (A) and the dissimilarity between groups (B).

Impacted sites: T3W1-Cg, T3W1-G. Transitional Sites: T3WS-C, T3WS-Cg, FS-C2. Reference sites: FS-C, FS-Cg, FS-G.

Cumulative species contributions cut off at 90%. Species codes shown in Table 3. The information shown in this table was used for

identifying potential indicator species.

B Impacted and Transitional Sites: Average Dissimilarity = 52.79 Species Impacted

Abundance Transitional Abundance

Average Dissimilarity

Dissimilarity/ SD

Percent Contribution

Cumulative Percent

GAHO 2.1 4.63 23.43 3.35 44.38 44.38

JOFL 0 1.17 12.34 3.14 23.38 67.76

FUCO 0.59 0.70 8.06 1.14 15.27 83.03

LUGO 0 0.33 3.69 0.64 7.00 90.03

Impacted and Reference Sites: Average Dissimilarity = 68.56

Species Impacted Abundance

Transitional Abundance

Average Dissimilarity

Dissimilarity/ SD

Percent Contribution

Cumulative Percent

JOFL 0 2.4 14.02 2.98 20.45 20.45

GAHO 2.1 4.19 11.86 2.23 17.30 37.75

FUCO 0.59 2.74 11.48 2.45 16.75 54.49

POLA 0 1.05 6.27 1.15 9.15 63.64

LESP 0 1.28 5.97 1.18 8.71 72.35

FUCR 0 0.67 3.06 1.24 4.46 76.81

HELI 0 0.66 2.51 0.65 3.67 80.48

LEMAC 0 0.40 2.12 0.64 3.09 83.56

ELEV 0 0.40 2.12 0.64 3.09 86.65

HEFO 0 0.52 2.00 0.65 2.92 89.56

LEMAR 0 0.47 1.81 0.65 2.63 92.20

Transitional and Reference Sites: Average Dissimilarity = 47.77

Species Impacted Abundance

Transitional Abundance

Average Dissimilarity

Dissimilarity/ SD

Percent Contribution

Cumulative Percent

JOFL 0.70 2.74 9.42 1.83 19.72 19.72

GAHO 4.63 4.19 5.65 1.36 11.82 31.54

FUCO 1.17 2.40 5.58 3.15 11.69 43.23

POLA 0 1.05 4.83 1.20 10.10 53.33

LESP 0.33 1.28 4.73 1.28 9.90 63.23

FUCR 0 0.67 2.52 1.27 5.27 68.50

PKKF 0.44 0.40 2.17 0.85 4.55 73.05

HELI 0 0.66 2.14 0.66 4.47 77.52

LUGO 0.33 0.33 1.93 0.78 4.04 81.56

LEMAC 0 0.40 1.70 0.66 3.57 85.12

ELEV 0 0.40 1.70 0.66 3.57 88.69

HEFO 0 0.32 1.70 0.66 3.56 92.24

A Impacted Sites: Average Similarity = 71.89 Species Average Abundance Average Similarity Similarity/SD Percent

Similarity Cumulative

Percent GAHO 2.1 71.89 - 100 100

Transitional Sites: Average Similarity = 63.39

Species Average Abundance Average Similarity Similarity/SD Percent Similarity

Cumulative Percent

GAHO 4.63 48.76 4.77 76.93 76.93

JOFL 1.17 14.62 3.81 23.07 100.00

Reference Sites: Average Similarity = 58.30

Species Average Abundance Average Similarity Similarity/SD Percent Similarity

Cumulative Percent

GAHO 4.19 23.43 3.35 38.69 38.59

JOFL 2.40 12.34 3.14 25.33 64.02

FUCO 2.74 8.06 1.14 23.08 87.10

LESP 1.28 3.69 0.64 5.01 92.12

-40-

Figure12. The above figure is an MDS ordination with superimposed raw abundance for Gambusia holbrooki.

Abundance for Gambusia holbrooki was highest in the transitional ‘C2’ site, followed by the reference sites.

Figure 13. The above figure is an MDS ordination with superimposed raw abundance data for Fundulus confluentus.

Abundance data for Fundulus confluentus was highest in reference sites followed by the transitional site ‘C2’.

-41-

Figure 14. The above figure is an MDS ordination with superimposed raw abundance data for Jordonella floridae.

Abundance data for Jordonella floridae was highest at reference sites, followed by transitional sites. Jordonella floridae

was absent in impacted sites.

Figure15. The above figure is an MDS ordination with superimposed raw abundance data for Lepomis spp. This species

was most abundance in reference sites ‘C’ and ‘Cg’, followed by transitional site ‘C2’. Lepomis spp. were absent in all

other sites during the sampling period.

-42-

Sampling Frequencies

Sampling data used to create the MDS ordination was then observed at various levels of

organization in an attempt to identify sampling frequencies that most effectively measure fish

communities in comparison to monthly sampling. Data was divided into three subsets using composite

data from various months in order to represent different sampling frequencies over the sampling period.

Subsets included every month (September- March), every other month (September, November, January,

March), and every third month (September, December, March) for comparison with the MDS ordination

representing the complete set of composite fish community data.

Figure16. The above figure is an MDS ordination with superimposed raw abundance data for Poecilia latipinna.

This species was only found in reference sites ‘C’ and ‘G’ and was absent in all other sites through the sampling

period.

-43-

Figure 17. The above figure is an MDS ordination including composite fish community data from the entire sampling

period including the months of September, October, November, December, January, February, and March. The MDS

ordination shows groupings of sites of various restoration condition and habitat type when sampled every month during

the period of inundation.

Figure 18. The above figure is an MDS ordination including composite fish community data from every other month of

sampling including the months of September, November, January, and March The MDS ordination shows groupings of

sites of various restoration condition and habitat types when sampled every other month during the period of inundation.

This ordination was observed in comparison to an ordination representing a sampling frequency of every month through

the period of inundation.

-44-

Figure 19. The above figure is an MDS ordination including composite fish community data from every third month of

sampling including the months of September, December, and March. The MDS ordination shows groupings of sites of

various restoration phases and habitat types when sampled every third month during the period of inundation. This

ordination was observed in comparison to an ordination representing a sampling frequency of every month during the

period of inundation.

Temporal Community Structure

MDS ordinations were produced from subsets of sampling data that were broken down

further into monthly composites of fish community data in an attempt to observe the time of year

most appropriate for collecting samples from a mature fish community. Monthly MDS

ordinations were observed in comparison with the composite fish community MDS ordination

over the entire sampling period for this study. Monthly MDS ordinations that most closely

resemble that of the MDS ordinations from the complete data set would provide an example of a

mature fish community from which to sample. It should be noted that the only months during

which every samplable site held enough water to support a fish community were the months of

October and November. All other months contained at least one site that did not hold enough

water to support a samplable fish community. The months of October and November are

represented in the MDS ordinations pictured below (Figure 20, Figure 21). Monthly MDS

ordinations for all other months and restoration phases may be found in Appendix D.

-45-

Figure 20. The above figure is an MDS ordination including fish community data from the month of October at all

samplable sites. The MDS ordination shows groupings of sites of various restoration phases and habitat types during the

month of October. The ordination was then observed in comparison to an MDS ordination representing all sampling sites

sampled through the entire sampling period.

Figure21. The above figure is an MDS ordination including fish community data from the month of November at all

samplable sites. The MDS ordination shows groupings of sites of various restoration phases and habitat types during the

month of October. The ordination was then observed in comparison to an MDS ordination representing all sampling sites

sampled through the entire sampling period.

-46-

Fish Community Structure

To observe patterns of fish community change throughout the period of inundation and among

sites of different restoration phases, abundance data was analyzed in a visual representation using

Microsoft Excel (Table 7). Community data was also represented in MDS ordinations showing the

relative abundance of the three species having the greatest impact on differences between sites of

different restoration phases (Gambusia holbrooki, Jordonella floridae, Fundulus confluentus) over the

sampling period. MDS ordinations for reference sites are pictured below (Figure 22, Figure 23, Figure

24).

Table 8. The above table shows relative abundance data for Gambusia holbrooki, Jordonella floridae, and Fundulus

confluentus over the entire sampling period with dry months represented in yellow. The table is divided into two

sampling methods including Breder traps and dip netting. The table was used to observe changes in abundance and

community structure throughout the entire sampling period from month to month.

Monthly relative abundance data for these three species having the greatest impact on

dispersion patterns through multivariate space were observed to identify temporal patterns of

Impacted Transitional Reference No Water

Dip NetBreder

Site September October November December January February March Site September October November December January February March

T3W1-Cg 7 16 T3W1-Cg 1 2

T3W2-C T3W2-C

T3W1-G 1 3 T3W1-G 1

T3W5-C 4 325 T3W5-C 45

T3W5-Cg 7 96 T3W5-Cg 3

T3W5-G T3W5-G

FS-C2 8 4 16 679 752 FS-C2 51 37

FS-C 7 111 23 413 FS-C 3 4 3 95

FS-Cg 2 23 9 4 95 16 FS-Cg 3 8 2

FS-G 2 36 34 6 FS-G 1 1 6

Mosquito Fish (Gambusia holbroki)

Site September October November December January February March Site September October November December January February March

T3W1-Cg T3W1-Cg

T3W2-C T3W2-C

T3W1-G T3W1-G

T3W5-C 1 T3W5-C

T3W5-Cg 2 T3W5-Cg

T3W5-G T3W5-G

FS-C2 1 FS-C2 2

FS-C 1 3 8 11 4 FS-C 1 1

FS-Cg 13 27 FS-Cg 4

FS-G 1 11 16 FS-G

Flagfish (Jordanella floridae)

Site September October November December January February March Site September October November December January February March

T3W1-Cg 2 T3W1-Cg

T3W2-C T3W2-C

T3W1-G T3W1-G

T3W5-C T3W5-C

T3W5-Cg T3W5-Cg

T3W5-G T3W5-G

FS-C2 6 11 FS-C2 3

FS-C 1 25 18 24 FS-C 1

FS-Cg 1 1 3 93 5 FS-Cg 3

FS-G 1 1 9 FS-G

Marsh Killifish (Fundulus confluentus)

-47-

changes in fish community structure through the dry down. Species abundance data for

Gambusia holbrooki, Jordonella floridae, and Fundulus confluentus showed general patterns of

increased abundance from inundation through the dry down. Gambusia holbrooki was the only

species ubiquitous to all sites where fish were captured. Earlier colonizers in reference and

transitional sites included Gambusia holbrooki and Jordonella floridae. Total abundance of these

species also varied among sites of varying restoration phases. Reference sites had a higher

abundance of all represented species throughout the period of inundation and especially during

sampling events occurring during the dry down.

Figure 22. The above figure is an MDS ordination representing reference sites of various habitat types sampled each

month (indicated by month codes 9,10,11,12,1,2,and 3) during the entire sampling period with superimposed relative

abundance data for Gambusia holbrooki for each represented month when sampling occurred.

-48-

Figure23. The above figure is an MDS ordination representing reference sites of various habitat types sampled each

month during the entire sampling period with superimposed relative abundance data for Jordonella floridae for each

represented month when sampling occurred.

Figure24. The above figure is an MDS ordination representing reference sites of various habitat types sampled each

month during the entire sampling period with superimposed relative abundance data for Fundulus confluentus for each

represented month when sampling occurred.

-49- Temporal Dispersion

Temporal trajectories were superimposed over monthly MDS ordinations of fish community data

for each restoration phase in order to observe temporal dispersion patterns through multivariate space

(Figure 25, Figure 26). Trajectories for restoration phases with sufficient data follow a similar dispersion

pattern through multivariate space over the period of inundation. Dissimilarity between consecutive

months for each restoration phase was observed through SIMPER analysis which is displayed in Table 8.

Reference sites show a decrease in dissimilarity (increase in similarity) from September until January

when the dissimilarity slightly increases. Impacted and Transitional sites do not show apparent patterns

of changing dissimilarity between consecutive months. Average dissimilarity between months was

lowest in impacted sites, increased in transitional sites, and was highest in reference sites. These

differences are displayed graphically on the MDS ordination trajectories and with a table of SIMPER

analysis dissimilarity values from month to month.

Figure25. The above figure is an MDS ordination of monthly composite fish community data in transitional sites with

superimposed trajectory to observe movement through multidimensional space and changes in community structure

through the period of inundation.

-50-

Figure26. The above figure is an MDS ordination of monthly composite fish community data in reference sites with

superimposed trajectory to observe movement through multidimensional space and changes in community structure

through the period of inundation.

Table 9. The table below shows dissimilarity values between consecutive months within various restoration phases and

average dissimilarity for each restoration phase through the entire sampling period to observe changes in community

structure through the period of inundation. An ‘X’ represents a lack of sufficient data to calculate dissimilarity values

due to the dry down of sampling locations.

Monthly Dissimilarity

Impacted Transitional Reference

September

x x 62.67

October

22.93 36.06 43.18

November

x 20.35 31.75

December

x 33.77 18.41

January

x 18.04 21.05

February

x x 19.49

March

Average Dissimilarity 22.93 27.06 32.76

-51-

Restoration Success

Temporal composite data for each of the sampled sites was analyzed using an MDS ordination

and a cluster analysis with SIMPER in order to observe fish communities at sites in each conditional

replicate and whether transitional sites undergoing restoration are moving from impacted conditions

towards minimally impacted reference conditions. The MDS ordination shows three groupings of sites of

the similar restoration condition. All sites displayed had <42% similarity overall. Using SIMPER

analysis impacted sites showed 72% similarity, transitional sites showed 63% similarity, and reference

sites showed 58% similarity. Cluster analysis showed impacted sites in a distinct grouping. A second

grouping contained two transitional sites and one reference site all having >60% similarity. The third

apparent grouping contained one transitional site and two reference sites that show >50% similarity with

sites of other conditions.

Figure 27. The above figure is a cluster analysis showing restoration phase and habitat type using Bray-Curtis similarity

from all samples. The cluster analysis shows two distinct groupings of sites with three additional sub-groupings. Major

groupings include a cluster of impacted sites, transitional sites ‘C’ and ‘Cg’, and reference site ‘G’ at about 50%

similarity. The second major cluster shows reference sites ‘C’ and ‘Cg’ as well as transitional site ‘C2’ at >50%

similarity. Sub groupings include impacted sites at >70% similarity in one group, transitional sites ‘C’ and ‘Cg’ with

reference site ‘G’ at >60% similarity in the second group, and transitional site ‘C2’ with reference sites ‘C’ and ‘Cg’ in a

third sub grouping.

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Figure28. The above figure is an MDS ordination of composite fish community data for each sampling site representing

various restoration phases and habitat types through the entire sampling period using Bray-Curtis similarity from all

sampling events. The ordination through multivariate space is based on similarity values and places points in a three-

dimensional multivariate space. The three dimensional image has been compressed into a 2D representation in order to

represent the multivariate ordination effectively.

Figure29. The above figure is an MDS ordination of composite fish community data for each sampling site representing

various restoration phases and habitat types through the entire sampling period including superimposed similarity

groupings of 40 percent, 50 percent, and 60 percent similarity. Impacted sites show a distinct grouping at 50 percent and

all transitional and reference sites group together at 40 percent similarity.

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DISCUSSION

The primary focus of this study was to evaluate the success of the restoration activities in the

Picayune Strand by obtaining and analyzing fish species richness and relative abundance in

relation to various phases of restoration. Fish communities provide a means for evaluating the

effects of changing hydrologic conditions on the ecological health of wetlands (Main et al.

2007). Therefore, the biological health of the various restoration phases in the Picayune Strand

was measured by accessing the status and trends of fish populations.

The fish species richness and relative abundance data collected via active and passive

sampling techniques varied among sites of different restoration phases. Total abundance was

highest in transitional sites and lowest in impacted sites, while species richness was highest in

reference wetlands and lowest at impacted sites. The higher species richness values within

reference wetlands were expected because the associated hydrology and habitat quality are

greater and more natural than that of the impacted and transitional sites. It should also be noted

that 12 of the 15 species identified in this study had a higher relative abundance than in sites of

both impacted and transitional restoration phases. These findings support previous research that

fish can serve as performance indicators to monitor hydrologic restoration (Main et al. 2007).

The results of the cluster analysis showed two major clusters with one cluster containing

two groups of <50% similarity (Figure 9). Of the two main clusters, the most similar cluster was

comprised of two reference sites (cypress and cypress gramminoid) and one transitional site

(cypress). The overall cluster analysis showed that the transitional restored sites grouped

primarily with natural reference sites, but shares <50% similarity with impacted un-restored

sites. It appears that restored sites are transitioning between impacted un-restored sites towards a

more natural reference condition.

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The non-metric MDS ordination showed distinct groupings of the sample locations

within natural reference wetlands, transitional recently restored areas, and impacted un-restored

areas as each restoration phase appeared close together in 2D space (Figure 29). SIMPER

analysis showed that Reference sites were the least similar (58 percent), transitional sites were

slightly more similar (63 percent), and impacted sites were the most similar (72 percent). This

may be a result of the low species richness found at impacted sites, and increasing species

richness in transitional and reference sites. SIMPER analysis also showed that impacted and

transitional sites were 53 percent dissimilar, impacted and reference sites were 69 percent

dissimilar, and transitional and reference sites were 48 percent dissimilar. This indicated that

transitional sites are more similar to reference sites than they are with impacted sites. This can

be observed on the MDS ordination as impacted sites tightly group in the upper left, transitional

sites group in the center, and reference sites group in the upper right of the multidimensional

space. This may indicate that transitional sites are moving towards reference conditions, at least

within multidimensional ecological space.

Breder trap and dip-net sampling provided data on the fish communities found in the

Picayune Strand which was identified as necessary data by the DOI (2004) Science Plan.

Sampling identified a total of 15 fish species throughout the Picayune and Fakahatchee Strands.

All 15 species were found in reference wetlands, six species were found within transitional

restored sites, and two species were found within impacted un-restored sites. Native species

found only in reference wetlands included the Everglades pygmy sunfish (Elassoma evergladei),

golden topminnow (Fundulus chrysotus), least killifish (Heterandria formosa), spotted sunfish

(Lepomis macrochirus), dollar sunfish (Lepomis marginatus), coastal shiner (Notropis

petersoni), and sailfin molly (Poecilia latipinna). Native species also found in transitional sites

included marsh killifish (Fundulus confluentus), mosquito fish (Gambusia holbrooki), Florida

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flagfish (Jordonella floridae), juvenile sunfish species (Lepomis sp. juv), and bluefin killifish

(Lucania goodei) of which only mosquito fish and marsh killifish were also found in impacted

un-restored sites. Exotic species found in reference sites included the pike killifish (Belonesox

belizanus) which was also found in transitional sites, Mayan cichlid (Cichlasoma urophthalmus),

and jewelfish cichlids (Hemichromis letourneuxi). Fish populations in the Everglades are driven

mostly by hydroperiod (Ruetz 2005). Two exceptions to this general pattern are Jordonella

floridae and Gambusia holbrooki as their primary mechanism for synchronous population

dynamics is dispersal due to their higher tolerance for low dissolved oxygen and harsh

conditions. Poecilia latipinna is another species that can tolerate low dissolved oxygen and

harsh water conditions, though they are not known for using active dispersal as a primary means

of recolonization. The majority of species rely on dry season refugia to repopulate inundated

areas through the wet season. It should be noted that Fundulus confluentus is also a rapid

colonizer of newly inundated areas through active dispersal (Trexler 2010).

Species found in sites of varying restoration phases can be associated with these species

specific dispersal patterns. Impacted sites contained only species that use active dispersal as a

recolonization strategy, indicating a lack of connected deep-water refugia to newly inundated

areas of the represented natural habitats (cypress, cypress gramminoid, gramminoid).

Transitional sites contained these species as well as species that require dry season refugia in

order to recolonize inundated areas including Lepomis species and Lucania goodei which may

indicate greater connectivity in the region resulting from recent restoration activities. Figure 30

shows habitat maps of the Picayune Strand from 1940 and from 1995 showing the changes in

connectivity and habitat composition as a result of the original development project (CERP

2004). Many of the areas that may have provided dry season refugia to fish species prior to the

original construction have been converted to areas that may not provide the same functions to

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fish communities as a result of altered hydrology. For example, one of the sites intended for this

study in an impacted un-restored area contained a vegetative community characteristic of a deep

cypress habitat, but did not retain enough water during the wet season to support a fish

community.

With these altered conditions, it is also important to consider environmental factors that

area associated with various restoration phases which could influence fish species richness and

relative abundance values. Differences in water quality parameters, hydroperiods, predator

prevalence, and water flows vary among restoration phases which may have contributed to the

fish community composition within phases. For example, the population density, size structure,

and relative abundance of fishes in South Florida wetlands are regulated by the annual duration

of uninterrupted flooding according to Ogden et al. (2005). Ogden goes one to explain that

fishes such as sunfishes, golden topminnows, and bluefin killifish are less represented in areas

with shorter periods of inundation. Loss of connectivity and sheetflow in the Picayune Strand

may explain the differences in fish community composition within sites of different restoration

phases. “Channelization and compartmentalization of the previous sheet-flow-driven system

have resulted in major changes in physical and hydrologic processes, resulting in far-reaching

effects on ecological processes and habitat.” (Ogden et al. 2005).

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Figure30. The figure above shows habitat maps of the Picayune Strand from 1940 and 1995 in order to observe the changes that have occurred over time as a result of hydrologic alterations. Various habitat types are represented using colors and patterns which have been superimposed onto a map of the Picayune Strand Restoration Project (COE & SFWMD 2004).

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To further examine some of the environmental factors driving change in fish

community structure and composition, a principle component analysis (PCA) was

conducted using environmental data collected during sampling events. Figure 31 shows

an MDS ordination of all sampling events for sites of each habitat and restoration phase

representing environmental data collected for each event. Variables included dissolved

oxygen, temperature, average depth, salinity, and average conductivity. Distributions of

sampling events on the MDS ordination appear to be separated along the axes of both

average depth and average conductivity. Impacted sites appear to group as a result of a

low average depth, while transitional and reference sites appear to group on either side of

both the average depth and average conductivity axes throughout the sampling period.

This ordination may indicate that average depth within sites may be the driving factor

influencing species abundance and relative abundance variations among site of various

restoration phases.

Figure31. The above figure is a principal component analysis including an MDS ordination of all sampling events of various habitats and restoration phases with a superimposed representation of environmental factors including dissolved oxygen, average depth, average conductivity, salinity, and temperature. Location of points in comparison to the various environmental factors represented by lines allows for the observation of which factors are driving the differences between sampling events.

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Potential Indicator Species

As hydrologic restoration continues in the Picayune Strand, fish can provide useful

information to monitor the changes in the ecological health of the areas affected (Main et al.

2007; Ceilley 2008). Hydrology is a major component in fish species abundance and relative

abundance as indicated by abundance data and principle component analysis. To further link the

effects of hydrologic restoration to fish community data, several potential indicator species have

been identified. Species that contributed the most to the dissimilarity between impacted,

transitional, and reference sites included Gambusia holbrooki, Fundulus confluentus, and

Jordonella floridae. All of these species are known to use active dispersal as a method of

recolonizing newly inundated areas (Trexler 2010; Main et al. 2007) and are often pioneer

species as they are more tolerant of harsh water conditions than many other species of fish found

in this study (Ruetz et al. 2005). Active dispersal requires connectivity between habitats and the

presence of these species may indicate that they are connected to adjacent areas containing

source populations. It should be noted that these species do not need a long period of inundation

in order to colonize a newly inundated area due to their tolerance and active method of

recolonization. Therefore, additional indicator species may be required in order to observe

trends in both connectivity and period of inundation in various habitats and restoration phases as

restoration continues. Species such as Fundulus chrysotus, Lucania goodei, Lepomis species,

and Elassoma evergladei have been identified as indicators of longer periods of inundation as

frequent dry downs appear to limit the density of these species (Ruetz et al. 2005; Ceilley 2008).

Heterandria formosa has also been noted for having a response to drought similar to that of

Fundulus chrysotus and Lucania goodei, but was found to have a weaker association with period

of inundation than the other species (Ruetz et al. 2005).

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Sampling Frequencies

When conducting ecological monitoring, it is important to consider sampling effort in

order to determine the resources necessary to effectively carry out monitoring activities.

“Ecologists have long sought out concise and cost-effective measures that can characterize

ecosystem conditions,” (Schiller 2001). Data collected via monthly monitoring during this study

was observed in various subsets in order to identify sampling frequencies that most effectively

and efficiently measure fish communities in comparison to monthly sampling. The data was

divided into subsets representing every other month (September, November, January, March),

every second month (October, December, February), and every third month (November,

February) and analyzed using non-metric MDS ordination plots. MDS ordinations were

observed against the composite data including all data collected from monthly fish sampling in

order to identify ordinations that most closely represented that of the complete data set. The

sampling frequency that most closely resembled the MDS ordination of the complete data set

was the subset including data from every second month (October, December, February) followed

by the subset that included data from every other month (September, November, January,

March). During the 2011/2012 wet season a sampling frequency of every other month most

closely resembled the complete data set as the MDS ordination groupings were the most similar

to that of the original. It should be noted that additional sampling would be necessary to

establish trend data that supports this sampling frequency as the most effective over several

seasons due to hydrologic pattern variations from season to season. An established sampling

frequency containing a smaller number of sampling events would significantly lower sampling

effort and provide a more concise and cost-effective means to measure ecosystem conditions

using fish communities.

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Temporal Sampling

In addition to identifying a sampling frequency that best represents the fish community

through the entire period of inundation, it is also important to identify the time of year when

sampling efforts most effectively measure a mature fish community. Subsets of fish data

collected during each sampling month were analyzed with non-metric MDS ordinations in order

to identify months that most closely resembled the MDS ordination including the entire data set.

Due to the short periods of inundation in impacted sites, the only month that contained fish data

from all sites was November. The month of November was recommended for mature sampling

by Ceilley (2008) and Duever (personal communication). The month of October contained data

from all sites except for the transitional cypress #2 site as no fish were trapped during that

sampling event. When compared with the MDS ordination containing data from all sampling

events, the ordination that most closely resembles the complete ordination groupings is the

month of October. Even though there was lack of complete data for the month of October, it was

determined that both October and November would provide fish community data that best

represents a mature fish community. Additional sampling would be required to establish these

months as the most effective for sampling a mature fish community as hydrologic patterns vary

from year to year.

Fish Community Structure

Fish community data was also used to observe how fish communities change through the

period of inundation. Monthly sampling data for each site and restoration phase was analyzed

with non-metric MDS ordinations and superimposed relative abundance data, and a visual

representation of total abundance data for the three fish species (Gambusia holbrooki, Fundulus

confluentus, Jordonella floridae) that had the greatest impact on ordinations identified using

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SIMPER analysis. It should be noted that data from impacted sites was not used in this analysis

as total abundance for each species was too small to observe change. Abundance data showed a

general pattern of increasing total abundance for all three species from inundation through the

dry-down. This pattern was also observed by DeAngelis et al. (2010), who stated that seasonal

fluctuations of water level in the Everglades allows for the expansion of small fishes over an

extensive flooded area during the wet season, and surviving small fishes then tend to be

concentrated in smaller water bodies when water levels fall during the dry season, making fish

biomass available to higher trophic levels such as wading birds. Abundance data also showed

that Gambusia holbrooki and Jordonella floridae were early colonizers of newly inundated areas

as they were documented early in the sampling period.

Temporal dispersion

To further examine patterns of fish community changes through the period of inundation,

temporal trajectories were superimposed over monthly MDS ordinations of fish community data

for transitional and reference restoration phases in order to observe temporal dispersion patterns

through multivariate space, and community changes over time (Figures 25-26). It should be

noted that impacted sites could not be shown with a trajectory due to lack of data resulting from

the dry-down. The trajectory for reference and transitional sites are used to create a visual of

month to month variation within sites over the entire sampling period and can be used in future

studies to observe trajectory patterns in multidimensional ecological space. To further examine

this pattern, SIMPER analysis was conducted to observe dissimilarity from month to month in

each restoration phase (Table 6). Average month-to-month dissimilarity between sites that

provided sufficient data was lowest within impacted sites (22.93 percent) and increased as

restoration progressed to transitional sites (27.06 percent), and finally reference sites (32.76

percent). Reference sites also show a pattern of decreasing dissimilarity from month-to-month

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through the dry-down with the exception of January-February. The pattern shows that the fish

community is becoming more similar among reference sites as fish species become concentrated

into smaller areas with decreasing water levels. The jump in dissimilarity may be a function of

this concentration of species into smaller areas with the dry-down, which resulted in the capture

of species that were previously un-documented. Transitional sites showed a similar pattern of

decreasing dissimilarity with an increase in dissimilarity occurring in December-January. This

difference may be a result of a shorter overall period of inundation than that of reference sites.

Impacted sites provided data for only two months during the sampling period which only

allowed for a single dissimilarity value between the months of October and November.

Increasing average dissimilarity values from impacted sites through reference sites may be a

result of a more diverse fish community that can be found in reference sites, and a recovering

fish community in transitional sites. This pattern may provide a reference for future studies on

changes in fish community structure though the dry-down, and can be used to assess restoration

activities in ephemeral wetlands.

Restoration Success

Fish species abundance and relative abundance data for all sites of each restoration phase

was analyzed to observe the effects of hydrologic restoration in the Picayune Strand after the

initial phases of restoration were completed. Cluster analysis and MDS ordinations were created

for composite fish data from sites of different habitats though each of the impacted un-restored

sites, transitional recently restored sites, and natural reference wetlands to observe whether fish

communities in areas undergoing restoration are moving from impacted conditions towards

minimally impacted reference conditions. The resulting figures (Figures 27-29) showed distinct

groupings among sites of similar restoration phases. It is important to note that transitional sites

and reference sites also grouped together in the MDS ordination at a minimum of 40 percent

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similarity and a maximum of 60 percent similarity. Impacted sites grouped together at 40

percent similarity, and only showed 20 percent similarity to groups of other restoration phases.

Groupings of transitional and reference sites show a shift from impacted conditions prior to

restoration towards a more natural condition, confirming that the hydrology of transitional sites

is successfully being restored as restoration efforts continue.

It should be noted that transitional site C2 showed fish community data that very closely

resembled reference wetland sites through the period of inundation. Transitional site C2 was

selected based on its proximity to Prairie Canal and known hydrologic impacts resulting from the

large development activities in the area. The site is located slightly south of the additional

transitional sites about one mile east of Prairie Canal. Transitional C2 exhibited a longer period

of inundation than other sites of the transitional restoration phase, which may be a result of its

location further downstream. Downstream areas tend to flood sooner and retain water longer

through the dry season due to water’s stacking effect moving upstream and being situated

slightly closer to sea level, which may have given rise to a more mature fish community and a

more rapid overall restoration of the site.

Cluster analysis shows transitional cypress gramminoid and cypress sites grouping with

the gramminoid reference site. Gramminoid habitats are generally known to have a shorter

period of inundation than that of both cypress gramminoid and cypress habitats, which may

indicate that transitional sites continue to show impairment from the original hydrologic

alterations in the Picayune Strand. Another potential reason for this could be the unrestored

areas to the west of the transitional sites that may be continuing to impact the hydrology of the

newly restored areas. Additionally, this finding may be a result of restoration “time lag”, which

is the time between restoration and recovery when newer restored areas may be more susceptible

to disturbance that can hinder the overall goals of restoration. We must recognize that

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restoration itself is a disturbance and the newly establishing systems need time to organize and

move toward more complex, stable, native communities. It is expected that through further

restoration of impacted areas to the west in addition to increased recovery time, transitional

habitat areas will continue to move towards more natural reference conditions. Continued fish

sampling will be required to observe these changes as restoration continues.

Future Research Opportunities

A multi-agency Restoration Coordination and Verification Team (RECOVER) was

established to support the implementation of CERP with science that can be integrated into

management components through research, monitoring, modeling, planning, assessment, and

adaptive management (Dixon 2009; DOI 2005). Fish community monitoring is a critical

component of RECOVER and was identified to be an important aspect of the adaptive

assessment process set forth in the goals of CERP (USGS 2004).

A major problem that conservation biology faces is the lack of baseline data that exists

which is needed to measure and assess population changes (Dixon 2009; Busby & Parmelee

1996). However, baseline fish data is available within the Picayune and Fakahatchee Strand

Preserve is available (Addison et al. 2006). Additional fish data was also collected during the

first phase of restoration (Ceilley 2008). Experimental design used during baseline studies and

follow up studies during restoration was replicated in this study, allowing for the comparative

studies within the Picayune and surrounding areas. By, using this data with additional

monitoring, temporal trends in species richness and abundance can be evaluated (Dixon 2009).

The previous studies within the Picayune Strand help to support the results of this study. When

reviewed in combination, these studies can make future fish studies stronger by evaluating trends

in fish community composition over time. Therefore, fish monitoring within the Picayune

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Strand should be continued in the future to further observe and document ecological responses to

the hydrologic changes (DOI 2005).

Future research can be improved by adapting techniques and sampling methods based on

experiences from this study and previous research. If additional fish surveys are conducted,

different sampling frequencies may be employed in maximize data quality and minimize

sampling effort by utilizing data from this study. Future restrictions in monetary support and

man-power may result in a minimal sampling opportunity which can be optimized by sampling

during the recommended time periods identified in this study.

Additional sampling sites are recommended for future sampling efforts in order to

minimize and offset the amount of sites chosen that may retain enough water to support a

samplable fish community in a given year. Further additional sites may also be added to

examine the effects of hydrologic restoration on fish communities in additional habitat types

such as hydric pine flatwoods and downstream habitats. The addition of downstream habitats

would provide an opportunity to observe fish community changes as a result of additional

environmental factors such as conductivity, salinity, and water flow. It is also recommended that

sampling is continued for the duration of inundation to provide a complete data set that includes

the entire period of inundation through dry-down. Sampling of source populations and dry

season refugia may also provide valuable data on hydrologic connectivity and dispersion of fish

species across the landscape post inundation. Additional research on indicator species is also

recommended to determine if dispersion is directly linked to hydrologic connectivity, restoration,

and/or dry season refugia in the Picayune Strand.

Access to sampling sites at the height of the wet season was difficult due to high water

and lack of a sufficient 4WD vehicle. It is recommended that a swamp buggy or access to

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additional forms of off road transportation is arranged prior to the onset of the wet season to

ensure access to sampling sites.

Since fish are only one part of the overall aquatic fauna affected by the restoration in the

Picayune Strand, future research should incorporate the collection of other aquatic fauna

including macroinvertebrates and anurans. Long-term monitoring is needed for fish and other

taxa to determine if a continued display of positive responses to hydrologic restoration over time

persists. Aquatic faunal research should continue through the process of restoration and after the

restoration is complete in the areas highlighted in this study as well as additional areas

undergoing hydrologic restoration in the Picayune. Future studies, complimented by this and

previous studies, will continue to document restoration progress and feasibility to improve future

monitoring efforts and inform decision making. This document could potentially aid in

justification for continued funding, support for funding and management decisions, and assist

with acquiring new funding sources within the Picayune Strand.

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CONCLUSIONS

After careful analysis and review of the fish data collected as part of this research project,

the following conclusions can be made:

1) Restoring hydrology within the Picayune by the plugging of Prairie Canal recovered

fish habitat and hydrologic connectivity which resulted in increased species richness

and relative abundance within restored areas.

2) There were significant differences in the distribution of fish species relative to the

restoration phases: specifically Gambusia holbrooki, Fundulus confluentus, and

Jordonella floridae appear to be common indicator species of hydrologic

connectivity. Fundulus chrysotus, Lucania goodei, and Elassoma evergladei were

also unique to reference sites and may also be used as potential indicator species.

3) Sampling frequencies of every other month provide a similar signal to that of monthly

sampling, and may be adequate for collection of fish community data which has

implications for sampling effort.

4) The months of October and November provided samples of a mature fish community

which indicates the most effective time of year for sampling events.

5) Fish community structure and abundance changes throughout the period of inundation

as fish are concentrated into smaller areas by the dry-down. These patterns may

provide a snapshot of overall abundance and identify pioneer species that are early

colonizers of newly inundated areas.

6) Reference wetlands become more similar through the period of inundation and dry-

down while average month to month similarity increases as restoration progresses.

7) Transitional recently restored sites are moving towards more natural reference

conditions as restoration continues and hydrology/connectivity improves.

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A-1

APPENDIX A

FISH SAMPLING DATA FORM

Picayune Strand Aquatic Fauna Field Data Sheet

Site ID___________________ Sky Condition_________________________

Date____________________ Air Temp:____________________________

Field Crew_______________ Wind:_______________________________

________________________ Inverts Collected: Yes No

Sampling Start Time_______________ Invert Start_________ Breder Start_________

Sampling Finish Time______________ Invert Finish________ Breder Finish________

DO:___________ Salinity:___________ pH:____________ SI:____________ Temp:________________

Trap No. Depth(cm) Species Count Total

Dipnet & Visuals for fish/amphibians

Species Number Comments

Comments:______________________________________________________________________________________________

________________________________________________________________________________________________________

________________________________________________________________________________________________________

B-1

APPENDIX B

FISH SAMPLING DATA

Appendix B-1. Species table with species codes

Family Genus Species Common Name Species Code

Lepisosteidae Lepisosteus platyrhincus Florida gar LEPL

Ictaluridae Ameiurus natalis Yellow bullhead AMNA

Callichthyidae Hoplosternum littorale Brown hoplo HOLI

Cichlidae Cichlasoma bimaculatum Black acara CIBI

Cichlidae Cichlasoma urophthalmus Mayan cichlid CIUR

Cichlidae Hemichromis letourneuxi Jewelfish Cichlid HELI

Cichlidae Oreochromis aureus Blue tilapia ORAU

Fundulidae Fundulus chrysotus Golden Topminnow FUCR

Fundulidae Fundulus confluentus Marsh Killifish FUCO

Fundulidae Lucania goodei Bluefin killifish LUGO

Poeciliidae Belonesox belizanus Pike Killifish PKKF

Poeciliidae Gambusia holbrooki Mosquitofish GAHO

Poeciliidae Heterandria formosa Least killifish HEFO

Poeciliidae Poecilia latipinna Sailfin molly POLA

Cyprinodontidae Jordanella floridae Flagfish JOFL

Centrarchidae Lepomis sp. (juv) Sunfish juv. LESP

Centrarchidae Lepomis gulosus Warmouth LEGU

Centrarchidae Lepomis macrochirus Bluegill LEMAC

Centrarchidae Lepomis marginatus Dollar sunfish LEMAR

Centrarchidae Lepomis microlophus Redear sunfish LEMI

Centrarchidae Lepomis punctatus Spotted sunfish LEPU

Centrarchidae Micropterus salmoides Largemouth bass MISA

Percidae Etheostoma fusiforme Swamp darter ETFU

Clariidae Clarias batrachus Walking catfish CLBA

Soleidae Trinectes maculatus Hogchoker TRMA

Cyprinidae Notropis petersoni Coastal shiner NOPE

Elassomatidae Elassoma evergladei Everglades pygmy sunfish ELEV

Loricariidae Pterygoplichthys sp. (juv) Ptero PTSP

B-2

Appendix B-2. Fish sampling data – Impacted Cypress Gramminoid

(9/21/2011 – 3/13/2011)

Species

Code

Impacted-

Cg-

9-21-11

Impacted-

Cg-

10-24-11

Impacted-

Cg-

11-10-11

Impacted-

Cg-

12-12-11

Impacted-

Cg-

1-13-12

Impacted-

Cg-

2-14-12

Impacted-

Cg-

3-13-11

LEPL 0 0 0 0 0 0 0

AMNA 0 0 0 0 0 0 0

HOLI 0 0 0 0 0 0 0

CIBI 0 0 0 0 0 0 0

CIUR 0 0 0 0 0 0 0

HELI 0 0 0 0 0 0 0

ORAU 0 0 0 0 0 0 0

FUCR 0 0 0 0 0 0 0

FUCO 0 0 2 0 0 0 0

LUGO 0 0 0 0 0 0 0

PKKF 0 0 0 0 0 0 0

GAHO 0 8 18 0 0 0 0

HEFO 0 0 0 0 0 0 0

POLA 0 0 0 0 0 0 0

JOFL 0 0 0 0 0 0 0

LESP 0 0 0 0 0 0 0

LEGU 0 0 0 0 0 0 0

LEMAC 0 0 0 0 0 0 0

LEMAR 0 0 0 0 0 0 0

LEMI 0 0 0 0 0 0 0

LEPU 0 0 0 0 0 0 0

MISA 0 0 0 0 0 0 0

ETFU 0 0 0 0 0 0 0

CLBA 0 0 0 0 0 0 0

TRMA 0 0 0 0 0 0 0

NOPE 0 0 0 0 0 0 0

ELEV 0 0 0 0 0 0 0

PTSP 0 0 0 0 0 0 0

B-3

Appendix B-3. Fish sampling data – Impacted Gramminoid

(9/21/2011 – 3/17/2011)

Species

Code

Impacted-

G-9-21-11 Impacted-

G-10-24-

11

Impacted-

G-11-10-

11

Impacted-

G-12-12-

11

Impacted-

G-1-13-12 Impacted-

G-2-14-12 Impacted-

G-3-17-12

LEPL 0 0 0 0 0 0 0

AMNA 0 0 0 0 0 0 0

HOLI 0 0 0 0 0 0 0

CIBI 0 0 0 0 0 0 0

CIUR 0 0 0 0 0 0 0

HELI 0 0 0 0 0 0 0

ORAU 0 0 0 0 0 0 0

FUCR 0 0 0 0 0 0 0

FUCO 0 0 0 0 0 0 0

LUGO 0 0 0 0 0 0 0

PKKF 0 0 0 0 0 0 0

GAHO 0 11 3 0 0 0 0

HEFO 0 0 0 0 0 0 0

POLA 0 0 0 0 0 0 0

JOFL 0 0 0 0 0 0 0

LESP 0 0 0 0 0 0 0

LEGU 0 0 0 0 0 0 0

LEMAC 0 0 0 0 0 0 0

LEMAR 0 0 0 0 0 0 0

LEMI 0 0 0 0 0 0 0

LEPU 0 0 0 0 0 0 0

MISA 0 0 0 0 0 0 0

ETFU 0 0 0 0 0 0 0

CLBA 0 0 0 0 0 0 0

TRMA 0 0 0 0 0 0 0

NOPE 0 0 0 0 0 0 0

ELEV 0 0 0 0 0 0 0

PTSP 0 0 0 0 0 0 0

B-4

Appendix B-4. Fish sampling data – Transitional Cypress Gramminoid

(9/21/2011 – 3/17/2011)

Species Code

Transitional -Cg- 9-21-11

Transitional -Cg- 10-25-11

Transitional -Cg- 11-17-11

Transitional -Cg- 12-12-11

Transitional -Cg- 1-13-12

Transitional -Cg- 2-14-12

Transitional -Cg- 3-17-12

LEPL 0 0 0 0 0 0 0

AMNA 0 0 0 0 0 0 0

HOLI 0 0 0 0 0 0 0

CIBI 0 0 0 0 0 0 0

CIUR 0 0 0 0 0 0 0

HELI 0 0 0 0 0 0 0

ORAU 0 0 0 0 0 0 0

FUCR 0 0 0 0 0 0 0

FUCO 0 0 0 0 0 0 0

LUGO 0 0 0 0 0 0 0

PKKF 0 0 0 0 0 0 0

GAHO 0 10 96 0 0 0 0

HEFO 0 0 0 0 0 0 0

POLA 0 0 0 0 0 0 0

JOFL 0 0 2 0 0 0 0

LESP 0 0 0 0 0 0 0

LEGU 0 0 0 0 0 0 0

LEMAC 0 0 0 0 0 0 0

LEMAR 0 0 0 0 0 0 0

LEMI 0 0 0 0 0 0 0

LEPU 0 0 0 0 0 0 0

MISA 0 0 0 0 0 0 0

ETFU 0 0 0 0 0 0 0

CLBA 0 0 0 0 0 0 0

TRMA 0 0 0 0 0 0 0

NOPE 0 0 0 0 0 0 0

ELEV 0 0 0 0 0 0 0

PTSP 0 0 0 0 0 0 0

B-5

Appendix B-5. Fish sampling data – Transitional Cypress

(9/21/2011 – 3/17/2011)

Species Code

Transitional -C- 9-21-11

Transitional -C- 10-25-11

Transitional -C- 11-17-11

Transitional -C- 12-12-11

Transitional -C- 1-13-12

Transitional -C- 2-14-12

Transitional -C- 3-17-12

LEPL 0 0 0 0 0 0 0

AMNA 0 0 0 0 0 0 0

HOLI 0 0 0 0 0 0 0

CIBI 0 0 0 0 0 0 0

CIUR 0 0 0 0 0 0 0

HELI 0 0 0 0 0 0 0

ORAU 0 0 0 0 0 0 0

FUCR 0 0 0 0 0 0 0

FUCO 0 0 0 0 0 0 0

LUGO 0 0 0 1 0 0 0

PKKF 0 0 0 0 0 0 0

GAHO 0 0 4 370 0 0 0

HEFO 0 0 0 0 0 0 0

POLA 0 0 0 0 0 0 0

JOFL 0 0 0 1 0 0 0

LESP 0 0 0 0 0 0 0

LEGU 0 0 0 0 0 0 0

LEMAC 0 0 0 0 0 0 0

LEMAR 0 0 0 0 0 0 0

LEMI 0 0 0 0 0 0 0

LEPU 0 0 0 0 0 0 0

MISA 0 0 0 0 0 0 0

ETFU 0 0 0 0 0 0 0

CLBA 0 0 0 0 0 0 0

TRMA 0 0 0 0 0 0 0

NOPE 0 0 0 0 0 0 0

ELEV 0 0 0 0 0 0 0

PTSP 0 0 0 0 0 0 0

B-6

Appendix B-6. Fish sampling data – Transitional Cypress 2

(9/30/2011 – 3/17/2011)

Species Code

Transitional -C2- 9-30-11

Transitional -C2- 10-27-11

Transitional -C2- 11-14-11

Transitional -C2- 12-7-11

Transitional -C2- 1-27-12

Transitional -C2- 2-14-12

Transitional -C2- 3-17-12

LEPL 0 0 0 0 0 0 0

AMNA 0 0 0 0 0 0 0

HOLI 0 0 0 0 0 0 0

CIBI 0 0 0 0 0 0 0

CIUR 0 0 0 0 0 0 0

HELI 0 0 0 0 0 0 0

ORAU 0 0 0 0 0 0 0

FUCR 0 0 0 0 0 0 0

FUCO 0 0 0 0 6 14 0

LUGO 0 0 0 0 0 0 0

PKKF 0 0 0 0 0 3 0

GAHO 0 8 4 16 730 789 0

HEFO 0 0 0 0 0 0 0

POLA 0 0 0 0 0 0 0

JOFL 0 0 0 1 0 2 0

LESP 1 0 0 0 0 0 0

LEGU 0 0 0 0 0 0 0

LEMAC 0 0 0 0 0 0 0

LEMAR 0 0 0 0 0 0 0

LEMI 0 0 0 0 0 0 0

LEPU 0 0 0 0 0 0 0

MISA 0 0 0 0 0 0 0

ETFU 0 0 0 0 0 0 0

CLBA 0 0 0 0 0 0 0

TRMA 0 0 0 0 0 0 0

NOPE 0 0 0 0 0 0 0

ELEV 0 0 0 0 0 0 0

PTSP 0 0 0 0 0 0 0

B-7

Appendix B-7. Fish sampling data – Reference Cypress Gramminoid

(9/15/2011 – 3/17/2011)

Species Code

Reference -Cg- 9-15-11

Reference -Cg- 10-13-11

Reference -Cg- 11-7-11

Reference -Cg- 12-5-11

Reference -Cg- 1-13-12

Reference -Cg- 2-7-12

Reference -Cg- 3-17-12

LEPL 0 0 0 0 0 0 0

AMNA 0 0 0 0 0 0 0

HOLI 0 0 0 0 0 0 0

CIBI 0 0 0 0 0 0 0

CIUR 0 1 0 0 0 0 0

HELI 0 14 0 1 0 0 0

ORAU 0 0 0 0 0 0 0

FUCR 0 0 0 1 0 0 0

FUCO 0 1 1 3 96 50 0

LUGO 0 1 0 0 0 0 0

PKKF 0 2 0 0 0 0 0

GAHO 2 23 9 43 103 18 0

HEFO 0 2 1 2 1 0 0

POLA 0 0 0 0 9 0 0

JOFL 0 0 0 13 31 0 0

LESP 0 4 3 2 0 0 0

LEGU 0 0 0 0 0 0 0

LEMAC 0 0 0 0 0 0 0

LEMAR 4 0 0 0 0 0 0

LEMI 0 0 0 0 0 0 0

LEPU 0 0 0 0 0 0 0

MISA 0 0 0 0 0 0 0

ETFU 0 0 0 0 0 0 0

CLBA 0 0 0 0 0 0 0

TRMA 0 0 0 0 0 0 0

NOPE 0 1 0 0 0 0 0

ELEV 0 0 0 0 0 0 0

PTSP 0 0 0 0 0 0 0

B-8

Appendix B-8. Fish sampling data – Reference Gramminoid

(9/15/2011 – 3/17/2011)

Species Code

Reference -G- 9-15-11

Reference -G- 10-13-11

Reference -G- 11-7-11

Reference -G- 12-5-11

Reference -G- 1-13-12

Reference -G- 2-14-12

Reference -G- 3-17-12

LEPL 0 0 0 0 0 0 0

AMNA 0 0 0 0 0 0 0

HOLI 0 0 0 0 0 0 0

CIBI 0 0 0 0 0 0 0

CIUR 0 0 0 0 0 0 0

HELI 0 0 0 0 0 0 0

ORAU 0 0 0 0 0 0 0

FUCR 0 0 0 0 0 0 0

FUCO 0 1 1 9 0 0 0

LUGO 0 0 0 0 0 0 0

PKKF 0 0 0 0 0 0 0

GAHO 3 37 34 66 0 0 0

HEFO 0 0 0 0 0 0 0

POLA 0 0 1 3 0 0 0

JOFL 1 11 0 16 0 0 0

LESP 0 0 0 0 0 0 0

LEGU 0 0 0 0 0 0 0

LEMAC 0 0 0 0 0 0 0

LEMAR 0 0 0 0 0 0 0

LEMI 0 0 0 0 0 0 0

LEPU 0 0 0 0 0 0 0

MISA 0 0 0 0 0 0 0

ETFU 0 0 0 0 0 0 0

CLBA 0 0 0 0 0 0 0

TRMA 0 0 0 0 0 0 0

NOPE 0 0 0 0 0 0 0

ELEV 0 0 0 0 0 0 0

PTSP 0 0 0 0 0 0 0

B-9

Appendix B-9. Fish sampling data – Reference Cypress

(9/30/2011 – 3/13/2011)

Species Code

Reference -C- 9-30-11

Reference -C- 10-27-11

Reference -C- 11-14-11

Reference -C- 12-7-11

Reference -C- 1-13-12

Reference -C- 2-7-12

Reference -C- 3-13-12

LEPL 0 0 0 0 0 0 0

AMNA 0 0 0 0 0 0 0

HOLI 0 0 0 0 0 0 0

CIBI 0 0 0 0 0 0 0

CIUR 0 0 0 0 0 0 0

HELI 0 0 0 0 0 0 0

ORAU 0 0 0 0 0 0 0

FUCR 0 0 0 0 0 0 1

FUCO 0 0 0 1 25 18 25

LUGO 0 0 0 0 0 0 0

PKKF 0 0 0 0 0 0 0

GAHO 0 0 3 7 115 206 508

HEFO 0 0 0 0 0 0 0

POLA 0 0 0 0 0 0 0

JOFL 0 1 0 3 9 11 5

LESP 2 1 1 0 1 2 13

LEGU 0 0 0 0 0 0 0

LEMAC 0 0 0 0 0 1 1

LEMAR 0 0 0 0 0 0 0

LEMI 0 0 0 0 0 0 0

LEPU 0 0 0 0 0 0 0

MISA 0 0 0 0 0 0 0

ETFU 0 0 0 0 0 0 0

CLBA 0 0 0 0 0 0 0

TRMA 0 0 0 0 0 0 0

NOPE 0 0 0 0 0 0 0

ELEV 0 0 0 0 0 0 2

PTSP 0 0 0 0 0 0 0

C-1

APPENDIX C

TOTAL FISH ABUNDANCE

Total Numbers of Fish - Breder Traps

Site September October November December January February March

Impacted Cg 7 18

Impacted Cg 0

Impacted G 1 3

Transitional C 0 4 327

Transitional Cg 7 98

Transitional G 0 0

Transitional C2 1 8 4 17 685 766

Reference C 2 2 1 11 145 55 455

Reference Cg 6 49 14 26 225 21

Reference G 3 48 36 34

Total Numbers of Fish - Dip Netting

Site September October November December January February March

Impacted Cg 1 2

Impacted Cg 0

Impacted G 1 0

Transitional C 0 0 45

Transitional Cg 3 0

Transitional G 0 0

Transitional C2 0 0 0 0 51 42

Reference C 0 0 3 0 5 3 100

Reference Cg 0 0 0 3 15 2

Reference G 1 1 0 6

Total Numbers of Fish - Composite

Site September October November December January February March

Impacted Cg 0 8 20 0 0 0 0

Impacted Cg 0 0 0 0 0 0 0

Impacted G 0 2 3 0 0 0 0

Transitional C 0 0 4 372 0 0 0

Transitional Cg 0 10 98 0 0 0 0

Transitional G 0 0 0 0 0 0 0

Transitional C2 1 8 4 17 736 808 0

Reference C 2 2 4 11 150 58 555

Reference Cg 6 49 14 29 240 23 0

Reference G 4 49 36 40 0 0 0

D-1

APPENDIX D

RESTORATION PHASES:

CLUSTER ANALYSIS AND MDS ORDINATIONS

Appendix D-1 Impacted Cluster Analysis and MDS Ordination

D-3

Appendix D-2 Transitional Cluster Analysis and MDS Ordination

D-3

Appendix D-3 Reference Cluster Analysis and MDS Ordination