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Home , Blue Eye Run, Ichthyomyzon, Little Neshannock Creek, Mountain brook lamprey, Northern brook lamprey, Ohio lamprey

The State University

The Graduate School

School of Forest Resources

AN EXAMINATION OF PETROMYZONTIDAE IN PENNSYLVANIA

A Thesis in

Wildlife and Fisheries Science

by

Shan Li

 2012 Shan Li

Submitted in Partial Fulfillment of the Requirements for the Degree of

Master of Science

May 2012

The thesis of Shan Li was reviewed and approved* by the following:

Jay R. Stauffer Distinguished Professor of Ichthyology Thesis Advisor

Paola C. Ferreri Associate Professor of Fisheries Management

Donna J. Peuquet Professor of GIScience

Jay R. Stauffer Distinguished Professor of Ichthyology Chair of Graduate Program

*Signatures are on file in the Graduate School

iii ABSTRACT

Lampreys are one of the two jawless vertebrate groups in . The adult’s life is shorter than the larval stage which is known as “ammocoete”. They are either in parasitic forms or non-parasitic forms. Parasitic adults attach to other species, rely on the blood or flesh of the hosts, and non-parasitic adults die after spawning. There is little historical data on lampreys because ammocoetes are filter feeders who are in the sediment, and captures of adults were all by chance. The objectives I achieved in this study were included: 1) Compiled all existing PA historical data (prior to 1990s) of lampreys and created database and distribution maps for each species; 2) Sampled historical sites for native lampreys in 2011 by using backpack designed for ammocoetes, documented changes in communities at the watershed scale; 3) Conducted substrate sampling at sites where ammocoetes were present, analyzed substrate size preferred by ammocoetes; 4) Identified collections to species, compared the current data and historical data to see the presence and absence of native lamprey species and value the changes of distributions.

In Pennsylvania, 7 species were found historically. They are aepyptera,

Lampetra appendix, bdellium, Ichthyomyzon fossor, Ichthyomyzon greeleyi,

Ichthyomyzon unicuspis, Petromyzon marinus, among which I. fossor and I. unicuspis are endangered, and other species are in concern. In 2011, 21 streams were inspected, 18 creeks were sampled by using an ammocoete backpack shocker designed in Dr. Stauffer’s lab, and 4 species-

Lampetra aepyptera, Lampetra appendix, Ichthyomyzon greeleyi and Petromyzon marinus were found in 14 creeks statewide. Lampreys were extinct at some sites because of flow regulation, channelization, poor water quality, and chemical treatments. In tributaries of Lake Erie, Sea

Lamprey treatment might be a major reason to the population declining of other native species.

Substrate size and water velocity are the most important factors determine the presence and absence of larval lampreys, so substrate samples were collected at 5 sites where ammocoetes

iv were present. Differences on size of the sediments were found between lamprey spots and non- lamprey spots. Ammocoetes prefer smaller sized substrate with particle diameter less than

0.25mm in shallow, warm waters with dissolved organic matter. This study elaborated the current status of different larval lamprey species and indicated that their distributional range is shrinking and there might be a declining trend of native lamprey species although abundance survey was not implemented.

v Table of Contents

LIST OF FIGURES ...... vii

LIST OF TABLES ...... ix

ACKNOWLEDGEMENTS ...... x

Chapter 1: Research Background and Motivation ...... 1

Life history of lampreys ...... 1 Systematic review of lampreys and their worldwide status ...... 4 Research objectives ...... 8 General approach ...... 9 Data resources ...... 10 Discussion ...... 11

Chapter 2: Characteristics of Native Lamprey Species in Pennsylvania ...... 13

Lampetra aepyptera (Least ) ...... 13 Characters ...... 13 Habitat and Biology ...... 13 Distribution ...... 14 Status ...... 14 Lampetra appendix (American Brook Lamprey) ...... 15 Characters ...... 15 Habitat and Biology ...... 15 Distribution ...... 16 Status ...... 16 Ichthyomyzon bdellium ( Lamprey) ...... 17 Characters ...... 17 Habitat and Biology ...... 17 Distribution ...... 18 Status ...... 18 Ichthyomyzon fossor () ...... 19 Characters ...... 19 Habitat and Biology ...... 19 Distribution ...... 19 Status ...... 20 Ichthyomyzon greeleyi () ...... 20 Characters ...... 20 Habitat and Biology ...... 21 Distribution ...... 21 Status ...... 21 Ichthyomyzon unicuspis () ...... 22 Characters ...... 22 Habitat and Biology ...... 22 Distribution ...... 23

vi Status ...... 23 Petromyzon marinus () ...... 24 Characters ...... 24 Habitat and Biology ...... 24 Distribution ...... 25 Status ...... 25 Discussion ...... 26

Chapter 3: Sampling of Lampreys ...... 28

Introduction ...... 28 Methods and materials ...... 31 Historical data integration ...... 31 Site selection ...... 32 Substrate collection ...... 34 Lamprey collection techniques ...... 35 Substrate drying and sieving ...... 37 Results ...... 38 Sampling of ammocoetes ...... 38 Analysis of substrate ...... 42 Minitab analysis of the sampling results ...... 43 Mood Median Test for ammocoetes sites and random sites...... 44 Discussion and conclusion ...... 46 Discussion ...... 46 Conclusion ...... 49

Bibliography ...... 81

Appendices ...... 86

Appendix A． Ichthyomyzon bdellium historical sites ...... 86 Appendix B. Ichthyomyzon fossor historical sites ...... 91 Appendix C. Ichthyomyzon greeleyi historical sites ...... 91 Appendix D. Ichthyomyzon unicuspis historical sites ...... 94 Appendix E. Lampetra aepyptera historical sites ...... 94 Appendix F. Lampetra appendix historical sites ...... 98 Appendix G. Petromyzon marinus historical sites ...... 107 Appendix H. 2011 ammocoete sampling sites in PA with historical collection information...... 109 Appendix I. Sampling results by species ...... 110

vii LIST OF FIGURES

Figure 1: Map of Pennsylvania’s major basins...... 51

Figure 2: Adult lamprey teeth structure. Petromyzon marinus disc. Made By Canadian Museum of Nature...... 51

Figure 3: Distribution of Lampetra aepyptera in PA...... 52

Figure 4: Distribution of Lampetra appendix in PA...... 53

Figure 5: Distribution of Ichthyomyzon bdellium in PA ...... 54

Figure 6: Distribution of Ichthyomyzon fossor in PA...... 55

Figure 7: Distribution of Ichthyomyzon greeleyi in PA ...... 56

Figure 8: Distribution of Ichthyomyzon unicuspis in PA ...... 57

Figure 9: Distribution of Petromyzon marinus in PA ...... 58

Figure 10: North American State/Province Conservation Status of L. aepyptera...... 59

Figure 11: North American State/Province Conservation Status of I. bdellium...... 60

Figure 12: North American State/Province Conservation Status of L. greeleyi...... 61

Figure 13: Lamprey Historical Sites (1933-2009) by species in Pennsylvania ...... 62

Figure 14: 2011 lamprey sampling sites in Pennsylvania...... 63

Figure 15: ETS-ABP electrofisher ...... 64

Figure 16: Smith-Root LR 24 electrofisher ...... 64

Figure 17: Ammocoete field container ...... 65

Figure 18: West Branch of White Clay Creek ...... 66

Figure 19: Lamprey sampling results in 2011 ...... 67

Figure 20: Lamprey substrate sampler ...... 68

Figure 21: Sieve series produced by Fisher Scientific Company ...... 68

Figure 22: Two different scales for substrate weighing...... 69

Figure 23: Normality test for random samples...... 69

viii Figure 24: Normality test for lamprey sites...... 70

Figure 25: Homogeneity of variance test for data of random samples and lamprey sites...... 70

Figure 26: Regression analysis for random samples ...... 71

Figure 27: Regression analysis for lamprey site ...... 72

ix LIST OF TABLES

Table 1: Distinctive characteristics in Petromyzontidae and the other two families ...... 73

Table 2: 2011 Lamprey sampling results in Pennsylvania, site with lamprey present ...... 73

Table 3: 2011 Lamprey sampling results in Pennsylvania, sites without lamprey ...... 73

Table 4: 2011 Lamprey sampling results in Pennsylvania, sites not assessable ...... 74

Table 5: Auxiliary lamprey collection from other field study conducted in 2011...... 74

Table 6: Length analysis of ammocoetes caught in 2011...... 74

Table 7: Comparison of historical species and current species at 19 sampled sites in PA, 2011 ...... 75

Table 8: Substrate sampling sites ...... 75

Table 9: Sampled creeks and sieved results for each spot of different creeks ...... 76

Table 10: Random site sample data analysis ...... 77

Table 11: Lamprey site sample data analysis ...... 78

x ACKNOWLEDGEMENTS

I would like to express my sincere appreciation to my adviser Dr. Jay R Stauffer for his continuous guidance and funding. It was him who brought me to the amazing field of wildlife and fishery in which I am most interested. His knowledgeable research experiences and rigorous research attitude benefit me for my entire study and research.

I greatly appreciate all my committee members for their help and support to my work.

Especially, I am very thankful to Dr. Paola C. Ferreri, Dr. Donna J. Peuquet from PSU, Robert

Criswell from Department of Natural Conservancy and Douglas Fischer for kind use of their data and research materials, without them I cannot get a good understanding of the historical record. I am also grateful to Dr. Margaret Brittingham for her lab equipment.

I would like to thank my labmates Rich Taylor, Rachel Yoder, Ben Lorson, Bill Hanson,

Sara Mueller, Cindy Nau, Shelly Pickett and 2011 WFS 497A class for their help in preparing of gears and the field work. I cannot finish my master project without their help.

My parents (Guanghui Li and Wenmei Wang), my uncle (Wenzhu Wang) and my boyfriend Cheng Luo are thanked for their support and patient and their continuous love, I am reminded all the time that the research is not all that important.

1

Chapter 1: Research Background and Motivation

Life history of lampreys

Lampreys (Petromyzontiformes) are of significant ecological, cultural and economic importance (Hardisty 1986a; Renaud 1997; Kelly and King 2001; Lucas and Baras 2001), and they play an important role in the health and balance of the fresh water and brackish water systems they inhabit. A definitive lamprey species, Mayomyzon pieckoensis was discovered from the mid-Pennsylvanian (about 280 million years ago) (Bardack and Zangerl 1968). Because lampreys have so many “primitive” characteristics and rely on a simple environment, this might show us what our ancestors were like when they had a simple life form before colonizing a more complicated environment.

The implication of the “primitiveness” of lampreys is presented by their simple habitat.

Sometimes people might regard lampreys as primitive due to the jawless condition, however they also rely on the prey. Larval lampreys have an efficient method of filter feeding, but perhaps the simplicity of their digestive system derived from their simple life style (Barrington 1961).

Although they have a hundred million year history, they are still abundant and probably are more plentiful than other fishes in many river systems. As larvae, they occupy important niches (Young

1935).

Parasitic lamprey prey on other fish species, such as salmonids, while lamprey eggs and larvaes are also preyed on by benthic fish species and birds, raccoons, etc. So they play an important role in fresh water systems.

Abundance and distribution of native lampreys have changed through time due to water quality degradation. Such degradation can be attributed to input of waste water, landscape alternation, such as construction of dams and roads, anthropogenic activities such as recreational

2 fishing or agricultural land and irrigational needs. Native lampreys in region appear to be declining regionally (Neave et al. 2007), and this decline may be associated with efforts to control the invasive Sea Lamprey using the chemical lampricide 3-trifluoromethyl-4-nitrophenol

(TFM) (Steeves 2003). As a result, native lampreys, specifically Ichthyomyzon fossor and

Ichthyomyzon unicuspis, have been given special conservation status in Great Lakes area. While multiple sources of lamprey data are available, no reliable distribution document of either species has been published due to the difficulties in capturing adults and erroneous identification of the ammocoetes. To understand the nature of the changes in their distribution and abundance, it is very important to address the historical patterns of each species.

Lampreys are Agnatha which is the sister clade of all Gnathostomata (jawed vertebrates).

They lack jaws and pelvic fins, and the dorsal fins have no dermal rays. They are well known by their -like body shape and two-stage life cycle, including an extended larval stage and for the non-parasitic species a relatively brief adult stage (Hardisty 1971). Instead of jaws both parasitic and non-parasitic adult lampreys may have a very well developed oral disc, with teeth on both tongue and disc which non-parasitic ones use to lift pebbles for their nests. In May and June, adults aggregate together in clean, freshwater streams to spawn. Adult lampreys build nests on pebbles and cobbles in headwater streams. After the eggs hatch, larval lampreys flow downstream to wherever silty substrate is found, usually it is shallow, clear and slowly flowing water. They can stay on this larval stage for up to seven years in Lake Erie, and even longer in other Great

Lakes, depend on different species, and then begin to transform into adult in August. There are two different forms as adult, parasitic form which needs to prey on other fishes and non-parasitic form which do not feed at all, just spawn and die within six months. Parasitic species attach and feed on different hosts, and this process may last for 1-2 years to grow bigger and mature before spawning, and they might be divided into two different categories: anadromous, which is to feed at sea, and those are restricted to river system. The non-parasitic species have less developed

3 tooth patterns, during transformation into the adult phase, digestive tract becomes non-functional and the gonads mature. All lampreys are semelparous, meaning that they die after they spawn for the first time.

Ammocoetes, the larval phase of lampreys have a completely different appearance and characteristics of adult lampreys. The most distinctive characteristic of ammocoetes’ behavioral pattern is the burrowing response. This process initiates by whip-like contractions of the tail, and then forcing the head vertically down to the substrate, their tails are sensitive to light, which could make sure they are fully buried in the substrate with tail laid horizontally over the surface

(Sawyer 1959). Once buried, the body is arched so as to bring the snout towards the surface

(Potter et al. 1970). Secretion from the gill slits and mouth build the wall of the tube in the substrate, so that the water could enter in. Instead of oral disc, ammocoetes have an oral hood at the entrance into their oral cavity which guarded by a ring of oral tentacle. Waters enter into their body through the tentacle, and the pumping action and contraction of their bronchial area help water pass out of the body through gill slits. They filter feed on detritus, algae, organic matter and grow for 3-7 years before their metamorphosis into adult lampreys. During the transformative period of 1-2 months, lampreys develop eyes which are absent for ammocoetes, tooth-bearing oral disc, fins and other adult characteristics.

Lampreys are one of the most primitive vertebrates. The early fossil record dates back to

280 million years ago, discovery of a definitive lamprey, Mayomyzon pieckoensis, from mid-

Pennsylvania. An assemblage of lampreys was also found recovered from a coal strip mine about

50 miles southwest of Chicago, (Bardack 1968). Richardson and Johnson (1971) indicated that the fossil may suggest these were deposited in a coastal deltaic area of fluctuating marine and freshwaters. No matter what standard is employed, biomass, global distribution, or survival through such a long period of time, there is no doubt that lampreys are among the most successful groups of animals. This success is not only owed to the protracted freshwater larval

4 stage, but also the parasitic life form out in the ocean for some species. The morphological changes that occur during metamorphosis are so radical that ammocoetes were not identified as the larval stage of lampreys until the middle of nineteenth century (Ohman 1971). Adult lampreys are preyed upon in fresh waters by other animals, and their eggs are preyed upon by a wide variety of fishes and birds (Renaud 1997). Some species such as tridentatus (Pacific

Lamprey) and camtschaticum () are eaten by Native Americans as food (Renaud 1997).

Although lampreys include both parasitic and non-parasitic forms (Nelson 2006), most lamprey species are non-parasitic. Because anadromous parasitic lampreys spend most of their life, usually 1 ½ ~2½years (Hardisty and Potter 1971) feeding in marine systems, they are often by-catch along with their hosts. For non-parasitic lampreys, life as adults is even shorter, only lasting 3-6 months without feeding. Therefore, examining the presence and absence of adult lampreys is not sufficient to document the status of the populations. All species of lampreys, no matter parasitic or non-parasitic all have a distinct larval life stage lasting from 3-7 years

(Hardisty and Potter 1971; Beamish and Austin 1985; Burr and Shasteen 2007; Smith 2011), burrowing into the substrate feeding on detritus, , and algae (Beamish and Austin 1985;

Sutton and Bowen 1994; Beamish and Lowartz 1996). Therefore, it is crucial to investigate habitat selection of ammocoetes.

Systematic review of lampreys and their worldwide status

Lampreys are now generally recognized as 3 different families: Geotriidae, Mordaciidae in Southern Hemisphere, and Petromyzontidae restricted in Northern Hemisphere. Within

Petromyzontidae, there are about 40 species in nine genera. Characteristics vary a lot between

Southern Hemisphere and Northern Hemisphere (Table 1).

5

Nine lineages have been identified by Creaser and Hubbs in 1922, which are

Ichthyomyzon, Petromyzon, Caspiomyzon, , Okkelbergia, Lampetra, Lethenteron,

Entosphenus, . Gill et al. (2003) recognized eight genera, and they were unable to comment on Okkelbergia. Most of what have known about lampreys all over the world is organized in a 5-volumn book- The Biology of Lampreys edited by Hardisty and Potter (1971-

1982). 8 of the 34 recognized lampreys were described by Vadim D. Vladykov and his colleagues.

Lampreys are declining throughout the world because of flow regulation, channelization, and poor water quality as well as chemical treatments (Close 2002). Habitat in spawning streams has been changed by stream regulation. Beamish and Northcote (1989) discovered that the constructions around Elsie Lake in British Columbia prevented young feeding adult Pacific

Lamprey from going to the sea and also impede the spawning lamprey reaching the spawning area. Most lamprey species are distributed in European countries and the population was reported declining recently. Now in 23 European countries, Lampetra fluviatilis, Lampetra planeri,

Petromyzon marinus are protected by law (Bern Conservation, Appendix III). In US, A lot of species are endangered or threatened national wide or at least in some states.

Species that are endangered or threatened include:

Ichthyomyzon bdellium (): endangered in Ohio (Ohio Department of

Natural Resources 1990); rare in Maryland (Miller 1972)

Eudontomyzon mariae (): endangered in (Witkowski

1992), sharply declining numbers in the and Moldavia (Pavlov et al. 1985)

Ichthyomyzon castaneus (): endangered in Nebraska and rare in Kansas

(Miller 1972); threatened in Iowa (Roosa 1977)

Ichthyomyzon fossor (Northern Brook Lamprey): endangered in Illinois (Illinois

Endangered Species Protection Board 1994); vulnerable in (Lanteigne 1992a)

6

Lampetra fluviatilis (): endangered in Europe (Lelek 1987); extinct in Swizerland (Pedroli et al. 1991)

I. greeleyi (Mountain Brook Lamprey): Threatened in (Burr et al. 1990)

L. appendix (American Brook Lamprey): Threatened in Iowa (Roosa 1977); Threatened in (Rohde et al. 1994)

The reasons for the endangered or threatened status of the above species are due to loss of spawning and larval habitats suffering pollution or alteration of streams in sand extraction, damming or irrigation projects (Renaud 1997). So examination of the population and habitat selection of different species is the first crucial step for lamprey protection, then after that we could investigate the degradation of their living environment.

Studies of lamprey status and their habitat preference by local native species were done in

Europe and other states in US. However less was found in Pennsylvania. One of my objectives is to conduct a survey of Pennsylvania native lampreys and their current status specifically to broad our knowledge.

According the historical data, seven species were reported from Pennsylvania in 3 genera: Least Brook Lamprey (Lampetra aepyptera), American Brook Lamprey (lampetra appendix), Ohio Lamprey (Ichthyomyzon bdellium), Northern Brook Lamprey (Ichthyomyzon fossor), Mountain Brook Lamprey (Ichthyomyzon greeleyi), Silver Lamprey (Ichthyomyzon unicuspis), and Sea Lamprey (Petromyzon marinus). Silver Lamprey is considered distinguished in Pennsylvania; Northern Brook Lamprey is the endangered species classified by PA Fish and

Boat Commission; the population of other species is also diminishing now.

The exotic parasitic Sea Lampreys - Petromyzon marinus might be a significant trigger that led to the degradation of the native lamprey population in Lake Erie drainage. In 1930s, the first invasive Sea Lamprey was introduced into Lake Erie, and in the next few years, it migrated into the other Great Lakes. In 1955, Great Lakes Fisheries Commission was charged to formulate

7 and implement a comprehensive program for the purpose of controlling Sea Lamprey population.

Mechanical and electrical weirs were used as well as the lampricide TFM (3-trifluoromethyl-4- nitrophenol), the most frequently used chemical poison. This chemical treatment decimated not only the invasive Sea Lamprey but also native lamprey species throughout the Great Lakes tributaries. The distribution of native lampreys has been altered, and their populations are declining, not only because of invasive Sea Lamprey, but also because of other anthropogenic influences (Close 2002). For example, hydropower dams can delay or restrict migrations of adults, and turbine entrainment or screen impingement can kill juvenile lampreys.

Because they are one of the most primitive species, their status has been static during the last million years and now their declining status is of concern. The purposes of this study are to clarify the status of native lamprey species, to examine the historical records of native species in

Pennsylvania, and also sample historical sites of lampreys to determine the current distributional patterns.

Lampreys are widely distributed in both Northern and Southern Hemispheres on the 20 ºC annual isotherm (Hardisty and Potter 1971). Although predominantly living in freshwater they can tolerate saline water as they were found in western Mediterranean (ca. 3.7%-3.9%) and

Caspian Sea (15 %) (Lanzing 1957). Generally, anadromous lampreys are larger than the non- anadromous forms and disperse widely: Petromyzon marinus occurs in North American, northern

European and Mediterranean watersheds while freshwater parasitic lampreys have a relatively restricted distribution, generally within a single large drainage basin, such as the three species in genus Ichthyomyzon which are limited in tributaries of the Gulf of Mexico, Gulf of St. Lawrence and Hudson Bay.

Lamprey systematics are based on the abundance, arrangement, and strength of the lingual and oral disc-tooth series, the numbers of myomeres (number of blocks of skeletal muscle tissue count from the last gill aperture and cloaca), and the degree of lateral line pigmentation

8

(Regan 1911; Creaser and Hubbs 1922; Vladykov and Follett 1958). Both anadromous and freshwater species are found in PA.

Native lampreys are distributed in the major drainage basins that traverse Pennsylvania

(Figure 1). Early collections by Dr. Cooper and Dr. Stauffer documented the diversity and abundance of lampreys in Pennsylvania and other states, and the specimens are stored in the Penn

State Fish Museum. Earliest lamprey specimen found in museum was in the 1930s. Surveys conducted by researchers and agencies from time to time provide evidence that 7 native species are now present or had at least once occurred in Pennsylvania. So one of my objectives was to assemble a database of each native lamprey species in Pennsylvania that spanned approximately the last 70 years, document the changes in lamprey community.

Research objectives

Given their unique life cycles and behaviors, their long history, it is very important to investigate their success of evolution, behavior and current status worldwide. The goal of this project is to study distribution and habitat preference of lampreys within the state of

Pennsylvania.

Unfortunately, few records of lampreys in the state of Pennsylvania exist. Most records are of larval forms because the adult stage is so short. Where records of adults exist, they were often taken by chance as they were attached to a host that was the target of study. Although more information is available for the larval phase, there is still a lack of information as ammocoetes are so well hidden in the substrates of freshwater streams, that it is difficult to find them and little efforts was put into developing effective sampling gears. As such, one of my objectives of this research was to assemble a database of historical records for each native lamprey species in

Pennsylvania based on available information that spanned approximately the last 70 years, in order to document the changes in lamprey community. Habitats and water qualities at those

9 historical sites where lampreys have not been found for decades may have altered which are not suitable for lampreys anymore, therefore in order to know their current distributional status, the key objective of this study was to examine the current distribution of native lampreys at the historical sites which have not been sampled for at least 20 years, and update their distribution.

This study also qualified substrate in which ammocoetes were found by conducting substrate samples at the sites where ammocoetes were present. Four research objectives were undertaken:

Compile all PA historical data (prior to 1980s) of lampreys and create database and distribution maps for each species.

Sample historical sites for native lampreys in 2011 by using backpack designed for ammocoetes which is the larvae form of lampreys, document changes in lamprey communities at the watershed scale.

Conduct substrate sampling at sites where ammocoetes are present, analyze substrate size and physical factors preferred by ammocoetes.

Identify collections to species, compare the current data and historical data to determine the presence and absence of native lamprey species and value the changes of distributions.

General approach

Geographic information system (GIS) is a powerful tool capable of displaying, organizing, analyzing spatial data (Isaak and Hubert, 1997), and it is becoming widely used in fisheries science. With the combination of the relational fish database and GIS, I could easily create maps of the geographic changes of the distribution. Attribute tables linked to maps in GIS system could also help to simplify the distribution changes through time. Moreover, the distribution data are more convenient to integrate with other land use data, facilitate a wide range of investigations (Angermeier and bailey 1992, Isaak and Hubert 1997). Current database could be easily imported into GIS system and it is more efficient to analyze various factors affecting the

10 distribution of native lamprey species through GIS. In my study, GIS is utilized to visualize the changes of the distribution, examine the status of each species and compare the historical and extent distributional patterns of indigenous lampreys. Fish records from the Pennsylvania Fish and Boat Commission; the Pennsylvania State University Fish Museum; Cornell University; PA

Natural Diversity Inventory Database were assembled to complete this research.

Data resources

For this study, I obtained historical fish records from: the Pennsylvania Wild Resource

Conservation Program; the Pennsylvania Fish and Boat Commission; the Pennsylvania State

University; Cornell University; Dr. Edwin L. Cooper; Dr. Jay R. Stauffer. The earliest data was from 1930s, but most data was after 1990s. Seven native lamprey species were found in the database: Least Brook Lamprey (Lampetra aepyptera), American Brook Lamprey (Lampetra appendix), Ohio Lamprey (Ichthyomyzon bdellium), Northern Brook Lamprey (Ichthyomyzon fossor), Mountain Brook Lamprey (Ichthyomyzon greeleyi), Silver Lamprey (Ichthyomyzon unicuspis), and Sea Lamprey (Petromyzon marinus). Most collections were adult lampreys captured with other host fish or during the spawning season in head waters. Because adults have distinctive tooth arrangements on the oral discs, identification of adult lampreys is well accepted.

For those ammocoetes collected with the adults at the same spot, the previous collectors compared other morphological keys of ammocoetes with the adults, and if they match, they put lampreys in the same jar and tagged them as the same species. For those ammocoetes collected without any adults, they were just identified to genus level based on the shape of their dorsal fins, colors and also the counting of the myomeres. In the database, locality, latitude and longitude, capture date, collector, numbers of individuals, and creek name were recorded (Appendix A-G).

11

Discussion

There were 568 valid historical collections of lampreys in my database (Appendix A-G), invalid records were those not identified to species or those did not have any locality information.

For data from the Penn State University Fish Collection, I was able to verify identifications in the museum. Information for the collection was on the labels inside fish jars. This indicated site record was valid. It is a reliable place if we want to check back later on. If missing data could not be obtained, I did not include them. Of the 568 records, most were reliable except the exact locality information was not available. For example, Ichthyomyzon bdellium was found in

Oswayo Creek in Potter Co. on 1996/8/5 by PFBC, but beyond that, no more details were offered.

In this circumstance, I pinpointed the site on the middle point of the creek inside the county, and extracted the lat/long from there. A lot of soft descriptions of localities were found in the database, such as “under the wood bridge” or “beside a barn”, which was very difficult to visualize on map or even they are no longer exist. In this case, if the site was among those ones I sampled in 2011, I checked on site and revised that to a solid description, otherwise I pinpointed at the closest point to the route in creek. The lat/long may not be accurate enough, but at least it indicated the presence and absence of the specific lamprey species in that stream. The typo on locality was another annoying problem while this was processing, sometimes it took hours to figure out the actual location, and later on data could be reviewed or validated by looking at recent collections. Again, since my objective was to look at the presence and absence of native lampreys in Pennsylvania, I did not summarize the abundance of each species. Although no abundance study was conducted, we could still predict their general declining status all across

Pennsylvania by looking at their presence and absence compared to historical data. From the distribution map of each species, I was able to see the overall status of each species, and detailed information could be inspected in GIS system, by clicking the site on the map, Data appears as

12 indicated in the Appendix. Thus GIS has an integrated output system which makes it easy to check overall distribution and specific field records of each site back and forth with one interface.

13

Chapter 2: Characteristics of Native Lamprey Species in Pennsylvania

Endangered, threatened, and candidate species will be mentioned. An endangered species is one that is at risk of extinction. Threatened species are any species which are vulnerable to endangerment in the near future. Candidate species are ones which could achieve endangered or threatened status in the near future. In first paragraph under each species, morphological characteristics were described, among which teeth structure of adult lamprey were the very important keys. Figure 2 clarified names of different teeth structure of adults. A distributional map was attached for each species after the description.

Lampetra aepyptera (Least Brook Lamprey)

Characters

Small non-parasitic lamprey with 2 deeply notched dorsal fins; 51-62 (usually 53-69) trunk myomeres. Poorly developed teeth; anterior intermediate teeth 7-23; lateral intermediate and posterior cirumoral teeth absent, lateral circumoral tooth groups 1-3 (usually 2); supraoral plate with single large tooth per end; infraoral cusps 5-9. Transverse lamina usually fleshy

(Jenkins and Burkhead 1994). Light tan to silver-gray above; yellow or white below; yellow or gray fins. Spawning adult darker grey to blackened. Adult size about 80-180mm TL. Maximum ammocoetes size 180mm TL (Page and Burr 1991).

Habitat and Biology

Least Brook Lampreys prefer clean, clear gravel riffles and runs of creeks and small rivers which contain significant deposits of mud, musk, sand, and detritus (Smith 2011). They

14 spawn from mid-March to early May in WV and MD (Schwartz 1959, Rhode et al. 1976). They move pebbles with their mouth to build their nests. The ratio has been observed as 1.38 males to 1 female in WV (Holbrook 1975). Adults die immediately after spawning. Larval stage exists for

5.4 years on average. Transformation begins in August. The diet of the ammocoetes is assumed to be zooplankton and vegetation.

Distribution

Lampetra aepyptera is distributed on the Atlantic Slope from Susquehanna River drainage in southeastern PA to the Neuse River drainage, NC; River basin from western PA to south central MO and northern AR, and south to northern AL; also inhabits the

Mobil Bay and Pascagoula River drainages in GA, AL, and MS (Page and Burr 1991). In western PA, this lamprey has been reported from the drainage in Allegheny,

Armstrong, Beaver, Butler, Fayette, , and Westmoreland counties. It also occurs in a small area of the Susquehanna River drainage in southern Chester County (Figure 3).

Status

Least Brook Lamprey is currently classified as a candidate species in PA (PFBC 2006).

During a 2003 survey, it was collected in 15 out of 19 historic waterways sampled, including as many as 35 individuals per station. Although insufficient data was available to compare population numbers between historic collections and those obtained during this survey. It is apparent that there has been little change in the Least Brook Lamprey's status over the last few decades (Criswell 2003). Most of collections were collected in the southwestern part of PA and a few in Chester County. The status is considered as secure now.

15

Lampetra appendix (American Brook Lamprey)

Characters

Small non-parasitic lamprey with deeply notched dorsal fin; and generally 63-74 trunk myomeres. Not well developed teeth; anterior intermediate teeth 10-28; lateral intermediate teeth absent; posterior circumoral teeth minute, usually forming a single row numbering 17-24; lateral circumoral tooth groups 3 per side, usually bicuspid. Transverse lamina with 7-11 cusps, medial one enlarged (Jenkins and Burkhead 1994). Generally gray, olive, or brownish above; usually becoming darkened as spawning season progresses; and light gray below; dorsal fin tan to yellow

(Page and Burr 1991).

Habitat and Biology

The American Brook Lamprey prefers small to median-sized creeks, and is less frequently observed in large streams and rivers. Clear water is preferred and adults choose sandy and gravelly riffles or runs to spawn, while ammocoetes prefer deposit of silt, mud and detritus

(Jemkins and Burkhead 1994). Spawning starts in April and lasts for more than one month.

Adults could be collected in PA as late as May 30 (E. Cooper, field notes). The water temperatures range is from 8.7-15.5 ºC. Average adult lamprey length or mass differed significantly among streams, but there is no significant difference between males and females.

The nest is an oval-shaped depression 150-180 mm long, 100-125 mm wide, and 25-50 mm deep

(Becker 1983). These lampreys spawn in groups of 2-14 individuals. A male attaches to the head

16 of a female, which is attached to a rock at the head of the nest. Adults die after spawning. Newly hatched larvae drift downstream, where they burrow in sand or silt detritus in runs and pools which have slow water velocity, and stay as ammocoetes for about five years (Smith et al. 1970;

Hubbs 1925).

Distribution

It is widely distributed in Atlantic, Great Lakes, and basins from St.

Lawrence River, Quebec, west to , south to Roanoke River system, ,

Tennessee River system, northern , and St. Francis and White river systems, Missouri and Arkansas; Martin River (Arctic basin), Northwest Territories, to Ugashik and Chatanika

River (Pacific basin), Alaska; uncommon (Page and Burr 1991). This species is restricted to the northwest and southeast corners of Pennsylvania, where it inhabits the following drainages –

Lake Erie, , Genessee River, and Delaware River. Has been collected from

Crawford, Elk, Erie, Forest, McKean, Potter, Venango, and Warren counties, as well as from the

White Clay Creek drainage in southern Chester County (Figure 4).

Status

Numerous records are present in northwestern part of PA and a few have been collected in Chester County, and the population has not fluctuated in the past decade. Currently, it is considered as secure; although it is classified as a candidate species by PFBC (2006).

17

Ichthyomyzon bdellium (Ohio Lamprey)

Characters

Parasitic lamprey with single slightly notched, undivided dorsal fin and bicuspid circumoral teeth. Trunk myomeres 53-62 (usually 55-61 in PA). Sharp, well-developed disc teeth; usually 2-3 supraoral teeth; infraoral cusps 7-10 (most frequently 8); teeth in circumoral row 19-

22 (21); teeth in anterior row 4-5 (4); teeth in lateral rows 7-10 (8); bicuspid circumorals 5-10 (8).

Transverse lingual lamina moderate-strongly bilobed. Adult size 124-259 mm TL (Hubbs and

Trautman 1937). Adult color slate-gray to yellowish-grey above; fading to lighter on belly; black spots (pigmented lateral line organs) present on upper half of body (Page & Burr 1991).

Habitat and Biology

When in parasitic phase, Ohio Lamprey occurs in medium streams and rivers, but may enter smaller streams to spawn. Unattached adults are found in riffles and runs with gravel, cobble, and rubble substrate. Ammocoetes live near debris in muddy pools and backwaters. Ohio

Lampreys spawn in mid-late May in PA, sometimes in the company of the smaller, non-parasitic

Mountain Brook Lamprey (Cooper 1983). Maturity occurs successfully at a minimum length of

200mm TL (Hubbs and Trautman 1937). Spawning occurs in a pit, usually in riffles with gravel and rubble substrate, in open areas or concealed under a large rock. Then in June, eggs hatch, and the larval form lasts about 4 years and one month. Transformation begins in late July or early

August, and lasts about 21 months. Adults die after spawning when they are about 6 years old.

Host fishes for all lampreys include paddlefish, carp, sucker, black bass, catfish, madtom, darter

18 and others (Jenkins and Burkhead 1993). In PA, Ohio Lampreys have been found attached on

Northern Hogsucker (Hypentelium nigricans), Redhorse Suckers (Moxostoma carinatum),

Smallmouth Bass (Micropterus dolomieu), (Sander vitreus), European Carp (Cyprinus carpio), and Rainbow Trout (Oncorhynchus mykiss) (Cooper 1983).

Distribution

It is distributed in the Ohio River basin from southwestern to northern IN and eastern IL, and south to northern AL (Page and Burr 1991). In PA, it appears in Allegheny River drainage, where it is most abundant in the northwestern section of state near the Great Lakes area

(Figure 5).

Status

This species is widely captured in northwestern PA throughout the Allegheny River drainage in Crawford, Venango, Erie and Warren counties. It was frequently collected in1960s and after 2000. In the 1970s, the population fluctuated, but fewer collections were conducted. It is currently listed as a candidate species by PFBC (2006). Cooper (1983) stated that the Ohio

Lamprey was in no immediate danger in PA.

19

Ichthyomyzon fossor (Northern Brook Lamprey)

Characters

Small, non-parasific lamprey with single, notched dorsal fin and unicuspid circumoral teeth. Usually 50-52 trunk myomeres. Weakly developed teeth and small, blunt disc teeth; diameter of oral disc smaller than body size; supraoral cusps 1-2 (usually 2); infraoral cusps 6-11; teeth in circumoral row 15-25; teeth in anterior row 1-3; teeth in lateral rows 2-6; bicuspid circumorals absent. Transverse lingual lamina strongly bilobed. Adult size 94-146 mm TL

(Hubbs and Trautman 1937). Adult color gray-brown on back and sides; belly whitish (Page and

Burr 1991).

Habitat and Biology

Northern Brook Lamprey inhabits small-medium sized streams. Substrates include sand, gravel, mud, and silt. Spawning has been observed only in mid-late May in PA. Ammocoetes stay in the larval period for 3-6 years. Transformation begins in August or September and is completed in December. Individuals may decrease in length by 10% after they finish transformation.

Distribution

It is found from the St. Lawrence River drainage in Quebec west through the Great lakes and northern Mississippi River basins to the Red River, Hudson Bay drainage, southern

20

Manitoba; also localized in the Ohio River basin of northwestern PA, western WA, eastern KY,

IN and OH, also Missouri River drainage in MO (Page and Burr 1991). In PA, Northern Brook

Lamprey is found in Conneaut Creek drainage in Crawford and Erie counties (Cooper 1983)

(Figure 6).

Status

The population of Northern Brook Lamprey is extremely rare in PA, with just a few records from Conneaut Creek and Temple Creek in county Erie and Crawford. Cooper (1983) collected a few individuals in Conneaut Creek during 1960s-1970s. Conneaut Creek, including the section occupied by this species, has been periodically treated with lampricide 3- triflouromethyl-nitrophenol (TFM) in an effort to control the invasive species sea lamprey

(Petromyzon marinus) in Lake Erie since 1986 (Klar and Young 2003). It is now regarded as the endangered species by PFBC (2006).

Ichthyomyzon greeleyi (Mountain Brook Lamprey)

Characters

Small, non-parasitic lamprey with a single, undivided slightly notched dorsal fin and bicuspid circumoral teeth present. 55-61 trunk myomeres. Moderately well-developed disc teeth; supraoral cusps 2-3 (rarely 4); infraoral cusps 7-12 (usually 8-10); teeth in circumoral row 19-24

(usually 21); teeth in anterior row 3-5 (usually 4); teeth in lateral rows 5-9 (usually 7-8); bicuspid circumorals 7-11 (typically 8). Transverse lingual lamina moderate-strongly bilobed. Adult size

21

105-161 (usually 110-150) mm TL (Hubbs and Trautman 1937). Color dark olive on back and sides, often mottled; belly whitish (Page and Burr 1991).

Habitat and Biology

Mountain Brook Lamprey prefers clear and clean high-gradient little runs. In PA, they frequently occur in stocked trout streams. Ammocoetes are found in the substrate of sand, mud, and debris in pools and backwaters. In PA, spawning occurs over gravel and sandy substrate in mid-late May (Raney 1939). Males and females construct pit-nest for spawning. Peak nest building and spawning happen in during mid-afternoon hours (Raney 1939). The adults die immediately after spawning (Schwartz 1959). Mountain Brook Lamprey sometimes spawns in the same nests as Ohio Lampreys (Cooper 1983). Larval period lasts for about 4-5 years, and the whole life cycle is 4-5 years. Transformation begins in August, and lasts for 100-140 days.

Distribution

It is localized in Ohio River drainage from southwestern New York to northern AL (Page and Burr 1991). The Pennsylvania distribution includes the Clarion and upper Allegheny River drainages, including French Creek, and in the Beaver River drainage (Figure

7).

Status

They are present in Crawford, Elk, Venango, Erie, Forest, Jefferson, Lawrence, Mckean,

Potter and Mercer counties which are generally northwestern part of PA. The adult is the only

22 life stage which can be identified to species with confidence, collections of the other stages could just be classified as Ichthyomyzon ammocoetes. Field surveys conducted in 2004-2005 by A.

Turner, Clarion University, and recorded Clarion River drainage as one of the unreported locations. Mountain Brook Lamprey is currently listed as threatened species by PFBC.

Ichthyomyzon unicuspis (Silver Lamprey)

Characters

Parasitic lamprey with single, undivided dorsl fin and unicuspid circumoral teeth. Trunk myomeres 47-55 (usually 49-52). Teeth on oral disk well developed; supraoral cusps 2 (1-4); infraoral cusps 5-11 (average 7.8); teeth in circumoral row 15-25 (average 19.2); teeth in anterior row 2-4 (usually 3); teeth in lateral rows 5-8 (usually 6-7), bicuspid circumorals 0 (rarely 1-2).

Transverse lingual lamina moderate-very strongly bilobed. Adult size 103-328 mm TL (mature adults 205-328mm) (Hubbs and Trautman 1937). Younger adults’ yellowish-tan, with light pigmentation on belly and dark on back, darker through spawning period (Page and Burr 1991).

Habitat and Biology

Feeding adults are found attached on larger fishes in large rivers and lakes. Adults migrate upriver to spawn in gravel riffles and runs; ammocoetes inhabit sandy, muddy pools which have organic debris that is free from clayey materials. Spawning occurs in April and May in Lake Erie tributaries when the water temperature reaches 10° C; the larval stage may last 4-7 years (Scott and Crossman 1973). Transformation begins in August and is finished in next spring.

Those metamorphosing in Ohio drift downstream and enter Lake Eire in late April, May, and

23

June (Trautman 1981). The parasitic lampreys have been found attached on Northern Pike,

Paddlefish, Common Carp, , White Suckers, and Catfish (Becker 1983).

Distribution

It is distributed from the St. Lawrence- Great Lakes basin from Quebec to southwest

Ontario and south through upper Mississippi and Ohio River basins to central ; also isolated records in MN, MS, and NE (Page and Burr 1991). It is recorded in Pennsylvania only rarely from a few Lake Erie tributaries (Figure 8).

Status

On 24 May 1987, PFBC personnel captured a specimen in a lamprey trap in Crooked

Creek, Erie County (PSU 1951). US Fish and Wildlife Service personnel have also reported a few collections from Erie County tributaries during the period 1980-1986. In 2004 a specimen was collected in Crook Creek, since it is an ammocoete, it could be misidentified. The population of Silver Lamprey declined significantly after 1900, before that abundant have been reported in the Ohio waters. It has not been clearly classified, but as figure 2-8 shows, it is obviously one of the rarest species in PA.

24

Petromyzon marinus (Sea Lamprey)

Characters

Large, parasitic lamprey with dorsal fin by a deep notch into two fins. Trunk myomeres

63-73. Curved and sharp teeth well developed; circumoral teeth 17-19; bicuspid circumoral teeth

8, supraoral cusps 2; infraoral cusps 6-10; lateral cusps 7-9. Transverse lamina bilobed with 14 cusps (Jenkins and Burkhead 1994). Ammocoetes gray or brownish on upper sides and back; paler ventrally; transformed individuals bluish-gray dorsally grading to white ventrally; purplish sheen on the sides; adults generally dark brown or black dorsally and lighter brown to yellow ventrally (Page and Burr 1991).

Habitat and Biology

Sea Lamprey prefer cool and deeper water in Great Lakes and ammocoetes burrow in silt and mud deposits in sluggish sections of streams filter feed on algae and protozoan (Moore and

Beamish 1973). Migrations begin from March and they may travel more than 460 km upstream to spawn. Spawning begins from May and reaches a peak in mid-late May in the Delaware drainage

(Bennett and Ross 1995). Spawning occurs in nests in riffles with sand or gravel substrate and the temperatures range from 10-26°C. Transformations of larvae to adults begin in July and completed by late October. Transformers include 100-180 mm TL for Great Lakes and 146-210 mm TL for Atlantic Slope populations (Manion and Stauffer 1970, Beamish and Medland 1988).

They stay in larvae stage for 4-5 years and they can live up to 8 years (Beamish and Medland

1988). They die after spawning event.

25

Distribution

It is distributed along the Atlantic Coast from Labrador to the Gulf of Mexico in FL, native to Lake Ontario, Lake Champlain, and several New York lakes, and introduced in other

Great Lakes; also Atlantic Coast of Europe and Mediterranean Sea (Page and Burr 1991, Bryan et al 2005). In PA, it is native to the Delaware and Susquehanna River drainages and introduced in the Lake Erie drainage, where we have taken it as far upstream in Conneaut Creek as

Conneautville, Crawford County (Fowler 1940, Cooper 1983, our data). We are aware of only one modern record from the Susquehanna River drainage - a 250 mm specimen was attached to a large rainbow trout (Oncorhynchus mykiss) caught by an angler from Harvey’s Lake, Luzerne

County on 24 May 1980 (E. Cooper field notes). Sea lampreys entered Lake Erie via the Welland

Canal , which was completed in 1829, but apparently did not move into the lake for nearly a century – the first Lake Erie record is from 1921 near Merlin, Ontario (Dymond 1922). They were reported in 1936, 1937, and 1946 from lakes Michigan, Huron, and Superior, respectively

(Applegate 1950, Smith and Tibbles 1980) (Figure 9).

Status

It is now regarded as a pest and has a negative impact on game fish stocks, especially

Lake Trout, Salvelinus namaycush. Control programs have reduced their population size. The Sea

Lamprey have been widely collected throughout Delaware drainage, while it is considered extirpated from the Susquehanna River in Pennsylvania as a result of the constructions of hydroelectric dams. A few records are present in the tributaries of Lake Erie.

26

Discussion

However, only looking at the distribution of native lamprey within the political boundaries of Pennsylvania is not good enough to evaluate their general status. Their rare distribution in Pennsylvania might be caused by the degradation of water quality or the fact that their range in PA is just a corner of their whole occurrence in US, which indicates that the limited distribution and abundance now is highly relied on the nearby geography and their historical distribution national wide or even international wide. By looking at Figure 9-Figure 13, we could see the current status of lampreys in other states in the . Least Brook Lamprey

(Figure 10) is secure in most middle-east states, such as Kentucky, Tennessee, Maryland, because

Pennsylvania is on the northeast corner of their whole distribution in the US, it makes sense that they are widely distributed in southwest PA and they are vulnerable because they are at the boundary of the range. In general, their status is pretty secure in US, large populations were found in Kentucky, Tennessee, Alabama, Missouri, Mississippi, and Maryland (NatureServe 2007).

Figure 11 indicated that distribution range of Ohio Lamprey is shrinking both within the state and outside the state which may indicate potential population decline. Secure status was found in

West Virginia, Kentucky, and Tennessee. In Pennsylvania, it was once occurred in Jefferson and

Indiana Co. which is not any more now (NatureServe 2003). Mountain Brook Lamprey is distributed in eastern states, and it is vulnerable in most of these states including the red colored states of New York, Ohio, , Alabama and orange colored states like Pennsylvania,

Virginia, Kentucky and indicate (NatureServe 2007). I. unicuspis were found in the upper Mississippi and Ohio River basins, before 1900, it was widely spread in the Ohio drainage within Pennsylvania. However, due to hydrological hydrological dams and other artificial constructions in stream impeded lampreys’ migration, the population declined dramatically.

27

Among the 34 lamprey species described in the Northern Hemisphere, ten are endangered, nine are threatened, Although various agencies and authors have investigated either adult lampreys or ammocoetes, not too many collections are recorded in either of these databases.

Because most of the adults are captured accidently with other fish, and few surveys of ammocoetes were conducted. Comparing historical records, I. fossor and I. unicuspis are very rare and they are under special conservation status. I. greeleyi is a threatened species; L. appendix and I. bdellium are both the candidate species (Candidate species are plants and animals for which the US Fish and Wildlife Service (FWS) has sufficient information on their biological status and threats to propose them as endangered or threatened under the Endangered Species Act). Among the factors that reduce the abundance of native lampreys, Sea Lamprey control efforts might be a very significant one in Erie tributaries. Pennsylvania is neighbored with Lake Erie. Sea Lamprey control program is executed by the Great Lakes Fishery Commission in 1955 and works with

Fisheries and Oceans Canada, the U.S. Fish and Wildlife Service, and the U.S. Army Corps of

Engineers. Sea Lampreys were treated with lampricide TFM (3-trifluoromethyl-4-nitrophenol) to kill larval Sea Lamprey in about 175 Great Lakes tributaries. In those areas where TFM is not affective, such as the St. Maryus River and other lentic areas, different lampricide-granular

Bayluscide-is used. The Great Lakes Fishery Commission has successfully reduced Sea Lamprey populations in the Great Lakes by 90%. Lampricides are not harmful to other fish and mammals, and a typical treatment takes 48-72 hours every 4 years several times, so the short lived invertebrates could recover between treatments. Unfortunately it is toxic to other lamprey species.

American Brook Lamprey and Silver Lamprey are distributed in Lake Erie tributaries also, so this program could wipe out any of these native lamprey species in the tributaries. On May 11th, 2011,

Conneaut Creek, Crawford Co. was visited right after the treatment was implemented. The water was yellowish; only one ammocoetes either L. appendix or P. marinus was collected while the local people told us that there were plenty of them before the treatment.

28

Chapter 3: Sampling of Lampreys

Introduction

A continuous intensive sampling of both ammocoete and adult lamprey is crucial to assess lamprey population status, lamprey fisheries management, and endangered lamprey conservation (Moser 2007). Over half of all native lamprey species are considered endangered or threatened in at least a certain area of their range (Renaud 1997). Populations of native lampreys are declining due to various reasons, such as habitat degradation or reduction as well as treatment to control Sea Lamprey in Lake Erie area. Much of the available data on lamprey distribution and abundance were collected during surveys for other fishes. Sampling adults is difficult. It requires preliminary study of migration times and spawning habitat of each species. Trapping of migrating lampreys is conducted in areas where they are concentrated, such as dams and weirs (Moser

2007). Finding lamprey nests also requires knowledge of spawning times and appropriate water quality (Takayama 2002; Jang and Lucas 2005). Therefore, insufficient biological information of adult lampreys is due to little knowledge of adults’ spawning habitat and maturation time.

Furthermore the status and distribution of ammocoetes are incomplete because they bury themselves in the sediment which makes the sampling completely different to adult sampling.

Multiple protocols and sampling strategies have been developed depending on different species, their life stage and sampling objectives. Most collecting methods were developed during efforts to eradicate the invasive Sea Lamprey in the Laurentian Great Lakes area (Braem and Ebel

1961). Morris (1987) designed a double-funnel trap- in which lampreys entered the trap accidentally while seeking an upstream passage. Philip and Saint (2004) also set up a portable Sea

Lamprey assessment trap designed by Schuldt and Heinrich (1982) below the DePere Dam, and the catches were enumerated several times per week. Slade et al. (2003) quantified larval Sea

Lamprey by using direct current backpack electrofishing in 5m2to 15m2 wadable areas. An

29 electric beams trawl, which combines a sled-mounted net, has been successfully developed for

American Brook Lamprey, Northern Brook Lamprey and Silver Lamprey (Mclain and Dahl

1968). Bergstedt and Genovese (1994) designed a deep water electrofishing device to stimulate emergence of larvae from the substrate. Lampreys spend 3-7 years as ammocoetes burrowed in the substrate. For parasitic species, the adult life of parasitic forms span about 1-2 years; for non- parasitic lampreys, it is only 3-6 months without any kind of feeding actions. Therefore, ammocoete sampling is much more important to know the distribution, because all ammocoete species spend most of their life in freshwater system, their presence and absence or distributional change may better indicate freshwater quality or stream structure change through time.

Classifying and quantifying habitat within the study area were the most important steps in assessing ammocoete distribution. As the ammocoete sampling going along, I realized that ammocoetes only present at certain type of substrate. From published papers, I found out many factors have contributed to the distribution and survival of ammocoetes. Typically, the perfect substrate for ammocoetes is comprised mud, silt, sand, clay, with a large percentage of organic material (F.W.H. Beamish and S. Lowartz, 1995; Hardisty 1944; Baxter, 1957; Schroll, 1959). So by inspecting these factors at historical sites, and comparing with published data, we could at least get an idea that whether the cause of declining is the habitat degradation or not. In Europe and other states in US, substrate evaluations of ammocoetes habitat for different species have been done. PH, water conductivity are all very important indicators of the abundance of ammocoetes (Moser 2007), Several studies have focused on different factors affect the distribution of ammocoetes, such as substrate grain size, organic content, water hardness, temperature, stream class, water velocity and permeability (hydraulic 2007; William, 1994).

Among these factors, substrate grain size and water velocity were the most important indicators of ammocoetes abundance (Sugiyama and Goto 2002). Most ammocoetes prefer small substrate size (<0.5mm); high organic content (10.8%) (William 1994). However other variables (e.g.

30 shallow water depth (0-30cm); slow water velocity (0-10cm) (Sugiyama 2001)) are all very important on larger spatial scales (Moser 2007). However in Pennsylvania, I did not see any relevant studies of substrate sampling. Therefore I conducted a survey in those streams that had ammocoetes present, and looked at the difference between the ammocoete spot and non- ammocoete spot to see the differences which may help us further more in concluding what kind of habitat they prefer. This information should allow researchers better predict the distribution of ammocoetes in Pennsylvania.

Particle size of substrate might be one of the most important factors. It not only influences the respiration of ammocoetes but also the substrate porosity. Manion and Mclean

(1971) did a research on Petromyzon marinus, found out that abundant of ammocoetes were in the substrate of which the particles are less than 0.5 mm. Kelso concluded that ammocoetes preferred substrate size between 0.2 and 2mm. Potter et al. (1986) identified chlorophyll a, macrophyte roots, and low –angle shading are very important characteristics in the distribution of

Australian lamprey larvae, australia. Water depth (Malmqvist 1980), water temperate and stream gradients influenced ammocoetes distribution (Baxter 1957). So an examination of the benthic habitat size is necessary to know ammocoetes’ distribution pattern since they are filter- eaters and live under the detritus.

My objectives were to sample ammocoetes at the historical sites in Pennsylvania which have not been sampled recently, to update the presence and absence status of native lamprey species in Pennsylvania, and evaluate substrate size to compare the benthic habitat with ammocoetes and without ammocoetes within the same water system.

31

Methods and materials

Historical data integration

I obtained the historical native lamprey sites from several different organizations as listed in next section, and I condensed all data into the Arc/Info 9.3 GIS Software, which I used to create the distributional thematic map. There were about 1000 collections of historical lamprey records in the database, among which only 567 collections were valid, which were those complete records, the other ones were missing any kind of locality information which could not be mapped in GIS system. Even among the 567 records, only a very small percentage had latitude and longitude data recorded. For those sites which have latitude and longitude information, I imported them directly; for those which only have locality information, I located the sites on Delorme and

Google Earth to generate the latitude and longitude data; for Cooper’s records from the

Pennsylvania State University Fish Museum, the majority of them only had catalog numbers in file, so I selected the specimens from the shelf based on the catalog number in the database, and transcribed the locality on the tag in jar and then extracted latitude and longitude data before imported them manually into the database. For example, there was one collection of Lampetra aepyptera in Dr. Cooper’s notes conducted in 1978 and there is no lat/long information in file, the locality was Hodgsons Run-tributary to Big Elk Creek, Chester Co., 1.5 mile SW of New

London, PA. So I located the town New London in Delorme, and I used the measuring tool and located the site in the creek which was 1.5 mile southwest of the town, then extracted the lat/long from Delorme while at the same time input it into the database. I verified their locations with the latest literatures describing the range if there is any found published. For those ones only have a creek name and county name, I located the point in the middle of the creek within the county. In

32 the following section, characters, preferred habitat and general biology of each native lamprey species in Pennsylvania were provided; distributions in US and status in PA of each species were also described.

Site selection

I retrieved the information of historical sites from multiple resources, both published and unpublished. For some sites, latitude and longitude data were reported in the field notes, however, for the majority of historical sites these data were lacking. For historical sites missing their latitude and longitude, I used site descriptions to locate the sites on Delorme. Once located, I extracted the latitude and longitude data from the digital map. When all of the historical sites were mapped (Figure 13), most of them were located in Allegheny River Drainage except for those with native Sea Lamprey. All the historical collections were from the 1930s to 2009 and were recorded in the database (Appendix A-G, species specific).

The endangered species Ichthyomyzon unicuspis and Ichthyomyzon fossor were rarely recorded in PA. Therefore I sampled the streams where they were present historically regardless of location. Because Ichthyomyzon fossor is an endangered species listed by Pennsylvania Fish and Boat Commission, the only two creeks in the database - Conneaut Creek in Crawford County and Temple Creek in Erie County (see detailed information in Figure 6; Appendix B) were sampled. Ichthyomyzon unicuspis is a rarely recorded species in PA, therefor Crooked Creek in

Erie County (Figure 8; Appendix D) was the only site where they were found during 1987 and

2004 respectively so it was sampled.

Among the native lamprey species in Pennsylvania, Lampetra appendix, Lampetra aepyptra, Petromyzon marinus and Ichthyomyzon bdellium are candidate species, Ichthyomyzon

33 greeleyi is threatened species. Historical sites with at least two different native lamprey species present before no matter what their status are right now were selected as the sampling sites. The historical sites were restricted in Allegheny River drainage, Ohio River drainage, the Delaware

River main branch, and a small area along the boundary of Susquehanna River drainage. Among sites which had only one species, those had not been sampled for at least 20 years were selected.

Due to the sampling complexity and cost, clustered sampling was applied. Each creek was regarded as a single site, because my goal was only to see the presence and absence in each creek;

I did not do any abundance sampling. So my sampling sites were: Raccoon Creek (Eire County), in which Petromyzon marinus and Lampetra appendix might potentially be collected; Oil Creek

(Crawford County) with Ichthyomyzon bdellium and Lampetra appendix present, East Branch of

Oil Creek (Crawford county) with Lampetra appendix and Ichthyomyzon greeleyi present; Blue

Eye Run (Warren County) with Ichthyomyzon bdellium and Lampetra appendix present;

Woodcock Creek (Crawford County) with Ichthyomyzon bdellium and Lampetra appendix present; Spring Creek (Warren County) with Lampetra appendix and Ichthyomyzon greeleyi present (Figure 14; Appendix H). Fourteen creeks with just one historical species were selected

(Figure 14; Appendix H). They were Elevenmile Creek and Mill Creek in Potter County, east branch of Tionesta Creek and south branch of Kinzua Creek in Mckean County, west branch of

White Clay Creek and Hodgeson Run in Chester County, Martin Run in Elk County, Bull Creek in Allegheny County, Crooked Creek in Indiana County, Shannon Run and Little Pucketa Creek in Westmoreland County, Raccoon Creek in Beaver County, Mountain Creek in Fayette County and Delaware River at Porterland. Historical species at each site and information of the collections were summarized in Appendix H.

In 2011, 21 streams were inspected, among which 18 were sampled to determine the current status and distribution of lampreys, because three of them were not accessible. I surveyed

34 as close as possible to the historical GPS coordinates. However, localities were selected based on accessibility and site conditions.

Substrate collection

In May and June 2011, substrate sampling was conducted at five sites (Table 8). At each site, the procedure was performed at a twenty-meter section, which the length was overlapped by or in between the ammocoetes substrate spots and the width was stream width. The goal of this design was to analyze and compare the differences between the ammocoete substrate and the nearby random substrate under the similar physical and chemical water system, such as the same stream gradient and water hardness. If there was a particularly “good” spot in the stream, where ammocoetes were abundant as sampling, it was considered to be a good substrate sampling site.

The creek needed to be fairly at least 5 meters with a gradual substrate change, cobble- pebble like substrate in center of the stream and sandy- silty sediment toward the banks so that ammocoetes could find more sandy and calm water to burrow down. In most cases, one side was a deep bank on which higher plants were present, and on the other side it is more like a sandy, low gradient bank. I did not sample the headwaters in which the width was less than three meters and ammocoetes were found cross the width of the stream.

Twenty meters of a substrate sampling section was lined up at each site which overlapped the spots where I found ammocoetes or in between two ammocoetes spots. Substrate at each of these ammocoetes spots was sampled. Then I drew a site map on paper and put grids with 1m by

1m units on top of the sampling area on the map. Three random units were selected, and one bucket of substrate was collected at the center of each unit by using the substrate sampler (Figure

20).

35

Lids were put on the buckets during the transportation. The sampler was 60 cm high for total length, 12 cm for neck length of the cylinder shaped aluminum tool with two handles at the side toward to the upper mouth. The diameter of the upper mouth was 36 cm, and it was necked down to 15 cm of the lower neck. For most of the ammocoetes spots, it was soft and sandy, so it was easy to drill the sampler in. when it reached the upper edge of the neck, I stopped in order to get the same amount of substrate at each site. Then, a shovel was used to excavate the substrate from the sampler into the bucket. However the substrate for the random sample was more like cobble and pebble which would stop the sampler on its way down, therefore, I had to pick the big rocks out and into the bucket using my hands. The substrate sample for each spot was put into a small bucket with a lid.

Lamprey collection techniques

Most ammocoete collections rely on the use of a backpack or shore-based electrofisher in streams with water depth less than 0.8m (Moser, 2007). For juvenile sampling, the backpack electro-fishing method in either single pass (Almeida and Quintella2002) or depletion sampling

(Pajos and Weise 1994) is most frequently used. It is a two stage method for both shallow water and deep water sampling. First, a 90-125 V direct current with a 10-25% duty cycle is applied at a slow rate of 2 pulses/s to bring ammocoetes up from the sediment followed by a skipped pulse to help emerge the ammocoetes. In shallow water, the second step is followed by capturing the ammocoetes in dip nets or seines. However in deep water it is coupled with trawls (Moser,

Butzerin and Dey. 2007). Other active capture gears and methods were applied too, such as using a shovel or suction dredge to pump out ammocoetes from the sediment (Kainua and Valtonen

1980; Beamish and Youson 1987; Ojutkangas 1995) or towing nets to collect migrating ammocoetes.

36

The backpack I used to collect ammocoetes was an ETS ABP 3(Engineering technical service) made by The Penn State University- Dr. Stauffer’s lab (Figure 15). It had only one channel and two probes with mesh on the diamond shaped hoods. By using this, it was easy to scoop the ammocoetes up as soon as they were seen. Electrofishing relies on two electrodes which deliver current into the water to stun fish. The current runs from the anode to the cathode.

One of the probes is anode and the other is cathode, so both of them have to be in the water to work. The backpack could be turned on by toggling the switch under the meter box. It has a pulsed DC system designed especially for ammocoete, and the voltage was set to be between 0-

125 volts, depended on different conductivity. A lot of the ammocoete sites were in low order streams; so, the conductivity was low and the resistance was high. Therefore, the voltage was set higher to get a higher current which range from 2-4 ampere. The duty cycle was about 20%, and the frequency was either 3 pulses/s to draw the ammocoetes out from the substrate or 30 pulses/s to shock them after them emerging from the sediment.

The other spare backpack was Smith-Root LR24 which has two channels and the frequencies were set up to the same value as the ETS ABP 3 (Figure 16). The probe is anode and tale is cathode. Burst of pulses could be selected by push the pulses type button, and it was set 2-

2. The 2-2 setting produced three pulses/s followed up by a skipped pulse, which is an appropriate frequency shocks ammocoetes up from the substrate. I found out that the ammocoetes were shooting up from the sediment and then trying to burrow themselves into the substrate with vigorous contractions of the tail (Hill and Potter 1970). Then right after any ammocoetes surfaced, the frequency was immediately switched to 30 pulses/s to immobilize the ammocoetes.

The ammocoetes were then moved to the buckets containing stream water.

Before each collection, I did a visual observation of the site. The water was not too high or too low, between 0.2-1m, I would conduct the backpack sampling irrespective of the substrate type. The physical conditions limited most sites to stream margins of sandy substrate. I walked

37 both the upstream and downstream portions of the historical sites and then selected a 100m-200m sampling section with a combination of different substrate types. I also sampled both the center of the streams and bank sides. I sampled a minimum of 100m if there were any ammocoetes found; if none were found in the first 100m, I extended my sampling length to 200m, either upstream or downstream of the historical sites.

All captured lampreys were kept and anesthetized using MS 222, preserved in 10% formalin in the field, and after one week, rinsed and transferred to 70% ethanol for permanent storage in the Penn State University Fish Museum. Lampreys have an eel-like body, and would bend when preserved in formalin, which hindered myomere counting under the microscope.

Therefore, I cut PVC pipes with diameters ranging from 10mm-20mm into pieces to fit in the plastic jars. At different sites, appropriate combination of pipes were bound together with zip ties and put in the jars filled with 10% formalin (Figure 17). Each individual was slid into a pipe, so that they would be straight. In the laboratory, adults were identified to species based on their teeth structure; ammocoetes can seldom be identified to species with confidence due to the lack of teeth development, so in most cases they were identified based on the number of trunk myomeres, blocks of muscle tissue along body, or the adults found at the same location were used as reference.

Substrate drying and sieving

Dried substrate was sieved to determine the substrate size by weight. In the laboratory, each bucket of substrate was dumped onto a baking tray and dried at 100 ºC for 24 hours.

Immediately after the substrate was taken out from the oven, it was sieved by using a set of standard series of sieves produced by Fisher Scientific Company (Figure 21). The 12 copper sieves stacked together and had a diameter of 21cm and a depth of 5cm. and the mesh size

38 decreased from top to the bottom. The aperture sizes, from top to bottom were 12.7mm, 8mm,

4mm, 3.35mm, 2.38mm, 2mm, 1.68mm, 1.4mm, 1.19mm, 1mm, 0.85mm, and 0.25mm.

The residual material after being dried contained cobbles, pebbles, debris, and silt-clay. I crushed the dried substrate in the tray and the contents were dumped on the top sieve. I also brushed off the sand that stuck on the big rocks into the sieves, so that when I picked up big ones it would have a minor influence on the result. I stirred the substrate with a stick, and shook the whole set of sieves very hard to let the substrate drop through the apertures, and each size class was weighed on a large mechanical scale when estimated to be more than 10g, or the a small electronic scale if the sample was less than 10g (Figure 22). Sieved fractions, as well as the material which passed through the last sieve were held by a plastic tray, and it was weighed along with the last tray as <0.25mm diameter material.

Classification of particle sizes was modified by dividing the substrate into different categories as following: cobble/pebble, >=8mm; gravel, 2mm-4mm; coarse sand, 1.4mm-2mm; median sand, 1mm-1.4mm; fine sand, <1mm.

Results

Sampling of ammocoetes

Twenty-one creeks were inspected. Lampreys were captured at fourteen sites within

Pennsylvania (Table 2; Figure 19); four creeks were sampled but no lampreys were captured

(Table 3; Figure 19). The three remaining sites were visited but not electrofished, either because of a high water level that was not suitable for backpack electrofishing, or low water with cobble substrate in which lampreys were unlikely to be present (Table 4; Figure 18). For example,

Raccoon Creek was very turbid and deep, thick vegetation covered half of the stream which was

39 inaccessible. Additionally, a historical site in the West Branch of White Clay Creek had its headwater on a private farm; The stream was only 1-2 meters wide with no clay or sand like substrate (Figure 18). Two auxiliary collections were provided by Rich Taylor in 2011. P. marinus was collected at two sites, Patrick Run, Erie County and Marshalls Creek, Monroe

County. (Figure 19; Table 5)

Three hundred and fifty-three individuals of 4 species, L. aepyptera, L. appendix, I. greeleyi, P. marinus were found in the field study, and lengths of each species were summarized.

(Table 6).

Many protocols have been developed for conservation or eradication of ammocoetes or adults (Moser and Dey 2007). Most of them were species specific which provided useful biological and ecological information of both adults and ammocoetes. Among these protocols, backpack electrofishing was widely utilized for ammocoete capture (Steeves 2003). Various environmental factors may have influences on collecting effectiveness. For ammocoetes sampling in tributaries of the Great Lakes, effectiveness is positively related to length of lampreys and negatively related to water depth and larval densities (Hill and Willis 1994; Hardin and Connor

1992; Steeves 2003). Capture rates also depended on the personnel’s experience and the time devoted to the sampling spots. At the first site, I did not collect any ammocoetes because I spent the most time shocking the middle of the channel. After a few trials, I realized ammocoetes were found in silty and shallow areas; Therefore, I put more efforts into sampling sandy, low water velocity areas. If there were any ammocoetes occurring in one spot, I would keep sampling back and forth until no additional lampreys were captured. A frequency of 3 pulses/ second frequency worked very well to bring ammocoetes out from the substrate, but the collector had to switch to higher frequency quickly before they burrowed themselves again. I discovered that 30 pulses/second might not be high enough to stun the fish, because a few ammocoetes escaped.

40

The observation at each historical site was very crucial. Although the GPS could locate the historical site very accurately, and the water chemistry was very similar within the stream, habitat type was still quite different from one spot to another because of the current’s effect, vegetation cover and flow direction. It would also change after high water level events. So, at each site, I walked through both upstream and downstream, figured out the best section in that stream and started to sample. For example at my first site, Oil Creek, nothing was found in the first 200m, however before we headed back, we found two spots where plenty of ammocoetes were emerging. Therefore, I observed at historical site for at least 500m and sampled at least

200m to conclude that there was no suitable substrate or there were no ammocoetes discovered.

A major problem in understanding the lamprey’s life history, behavior, and conservation status is the identification of ammocoetes to species. For example, most Ichthyomyzon species were considered indistinguishable as ammocoetes (Hubbs and Trautman 1937) until a few key characteristics were developed based on individual sizes, lotic habitat preference, diagnostic genetic, meristic, morphometric and pigmentation characteristics (Vladykov and Kott 1980;

Lanteigne 1981; Fuiman 1982). Taxonomic keys with respect to the classification of ammocoetes, such as restriction fragment length polymorphisms (RFLP) have been widely used and successfully applied to distinguish Chestnut Lampreys from Silver Lampreys and Northern Brook

Lampreys (Mandrak et al. 2002). However it is impossible to do genetic identification in the field, so external characteristics such as pigmentation and morphometric keys are used frequently. A lot of ammocoetes continue to be difficult to identify, even though there are differences in the numbers of trunk myomeres which can be observed under microscope, some overlaps of which of different species could still cause confusion. Other researchers are trying to use other common characteristics to solve this confusion.

Mandrak (2002), who was trying to distinguish Chestnut Lamprey (I. castaneus), Silver

Lamprey (I. unicuspis), and Northern Brook Lamprey (I. fossor) found in the Great Lakes Basin

41 based identification on pigmentation of the lateral line and caudal fin, Vladykov and Kott (1980) found out that ammocoetes of Silver Lamprey have more pigmentation on the caudal fin and head than Northern Brook Lamprey; Lanteigne (1988) noticed that Northern Brook Lamprey has a narrow band of pigmentation on its dorsal fin and extending to the branchial pores.

I used numbers of trunk myomeres, dorsal fins and different geographic ranges to identify the ammocoetes. Unfortunately, Silver Lamprey has 49-52 trunk myomeres while Northern

Brook Lamprey have 50-52 trunk myomeres; Mountain Brook Lamprey (I. greeleyi) have 57-60 myomeres while Least Brook Lamprey (L. aepyptera) have 52-59 myomeres. Although they could theoretically be distinguished by numbers of dorsal fins, when the ammocoetes were 3 inches or less, it was difficult to tell the beginning of the myomeres and how deep the dorsal fin was notched. There are some overlaps in the numbers of myomeres between species, but I could at least narrow them down to one or two species based on the historical range.

There were only two collections for which I was not sure about identification. One had only one individual which was found in Conneaut Creek, Crawford County. Bacause L. appendix,

P. marinus and I. fossor were all in the historical record; my myomere counting result was within both ranges of L. appendix and P. marinus. The other collection contained individuals found in

Hodgeson Creek. The only historical species was P. marinus, but 24 individuals with about 55 myomeres were discovered at the same site, I identified them as L. aepyptera, because of the nearby distribution of Least Brook Lamprey. To prove my identification, I will go back and collect adults for my identification next year.

Historical and current species at 19 sampled sites were compared in Table 7. Among 7 sites which had 2 or 3 species present historically, only the E. Branch of Oil Creek and Spring

Creek had 2 species present as historical data; three creeks-Blue Eye Run, Crooked Creek in Erie

Co. and Conneaut Creek had only 1 species occur. None were discovered in Oil Creek,

Woodcock Creek and Raccoon Creek in Eire Co. Among the streams that had only 1 species

42 historically, no lampreys were discovered in Mill Creek, Little Pucketa Creek, West Br. of White

Clay Creek and Raccoon Creek State Park. P. marinus was historically found in Hodgeson Creek, but L. aepyptera was also found in my study. This was not surprising, because nearby creeks had

L. aepyptera present, so as long as Hodgeson Creek had suitable substrate and no geological or human barrier was found, migration was predictable. Although I did not conduct any complete sample to predict the quantity of each species, we could see their declining trend by looking at the presence and absence data. However, I want to clarify that the presence and absence data were based on my one and a half month field study. I only walked through each creek less than 500m, and sampling was done within 200m. If no lampreys were collected, the site was classified as no individuals found in the creek. Individuals could possibly be found in other section of the same creek, but since I was sampling historical sites, they should have higher possibility of lampreys present.

Analysis of substrate

Both lamprey and random substrate samples were collected at five sites, and they were dried, sieved and weighed at Dr. Stauffer’s fish laboratory (Table 9). The sampling locations were listed in Table 8, and following abbreviations were clarified:

“Rs” represents Random sample (starting transact), “Lu” represents Lamprey site (upstream).

“Rm” represents Random sample (mid transact), “Lm” represents Lamprey site (midstream).

“Re” represents Random sample (ending transact), “Ld” represents Lamprey site (downstream).

Two general patterns of sites were observed. For each creek except Elevenmile Creek in

Potter County, both ammocoete spots and random spots were sampled. Elevenmile Creek was only about 2-3 meters wide, and it was all sandy substrate across the creek, therefore, I only

43 sampled the ammocoete spots. Table 10 is all of the random sample data, I calculated the probability of each sieve size by the given weight, and then placed the mean substrate size for each set of data on the right-most column. The mean value for each column was summarized at the bottom. Particle size composition of all ammocoete sites varied, but it can be seen that, for randomly picked samples, the 12.7mm sieve contributes majority of the entire substrate weight, which is 56%. The other sieves all take a proportion less than 10%. The average substrate size is about 8.5mm.

Substrate remaining on each sieve was weighed and the percentage taken from the whole weight was calculated for every substrate sample (Table 11). The probabilities of the first few weights are either 0 or very small percentile. Median sand typically contributes more than the coarse sediment. Fine sand especially <0.25mm constitutes 36%-99% with mean value 68%, which means the large amount of fine silt-clay substrate is an essential factor of the presence and absence of ammocoete in creek. For substrate size, compared to 8.5mm of random sample,

1.95mm is much smaller which indicates the same conclusion.

Minitab analysis of the sampling results

To verify the effectiveness of the sampling for both random samples and lamprey site samples, as well as getting a deeper insight of what the data tells us, Minitab was utilized to perform a series of analysis on the collected data, as following.

First I tested the normality of the sampled data. Normality test results for the mean substrate sizes of random samples and lamprey sites are performed, as in Figure 23 and Figure 24 respectively.

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It can be seen that the mean sieve size for random samples is normally distributed (P-

Value greater than 0.05), while for lamprey sites, the mean sieve size is not normally distributed.

This is because that 3 points far away from the others in the dataset. Because the sample size was not too large, I preferred not to delete these points because it may represent the differences among species or other factors may potentially affect the substrate size.

For these two groups of data which are not both normally distributed, we can use the

Levene’s Test to test the homogeneity of variance of the two groups, as in Figure 25. Since the resulted P-Value (0.859) is greater than 0.05, it can be concluded that there is no statistical differences in the variances between the two groups of data.

Since the data is not normally distributed, I performed the Mood’s Median Test to compare the median value of the two groups, as in the following.

Mood Median Test for ammocoetes sites and random sites.

Individual 95.0% CIs group N<= N> Median Q3-Q1 ------+------+------+--

1 11 0 0.45 1.91 (+------) 2 1 11 8.25 1.56 (-+---) ----+------+------+------2.5 5.0 7.5 Overall median = 4.83

Chi-Square = 19.33 DF = 1 P = 0.000

A 95.0% CI for median(1) - median(2): (-8.59,-6.63)

The null hypothese is the population medians for both ammocoetes sites and random sites are the same. Since the P-Value is less than 0.001, there is significant difference between the median values of the two groups, as can be easily seen from the results above. So, we can conclude that the lamprey site samples and random samples have large differences in terms of

45 substrate size. Therefore, we can recognize and tell whether a site has lamprey or not based on the substrate size.

The next question would be, for different substrate sizes, what are the probabilities for either random samples or lamprey group samples. Therefore, I did regression analysis on the probability versus substrate size for both groups.

For random samples, the quadratic gives satisfactory regression results as in Figure 26.

The regression result reveals substrate size is very significant predictor (P<0.05) and the regression model is statistically significant. Therefore we can utilize the model below to predict the probability of a certain substrate size in a random sample (probability is calculated as the proportion of the unit weight by the total weight):

Probability = 0.0699-0.0356*Substrate Size+0.0057*Substrate Size^2 (95% CI)

For lamprey site samples, the data are more difficult for regression analysis. I tried the cubic regression with result shown in Figure 27.

The P-Value is greater than 0.05, which indicates that the regression factors are not very significant predictors, but this model can still give us some insight to the probability of different substrate sizes on the lamprey site.

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Discussion and Conclusion

Discussion

On each map I created (Figure 3- Figure 9), historical sites were marked with red solid circles, and the numbers beside were corresponding to the detailed information of collections in

Appendix A-G, information such as the collection date and collectors could be looked up in appendix. The overview window on each map visualized the overall distribution of each species.

Three out of twenty-one sites were not accessible most likely because the water level was too high or the riparian zone was very bushy that making the historical site not assessable, and in this case the substrate were not ideally for ammocoetes, such as Raccoon Creek was too turbid and deep so there was no shallow, calm and warm swamp for ammocoetes. I would assume that the chances of finding ammocoetes at these three sites- West Branch of White Clay Creek in Chester

Co., Woodcock Creek in Crawford Co., Raccoon Creek State Park in Beaver Co. was very small.

For those sites without any lampreys found: Oil Creek, Crawford Co., Raccoon Creek, Erie Co.,

Mill Creek, Potter Co., Little Pucketa Creek, Westmoreland Co., the sampling length was at least

200 meters long with potential lamprey substrate type existed in between the starting and ending point. Although I would not be able to conclude that there were no ammocoetes in creek by just sampling 200 meters, the fact that there were fewer lampreys present at the historical sites than in the past may indicate a declining trend of the population. There was no way to sample the whole stream, but the spots where ammocoetes were found have a higher probability to support ammocoetes than other locations where lampreys have never been found before, so the statement of their potential declining could be established. However substrate alters very quickly according to the temperature and precipitation. In May 2011 we captured quite a few ammocoetes in

47

Crooked Creek, Erie Co. One month later in June we sampled the site again, but the substrate changed a lot, and the sandy substrate was replaced by algae, there was less oxygen in the creek and water had risen up over the riparian zone, so those good spots even one month ago was gone.

Abundance sampling was required to assess the population alteration at those sites had ammocoetes historically and contemporarily, but it was very difficult to estimate the population by only completely sampling one section of creek because the available substrate may not be representative of the rest creek which was not observed. However I found that the number of species declined as time went by, e.g. Conneaut Creek, Crawford Co. only had one species as well as Blue Eye Run, Warren Co. instead of multiple species as was found historically, in this case either competition existed between species while available substrate areas were shrinking or the total population was declining because of various reasons such as the degradation of water quality or the survival rate of lamprey eggs.

Other factors like spawning habitat and water quality degradation could also affect the inland distribution. An ammocoete burrows by inserting its head into the substrate vertically with vigorous contractions of the body. This process is combined by a quick inserting of its anterior part and a gradually inserting of the posterior part. But when being shocked gently, it was very fast drilling down, mucous secretion from the larvae endostyle line the burrow and structure the sandy tube. When scooped up the ammocoetes in the drilling process by the diamond shaped mesh probe, we usually dug down a few centimeters more to the substrate. One of the most important factors that influence their suitable habitat is the particle size, and of course fine sediment and little debris was optimum material since they represent most percentage of the substrate. However an over-abundance of silt/clay particles could keep oxygen away by clogging their gill lamellae (Beamish & Jebbink 1994). Unfortunately the finest mesh size was 0.25 mm in my study, anything smaller than that was put in the 0.25mm column which included a large proportion of the total particles in the last column. Smaller mesh may be required for any

48 subsequent studies to narrow down the preferences of particles sizes of each ammocoete species.

The habitat used by ammocoetes were always found in eddies and backwaters, either toward the edge of vegetation roots or behind natural obstructions, like trunks or sand pond, where the water velocity was slower than the main stem of the creek and the organic matter accumulates enough to feed ammocoetes. To physically define the water velocity range is a little bit difficult, because of the fact that it varies with the different depth of the creeks and different time period of the collection. While most of the ammocoetes are found toward the bank, very shallow area, usually

>2.5 cm but <7.8 cm deep (Kelso, 1993), it is not always the case, some of them were found in deep water such as main rivers and lakes (Applegate, 1950). It is stated in Schroll’s (1959) research that average water surface velocity for Lampetra planeri and Eudontomyzon danfordi is

0.5m/s, Thomas (1962) and his group indicated that the maximum flow rate is no more than 0.6-

0.8 m/s. In Schroll’s (1959) and Baxter’s (1957) study, it was found that “ammocoete beds”

(Baxter 1957) were found in partly shady area, which provides them a stable microenvironment.

But in sampling creeks I found that, a lot of “ammocoetes beds” were in an open-air area, and the water temperature was warmer than areas in the shade. Schroll (1959) suggested that ammocoetes of L. planeri prefered water temperature to be about 12 ºC; while from Reynolds and Casterlin’s

(1978) lab experiment, 10-19 ºC would be more preferred. Other studies have attempted to define the substrate selection of ammocoetes, and there is not too much difference among species.

Schroll (1959) and Thomas (1962) studied the relationship between the substrate selection and parameters such as O2, CO2, pH, hardness, particle size and organic content of the substrate, temperature, flow volumes and water velocity, and only found significant relationship between water velocity, substrate hardness and ammocoete abundance.

Water parameters were recorded at some ammocoete sites, temperature was from 12.6

ºC - 17.3 ºC, and bottom flow for the ammocoete spot was ranged from -0.01-0.01m/s while 6/10 flow was between 0.01-0.12 m/s. Water depth was very shallow which was from 1- 20cm toward

49 the riparian zone in small creeks. Because of the gear availability and human efforts limitation, not all sites were measured, however the data above indicated that water was pretty calm and shallow compared to the adult lamprey habitats. This might be correlated with their oxygen uptake underneath of the sandy substrate which depends on the amount of dissolved oxygen.

Only one adult was captured together with ammocoetes, I not only sampled those calm areas near shore with fine substrate using a combined frequencies of 3 pulses/s and 30 pulses/s but also sampled the middle channel by using 30 pulses pulses/s all the way up and down, no adults were found separately. The causes could be either too low frequencies and voltage because adults were collected by another crew in our lab aimed for trout by using higher frequencies or it was too late for spawning adult lampreys based on the suitable temperature for them in the literature, and it is usually from late March to early June depends on the latitude. In spring 2012, we are going to sample those creeks with lampreys present and focus our capture methods toward spawning adult lampreys. This will help to verify the identification, because the counting of myomeres and observation of their dorsal fin might not be accurate enough while also providing data to check if there are any more spawning lampreys or not in the same stream. Predators of lamprey eggs will also be captured and analyzed to see the composition of their living communities and competition between them. To solve the confusion in 2011, two different backpacks with different frequencies and voltage settings will be utilized to verify the effectiveness of the ammocoete backpack.

Conclusion

Seven native lamprey species were found historically in Pennsylvania based on data from the Pennsylvania Wild Resource Conservation Program; the Pennsylvania Fish and Boat

50

Commission (PFBC); the Pennsylvania State University; Cornell University; Dr. Edwin L.

Cooper and Dr. Jay R. Stauffer. They are Lampetra aepyptera, Lampetra appendix, Ichthyomyzon bdellium, Ichthyomyzon fossor, Ichthyomyzon greeleyi, Ichthyomyzon unicuspis, Petromyzon marinus. Lampetra aepyptera was found in western PA in the Ohio River drainage in Allegheny,

Armstrong, Beaver, Butler, Fayette, Indiana, and Westmoreland counties. Lampetra appendix is restricted to the northwest and southeast corners of Pennsylvania, where it inhabits the following drainages – Lake Erie, Allegheny River, Genesee River, and Delaware River. It has been collected from Crawford, Elk, Erie, Forest, McKean, Potter, Venango, and Warren counties, as well as from the White Clay Creek drainage in southern Chester County (Figure 4). Ichthyomyzon bdellium appears in the Allegheny River drainage, where it was most abundant in the northwestern section of the state near the Great Lakes area (Figure 5).

In Pennsylvania, Ichthyomyzon fossor was found in the Conneaut Creek drainage in

Crawford and Erie Counties (Cooper 1983), and it is an endangered species listed by PFBC

(Figure 6). The Pennsylvania distribution of Ichthyomyzon greeleyi includes the Clarion and upper Allegheny River drainages, and also the French Creek, and the Neshannock Creek in the

Beaver River drainage (Figure 7). Ichthyomyzon unicuspis was one of the rarest species in PA. It was only recorded in Crooked Creek, Erie Co. (Figure 8). In Pennsylvania, Sea Lamprey is native to the Delaware and Susquehanna River drainages and introduced in the Lake Erie drainage

(Figure 9).

Lampreys were captured at 14 sites; four creeks were sampled but no lampreys were found; three sites were not accessible. In total, 353 individuals of four species were found in my study in May and June, 2011, these species include Lampetra aepyptera, Lampetra appendix,

Ichthyomyzon greeleyi and Petromyzon marinus. Sampling results by species and collections are in Appendix I.

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No endangered species Ichthyomyzon unicuspis or Ichthyomyzon fossor was found in my study, which may indicate their limited population. Historical species and current species at 19 sampled sites were compared in Table 7. Among 7 sites which had 2 or 3 species present historically, 2 different species were collected simultaneously in E. Branch of Oil Creek and

Spring Creek; Only one species was found in three other creeks-Blue Eye Run, Crooked Creek in

Erie Co. and Conneaut Creek. No lamprey species was discovered in Oil Creek, Woodcock Creek or Raccoon Creek in Eire Co. Among those ones only with 1 species present historically, no lampreys were discovered in Mill Creek, Little Pucketa Creek, West Br. of White Clay Creek and

Raccoon Creek State Park.

The sieved results revealed that ammocoetes prefer small-sized particles < 0.25mm with resolved organic matters; substrate collected from non-ammocoete sites were between 0.85mm-

12.7mm, which was significantly different than the former one.

More information on the distribution of lampreys in Pennsylvania is essential both to confirm the representativeness of their historical distributions already altered and to provide a new distribution map and potential water quality degradation trend. In a lot of lamprey surveys, juvenile lampreys were not identified to species, this is a very important fact we need to consider when compiling other studies. It would be extremely beneficial if a quick reliable field identification method were available to permit the identification of juveniles to species. Routine river survey work from institutions and organizations and by-catch collections are suggested to be put in a well-designed database in GIS system based on fundamental distribution knowledge of each species, which could visualize the on-going change of their dispersion on map. And the interface of GIS will be a fabulous way to check the table of detailed information and distribution map since it would be very accessible.

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Overall, L. appendix, L. aepyptera, and I. greeleyi are widely present in Pennsylvania because of their historically wide distribution. Invasive P. marinus is believed to be distributed in

Lake Erie tributaries whereas native P. marinus is distributed in the Delaware River drainage.

Treatment using lamprecide is implemented every few years, and adult Sea Lampreys were captured in Marshall’s Creek, Delaware River drainage. I. fossor and I. unicuspis are of particular concern. They are both suspected to be distributed in Lake Erie drainage. I. fossor is an endangered species in Pennsylvania while the status of I. unicuspis in Pennsylvania has not been proved. Therefore the population of native lamprey species in Lake Erie River drainage is declining due to the chemical treatment program for Sea Lamprey. Most of the lamprey populations are in Allegheny River drainage and Ohio River drainage and their status is secure. L. aepyptera and L. appendix abundance could even be increasing because they are further away from civilizations in head waters. Their population cannot be predicted because abundance surveys have not been done by any agencies or individual researchers in PA. It is difficult to capture them all because they all hide under the muddy substrate, and substrate type cannot be known until an actual site observation carried out. Even if there is suitable substrate present it does not guarantee their presence, which depends on the geographical approach of different species. However, they prefer to spawn in clear and shallow middle channel with faster water velocity and high dissolved oxygen, so it might be a sign of degraded water quality if they are absent in spawning season. In Pennsylvania, few surveys about lampreys are done, most of the lamprey collections were by-catch. Therefore, knowing lamprey’s presence and absence not only casts light on the status of lampreys in Pennsylvania, but also enlightens us on the water quality of freshwater systems. Subsequent projects are needed to elaborate broader range of lampreys in

Pennsylvania.

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Figure 1: Map of Pennsylvania’s major drainage basins. Six major watersheds: the Susquehanna River, Lake Erie, the Genesee River, the Ohio River, the Delaware River and the Potomac River.

Figure 2: Adult lamprey teeth structure. Petromyzon marinus disc. Made By Canadian Museum of Nature.

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Figure 3: Distribution of Lampetra aepyptera in PA. Historical data was from 1933-2009. Red circles indicate historical sites; blue lines are Pennsylvania major rivers; grey lines are boundary of major river basins. Overall distribution of each species in Pennsylvania was shaded in the inset window. Least Brook Lamprey aggregated in the southwest of PA. Detailed data for historical sites are provided in Appendix E.

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Figure 4: Distribution of Lampetra appendix in PA. Historical data was from 1959-2009. Red circles indicate historical sites; blue lines are Pennsylvania major rivers; grey lines are boundary of major river basins. Overall distribution of each species in Pennsylvania was shaded in the inset window. American Brook Lamprey aggregated in the northwest of PA and also in the southeast corner of PA. Detailed data for historical sites are provided in Appendix F.

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Figure 5: Distribution of Ichthyomyzon bdellium in PA. Historical data was from 1965-2009. Red circles indicate historical sites; blue lines are Pennsylvania major rivers; grey lines are boundary of major river basins. Overall distribution of each species in Pennsylvania was shaded in the inset window. Ohio Lamprey aggregated in Allegheny River Drainage. Detailed data for historical sites are provided in Appendix A.

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Figure 6: Distribution of Ichthyomyzon fossor in PA. Historical data was from 1968, 1975, 2003, and 2008 in the database. Red circles indicate historical sites; blue lines are Pennsylvania major rivers; grey lines are boundary of major river basins. Overall distribution of each species in Pennsylvania was shaded in the inset window. Northern Brook Lamprey was found in Temple Creek, Erie Co. and Conneaut Creek, Crawford Co. Detailed data for historical sites are provided in Appendix B.

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Figure 7: Distribution of Ichthyomyzon greeleyi in PA. Historical data was from 1934-2009. Red circles indicate historical sites; blue lines are Pennsylvania major rivers; grey lines are boundary of major river basins. Overall distribution of each species in Pennsylvania was shaded in the inset window. Mountain Brook Lamprey aggregated in the northwest of, Ohio drainage and Allegheny drainage. Detailed data for historical sites are provided in Appendix C.

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Figure 8: Distribution of Ichthyomyzon unicuspis in PA. Historical data was from 1987 and 2004. Red circles indicate historical sites; blue lines are Pennsylvania major rivers; grey lines are boundary of major river basins. Overall distribution of each species in Pennsylvania was shaded in the inset window. Silver Lamprey was only found in Crooked Creek, Erie Co. Detailed data for historical sites are provided in Appendix D.

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Figure 9: Distribution of Petromyzon marinus in PA. Historical data was from 1975-2009. Red circles indicate historical sites; blue lines are Pennsylvania major rivers; grey lines are boundary of major river basins. Overall distribution of each species in Pennsylvania was shaded in the inset window. Sea Lamprey aggregated in Erie drainage, Delaware drainage and a little corner there Chester Co. Individuals found in Lake Erie are all invasive Sea Lamprey, and those in Delaware River drainage are native Sea Lamprey. Detailed data for historical sites are provided in Appendix G.

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Figure 10: North American State/Province Conservation Status of L. aepyptera. Map by NatureServe (September 2007).

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Figure 11: North American State/Province Conservation Status of I. bdellium. Map by NatureServe (June 2003).

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Figure 12: North American State/Province Conservation Status of L. greeleyi. Map by NatureServe (September 2007).

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Figure 13: Lamprey historical sites (1933-2009) by species in Pennsylvania. Dots with different colors indicate different species. Most of them are located in west Pennsylvania, and Allegheny River drainage.

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Figure 14: 2011 lamprey sampling sites in Pennsylvania. The sampling was designed for ammocoetes by using the ammocoete backpack, but adults was also aimed to by using 30 pulses/ second frequency. They were sampled from May to early June. Detailed information for each site and the expected species are provided in Appendix H.

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Figure 15: ETS-ABP electrofisher. Left is the backpack panel, voltage range, duty cycles and frequency could all be adjusted, and right hand side are the two probes, one is anode, the other is cathode.

Figure 16: Smith-Root LR 24 electrofisher

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Figure 17: Ammocoete field container. Different sized PVC tubes were bounded together by zip tiles and put in the plastic jars, so that the ammocoetes could be straight laid in the tube.

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Figure 18: West Branch of White Clay Creek, visited on June, 2011. Very narrow, historically Lampetra appendix was found, but at both upstream and downstream there was no suitable ammocoete substrate.

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Figure 19: Lamprey sampling results in 2011. The green solid dots were where I discovered lampreys, which were labeled from 1-14, See Table2 for detailed information. Hollow points were sites I sampled but did not find any lampreys, see Table 3 for detailed information. Circle with cross were sites not accessible, see Table 4 for detailed information. Purple points-22, 23 were auxiliary data from other field study, see Table 5 for detailed information. Detailed collection data by species is provided in Appendix I.

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Figure 20: Lamprey substrate sampler. The sampler was 60 cm high for total length, 12 cm for neck length of the cylinder shaped aluminum tool with two handles at the side toward to the upper mouth. The diameter of the upper mouth was 36 cm, and it was necked down to 15 cm of the lower neck.

Figure 21: Sieve series produced by Fisher Scientific Company. The 12 copper sieves stacked together and had a diameter of 21cm and a depth of 5cm. and the mesh size decreased from top to the bottom. The aperture sizes, from top to bottom were 12.7mm, 8mm, 4mm, 3.35mm, 2.38mm, 2mm, 1.68mm, 1.4mm, 1.19mm, 1mm, 0.85mm, and 0.25mm.

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Figure 22: Two different scales for substrate weighing. Large mechanical scale (right) when weights were estimated to be more than 10g, or small electronic scale (left) if the sample was less than 10g.

Figure 23: Normality test for random substrate samples.

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Figure 24: Normality test for lamprey substrate samples.

Figure 25: Homogeneity of variance test for data of random substrate samples and lamprey substrate sites.

73

Figure 26: Regression analysis for random samples.

74

Figure 27: Regression analysis for lamprey substrate samples.

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Table 1: Distinctive characteristics in Petromyzontidae and the other two families

Petromyzontidae Geotriidae Mordaciidae

Snout and disc region of sexually mature males slightly enlarged greatly enlarged greatly enlarged

Gular pouch of mature male of anadromous species Never conspiuous Well developed Well developed

Changes in dentition during spawning run Relatively slight Radical in males Radical in both

First and second dorsal fins in forms approaching maturity Continuous Separate Separate

Fresh water parasitic species Several None None

Table 2: 2011 lamprey sampling results in Pennsylvania, site with lamprey present. Ammo. ID CREEK COUNTY Coll. No. LATITUDE LONGITUDE No. 1 E. Branch of Oil Creek Crawford SLL-2-11 41.75685833 -79.735675 27 2 Spring Creek Warren SLL-3-11 41.85499 -79.54156167 4 3 Blue Eye Run Warren SLL-4-11 41.84833333 -79.42833333 8 4 Crooked Creek Erie SLL-5-11 41.993645 -80.41923833 4 5 Conneaut Creek Crawford SLL-7-11 41.75846333 -80.37145667 1 6 Martin Run Mckean SLL-9-11 41.600675 -78.90640667 53 7 Elevenmile Creek Potter SLL-13-11 41.94657167 -78.070145 18 8 Shannon Creek Westmoreland SLL-14-11 40.37173167 -79.079835 5 9 Mountain Creek Fayette SLL-15-11 39.77792833 -79.82420333 31 10 Bull Creek Allegheny SLL-16-11 40.61492 -79.76466667 2 11 Crooked Creek Indiana SLL-18-11 40.667215 -79.08959667 49 12 Hodgeson Creek Chester SLL-19-11 39.747095 -75.86673833 52 13 S. Branch of Kinzua Creek Mckean SLL-11-11 41.708618 -78.820312 11 14 E. Branch of Tionesta Creek Mckean SLL-10-11 41.688442 -78.92524 16 Total: 281 Table 3: 2011 lamprey sampling results in Pennsylvania, sites without lamprey. ID CREEK COUNTY Coll. No. LATITUDE LONGITUDE 15 Oil Creek Crawford SLL-1-11 41.73323833 -79.76863 16 Raccoon Creek Erie SLL-6-11 41.96507333 -80.45985833 17 Mill Creek Potter SLL-12-11 41.75362167 -77.94954333 18 Little Pucketa Creek Westmoreland SLL-17-11 40.57335167 -79.748135

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Table 4: 2011 lamprey sampling results in Pennsylvania, sites not assessable. ID CREEK County LATITUDE LONGITUDE 19 West Branch of White Clay Creek Chester 39.769033 -75.824286 20 Woodcock Creek Crawford 41.689483 -80.0518 21 Raccoon Creek State Park Beaver 40.507116 -80.3556

Table 5: Auxiliary lamprey collection from other field study conducted in 2011. Ammo. ID CREEK COUNTY Coll. No. LATITUDE LONGITUDE No. Species 22 Marshalls Creek Monroe RBT-014-11 40.99859 -75.13886 8 P. marinus 23 Patrick Run Erie RBT-006-11 41.83134 -79.67471 69 P. marinus

Table 6: Length analysis of ammocoetes caught in 2011. standard Lamprey species N Mean Range Minimum Maximum deviation mean +- 95% CI I. greeleyi 23 75.742 104.83 33.07 137.9 38.353 (59.16, 92.33) P. marinus 36 98.38 131.21 48.83 180.04 36.79 (85.93, 110.82) L. aepyptera 109 82.7 135.39 31.79 167.18 29.19 (77.15, 88.24) L. appendix 183 131.69 177.22 30.16 207.38 44.27 (125.23, 138.15)

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Table 7: Comparison of historical species and current species at 19 sampled sites in PA, 2011. Creek Co. Historical sp. Current sp. E. Branch of Oil Creek Crawford L. appendix/I. greeleyi L. appendix/I. greeleyi Spring Creek Warren L. appendix/I. greeleyi L. appendix/I. greeleyi Blue Eye Run Warren I. bdellium/L. appendix L. appendix Crooked Creek Erie I. unicuspis L. appendix Conneaut Creek Crawford L. appendix/P. marinus/I. fossor L. appendix?P. marinus? Martin Run Mckean L. appendix L. appendix Elevenmile Creek Potter L. appendix L. appendix Shannon Creek Westmoreland L. aepyptera L. aepyptera Mountain Creek Fayette L. aepyptera L. aepyptera Bull Creek Allegheny L. aepyptera L. aepyptera Crooked Creek Indiana L. aepyptera L. aepyptera Hodgeson Creek Chester P. marinus L. aepyptera/P. marinus S. Branch of Kinzua Creek Mckean L. appendix L. appendix E. Branch of Tionesta Creek Mckean L. appendix L. appendix Oil Creek Crawford I. bdellium/L. appendix None Raccoon Creek Erie P. marinus/L. appendix None Mill Creek Potter L. appendix None Little Pucketa Creek Westmoreland L. aepyptera None W. Branch of White Clay Creek Chester L. appendix None Woodcock Creek Crawford L. appendix/I. bdellium None Raccoon Creek State Park Beaver L. aepyptera None

Table 8: Substrate sampling sites.

1 2 3 4 5 Creek: Martin Run Creek: Crooked Creek: Elevenmile Creek: Mountain Creek: Crooked Creek County: Mckean County: Indiana County: Potter County: Fayette County: Erie Latitude: 41°36.0405'N Latitude: 40°40.0329'N Latitude: 41°56.7943'N Latitude: 39°46.6757'N Latitude: 41°59.6187'N Longitude: 78°54.3844'W Longitude: 79°5.3758'W Longitude: 78°4.2078'W Longitude: 79°49.4522'W Longitude: 80°25.1543'W Date: 5/25/2011 Date: 6/2/2011 Date: 5/26/2011 Date: 6/1/2011 Date: 5/11/2011

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Table 9: Sampled creeks and sieved results for each spot of different creeks.

Sieve Size 12.7 8 4 3.35 2.38 2 1.68 1.4 1.19 1 0.85 0.25 (mm) Location Substrate Weight for Different Sieve Size (g) Rs 637 340 626 12 388 104 69 77 36 34 32 752 Rm 1439 377 388 8 197 54 49 42 27 23 21 277 Re 1563 290 293 5 197 60 45 43 33 29 39 287 1 Lu 0 0 1.8 0.155 1.283 1.04 0.8604 1.633 1.1729 1.4517 1.0305 1085 Lm 0 0 0 3 65 74 20 20 18 15 18 1570 Ld 0 0 0 0 1.333 1.788 1.58 2.875 2.9388 4.5841 8.1482 1226 Lu 0 0 41 6 273 158 146 153 122 131 130 653 2 Ld 0 0 0 0 0 1.25 1.0309 1.012 1.0315 0.9033 0.9367 778 Lu 1000 373 382 10 208 73 70 100 76 90 110 1756 Ld 325 102 201 6 183 86 64 86 66 64 65 2123 3 Rs 1179 367 216 8 267 74 47 59 43 34 23 394 Rm 1523 231 178 5.887 145 54 36 35 25 19 18 98 Re 1393 198 183 7.375 163 63 39 48 27 21 13 193 Lu 0 15.4 42 2.745 36 13 15 19 12 17 21 1930 Ld 0 0 0 0 0 0.736 1.894 1.629 1.662 1.736 2.159 1665 4 Rs 1897 233 343 9 231 74 59 70 48 40 44 446 Rm 1407 200 247 4 152 52 43 50 38 30 32 296 Re 2919 62 62 2.45 45 13 10 10.52 8.0998 5.6441 4.7205 14 Lu 782 377 478 8.459 219 73 66 89 83 101 170 1507 Ld 0 0 0.73 0 0.47 4.695 6.8497 14.54 18.06 29.472 63 2708 5 Rs 1052 162 230 5.331 127 41 32 39 30 26 48 229 Rm 2222 16 264 4 165 45 31 28 17 10 9 52 Re 1729 468 511 7.813 234 64 49 51 50 40 78 460

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Table 10: Random site sample data analysis.

Random Sample Results Mean Locat Sieve Substrat Sum ion size 12.7 8 4 3.35 2.38 2 1.68 1.4 1.19 1 0.85 0.25 e Size (mm) (mm) W (g) 637 340 626 12 388 104 69 77 36 34 32 752 3107 4.8282 P 0.21 0.11 0.2 0.004 0.12 0.033 0.022 0.0248 0.012 0.011 0.01 0.242 1 W (g) 1439 377 388 8 197 54 49 42 27 23 21 277 2902 1 8.1772 P 0.5 0.13 0.13 0.003 0.07 0.019 0.017 0.0145 0.009 0.008 0.007 0.095 1 W (g) 1563 290 293 5 197 60 45 43 33 29 39 287 2884 8.4108 P 0.54 0.1 0.1 0.002 0.07 0.021 0.016 0.0149 0.011 0.01 0.014 0.1 1 W (g) 1179 367 216 8 267 74 47 59 43 34 23 394 2711 7.3583 P 0.43 0.14 0.08 0.003 0.1 0.027 0.017 0.0218 0.016 0.013 0.008 0.145 1 W (g) 1523 231 178 5.887 145 54 36 35 25 19 18 98 2368 3 9.533 P 0.64 0.1 0.08 0.002 0.06 0.023 0.015 0.0148 0.011 0.008 0.008 0.041 1 W (g) 1393 198 183 7.375 163 63 39 48 27 21 13 193 2348 8.8533 P 0.59 0.08 0.08 0.003 0.07 0.027 0.017 0.0204 0.011 0.009 0.006 0.082 1 W (g) 1897 233 343 9 231 74 59 70 48 40 44 446 3494 8.1565 P 0.54 0.07 0.1 0.003 0.07 0.021 0.017 0.02 0.014 0.011 0.013 0.128 1 W (g) 1407 200 247 4 152 52 43 50 38 30 32 296 2551 4 8.3319 P 0.55 0.08 0.1 0.002 0.06 0.02 0.017 0.0196 0.015 0.012 0.013 0.116 1 W (g) 2919 62 62 2.45 45 13 10 10.52 8.1 5.644 4.721 14 3156 12.042 P 0.92 0.02 0.02 8E-04 0.01 0.004 0.003 0.0033 0.003 0.002 0.001 0.004 1 W (g) 1052 162 230 5.331 127 41 32 39 30 26 48 229 2021 8.0376 P 0.52 0.08 0.11 0.003 0.06 0.02 0.016 0.0193 0.015 0.013 0.024 0.113 1 W (g) 2222 16 264 4 165 45 31 28 17 10 9 52 2863 5 10.493 P 0.78 0.01 0.09 0.001 0.06 0.016 0.011 0.0098 0.006 0.003 0.003 0.018 1 W (g) 1729 468 511 7.813 234 64 49 51 50 40 78 460 3742 7.7214 P 0.46 0.13 0.14 0.002 0.06 0.017 0.013 0.0136 0.013 0.011 0.021 0.123 1 W (g) 1580 245 295 6.571 193 58.17 42.42 46.043 31.84 25.97 30.14 291.5 2846 Mean 8.4704 P 0.56 0.09 0.1 0.002 0.07 0.02 0.015 0.0162 0.011 0.009 0.011 0.102 1

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Table 11: Lamprey site sample data analysis.

Lamprey Site Results Mean Locati Sieve Substrate Sum on size 12.7 8 4 3.35 2.38 2 1.68 1.4 1.19 1 0.85 0.25 Size (mm) (mm) W (g) 0 0 1.802 0.155 1.28 1.04 0.86 1.63 1.173 1.45 1.031 1085 1095 0.266166 P 0 0 0.002 1E-04 0 9E-04 8E-04 0 0.001 0 9E-04 0.99 1 W (g) 0 0 0 3 65 74 20 20 18 15 18 1570 1803 1 0.454004 P 0 0 0 0.002 0.04 0.041 0.011 0.01 0.01 0.01 0.01 0.87 1 W (g) 0 0 0 0 1.33 1.788 1.58 2.87 2.939 4.58 8.148 1226 1249 0.268111 P 0 0 0 0 0 0.001 0.001 0 0.002 0 0.007 0.98 1 W (g) 0 0 41 6 273 158 146 153 122 131 130 653 1813 1.190982 P 0 0 0.023 0.003 0.15 0.087 0.081 0.08 0.067 0.07 0.072 0.36 1 2 W (g) 0 0 0 0 0 1.25 1.031 1.01 1.032 0.9 0.937 778 784 0.258971 P 0 0 0 0 0 0.002 0.001 0 0.001 0 0.001 0.99 1 W (g) 1000 373 382 10 208 73 70 100 76 90 110 1756 4248 4.439049 P 0.235 0.088 0.09 0.002 0.05 0.017 0.016 0.02 0.018 0.02 0.026 0.41 1 3 W (g) 325 102 201 6 183 86 64 86 66 64 65 2123 3371 2.174904 P 0.096 0.03 0.06 0.002 0.05 0.026 0.019 0.03 0.02 0.02 0.019 0.63 1 W (g) 0 15.36 42 2.745 36 13 15 19 12 17 21 1930 2123 0.468756 P 0 0.007 0.02 0.001 0.02 0.006 0.007 0.01 0.006 0.01 0.01 0.91 1 4 W (g) 0 0 0 0 0 0.736 1.894 1.63 1.662 1.74 2.159 1665 1675 0.255988 P 0 0 0 0 0 4E-04 0.001 0 1E-03 0 0.001 0.99 1 W (g) 782 377 478 8.459 219 73 66 89 83 101 170 1507 3953 4.176459 5 P 0.198 0.095 0.121 0.002 0.06 0.018 0.017 0.02 0.021 0.03 0.043 0.38 1 W (g) 0 0 0.731 0 0.47 4.695 6.85 14.5 18.06 29.5 63 2708 2846 0.290533 P 0 0 3E-04 0 0 0.002 0.002 0.01 0.006 0.01 0.022 0.95 1 W (g) 175.6 72.28 95.54 3.03 82.3 40.54 32.77 40.7 33.49 38 49.11 1417 2080 Mean 1.953376 P 0.084 0.035 0.046 0.001 0.04 0.019 0.016 0.02 0.016 0.02 0.024 0.68 1

81

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86

Appendices

Appendix A． Ichthyomyzon bdellium historical sites

INDIVIDUA NO CO LOCATION LAT LON DATE COLLECT DEPOSIT NO RET L CREEK CED RLR JSL SEW 1 Warren Allegheny R along Rte 62, 2.3 rd mi S of junction with Rte 6 41.81290 -79.27805 6/11/85 JMB 1650 Allegheny River

2 Potter Allegheny R. ~560m D-stream from Reed Run 41.75525 -78.11045 2008/6/20 PSU/IBI PFBC, PSU 15 Allegheny River

3 Warren Allegheny R., Hosp Warren, PA, FWS shocker 41.84413 -79.15520 1973/7/19 1491 1 Allegheny River

4 Venango Allegheny River - 560m downstream of Reed Run 41.40433 -79.79573 6/20/2008 4669 15 Allegheny River

5 Potter Allegheny River - about 2 miles NW of Port Allegheny off T379 41.85261 -78.32227 8/27/2009 4874 2 Allegheny River alleghen 6 y Allegheny River - L&D 3 at Springdale 40.53666 -79.81368 1968/9/18 ELC ELC0547 1 1 Allegheny River

7 Potter Allegheny River - TR405 bridge in Roulette 41.77881 -78.16406 2009/5/27 RWC09023 PSU10088 2 Allegheny River

8 McKean Allegheny River - TR428 bridge in Port Allegheny 41.80774 -78.28815 2009/5/27 RWC09020 PSU10086 3 Allegheny River

9 Venango Allegheny River @ Reno. 41.41515 -79.76782 2004/6/7 RWC 9 0 Allegheny River

10 Venango Allegheny River @ Reno. 41.41515 -79.76782 2004/7/9 RWC 2 Allegheny River

11 McKean Allegheny River at bridge in Turtle Point, PA 41.86773 -79.34332 1988/4/13 2040 2 Allegheny River BDL-08- 12 Forest Allegheny River Benthic Trawling 41.54144 -79.43921 2008/8/30 Benjamin D. Lorson 200 1 Allegheny River BDL-08- 13 Venango Allegheny River Benthic Trawling 41.46091 -79.58792 2008/8/31 Benjamin D. Lorson 205 1 Allegheny River BDL-08- 14 Venango Allegheny River Benthic Trawling 41.22720 -79.75372 2008/9/13 Benjamin D. Lorson 222 1 Allegheny River BDL-08- 15 Venango Allegheny River Benthic Trawling 41.20872 -79.72497 2008/9/13 Benjamin D. Lorson 224 1 Allegheny River BDL-08- 16 Venango Allegheny River Benthic Trawling 41.17423 -79.69539 2008/9/14 Benjamin D. Lorson 225 1 Allegheny River alleghen 17 y Allegheny River, Allegheny Co., Lock and Dam #3 at Springdale 40.53872 -79.81627 9/18/68 PSU-Cooper-547 547 1 1 Allegheny River

18 McKean allegheny River, bridge at Turtle point, Mckean Co. PA 41.86715 -78.34350 2005/9/23 4055 1 Allegheny River

19 Warren Allegheny River, Warren Co. PA 41.83465 -79.21023 1965/7/28 135 many Allegheny River

20 Warren Allegheny River, Warren Co., at Starbrick 41.83465 -79.21023 7/3/73 PSU-Cooper-1593 1593 2 2 Allegheny River

21 Warren Allegheny River, Warren Co., Buckaloons to Starbrick 41.83499 -79.21093 11/18/71 PSU-Cooper-1595 1595 4 4 Allegheny River

22 Warren Allegheny River, Warren Co., near Glade Run (Warren Pa.) 41.82646 -79.12490 6/9/80 PSU-Cooper-2190 2190 1 1 Allegheny River

23 Warren Allehgeny River, Warren Co., near Hemlock Eddy 41.84637 -79.06107 6/19/73 PSU-Cooper-1577 1577 0 0 Allegheny River

87

24 McKean Bell Run, Potter Co., near Myrtle and Shinglehouse, Pa. 41.97874 -78.23586 6/1/80 PSU-Cooper-2197 2197 1 1 Bell Run

25 Potter Bell Run,(twl to Oswayo Cr.) Potter Co. PA 41.98210 -78.23322 1980/6/1 2197 1 Bell Run

26 Warren Blue Eye Run 41.85833 -79.46306 1977/6/16 PFBC Blue Eye Run

27 Warren Blue Eye Run 41.83083 -79.43833 1977/6/17 PFBC Blue Eye Run

28 Warren Brokenstaw and coffee Creek Confluence, Warren Co. 41.93979 -79.58201 1985/6/2 1637 1 Brokenstraw Cr. PSU -Stauffer-JMB 29 Warren Brokenstraw and Coffee Cr confluence in Columbus, PA 41.93979 -79.58201 6/2/85 85.30 JMB 85.30 0 0 Brokenstraw Cr. Brokenstraw Cr. at confluence of Blue Eye Run at Rt 27 bridge W of PSU-Stauffer-JMB 30 Warren Pittsfield, PA 41.81873 -79.42914 6/2/85 85.28 JMB 85.28 0 0 Brokenstraw Cr.

31 Warren 41.84639 -79.28056 2005/5/31 PFBC Brokenstraw Cr.

32 Warren Brokenstraw Creek - above railroad bridge near mouth 41.83752 -79.26435 2005/7/1 CLU516 PSU3990 1 Brokenstraw Cr.

33 Warren Brokenstraw Creek - at mouth 41.83111 -79.26102 2005/8/9 CLU522 PSU3996 1 Brokenstraw Cr. Brokenstraw Creek - downstream replicate, about 1 mile South of Horn 34 Warren Siding 41.83166 -79.46474 8/27/2009 4875 1 Brokenstraw Cr.

35 Warren Brokenstraw Creek - just above mouth 41.83527 -79.25727 2005/6/8 RWC05047 PSU4036 3 1 Brokenstraw Cr. Brokenstraw Creek - upstream of targeted site, 1 mile South of Horn 36 Warren Siding 41.83582 -79.46593 8/27/2009 4876 2 Brokenstraw Cr.

37 Warren Brokenstraw Creek at Confluence of blueeye Run at 27 Bridge 41.81873 -79.42914 1985/6/2 1606 1 Brokenstraw Cr.

38 Warren Brokenstraw Creek at mouth juction with Allengheny River 41.83482 -79.25893 1965/7/27 129 many Brokenstraw Cr.

39 Warren Brokenstraw Creek, at mouth, Warren Co. 41.83482 -79.25893 7/1/65 PSU-Cooper-123 123 175 175 Brokenstraw Cr.

40 Warren Brokenstraw Creek, Warren Co. 1st riffle, 100 yds. above mouth 41.83445 -79.25777 5/21/65 PSU-Cooper-126 126 17 17 Brokenstraw Cr.

41 Warren Brokenstraw Creek, Warren Co., 1st riffle, 100 yds. above mouth 41.83441 -79.25771 5/24/65 PSU-Cooper-127 127 62 62 Brokenstraw Cr. Brokenstraw Creek, Warren Co., Floated boat 1.7miles above little 42 Warren brokenstraw mouth downstram for one mile 41.83525 -79.35607 1965/7/27 133 many Brokenstraw Cr.

43 Warren Brokenstraw Creek, Warren Co., mouth of L B down stream 41.83287 -79.38357 1965/5/24 128 many Brokenstraw Cr. Brokenstraw Creek, Warren Co., mouth of Little Brokenstraw upstream 44 Warren 100y. 41.83238 -79.38417 1965/7/27 132 many Brokenstraw Cr. Brokenstraw downstream to first cement, youngsville-tag ripped and hard 45 Warren to read 41.84825 -79.29567 1965/7/27 130 many Brokenstraw Cr.

46 Warren Conewango Creek - at North Warren 41.87168 -79.14403 2009/5/26 RWC09017 PSU10084 7 Conewango Cr. Crawfor PSU-Stauffer-JMB 47 d E Br of Oil Cr. at Rte 8 bridge in Centerville, PA 41.73488 -79.76319 6/18/85 85.51 JMB 85.51 0 0 E Br of Oil Cr. Crawfor 48 d E Br Oil Creek - 2 mi NE of Centerville (spawning w/8 I. greeleyi) 41.70647 -79.78100 1975/5/22 ELC ELC1524 4 4 E Br of Oil Cr. Crawfor 49 d E. Br. Oil Creek - bridge on Britton Run Rd. 41.75645 -79.73910 2009/5/24 RWC09011 PSU10079 4 E Br of Oil Cr. Crawfor 50 d East Branch of Oil Creek, Crawford Co., 2 mi. NE of Centerville 41.77679 -79.72122 5/22/75 PSU-Cooper-1524 1524 4 4 E Br of Oil Cr. Crawfor East Branch Oil 51 d East Branch Oil Creek 41.74694 -79.75556 1977/6/21 PFBC Creek Crawfor East Branch Oil 52 d East Branch Oil Creek 41.78333 -79.71278 1977/6/22 PFBC Creek

53 Potter Fishing Creek, Potter Co., 100 yards from mouth at Roulette 41.78165 -78.16516 5/0/75 PSU-Cooper-1523 1523 3 3 Fishing Creek Crawfor PFBC, 54 d French Ck. At Venango, D-stream of bridge 41.76916 -80.11191 2008/6/2 PSU/IBI DEP, CalU 82 French Cr.

88

PSU-Stauffer-JRS 55 Erie French Cr at 6N bridge W of Mill Village, PA 41.88223 -79.99958 5/12/87 87.22 JRS 87.22 0 0 French Cr. PSU-Stauffer-RLR 56 mercer French Cr at bridge in Calrton, PA 41.47144 -80.01868 5/20/87 87.17A 1863 0 3 French Cr. PSU-Stauffer-MJG 57 Venango French Cr downstream of Takitezy, PA 41.41235 -79.92132 5/30/91 91.36 MJG 91.36 0 0 French Cr. PSU-Stauffer-MJG 58 Venango French Cr in Utica, PA 41.43762 -79.95574 5/29/91 91.32 2516 0 0 French Cr. Crawfor PSU-Stauffer-JMB 59 d French Cr on N side of Venango, PA 41.77632 -80.10701 4/22/85 85.3 JMB 85.3 0 0 French Cr. PSU-Stauffer-MJG 60 Venango French Cr. about 0.5 mi below Utica, PA 41.43354 -79.95041 5/29/91 91.35 MJG 91.35 0 0 French Cr. Crawfor 61 d French Cr. In Meadville 41.68417 -80.17167 1989/9/9 2234 1 French Cr. French Cr. Rt 6 bridge 100 yards E of junction with Rt 19 near Mill Village, PSU -Stauffer-JMB 62 Erie PA 41.88223 -79.99958 7/2/85 85.82 JMB 85.82 0 0 French Cr. French Cr. Rt 6n Bridge 100 yrds E of junction w/ Rt 19, W of Mill Village, PSU-Stauffer-JMB 63 Erie PA 41.88223 -79.99958 7/1/85 85.73 JMB 85.73 0 0 French Cr.

64 Erie French Creek 41.58861 -80.14917 2005/6/6 PFBC French Cr.

65 Erie French Creek 41.40861 -79.85500 2005/6/9 PFBC French Cr.

66 Erie French Creek - along Dutchtown Rd. 41.85222 -79.97801 2008/6/20 RWC08028 1 French Cr. Crawfor 67 d French Creek - at Venango 41.76978 -80.10799 2005/6/29 CLU513 PSU3987 1 French Cr. Crawfor 68 d French Creek - at Venango 41.77148 -80.10773 2006/6/11 RWC06003 BPEF 1 French Cr. Crawfor 69 d French Creek - at Venango 41.77242 -80.10792 2007/5/21 RWC07039 PSU10027 4 4 French Cr.

70 Venango French Creek - downstream from Utica 41.42495 -79.93513 2005/7/5 CLU517 PSU3991 1 French Cr.

71 Erie French Creek - near Erie/Crawford county line, off Dutchman Rd. 41.85175 -79.97760 8/26/2009 4898 11 French Cr. Crawfor 72 d French Creek - PFBC Meadville Access, 3 mi S of Meadville. 41.58927 -80.14965 2003/7/1 RWC 1 0 French Cr. Crawfor 73 d French Creek @ Venango 41.75702 -80.11217 1999/5/19 RWC 1 French Cr.

74 Erie French Creek at 6 north bridge in Mill Village, PA (evening) 41.88223 -79.99958 1987/5/12 1852 3 French Cr.

75 Erie French Creek at 6N bridge w. of mill village, PA(night) 41.88223 -79.99958 1987/5/12 1856 1 French Cr. Crawfor 76 d French Creek at bridge in Venango, Crawford Co. 41.77187 -80.10775 1987/9/27 1938 1 French Cr. Crawfor 77 d French Creek at bridge in Venango, Crawford Co. 41.77187 -80.10765 1988/7/26 2080 2 French Cr. Crawfor 78 d French Creek at bridge in Venango, PA 41.77187 -80.10775 1987/5/13 1853 1 French Cr. Crawfor 79 d French Creek at Cochranton 41.51863 -80.05412 1991/5/7 2621 1 French Cr. Crawfor 80 d French Creek at Crawford Co. 41.77187 -80.10775 1988/7/26 2047 2 French Cr. Crawfor 81 d French Creek at Rt. 173 bridge in Cochranton, PA 41.51847 -80.05388 1986/8/4 1832 1 French Cr. Crawfor 82 d French Creek at Venango 41.77187 -80.10775 1992/6/25 2630 3 French Cr. Crawfor 83 d French Creek at Venango 41.77148 -80.10773 1989/7/25 2226 1 French Cr. Crawfor 84 d French Creek at Venango - 400m downstream of bridge 41.76968 -80.11117 6/2/2008 4611 66 French Cr. Crawfor 85 d French Creek in Cochranton 41.51863 -80.05412 1989/9/9 2242 1 French Cr.

89

Crawfor 86 d French Creek in Cochranton 41.51863 -80.05412 1989/9/9 2244 1 French Cr. Crawfor 87 d French Creek in meadville 41.68417 -80.17167 1989/7/17 2223 2 French Cr. Crawfor 88 d French Creek in venango 41.77242 -80.10792 1989/9/9 2236 2 French Cr.

89 Venango French Creek, 0.6 km downstream of utica, PA 41.42912 -79.94643 1971/5/20 2544 2 French Cr. Crawfor 1998/10/2 90 d French Creek, 1.1 km S of Cochranton. 41.50423 -80.04158 4 CWB PSU 1 1 French Cr. Crawfor 91 d French Creek, Crawford Co. PA 41.77187 -80.10775 1990/6/28 2445 1 French Cr. Crawfor 92 d French Creek, Crawford Co. PA at Cochranton, PA 41.51863 -80.05412 1990/7/21 2383 1 French Cr. Crawfor 93 d French Creek, Crawford Co., at Saegerstown 41.71993 -80.15207 9/26/69 PSU-Cooper-316a 316a 1 1 French Cr. Crawfor 94 d French Creek, Crawford Co., mouth of Little Sugar Creek, at Cochranton 41.51933 -80.05502 6/1/66 PSU-Cooper-317 317 5 5 French Cr.

95 Erie French Creek, downstr. Of RT6N bridge 2 mi W of Mill Village. 41.88133 -79.99933 2002/7/7 RWC 1 French Cr.

96 Erie French Creek, Erie CO. 2km N of New Ireland 41.89790 -79.93425 6/2/1992 2602 1 French Cr.

97 Erie French Creek, Erie Co., 1 mile west of Mill Village 41.88232 -79.99956 6/2/66 PSU-Cooper-319 319 1 1 French Cr.

98 Erie French Creek, Erie Co., 2 mi. W. of Mill Village 41.88957 -79.99393 6/26/75 PSU-Cooper-1549 1549 10 10 French Cr.

99 Erie French Creek, Rt 6 Bridge, 100 y east of junction R 19 41.88223 -79.99958 1985/7/2 1599 2 French Cr.

100 Venango French Creek, Venango Co. 41.77242 -80.10792 1991/5/30 2514 2 French Cr.

101 Venango French Creek, Venango Co., trib. to Allegheny River, 2 miles above Utica 41.45544 -79.98519 8/5/68 PSU-Cooper-606 606 1 1 French Cr. Little Brokenstraw 102 Warren Little Brokenstraw Creek - .5 mi W of Lottsville 41.95168 -79.44316 2009/5/26 RWC09015 PSU10082 7 Cr. Little Brokenstraw 103 Warren Little Brokenstraw Creek - TR 539 (Valastiak Rd) bridge NW of Lottsville 41.96889 -79.46301 2009/5/26 RWC09016 PSU10083 1 Cr. Little Brokenstraw 104 Warren Little Brokenstraw Creek, 1/2 mi W of Lottsville (spawning w/6 I. Greeleyi) 41.95153 -79.44330 1975/5/27 PSU-Cooper-1548 ELC1548 4 4 Cr. Little Brokenstraw 105 Warren Little Brokenstraw Creek, Warren Co., at Pittsfield, under highway bridge 41.83272 -79.38419 7/12/65 PSU-Cooper-141 141 38 38 Cr. Little Brokenstraw, Warren Co., mouth to road bridge at Pitts field 100 106 Warren yards 41.83435 -79.38390 1965/7/27 131 many Little Brokenstraw Little Brokenstraw, Warren Co., mouth to road bridge at Pitts field 100 107 Warren yards 41.83435 -79.38390 1965/7/27 134 many Little Brokenstraw

108 Venango Little Sandy Creek 41.38583 -79.93556 1994/6/8 PFBC Little Sandy Creek Little Brokenstraw 109 Venango Little Sandy Creek - at SR 62 bridge E of Polk 41.37392 -79.91998 2007/5/23 RWC07044 PSU10029 6 3 Cr. Little Brokenstraw 110 Venango Little Sandy Creek - SR 62 bridge near Polk 41.37392 -79.91998 2009/5/24 RWC09013 13 Cr. Crawfor Little Brokenstraw 111 d Little Sugar Creek, just above mouth @ Cochranton. (33 FOUND DEAD) 41.52033 -80.05410 1999/5/20 RWC PSU 36 33 Cr. Armstro CalU, 112 ng Mahoning Ck. ~1.75mi SW of Putneyville PA. 40.93480 -79.33960 2008/7/22 PSU/IBI PFBC, DEP 1 Mahoning Cr. Jefferso 113 n Mahoning Creek - appx. 1.75 miles southwest of Putneyville, PA 40.92255 -79.01090 7/22/2008 4717 1 Mahoning Creek Crawfor 114 d Middle oil Creek, Crawford Co. Upstream from centerville 41.70647 -79.78100 1972/7/25 1448 many Middle oil Creek

115 Potter North fork, Hotston R., Potter Co. PA 41.51847 -80.05388 1974/6/15 1680 many North fork Crawfor 116 d Oil Creek 41.78972 -79.81417 1977/6/20 PFBC Oil Creek

90

Crawfor 117 d Oil Creek 41.76750 -79.79222 1977/6/21 PFBC Oil Creek Crawfor 118 d Oil Creek - 2 mi NE of Centerville. Ammocete. 41.70647 -79.78100 1967/8/7 ELC ELC0465 1 Oil Creek Crawfor 119 d Oil Creek - along Mystic Park, 1.5 km South of Centerville 41.68731 -79.78477 6/30/2009 4881 3 Oil Creek

120 Venango Oil Creek - riffle below SR 8 bridge N of Rouseville 41.47732 -79.69180 2007/5/24 RWC07048 PSU10032 7 7 Oil Creek Oil Creek at Oil Creek State Park, just upstream of mouth of Pioneer Run PSU-Stauffer-JRS 121 Venango at end of route 574 41.52937 -79.66531 5/7/90 90.21 2346 1 1 Oil Creek PSU-Stauffer-JRS 122 Venango Oil Creek at SR 1004 bridge, at Oil Creek State Park HQ 41.51823 -79.67881 8/1/90 90.76 2404 1 1 Oil Creek Crawfor 123 d Oil Creek below Centerville Crawford Co. 41.73233 -79.76883 1972/7/24 1449 2 Oil Creek Crawfor 124 d Oil Creek, Crawford Co., 1/2 mi. upstream from rte 27 bridge 41.63042 -79.69364 8/4/77 PSU-Cooper-2014 2014 0 0 Oil Creek Crawfor Oil Creek, Crawford Co., at Lincolnville, trib. to Allegheny, 2 miles NW of 125 d Centerville 41.79545 -79.84060 8/7/67 PSU-Cooper-465 465 1 1 Oil Creek

126 Potter Oswayo Creek 41.92111 -78.10972 1996/8/5 PFBC Oswayo Creek

127 Potter Oswayo Creek - SR44 bridge at Millport 41.92418 -78.11962 2009/5/27 RWC09021 PSU10087 4 Oswayo Creek

128 Potter Oswayo Creek - T372 bridge at Sharon Center 41.93681 -78.14554 2008/5/29 RWC08025 PSU10041 1 Oswayo Creek Oswayo Creek - TR376 bridge at Sharon Center (attached to 129 Potter Onchorynchus mykiss) 41.93647 -78.44362 2005/6/7 RWC05037 PSU4028 1 1 Oswayo Creek

130 McKean Oswayo Creek - TR438 bridge crossing N of Myrtle 41.98613 -78.24215 2005/5/17 RWC05034 PSU4027 1 1 Oswayo Creek

131 Potter Oswayo Creek, Potter Co., at Coneville, Pa. 41.90295 -78.06693 5/26/77 PSU-Cooper-1705 1705 1 1 Oswayo Creek

132 Potter Oswayo Creek, Potter Co., at Sharon Center 41.93667 -78.14588 5/30/75 PSU-Cooper-1521 1521 2 2 Oswayo Creek

133 Warren Pine Creek 41.64972 -79.53194 1977/6/29 PFBC Pine Creek PSU-Stauffer-JMB 134 McKean Potato Cr. at bridge in Crosby, PA 41.74492 -78.39279 7/5/85 85.95 1603 0 0 Potato Cr.

135 McKean Potato Creek, at rt. 146 bridge. 41.70753 -78.39070 1993/5/22 RWC PSU 1 1 Potato Cr.

136 McKean Potato Creek, McKean Co., 3 mi. S. of East Smethport 41.78097 -78.41550 5/30/75 PSU-Cooper-1522 1522 2 2 Potato Cr.

137 Venango Sandy Ck. Below Rt.8, ~1.6km d-stream of Pecan 41.32710 -79.87620 2008/6/3 PSU/IBI CalU 5 Sandy Creek

138 Venango Sandy Creek 41.32722 -79.84750 2005/7/12 PFBC Sandy Creek

139 Venango Sandy Creek - below rte 8, approx. 1.6 km downstream from Pecan 41.35017 -79.88450 6/3/2008 4708 5 Sandy Creek

140 Venango Sandy Creek - old Rt 8 (SR3013) bridge at Pecan 41.33450 -79.88705 2009/5/24 RWC09014 7 Sandy Creek

141 Venango Sandy creek - SR 965 bridge 1 mi SW of Polk 41.35485 -79.94020 2007/5/24 RWC07046 PSU10031 1 1 Sandy Creek

142 Venango Sandy Creek - SR3013 (Old Rt 8) bridge at Pecan 41.33450 -79.88657 2007/5/23 RWC07043 PSU10028 3 3 Sandy Creek

143 Venango Sandy Creek - SR965 bridge 1 mi SW of Polk 41.35485 -79.94020 2009/5/24 RWC09012 PSU10080 2 Sandy Creek

144 Venango Sandy Creek, Venango Co. at Pecan 41.33485 -79.88701 6/27/75 PSU-Cooper-1560 1560 2 2 Sandy Creek Sandy Creek, Venango Co., trib. to Allegheny R. 1/2 mile above mouth 145 Venango below Franklin 41.32696 -79.84934 6/1/66 ELC 321 Sandy Creek

146 Venango Sugar Creek 41.53333 -79.85806 1996/6/6 PFBC Sugar Creek Crawfor Woodcock Creek at Rt 198 bridge upstream of Woodcock Lake, Crawford 147 d Co. PA 41.68917 -80.05250 1991/5/8 2635 1 Woodcock Creek

91

Appendix B. Ichthyomyzon fossor historical sites

NO NO CO LOCATION LAT LON DATE DEPOSIT RET INDIVIDUAL CREEK

1 crawford Conneaut Creek, Crawford Co., 1 mi. N. of Conneautville 41.7740947 -80.3808416 1975/4/29 PSU-Cooper-1520 3 6 Conneaut Creek Conneaut Creek, Crawford Co., trib. to Lake Erie, off route 2 crawford 18, North of Conneautville 41.7650483 -80.3763741 1968/8/14 PSU-Cooper-601 3 1 Conneaut Creek

3 Erie Temple Creek - Bessemer St. bridge in Albion 41.90063 -80.35738 2003/5/23 RWC10066 3 1 Temple Creek

4 Erie Temple Creek - Bessemer St. bridge in Albion 41.90063 -80.35738 2008/7/23 RWC08043 3 6 Temple Creek

5 Erie Temple Creek just upstream of mouth in Albion 41.900833 -80.3556667 2003/5/22 RWC03045 3 1 Temple Creek

Appendix C. Ichthyomyzon greeleyi historical sites

NO INDIVID NO CO LOCATION LAT LON DATE COLLECT DEPOSIT RET UAL CREEK

1 Mckean Allegheny River at Port Allegheny 41.80774 -78.28815 1975/5/30 ELC ELC1527 2 2 Allegheny River

2 Mckean Allegheny River, McKean Co., at Port Allegheny 41.80777197970 -78.28822253360 5/30/75 PSU-Cooper 1527 2 2 Allegheny River

3 Elk Bear Creek - Below Spring Creek Rd bridge 41.3949001 -78.82270615 2005/5/13 CLU507 PSU3981 1 Bear Creek

4 Elk Bear Creek - For Svc Rd 135 bridge. - 1 adult - 3 amms. 41.46705991 -78.85125809 2005/5/13 CLU506 PSU3980 4 Bear Creek UMMZ1090 5 Venango Big Sandy Creek - near Franklin on Rt. 8. 41.326617 -79.872867 1935/3/23 Raney 84 1 1 Big Sandy Creek

6 Warren Brokenstraw Creek 41.99975 -79.6223 23886 PSU-Cooper PSU128 2 Brokenstraw Creek

7 Elk Clarion River - Portland Mills canoe launch - 6 adult 1 amm. 41.36642106 -78.82843857 2005/5/26 CLU no sp 7 Clarion River

8 Elk Clarion River - upstream of Arroyo bridge - 1 adult 1 amm. 41.38790617 -78.88069316 2005/5/26 CLU509 PSU3983 2 Clarion River

9 Elk Clarion River - upstream of Shanley Rd. bridge 41.38336267 -78.93634226 2005/5/26 CLU508 PSU3982 2 Clarion River

10 Crawford E. Br. Oil Creek - bridge on Britton Run Rd. 41.75645 -79.7391 2009/5/24 RWC09011 PSU10079 1 E. Br. Oil Creek East Branch of Oil Creek, Crawford Co., 2 mi. NE of Centerville 11 Crawford (spawning w/4 I. bdellium) 41.772667 -79.729 5/22/75 ELC 1524 E. Br. Oil Creek

12 Forest East Branch Spring Creek 41.4613 -78.9693 1963/6/18 ELC0065 3 3 E. Br. Spring Creek

92

Fishing Creek - 100 yds upstr from mouth at Roulette.(same 13 Potter nest w/3 I. Bdellium) 41.78164866530 -78.16515786990 1975/5/30 ELC 1523 8 8 Fishing Creek

14 Crawford Fivemile Creek - SR 89 bridge 2.5 mi NNW of Buell Corners. 41.7842167 -79.685833 2003/4/28 RWC PSU3727 4 3 Fivemile Creek

15 Erie French Creek - Tanner Rd bridge NW of Wattsburg 42.01535 -79.78252 2008/5/25 RWC08021 PSU10044 14 French Creek

16 Crawford French Creek at Venango, just off Rt. 19. 41.7570167 -80.1121667 1948/5/27 ELC1615 1 1 French Creek

17 Crawford French Creek at Venango, Pa 41.7570167 -80.1121667 5/22/1989 PSU2222 1 French Creek

18 Erie French Creek at Wattsburg 41.9995 -79.8121667 9/19/31 JR Geeley UMMZ 32 French Creek

19 Erie French Creek, 1 mi above (east of) Wattsburg 42.0101667 -79.788833 5/30/31 JR Greeley UMMZ 99 French Creek CL Hubbs & 20 French Creek, about 1 mi E of Wattsburg and about 3 mi from MB Erie NY state line 42.0101667 -79.788833 5/13/34 Trautman UMMZ 94 French Creek

21 Crawford French Creek, Crawford Co., at Venango, Pa. 41.77350534580 -80.10665260260 5/27/48 PSU-Cooper 1615 1 1 French Creek

22 Erie French Creek, trib. of Allegheny River, near Wattsburg 42.0131667 -79.783833 9/20/28 PA Webb UMMZ 30 French Creek Little Brokenstraw Creek - TR 539 (Valastiak Rd) bridge NW of Little Brokenstraw 23 Warren Lottsville 41.96889 -79.46301 2009/5/26 RWC09016 PSU10083 3 Creek Little Brokenstraw Creek, 1/2 mi W of Lottsville (spawning w/4 PSU-Cooper- Little Brokenstraw 24 Warren I. Bdellium) 41.95152642320 -79.44329708810 1975/5/27 1548 ELC1548 6 6 Creek Little Neshannock 25 mercer Little Neshannock Cr, near New Wilmington(Lawrence Co.) 41.1513333 -80.3131667 5/26/35 EC Raney UMMZ 150 Cr. Little Neshannock Cr., 2 mi. N of New Wilmington(Lawrence ECR Little Neshannock 26 mercer Co.) 41.1513333 -80.3131667 5/17/35 Cornell 35MAY17 1 1 Cr. Little Neshannock Cr., 2 mi. N of New Wilmington(Lawrence EC Raney & Little Neshannock 27 mercer Co.) 41.1513333 -80.3131667 5/17/35 J Fleck ECR 35MAY17 Cr. Little Neshannock Cr., 2 mi. N of New Wilmington(Lawrence ECR Little Neshannock 28 mercer Co.) 41.1513333 -80.3131667 5/19/35 Cornell 35MAY19 2 2 Cr. Little Neshannock Cr., 2 mi. N of New Wilmington(Lawrence Little Neshannock 29 mercer Co.) 41.1513333 -80.3131667 5/19/35 EC Raney ECR 35MAY19 Cr. Little Neshannock Creek, 1 mi E of New Wilmington(Lawrence Little Neshannock 30 lawrence Co.) 41.123333 -80.3211667 5/25/35 EC Raney UMMZ 134 Cr. Little Neshannock Creek, 2 mi N of New Wilminton(Lawrence Little Neshannock 31 mercer Co.) 41.1513333 -80.3131667 5/19/35 EC Raney UMMZ 151 Cr. Little Neshannock Creek, Lawrence Co., near Mercer- Little Neshannock 32 lawrence Lawrence Co. line 41.12725456520 -80.32124154020 6/14/75 PSU-Cooper 1530a 11 11 Cr.

33 Venango Little Sandy Creek - near Franklin 41.379333 -79.9205 1934/5/26 Raney CU5771 1 1 Little Sandy Creek UMMZ1068 34 Venango Little Sandy Creek - near Polk 41.3722167 -79.9214167 1934/5/26 Raney 36 1 1 Little Sandy Creek

35 Venango Little Sandy Creek - SR 62 bridge near Polk 41.37392 -79.91998 2009/5/24 RWC09013 PSU10081 4 Little Sandy Creek Edward C. 36 Venango Little Sandy Creek, near Polk 41.3722167 -79.9214167 5/26/34 Raney UMMZ 172 Little Sandy Creek Westmorel 37 and Loyalhanna Creek - SR 982 bridge S of Latrobe 40.2931 -79.37147 2009/5/22 RWC09001 PSU10074 1 Loyalhanna Creek Westmorel 38 and Loyalhanna Creek - SR711 bridge in Ligonier 40.24111 -79.24007 2009/5/22 RWC09002 PSU10075 5 Loyalhanna Creek

39 Lawrence Ltl. Neshannock Ck - SR208 bridge 2 mi E of New Wilmington 41.12244 -80.32107 2009/5/22 RWC09003 PSU10076 30 Ltl. Neshannock Ck

40 lawrence Ltl. Neshannock Ck., 1 mi. E of New Wilmington. 41-07-22/80- 41.123333 -80.3211667 2002/6/3 RWC PSU 2 2 Ltl. Neshannock Ck.

93

19-16

Ltl. Neshannock Ck., 1 mi. E of New Wilmington. 41-07-22/80- 41 mercer 19-16 41.123333 -80.3211667 2000/5/12 RWC PSU3400 1 1 Ltl. Neshannock Ck.

42 mercer Ltl. Neshannock Ck., near Mercer-Lawrence Co. line 41.1274 -80.321 1975/5/14 ELC1530 8 8 Ltl. Neshannock Ck.

43 mercer Ltl. Neshannock Ck.,near Wilmington Junction 41.12325 -80.32105 1975/5/14 ELC1530 3 3 Ltl. Neshannock Ck.

44 Crawford Muddy Creek - SR 77 bridge at Little Cooley 41.74309 -79.88864 2009/5/24 RWC09010 PSU10078 2 Muddy Creek

45 Crawford Muddy Creek, Crawford Co., at Little Cooley 41.74273698950 -79.88903936430 5/22/75 PSU-Cooper 1526 3 3 Muddy Creek

46 Jefferson N Fk Redbank Creek - Moore Bridge Rd. bridge 41.229183 -79.049533 2007/5/24 RWC07049 PSU10033 2 2 N Fk Redbank Creek EA & AM 47 mercer Neshannock Cr., 1 mi. S of Mercer 41.21525 -80.23215 5/13/39 Lachner EAL 026 Neshannock Cr. Oswayo Creek - at Sharon Center. (adults spawning with 2 48 Potter adult I. Bdellium) 41.0271333 -78.373967 1975/5/30 ELC ELC1521 12 12 Oswayo Creek Oswayo Creek - at Sharon Center.(1 adult found dead, dead 49 Potter and dying found upstream) 41.0271333 -78.373967 1977/5/26 ELC ELC1521a 1 1 Oswayo Creek

50 Potter Oswayo Creek - SR44 bridge at Millport 41.92418 -78.11962 2009/5/27 RWC09021 PSU10087 9 Oswayo Creek

51 Potter Oswayo Creek - T372 bridge at Sharon Center 41.93681 -78.14554 2008/5/29 RWC08025 PSU10041 2 Oswayo Creek Oswayo Creek at Coneville (spawning over -many dead 52 Potter specimens, few dying & alive) 41.90295346150 -78.06692993960 1977/5/26 ELC ELC1705 2 2 Oswayo Creek Oswayo Creek at Millport (spawning over -many dead 53 Potter specimens, few dying & alive) 41.92423737570 -78.11977787340 1977/5/26 ELC ELC1706 3 3 Oswayo Creek

54 Erie S Br French Creek - Concord Rd (SR2019) bridge at Concord 41.89367 -79.73524 2008/5/25 RWC08022 PSU10043 16 S. Br. French Creek UMMZ1081 S. Br. Ltl Sandy 55 Venango S Br Ltl Sandy Creek - near town of Pearl 41.29795 -79.9496 1935/5/29 Raney 04 3 3 Creek S. Br. Ltl Sandy 56 Venango S Branch Little Sandy Creek, near town of Pearl 41.29795 -79.9496 5/29/35 EC Raney UMMZ 174 Creek S. Br. of French 57 Erie S. Branch of French Creek, Erie Co., 1 mi. S. of Elgin, Pa. 41.89334668000 -79.73561884330 5/29/75 PSU-Cooper 1528 3 3 Creek Sandy Creek, Venango Co., trib. to Allegheny R. 1/2 mile 58 Venango above mouth below Franklin 41.32696368430 -79.84933869430 6/1/66 PSU-Cooper 321 1 1 Sandy Creek South Branch French Creek 1 mi. S of Elgin. Specimens found 59 Erie dead 41.8933667 -79.73565 1975/5/29 ELC1528 3 3 S. Br. French Creek Spring Creek - 1.6 mi SW of village of Spring Creek (on the 60 Warren Carlisle farm) -photo 41.855067 -79.539967 2005/5/30 Lbush PSU4069 1 Spring Creek

61 forest Spring Creek - above confluence of E Br - 1 adult - 4 amms 41.52079908624 -79.00313096634 2004/5/13 CLU no sp 5 Spring Creek

62 forest Spring Creek - above Lamonville Rd - 2 adults - 1 amm. 41.46333722482 -78.96780093182 2004/5/13 CLU503 PSU3977 3 Spring Creek

63 forest Spring Creek - Duhring Road (TR 403) - 3 adults 41.49795152738 -78.99656126505 2004/5/13 CLU502 PSU3976 3 Spring Creek

64 Elk Spring Creek - SR3002 bridge N of Hallton 41.416533 -78.61425 2006/4/18 RWC06001 BPEF 24 Spring Creek

65 forest Spring Creek, approx. 1 mi below Duhring 41.50373990360 -78.99959036610 1963/6/27 ELC0033 1 1 Spring Creek UMMZ1023 trib of Pymatuning 66 Crawford trib of Pymatuning Swamp 41.559467 -80.4992 0/0/0000 P Webb 90 1 1 Swamp

67 Erie W Br French Creek - Knoyl Rd. bridge SW of Little Hope 42.08171 -79.85028 2008/5/25 RWC08020 PSU10045 3 W. Br. French Creek

68 Elk W Branch Millstone Cr - 200 m above confl w E Branch 41.37360015 -79.08781462 2005/5/9 CLU504 PSU3978 1 W. Br. French Creek

94

W Branch Spring Creek - above confluence of E Br. 1 adult - 4 69 forest amms. 41.52079909 -79.00313097 2004/5/13 CLU501 PSU3975 5 W. Br. French Creek

70 Erie W. Branch French Creek 42.08171 -79.85028 27536 PSU-Cooper PSU1529 many W. Br. French Creek W. Branch of French Creek, Erie Co., at Little Hope Highway, 71 Erie rte 89 crossing 42.10228232750 -79.82198793030 5/15/75 PSU-Cooper 1529 12 12 W. Br. French Creek

72 Crawford Woodcock Creek - 1 mi SE of Saegertown 41.71027437900 -80.13907737880 1975/5/22 ELC ELC454 6 6 Woodcock Creek

73 Crawford Woodcock Creek, Crawford Co., 1 mi. SE of Saegertown 41.71027437900 -80.13907737880 5/0/75 PSU-Cooper 1525 6 6 Woodcock Creek

Appendix D. Ichthyomyzon unicuspis historical sites

NO CO LOCATION LAT LON DATE COLLECT DEPOSIT NO RET INDIVIDUAL CREEK 1 Erie Crooked Creek 41.9888333 -80.411 2004/6/4 Cannon 1 Crooked Creek

2 Erie Crooked Creek - in lamprey trap. 41.9888333 -80.411 1987/5/24 PFC PSU1951 1 1 Crooked Creek

Appendix E. Lampetra aepyptera historical sites

NO INDIVID NO CO LOCATION LAT LON DATE COLLECT DEPOSIT RET UAL CREEK Bull Creek - upstr. Fr. Upper edge of 850' culvert below new 1 Allegheny concrete br bel. Golf course 40.61445 -79.75925 0/0/1980 ELC CU7006 1 Bull Creek PSU- 2 Allegheny Bull Creek and Little Bull Creek, Allegheny Co. 40.610974 -79.756423 8/10/81 Cooper 2327 2 2 Bull Creek Bull Creek- below old red iron br @ pipeline downstr from Ira 3 Allegheny Woods Golf Course 40.615633 -79.760767 1980/8/21 ELC2299 1 Bull Creek Bull Creek, Allegheny Co., 8 stations from Tarentum to headwaters PSU - 4 Allegheny of both branches 40.615045 -79.759903 6/0/79 Cooper 2232 5 5 Bull Creek

5 Armstrong Cornplanter Run (reported as I. greeleyi) 40.754722 -79.676667 1977/7/7 PFBC Cornplanter Run

6 Armstrong Crooked Creek - no specific location - Clarion University survey 40.676667 -79.475067 1983/5/21 ClarionU 2 Crooked Creek

7 Armstrong Trib. "small brook flowing into Plum Creek in Plumcreek Twp." 40.706833 -79.336633 1963/4/12 CU64843 5 Plum Creek

8 Beaver Raccoon Creek 40.507116 -80.3556 0/0/1947 CU64854 1 Raccoon Creek

9 Beaver Raccoon Creek State Park 40.507116 -80.3556 1956/4/28 CU64853 2 Raccoon Creek

10 Beaver Traverse Creek 40.503367 -80.423887 0/0/1933 CU64827 1 Traverse Creek

11 Beaver Traverse Creek - Raccoon Creek State Park Rt 18 1st bridge 40.503367 -80.423887 1996/8/13 PSU PSU3078 7 7 Traverse Creek

95

12 Beaver Traverse Creek - Rt 30, Raccoon Creek State Park 40.509417 -80.366383 1996/8/13 PSU PSU3085 52 52 Traverse Creek

13 Butler Buffalo Run - Fennelton 40.91489167 -79.7057 1960/4/24 CU64844 1 Buffalo Run

14 Butler Cornplanter Run - at Moorehead Rd. bridge. 40.765117 -79.6959 2003/5/20 RWC PSU3710 1 1 Cornplanter Run

15 Butler Fennelton, Buffalo Run 40.86841658840 -79.72755926140 4/24/60 Cornell Z64844 1 1 Buffalo Run

16 Butler Little Buffalo Run - 1 mi ESE of Fenelton along Fenelton Rd. 40.863833 -79.7106 2003/5/20 RWC PSU3702 23 2 Little Buffalo Run

17 Butler Little Buffalo Run - Clearfield Rd., Fenelton 40.868367 -79.726333 1996/8/12 PSU PSU3086 37 37 Little Buffalo Run E.Br. Big Elk Creek, @ Old Rt 1 (Balt. Pike) bridge near 18 chester Forrestville. 39.82234298870 -75.89734383410 2002/10/2 RWC PSU3769 10 1 E.Br. Big Elk Creek East Branch of Big Elk Creek, Chester Co., near Forrestville at Old PSU- 19 chester rte 1 bridge 39.82234298870 -75.89734383410 3/24/77 Cooper 1739 1 1 E.Br. Big Elk Creek

20 chester Hodgsons Run - at SR 841, 1 mi SE of Oxford 39.74636667 -75.8666333 1996/9/29 PSU PSU3104 50 50 Hodgsons Run

21 Hodgsons Run - trib to Big Elk Creek on PA Rt 841, 3 mi S of New chester London 39.7415 -75.862833 1978/4/14 ELC2054 4 Hodgsons Run

22 Hodgsons Run (trib. to Big Elk Creek), Chester Co., 1.5 mi. SW of PSU- chester New London, Pa. 39.75491400370 -75.87273594440 5/14/78 Cooper 2053 1 1 Hodgsons Run

23

chester Hodgsons Run, @ SR841 bridge. 39.74636667 -75.8666333 2002/10/2 RWC PSU3746 5 2 Hodgsons Run

24 PSU- chester Hodgsons Run, Chester Co., at Rt. 841 crossing 39.74619204020 -75.86697030440 4/28/83 Cooper 2399 1 1 Hodgsons Run

25 Tril to Big ELK Creek, Chesta Co. PA on Pa Rte 841. 3 mile s of chester New Londa, PA 39.7415 -75.862833 1978/4/14 ELC 2054 5 Big ELK Creek

26 fayette Mountain Creek - 2 mi SE of Smithfield 40.781833 -79.795167 1996/10/12 PSU PSU3088 21 21 Mountain Creek

27 fayette Mountain Creek - at bridge in Ruble Mill. 40.781833 -79.795167 2003/3/17 RWC PSU3703 9 2 Mountain Creek

28 fayette Mountain Creek 2 mi S of Smithfield near Ruble 40.781833 -79.795167 1968/7/15 ELC0669 6 Mountain Creek Mountain Creek, Fayette Co., trib. to Georges Creek, Monongahela PSU - 29 fayette R., 2 miles S. of Smithfield 39.77774544010 -79.82424055470 7/15/68 Cooper 669 6 6 Mountain Creek Mountain Creek, Fayette Co., trib. to Georgia Creek, Monongahela PSU- 30 fayette R. at Ruble, same at #669 39.78144795750 -79.79515245320 5/24/69 Cooper 691 34 34 Mountain Creek

31 Indiana Anthony Run - SR 3039 bridge SE of Shelocta 40.646533 -79.279917 2007/4/3 RWC07004 PSU10020 26 2 Anthony Run

32 Indiana Cherry Run, off SR2005 200m upstream of confluence w/Crooked Dk. 40.67330000000 -79.45800000000 2008/6/3 PSU/IBI CalU 1 Cherry Run

33 Indiana Crooked Creek - collected at 4 stations in headwaters 40.693 -79.0825 6/0/1980 ELC2230 52 Crooked Creek

34 Indiana Crooked Creek - confluence of Pine Run 40.72255 -79.102383 0/0/1981 PFBC ? Crooked Creek

35 Indiana Crooked Creek - int. SR286 & SR1005 approx. 3 mi W of Clymer 40.6627 -79.059567 2003/3/17 RWC PSU3701 5 2 Crooked Creek

36 Indiana Curry Run - SR3022 birdge SE of Shelocta 40.651683 -79.2717 2007/4/4 RWC07012 PSU10021 7 2 Curry Run

96

37 Indiana Little Yellow Creek - at SR 422 bridge. 40.561683 -78.946117 2003/4/24 RWC PSU3707 1 1 Little Yellow Creek

38 Indiana Little Yellow Creek 240 m downstream from T-904 birdge 40.581111 -78.915 1984/6/26 PFBC Little Yellow Creek

39 Indiana N Br Plum Creek-int SR 85 & SR 210 at Plumville 40.786767 -79.207633 2007/4/3 RWC07007 PSU10036 5 1 Plum Creek

40 Indiana Repine Run - along TR 916, 2 mi NE of Pine Flats. 40.66675 -78.885867 2003/3/18 RWC PSU3704 15 2 Repine Run

41 Indiana Repine Run - site 01 at TR 916 crossing 40.666667 -78.88595 1987/7/14 PFBC Repine Run

42 Indiana S Br Two Lick Creek - downstream of SR2014 bridge E of Wandin 40.6746 -78.944317 2007/6/14 RWC07053 PSU10035 17 2 Two Lick Creek

43 Indiana S. Br. Plum Creek - 1.3 mi SW of Five Points 40.721 -79.24275 2007/4/3 RWC07006 PSU10022 27 2 Plum Creek

44 Indiana Sides Run 40.668611 -78.898611 1987/7/14 PFBC Sides Run

45 Indiana Sides Run 40.661944 -78.896667 1987/7/14 PFBC Sides Run

46 Indiana Sides Run - at SR 580 bridge 2 mi E of Pine Flats. 40.66885 -78.898483 2003/3/18 RWC PSU3709 4 2 Sides Run Trib to Crooked Creek - along SR 1005 S of SR286 int. from mouth 47 Indiana 200m upstream 40.653933 -79.062617 2003/3/17 RWC PSU3700 8 2 Crooked Creek

48 Indiana Yellow Creek - upstream of TR742 (Yellow Creek Rd.) bridge 40.593217 -78.989483 2007/6/14 RWC07052 PSU10034 2 2 Yellow Creek Westmorela 49 nd 5 mi SE of Ligonier (Powdermill Run) 40.147133 -79.268517 1956/4/15 CU64847 1 Powdermill Run Westmorelan Fourmile Run - 50m downstream from LR 64060 bridge (reported 50 d as I. greeleyi) 40.24374 -79.287384 1977/6/15 PFBC FourMile Run Westmorela 51 nd FourMile Run - bridge upstream of Donegal Lake. 40.146667 -79.370667 2003/4/24 RWC PSU3711 2 1 FourMile Run Westmorela Fourmile Run, Westmoreland Co., headwaters above Donegal PSU- 52 nd Lake 40.12406358510 -79.37055109200 6/7/78 Cooper 2066 9 9 FourMile Run Westmorela Fourmile Run, Westmoreland Co., trib. to Loyalhanna Cr., 2 miles PSU- 53 nd above mouth 40.23428960370 -79.29717584890 8/15/67 Cooper 431 6 6 FourMile Run Westmorela 54 nd Haymaker Run - along SR 4033 in Murrysville. 40.45545 -79.666567 2003/4/24 RWC PSU3708 11 2 Haymaker Run Westmorela 55 nd Haymaker Run - School Rd. at Murrysville 40.449167 -79.6707 1996/8/10 PSU PSU3084 8 8 Haymaker Run Westmorela 56 nd Haymaker Run at Murraysville,PA 40.42961323680 -79.69820657300 1977/8/21 ELC 1750 1 Haymaker Run Westmorela Haymaker Run, Westmoreland Co., at F.M. Sloan Elementary PSU- 57 nd School, about 4 mi. upstream from Murrysville, Pa. 40.46318107800 -79.63619866980 5/28/78 Cooper 2055 1 1 Haymaker Run Westmorela PSU- 58 nd Haymaker Run, Westmoreland Co., at Murraysville 40.42961323680 -79.69820657300 4/21/77 Cooper 1750 1 1 Haymaker Run Westmorela PSU- 59 nd Haymaker Run, Westmoreland Co., near Murrysville 40.42961323130 -79.69820694120 9/3/70 Cooper 1248 7 7 Haymaker Run Westmorela 60 nd Hendricks Creek - SR 711 br. 1 mi SW of W Fairfield 40.336616 -79.135467 1996/8/10 PSU PSU3076 56 56 Hendricks Creek Westmorelan 61 d Hendricks Creek (Reported as I. greeleyi) 40.336666 -79.137779 1977/8/30 PFBC Hendricks Creek Westmorela 62 nd Hendricks Creek along SR 711 2 mi SW of West Fairfield. 40.324833 -79.131833 2003/3/18 RWC PSU3714 22 2 Hendricks Creek Westmorela Hendricks Run, Westmoreland Co., at Pa. rte 711 crossing, 1 mi. PSU- 63 nd SW of West Fairfield, Pa. 40.33514760380 -79.13555266720 5/29/78 Cooper 2057 1 1 Hendricks Run Westmorela Little Pucketa 64 nd Little Pucketa Creek near New Kensington 40.57782678420 -79.73156083030 4/0/1936 Cornell CU5607 1 1 Creek

97

Westmorela Little Pucketa Creek, 2.5 mi up stream from the Allegheny River, GO UMMZ Little Pucketa 65 nd New Kensington 40.575433 -79.74225 4/29/34 Hollibaugh 26.1 Creek Westmorela 66 nd Loyalhanna Creek - SR711 bridge in Ligonier 40.24111 -79.24007 2009/5/22 RWC09002 PSU10075 3 Loyalhanna Creek Westmorela 67 nd Loyalhanna Creek, along SR 30 west of Ligonier 40.24939 -79.25348 2008/4/18 RWC08005 PSU10038 41 Loyalhanna Creek Westmorela 68 nd Macks Run - along TR984, .5 mi NE of Waterford. 40.259267 -79.15955 2003/4/24 RWC PSU3705 35 2 Macks Run Westmorelan Mill Creek - 270 m below McKelvey Rd bridge (T617) (reported as 69 d I. greeleyi) 40.259167 -79.191667 1980/7/7 PFBC Mill Creek Westmorela 70 nd Mill Creek - downstream of T617 bridge, just W of Oak Grove. 40.255367 -79.21125 2003/4/24 RWC PSU3712 8 2 Mill Creek Westmorela Powdermill Run - along SR 381 at Powdermill Nature Center, 2 mi. 71 nd S of Rector. 40.159267 -79.2744 2003/4/24 RWC PSU3713 9 2 Powdermill Run Westmorela 72 nd Powdermill Run - PNR - 2 mi. S of Rector 40.148333 -79.269333 1958/5/24 CU64851 1 Powdermill Run Westmorela 73 nd Powdermill Run - PNR - 2 mi. S of Rector 40.148333 -79.269333 1959/4/23 CU43615 2 Powdermill Run Westmorela 74 nd Powdermill Run - PNR - 2 mi. S of Rector 40.148333 -79.269333 1959/4/23 CU64850 1 Powdermill Run Westmorela 75 nd Powdermill Run - PNR - 2 mi. S of Rector 40.148333 -79.269333 1963/4/20 CU64849 5 Powdermill Run Westmorela 76 nd Powdermill Run - PNR - 2 mi. S of Rector 40.148333 -79.269333 1968/4/12 CU64852 3 Powdermill Run Westmorelan 77 d Shannon Run (reported as I. greeleyi) 40.372398 -79.082901 1977/8/29 PFBC Shannon Run Westmorela PSU- 78 nd Shannon Run, Westmoreland Co., near New Florence 40.37172762720 -79.08089832770 4/30/75 Cooper 1518 9 9 Shannon Run Westmorela Shannon Run, Westmoreland Co., Pa. rte 711 crossing at New PSU- 79 nd Florence 40.37172763940 -79.08089703990 5/29/78 Cooper 2056 2 2 Shannon Run Westmorela 80 nd Snyders Run - N of SR 259 bridge 40.3375 -79.19055 1996/8/10 PSU PSU3089 31 31 Snyders Run Westmorela Snyders Run 1/4 mi upstr fr PA Rt 259 xing along SR 1067. Approx 81 nd 6.5 mi NNW Ligonier. 40.331867 -79.193217 2003/3/18 RWC PSU3715 33 2 Snyders Run Westmorela Snyders Run, Westmoreland Co., 1/4 mi. upstream from Pa. rte PSU- 82 nd 259 crossing 40.34950129270 -79.15473388800 5/29/78 Cooper 2058 2 2 Snyders Run Westmorela Tubmill Creek - at SR1011 bridge above mouth of Hendricks Ck, 3 83 nd mi SE of Bolivar. 40.369083 -79.13545 2003/3/18 RWC PSU3706 17 2 Tubmill Creek Westmorelan 84 d Tubmill Creek (reported as I. greeleyi) 40.368889 -79.135556 1977/8/31 PFBC Tubmill Creek Westmorelan 85 d Tubmill Creek (reported as L. appendix) 40.383471 -79.147557 2007/8/14 PFBC Tubmill Creek Westmorela 86 nd Tubmill Creek at jct of Tubmill & Hendricks Creek 40.3710833 -79.1374833 1967/8/15 ELC0421 12 Tubmill Creek Westmorela 87 nd Tubmill Creek below Tubmill Reservoir 40.331633 -79.089 1975/4/30 ELC1519 19 Tubmill Creek Westmorela 88 nd Tubmill Creek, at junction of tTbmill and Handrick Creek 40.3710833 -79.1374833 1961/8/15 ELC 421 many Tubmill Creek Westmorela PSU- 89 nd Tubmill Creek, Westmoreland Co., just below Tubmill Reservoir 40.33114983000 -79.08897048240 4/30/75 Cooper 1519 19 19 Tubmill Creek

90 Butler Buffalo Creek 40.946389 -79.744444 2007/4/11 PFBC Buffalo Creek Westmorela 91 nd Fourmile Run 40.209389 -79.324287 2006/4/3 PFBC Fourmile Run

98

92 Indiana Laurel Run 40.575863 -78.993919 2006/4/14 PFBC Laurel Run Westmorela 93 nd Tubmill Creek (reported as L. appendix) 40.383471 -79.147557 2007/8/14 PFBC Tubmill Creek

Appendix F. Lampetra appendix historical sites

NO INDIVI NO CO LOCATION LAT LON DATE COLLECT DEPOSIT RET DUAL CREEK 41.68666 1 Potter Allegheny Portage Creek - 2 mi N of Keating Summit 7 -78.201333 1996/9/23 PSU PSU3087 9 9 Allegheny Portage Creek

2 McKean Allegheny Portage Creek - TR404 bridge near Wrights. 41.7446 -78.247933 2005/5/4 RWC05008 PSU4010 10 4 Allegheny Portage Creek 41.76516 3 McKean Allegheny Portage Creek - unmarked road S of Johnston 7 -78.264333 1996/9/22 PSU PSU3092 1 1 Allegheny Portage Creek 41.80765 4 McKean Allegheny River 9 -78.288109 2006/6/12 PFBC Allegheny River

5 McKean Allegheny River 41.86758 -78.34333 2006/6/13 PFBC Allegheny River 41.75781 6 Potter Allegheny River - 1.5 mi W of Coudersport 7 -78.052467 2005/5/4 RWC05009 PSU4011 15 2 Allegheny River 41.75781 7 Potter Allegheny River - 1.5 mi W of Coudersport 7 -78.052467 2005/6/7 RWC05009 PSU4011 1 1 Allegheny River

8 Potter Allegheny River - Avenue A bridge in Coudersport 41.79085 -78.01585 2005/5/18 RWC05039 PSU4031 22 2 Allegheny River

9 McKean Allegheny River - bridge at Turtlepoint 41.8677 -78.3433 1988/4/13 PSU PSU2040 1 1 Allegheny River

10 McKean Allegheny River - bridge at Turtlepoint 41.86715 -78.3435 2005/9/23 RWC05081 PSU4055 1 1 Allegheny River 41.75781 11 Potter Allegheny River - covered bridge 1.5m W of Coudersport 7 -78.052467 2006/9/27 RWC06030 BPEF 3 Allegheny River

12 Potter Allegheny River - covered bridge along SR6 W of Coudersport 41.75781 78.05235 2008/5/29 RWC08024 6 Allegheny River 41.87086 13 McKean Allegheny River - Rt 155, village of Turtlepoint 7 -78.338267 1996/9/23 PSU PSU3098 2 2 Allegheny River

14 Potter Allegheny River - TR405 bridge in Roulette 41.77881 -78.16406 2009/5/27 RWC09023 15 Allegheny River

15 McKean Allegheny River - TR428 bridge in Port Allegheny 41.80774 -78.28815 2009/5/27 RWC09020 2 Allegheny River 2002/10/1 16 McKean Allegheny River @ TR 428 bridge in Port Allegheny. 41.80774 -78.28815 0 RWC/PSU 3 3 Allegheny River - 17 Allegheny River, Potter Co., 1 mi. upstream from junction of Pa. 41.82147 77.97134756 PSU- Potter highways 44 and 49, 3 mi. NE of Coudersport 063780 740 5/0/75 Cooper 1539 10 10 Allegheny River - 18 41.85206 77.87959053 PSU- Potter Allegheny River, Potter Co., at Colesburg, 8 mi. NE of Coudersport 153020 570 7/29/71 Cooper 1376 1 1 Allegheny River - 19 41.77750 78.15417328 PSU- Potter Allegheny River, Potter Co., at Roulette 008140 090 5/2/70 Cooper 1179 3 3 Allegheny River

99

41.81084 20 Warren Andrews Run 1 -79.386238 2005/6/1 PFBC Andrews Run 41.58555 21 Forest Beaver Run 6 -79.3225 2001/6/5 PFBC Beaver Run

22 McKean Bell Run 41.93786 -78.22866 2007/4/11 PFBC Bell Run 41.32661 23 venango Big Sandy Ck - under Rt 8 bridge 7 -79.872867 1996/7/30 PSU PSU3080 7 7 Big Sandy Cr. 41.87611 24 Warren Blue Eye Run 1 -79.483889 1977/6/8 PFBC Blue Eye Run 41.82567 25 Warren Blue Eye Run - about 1 mi from mouth 8 -79.434483 1975/7/8 ELC1552 12 Blue Eye Run

26 Warren Blue Eye Run 3 mi W of Pittsfield 41.8312 -79.439067 1973/7/19 ELC1587 11 Blue Eye Run

27 Warren Blue Eye Run, .8 mi. from mouth, 3 mi WSW of Pittsfield 41.8216 -79.432333 2002/9/12 RWC PSU 1 1 Blue Eye Run

28 Warren Blue Eye Run, 1.3 mi. from mouth, 2.8 mi W of Pittsfield 41.82985 -79.436983 2002/9/12 RWC PSU 8 1 Blue Eye Run

29 Warren Blue Eye Run, 3 mi. from mouth, 4 mi WNW of Pittsfield 41.847 -79.453 2002/9/12 RWC PSU 1 1 Blue Eye Run 41.68766 30 McKean Brewer Run 120 yards upstream of T362 ( E. Valley Rd.) 7 -78.401167 2002/5/29 PSU PSU3608 1 Brewer Run - 31 41.81479 79.75939712 PSU- Crawford Britton Run, Crawford Co., 6 mi. N. of Centerville 961320 240 5/6/75 Cooper 1545 15 15 Britton Run 41.79478 32 Crawford Britton Run, Crawford Co., middle section 3 -79.739317 7/25/72 ELC 1445 Britton Run Brokenstraw Creek - Mill Rd. crossing at NY state line. 41-59.985/79- 33 Warren 37.338 41.99975 -79.6223 2005/7/21 Dcarlson 1 Brokenstraw Creek 41.67833 34 warren Caldwell Creek 3 -79.5825 1997/7/31 PFBC Caldwell Creek 41.65277 35 warren Caldwell Creek 8 -79.609444 1997/7/31 PFBC Caldwell Creek - 36 41.69989 79.55217496 PSU- warren Caldwell Creek, Warren Co., 2nd bridge above Selkirk Station 739000 810 9/7/67 Cooper 439c 7 7 Caldwell Creek - 37 Caldwell Creek, Warren Co., trib to Pine Creek, 1st bridge above Selkirk 41.68602 79.57540648 PSU- warren Station 079670 560 10/21/67 Cooper 439a 1 1 Caldwell Creek 41.65194 38 warren Campbell Creek 4 -79.529722 2001/6/27 PFBC Campbell Creek 41.82805 39 McKean Chappel Fork 6 -78.791667 1999/6/7 PFBC Chappel Fork 41.81777 40 McKean Chappel Fork 8 -78.835833 1999/6/7 PFBC Chappel Fork - 41 41.81693 78.85521985 PSU- McKean Chappel Fork, McKean Co. trib to Kinzua Creek, 5 mi. west of Marshburg 367400 290 8/12/69 Cooper 18a 1 1 Chappel Fork - 42 41.63380 78.99562562 PSU- warren Cherry Run, Warren Co., above USFS rte. 445, trib. to S. Br. Tionesta 939550 050 8/26/69 Cooper 6b 1 1 Cherry Run

43 Warren Cold Spring Brook - Still propoerty of SR 4007, 1 mi S of Pine Valley 41.95315 -79.525133 2005/5/5 RWC05015 PSU4014 2 2 Cold Spring Brook 2002/10/1 44 McKean Cole Creek @ SR 46 bridge just S of Farmers Valley. 41.85875 -78.447867 0 RWC/PSU PSU 2 2 Cole Creek

100

41.72466 45 McKean Colegrove Brook - upstream of SR 46 bridge at Colegrove 7 -78.38245 2005/5/6 RWC05020 PSU4017 7 4 Colegrove Brook - 46 41.75811 80.37158656 Crawford Conneauat Cr. At 198 Bridge 241770 090 1985/5/30 PSU PSU1634 1 Conneauat Cr. - 47 41.75811 80.37158656 PSU- JMB Crawford Conneaut Cr at PA Rte 198 bridge W of Conneautville, PA 241770 090 6/11/85 Stauffer 85.14 0 0 Conneauat Cr. 41.68472 48 Crawford Conneaut Creek - Agnew Rd. bridge 2 mi NW of Harmonsburg. 2 -80.340833 2003/4/28 RWC03026 PSU4004 5 3 Conneauat Cr.

49 Crawford Conneaut Creek - along Canal Rd just N of Wind Rd. bridge 41.7167 -80.34929 2008/5/23 RWC08007 36 Conneauat Cr.

50 Crawford Conneaut Creek - along Canal Rd just N of Wind Rd. bridge 41.7167 -80.34929 2008/7/23 RWC08041 35 Conneauat Cr. 41.72918 51 Crawford Conneaut Creek - at Fish Rd. bridge 3 -80.355233 2005/7/15 RWC05058 PSU4046 14 4 Conneauat Cr. 41.70666 52 Crawford Conneaut Creek - downstream of bridge in Dicksonburg. 7 -80.351667 2003/8/19 RWC03074 PSU4007 8 4 Conneauat Cr. 41.72918 PSU1002 53 Crawford Conneaut Creek - Fish Rd. bridge, Crawford Creek 3 -80.355233 5/20/2007 PSU 5 2 Conneauat Cr.

54 Crawford Conneaut Creek - just N of Dicksonburg 41.71085 -80.350767 2005/5/16 RWC05028 PSU4023 2 2 Conneauat Cr. - 55 41.68467 80.34158164 PSU- Crawford Conneaut Creek, Crawford Co., 5 mi. S. of Conneautville 467690 470 4/29/75 Cooper 1535 57 57 Conneauat Cr. - 56 41.66570 80.37211635 PSU- Crawford Conneaut Creek, Crawford Co., 8 mi. S. of Conneautville 696890 890 4/29/75 Cooper 1511 1 1 Conneauat Cr. - 57 Conneaut Creek, Crawford Co., trib. to Lake Erie, off route 18, North of 41.76504 80.37637410 Crawford Conneautville 834770 870 8/14/68 ELC 601 Conneauat Cr. PSU1005 58 Erie Darrow Brook - Wilson Rd. bridge near NY boundary 42.10429 -79.76725 2008/8/4 RWC08050 4 10 Darrow Brook - 59 42.10044 79.76219304 PSU- Erie Darrow Brook, Erie Co., at Pa.-NY state line, east of Little Hope 339440 020 6/15/75 Cooper 1533 2 2 Darrow Brook - 60 41.68824 78.92524023 PSU- McKean E. Branch Tionesta route 133 at picnic area 273040 300 8/20/69 Cooper 3a 7 7 E. Branch Tionesta

61 Erie E.Br. Conneaut Creek - Gage Rd, Albion. 41.90145 -80.359417 2003/5/22 RWC03045 PSU4005 8 4 E.Br. Conneaut Creek 41.77266 62 Crawford E.Br. Oil Creek - upstr. Of Britton Run Rd. bridge, 2 mi NE of Centerville. 7 -79.729 2003/4/28 RWC PSU3728 1 1 E.Br. Oil Creek

63 McKean East Branch of Potato Creek, at Rt. 46 Bridge (farthest upstream) 41.655 -78.3697 2002/6/10 PSU PSU3599 1 E. Br. of Potato Creek - 64 41.63580 78.85028523 PSU- East Branch of Tionesta McKean East Branch of Tionesta Creek, above road bridge at Jojo, PA 576720 260 8/20/69 Cooper 12a 15 15 Creek East Branch Oil Creek, Crawford Co., .5 mi. down from mouth of Brannon 65 Crawford Run 41.764 -79.728167 7/20/72 ELC 1446 E.Br. Oil Creek 41.79555 66 Warren East Branch Spring Creek 6 -79.541667 1977/9/12 PFBC East Branch Spring Creek 41.84249 67 McKean East Branch Tunungwant Creek 9 -78.642502 2005/7/25 PFBC East Branch Spring Creek 41.65111 68 Warren East Hickory Creek 1 -79.326667 2004/7/19 PFBC East Hickory Creek

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41.93222 69 Potter Elevenmile Creek 2 -78.096667 1978/7/25 PFBC Elevenmile Creek 41.80944 70 Potter Fishing Creek 4 -78.143889 1977/7/27 PFBC Fishing Creek

71 Potter Fishing Creek 41.79 -78.155 1977/7/27 PFBC Fishing Creek

72 Potter Fishing Creek - at mouth in Roulette 41.78185 -78.1653 2005/5/18 RWC05040 PSU4032 45 4 Fishing Creek

73 Potter Fishing Creek - at mouth in Roulette 41.78021 -78.16548 2009/5/27 RWC09024 12 Fishing Creek

74 Potter Fishing Creek - at Rt 341 in Roulette 41.838 -78.126283 1996/9/27 PSU PSU3105 137 137 Fishing Creek 41.79941 75 Potter Fishing Creek - SR 2004 bridge below West Branch 7 -78.147717 2005/5/4 RWC05013 PSU4012 8 2 Fishing Creek - 76 Five Mile Creek, Crawford Co., just below rte 89, about 10 mi. N. of 41.78429 79.69254980 PSU- Crawford Titusville 023810 850 4/27/77 Cooper 1751 2 2 Five Mile Creek 41.78416 77 Crawford Fivemile Creek 7 -79.685833 2006/6/20 PFBC Fivemile Creek 41.78421 78 Crawford Fivemile Creek - SR 89 bridge 2.5 mi NNW of Buell Corners. 7 -79.685833 2003/4/28 RWC 1 1 Five Mile Creek 41.78421 79 Crawford Fivemile Creek - SR 89 N of Titusville 7 -79.685833 1996/7/31 PSU PSU3090 175 175 Five Mile Creek - 80 41.78682 79.66074617 PSU- Crawford Five-mile Creek, Crawford Co. 837030 050 7/20/72 Cooper 1442 1 1 Five Mile Creek - 81 41.78634 79.68029788 PSU- Crawford Fivemile Creek, Crawford Co., 4 mi. S. of Spartansburg 561140 240 5/5/75 Cooper 1547 7 7 Five Mile Creek - 82 41.75472 79.01180118 PSU- warren Four Mile Run, Warren Co., about 4 mi. N. of Sheffield 038400 820 5/13/77 Cooper 1743 7 7 Four Mile Run - 83 41.72504 79.05490552 PSU- warren Four mile Run, Warren, Co., below Flat Run, North of rte. 6 972660 090 8/25/69 Cooper 35a 9 9 Four Mile Run 41.98777 84 Potter Genesee River 8 -77.8675 1979/5/31 PFBC Genesee River - 85 41.99151 77.86793629 PSU- RLR Potter Genesee River in Genesee, PA 484270 610 5/12/88 Stauffer 88.24 1 1 Genesee River - 86 41.98875 77.86826359 PSU- Potter Genesee River, Potter Co., at Genesee 854760 140 5/2/75 Cooper 1541 11 11 Genesee River - 87 41.94207 77.81585037 PSU- Potter Genesee River, Potter Co., below West Bingham, bridge into farm 757850 920 9/24/69 Cooper 746 1 1 Genesee River PSU1006 88 Erie Hare Creek - Scioto Rd. bridge E of Corry 41.92944 -79.62905 2008/11/2 RWC08068 0 2 Hare Creek - 89 Haven's Brook, McKean Co., at Rte 46 crossing, 1 mi. N. of 41.63426 78.35749414 PSU- McKean McKean/Carman Co. line 581240 580 8/9/78 Cooper 2135 4 4 Haven's Brook 41.63406 90 McKean Havens Run - State Game Lands 30, upstream of SR 46 bridge. 7 -78.3576 4/31/2003 RWC 2 Havens Run 41.63406 91 McKean Havens Run, SGL 30, upstr of SR46 bridge. 7 -78.3576 2002/6/10 RWC PSU 1 1 Havens Run

102

41.63406 92 McKean Havens Run, SGL 30, upstr of SR46 bridge. 7 -78.3576 4/31/2003 RWC/PSU 8 2 Havens Run sight - 93 venango Justus Lake, at mouth of unnamed trib across from park office. 41.47565 -79.765917 1999/5/1 photo 4 0 Justus Lake - 94 41.86839 78.79411307 PSU- Kavanaugh Branch of Sugar McKean Kavanaugh Branch of Sugar Run, McKean Co. 123310 980 8/12/69 Cooper 1a 1 1 Run 41.77861 95 McKean Kinzua Creek 1 -78.841111 1999/6/14 PFBC Kinzua Creek 41.77176 96 McKean Kinzua Creek - Rt 321, Red Bridge 7 -78.881583 1996/8/1 PSU PSU3091 3 3 Kinzua Creek - 97 41.77420 78.85175118 PSU- McKean Kinzua Creek, McKean Co., above USFS route 150 bridge 002290 120 8/13/69 Cooper 286f 4 4 Kinzua Creek - 98 41.77990 78.80776639 PSU- McKean Kinzua Creek, McKean Co., below Libby Cr. 075000 470 7/11/64 Cooper 286e 4 4 Kinzua Creek

99 McKean Knapp Creek - SR1011 bridge N of W. Eldred 41.9627 -78.400233 2005/5/17 RWC05033 PSU4026 13 3 Knapp Creek - 100 41.87292 80.13288124 PSU- Erie L. Conneauttee Creek, Erie Co., trib. to French Cr., South of route 6N 014320 240 5/15/71 Cooper 581a 21 21 L. Conneauttee Creek - 101 41.85168 78.67630317 PSU- McKean Lewis Run, McKean Co. 426580 850 4/1/64 Cooper 348 2 2 Lewis Run

102 Erie Lilley Run - at Shawver property along Elgin Rd 41.87209 -79.73539 2008/6/21 RWC08035 3 Lilley Run PSU1005 103 Erie Lilly Run - downstream of SR2019 (Concord Rd. bridge 41.87863 -79.73804 2008/5/25 RWC08023 1 1 Lilley Run 41.96898 104 Warren Little Brokenstraw Creek - TR 539 bridge NW of Lottsville 3 -79.463433 2005/5/5 RWC05017 PSU4016 1 1 Little Brokenstraw Creek Little Conneautee Creek - Kinter Hill Rd (SR3022) bridge E of Mt. PSU1004 105 Erie Pleasant 41.86392 -80.05729 2008/6/20 RWC08029 6 2 Little Conneautee Creek 41.87777 106 Erie Little Conneauttee Creek 8 -80.054167 1982/7/13 PFBC Little Conneauttee Creek 41.86388 107 Erie Little Conneauttee Creek 9 -80.0575 2006/8/4 PFBC Little Conneauttee Creek 41.55027 108 venango Little Sugar Creek 8 -79.796667 1977/8/31 PFBC Little Sugar Creek - 109 Little Sugar Creek, Venango Co., 2.25 miles above mouth of Prather 41.60153 79.79353094 PSU- venango Creek 794490 930 8/30/65 Cooper 262 1 1 Little Sugar Creek - 110 Little Sugar Creek, Venango Co., trib. to E. Br. Sugar Cr., near 41.56631 79.86263777 PSU- venango Wallaceville, route 428 468100 530 8/6/68 Cooper 617 1 1 Little Sugar Creek 41.89972 111 Potter Ludington Run 2 -77.800833 1979/5/29 PFBC Ludington Run

112 Potter Ludington Run 4th bridge upstream on SR 1009 41.9387 -77.8125667 2003/6/18 PSU PSU3661 1 Ludington Martin Creek - at delayed harvest access along rt. 6, 3.5 km West of 41.78849 113 McKean Smethport 7 -78.484429 7/1/2009 PSU PSU4847 4 Martin Creek - 114 Martin Run, Elk Co., trib to S. Branch of Tionesta, at end of F.S. route 41.60458 78.89940879 PSU- elk 195 534580 360 8/20/69 Cooper 42b 28 28 Martin Run

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41.95777 Middle Branch Genesee 115 Potter Middle Branch Genesee River 8 -77.856944 1979/5/30 PFBC River 41.90416 Middle Branch Genesee 116 Potter Middle Branch Genesee River 7 -77.840833 1979/5/30 PFBC River - 117 41.97071 77.85772046 Middle Branch Genesee Potter Middle Branch Genesee River - off Rt 449 1.5 mi N of Harmontown 424970 640 1996/10/1 PSU PSU3098 1 1 River - 118 41.97071 77.85772046 PSU- Middle Branch Genesee Potter Middle Branch Genesee River, Potter Co., 1 1/2 N. of Harmontown 424970 640 7/29/71 Cooper 1375 6 6 River - 119 41.75343 77.94988743 PSU- Potter Mill Creek, Potter Co., at Sweden Valley 725920 720 6/2/75 Cooper 1540 2 2 Mill Creek - 120 41.75343 77.94988743 PSU- Potter Mill Creek, Potter Co., at Sweden Valley 725920 720 7/29/71 Cooper 1395 9 9 Mill Creek

121 Forest Minister Creek 41.63 -79.159167 2003/9/16 PFBC Minister Creek 41.63000 122 Forest Minister Creek 1 -79.159164 2005/8/15 PFBC Minister Creek - 123 41.62056 79.15329902 PSU- Forest Minister Creek, Forest Co., trib. to Tionesta Creek, 1/2 mile above mouth 092990 070 8/20/69 Cooper 705 11 11 Minister Creek - 124 No name trib., Middle Branch of White Clay Creek, Chester Co., 1st trib. 39.79610 75.82612061 PSU- Middle Branch of White Clay chester upstream from Pa. rte 841 908290 120 5/12/78 Cooper 2049 1 1 Creek

125 Crawford Oil Creek, Crawford Co., 1/2 way between Linconville and Riceville 41.78435 -79.8100833 7/24/72 ELC 1447 Oil Creek - 126 41.79372 79.83544631 PSU- Crawford Oil Creek, Crawford Co., at Lincolnville 210320 980 5/6/75 Cooper 1544 7 7 Oil Creek - 127 41.90295 78.06692993 PSU- Potter Oswayo Cr., Potter Co., near Coneville, trib. to Allegheny 346150 960 9/24/69 Cooper 751 18 18 Oswayo Creek 41.90916 128 Potter Oswayo Creek 7 -78.092778 1978/7/27 PFBC Oswayo Creek

129 Potter Oswayo Creek - mouth of S. Br. Oswayo Ck. At Coneville 41.90517 -78.06745 2009/5/27 RWC09022 5 Oswayo Creek 41.90821 130 Potter Oswayo Creek - SR244 at Coneville 667 -78.0568 1996/9/29 PSU PSU3102 12 12 Oswayo Creek 41.90963 131 Potter Oswayo Creek - TR351 bridge at Clara 3 -78.093333 2005/5/18 RWC05038 PSU4030 45 3 Oswayo Creek 41.93646 132 Potter Oswayo Creek - TR376 bridge at Sharon Center 7 -78.14565 2005/5/18 RWC05037 PSU4029 16 2 Oswayo Creek - 133 41.91979 77.97162967 PSU- Potter Oswayo Creek, Potter Co., 1 mi. SW of Oswayo 765190 810 5/21/75 Cooper 1531 8 8 Oswayo Creek - 134 41.92423 78.11977787 PSU- Potter Oswayo Creek, Potter Co., at Millport, Pa. 737570 340 5/26/77 Cooper 1706 1 1 Oswayo Creek

135 Potter Oswayo Creek, Potter Co., near Sharon Center, trib. to Allegheny 41.9255 -78.122433 9/24/69 ELC 747 Oswayo Creek 41.41433 136 venango Patchel Run - Rt 322 1 mi NW of Rocky Grove 3 -79.85485 1996/8/19 PSU PSU3077 9 9 Patchel Run 41.41891 - PSU- 137 venango Patchel Run, Venango Co., 2 mi. NW of Rocky Grove, at rte 322 crossing 783840 79.87560894 4/27/77 Cooper 1752 5 5 Patchel Run

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930

41.94828 138 warren Pine Valley Creek - SR 6 bridge, State Game Lands 306. 3 -79.524033 2005/5/5 RWC05016 PSU4015 2 2 Pine Valley Creek 41.71037 139 McKean Potato Creek - downstream rt. 146 bridge. 1 km South of Hamlin Twp. 9 -78.391183 7/1/2009 PSU PSU4873 15 Potato Creek 41.78133 140 McKean Potato Creek - T377 off Rt. 46 3 -78.415333 1996/9/22 PSU PSU3093 1 1 Potato Creek 41.86088 141 McKean Potato Creek - T385 (Valley Rd.) bridge near int w/SR46/446 3 -78.440033 2006/9/27 RWC06033 BPEF 1 Potato Creek 41.70744 142 McKean Potato Creek - upstream of rt. 146 bridge, 1 km South of Hamlin Twp 8 -78.39101 7/1/2009 PSU PSU4872 4 Potato Creek - PSU- 143 41.78096 78.41549577 Cooper- McKean Potato Creek, McKean Co., 3 mi. S. of East Smethport 850980 620 5/30/75 1522 1522 Potato Creek - 144 41.63314 79.30838551 PSU- warren Queen Creek, Warren Co., trib. to Hickory Cr., Allegheny R. 376210 820 8/14/69 Cooper 701 7 7 Queen Creek 41.98888 145 Erie Raccoon Creek - just upstr. From mouth (Lake Erie). 9 -80.480833 2003/5/22 RWC PSU3719 1 1 Raccoon Creek - 146 41.71287 78.80924747 McKean S Br Kinzua Creek 673080 840 1969/8/13 ELC2a 6 6 S. Br. Kinzua Creek PSU1008 147 McKean S. Br. Cole Creek - TR349 bridge (Bordell Rd.) 41.85302 -78.51226 2009/5/27 RWC09019 5 6 S. Br. Cole Creek - 41.71287 78.80924747 PSU- 148 McKean S. Br. of Kinqua Creek 673080 840 8/13/69 Cooper 2a 6 6 S. Br. Kinzua Creek - 41.71287 78.80924747 PSU- 149 McKean S. Br. of Kinzua Creek 673080 840 8/13/69 Cooper 2a 6 6 S. Br. Kinzua Creek - 150 41.71204 78.81608589 PSU- McKean S. Br. of Kinzua Creek about 1/4 mile above mouth of Hubert Run 183020 030 8/21/69 Cooper 2b 3 3 S. Br. Kinzua Creek - 151 41.71204 78.81608589 PSU- McKean S. Br. of Kinzua Creek about 1/4 mile above mouth of Hubert Run 183020 030 8/21/69 Cooper 2b 3 3 S. Br. Kinzua Creek - 152 41.63101 78.99825252 PSU- warren S. Br. Tionesta, Warren Co. below USFS rte. 148 bridge 859870 190 8/26/69 Cooper 6a 1 1 S. Br. Tionesta 41.79268 153 McKean Sartwell Creek - SR 4001 4 mi E of Port Allegany 33 -78.205867 1996/9/27 PSU PSU3100 6 6 Sartwell Creek - 154 41.79439 78.20507417 PSU- Potter Sartwell Creek, Potter Co., near Burtville, at mouth of creek 159600 870 5/2/75 Cooper 1542 19 19 Sartwell Creek 41.71833 155 Crawford Shirley Run - Vrooman Rd. N of Titusville 3 -78.6501667 1996/9/22 PSU PSU3099 14 14 Shirley Run - 156 41.71617 79.65523476 PSU- Crawford Shirley Run, Crawford Co., 1 mi N. and 1.5 mi. E. of Cloverdale Corners 693600 950 7/19/72 Cooper 1436 11 11 Shirley Run - 157 41.69058 79.66974452 PSU- Crawford Shirley Run, Crawford Co., 5 miles N. of Titusville trib. to Thompson Cr. 573780 700 8/8/67 Cooper 460 1 1 Shirley Run - 158 41.70580 79.66343870 PSU- Crawford Shirley Run, Crawford Co., 500' East of Cloverdale Corners 392300 820 7/18/72 Cooper 1437 15 15 Shirley Run

105

- 159 41.74805 79.06946109 PSU- warren Sixmile Run, Warren Co., Lower section near railroad bridge at Tiona, PA 510950 970 8/1/63 Cooper 7 10 10 Sixmile Run 41.79111 160 McKean Skinner Creek - along TR488 on State Game Lands 61. 7 -78.300367 2005/5/6 RWC05025 PSU4021 1 1 Skinner Creek - 161 41.80248 78.28594471 PSU- McKean Skinner Creek, trib. to Portage Creek, 500' below bridge above mouth 462440 460 8/16/63 Cooper 28 2 2 Skinner Creek South Branch Cole Creek - upstream of TR349 bridge at Bingham Pump 162 McKean Sta. 41.8529 -78.512033 2005/5/6 RWC05023 PSU4020 25 4 S. Br. Cole Creek - 163 South Branch of French Creek, Erie Co. at rte. 89 crossing 1 mile south 41.90916 79.70525200 PSU- Erie of route 6 876700 720 6/2/66 Cooper 320 5 5 S. Br. of French Creek

164 Warren Spring Creek 41.8625 -79.517222 1997/8/20 PFBC Spring Creek - 165 41.87351 78.83092249 PSU- JMB McKean Sugar Run at Rt 321 bridge 064700 080 6/11/85 Stauffer 85.36 0 0 Sugar Run PFBC, 166 Warren Sugar Run, ~850m upstream of Allegheny Reservoir 41.88449 -78.88000 2008/6/3 PSU/IBI PSU 1 Sugar Run PFBC, 167 Warren Sugar Run, ~850m upstream of Allegheny Reservoir 41.88449 -78.88000 2008/6/3 PSU/IBI PSU 1 Sugar Run 4,190,06 168 Erie Temple Creek - Bessemer St. bridge in Albion 3 -80.35738 2008/5/24 RWC08015 9 Temple Creek PSU1004 169 Erie Temple Creek - Bessemer St. bridge in Albion 41.90063 -80.35738 2008/7/23 RWC08043 7 67 Temple Creek

170 Erie Temple Creek - bridge at Wellsburg 41.9008 -80.349383 2007/5/20 RWC07037 3 3 Temple Creek 41.90078 171 Erie Temple Creek - SR3006 bridge N of Albion 3 -80.35725 2005/5/16 RWC05030 PSU4024 12 12 Temple Creek MA 172 Shellgren, 41.71568 DG warren Tionesta Cr. below bridge at Saybrook, Sheffield Quad 3 -79.049083 7/27/59 Badarick MAS 024 Tionesta Cr. MA 173 Shellgren, DG forest Tionesta Cr., 1 mi. below Lynch around island, Sheffield Quad 41.6763 -79.0366 7/28/59 Badarick MAS T27 Tionesta Cr. - 174 Trib to West Branch White Clay Creek, Chester Co., first trib. crossing 39.76903 75.82428595 PSU- chester Pa. rte 841, at Schoolhouse Road 277280 370 5/12/78 Cooper 2052 2 2 W. Br. White Clay Creek 41.72833 175 Crawford Trib. To Oil Creek -Old Grade Rd. xing .5 mi from Clappville. 3 -79.7680556 2003/8/18 RWC03066 PSU4006 2 1 Oil Creek - 176 42.03352 80.26715008 PSU- Erie Trout Run, Erie Co., near mouth at Avonia, Pa. 841570 250 4/19/76 Cooper 1620 3 3 Trout Run

177 McKean Tunungwant Creek 41.96285 -78.636 2007/6/19 PFBC Tunungwant Creek 41.99666 178 McKean Tunungwant Creek 7 -78.623722 2007/6/19 PFBC Tunungwant Creek - 179 41.72276 78.92925006 PSU- McKean Two Mile Run, McKean Co., 5 mile N.W. Kane on old route 6 841110 580 8/20/69 Cooper 64a 8 8 Two Mile Run - 180 41.71257 78.98400182 PSU- warren Two Mile Run, Warren Co., 1 mile below Roystone, along RR 539790 080 8/26/69 Cooper 64b 7 7 Two Mile Run

106

- 181 41.72002 78.97058410 PSU- warren Two Mile Run, Warren Co., just above Roystone Station, near Sheffield 462660 920 5/15/77 Cooper 1744 3 3 Two Mile Run 41.42573 PSU1003 182 venango Twomile Run - 1.5 mi upstream of mouth and SR8/62 3 -79.786033 2007/5/23 RWC07045 0 9 2 Two Mile Run

183 warren Twomile Run - FH 258 & Rt 6 N of Roystone 41.7367 -78.9636667 1996/9/21 PSU PSU3097 1 1 Two Mile Run 41.72423 184 warren W Br Tionesta Creek - at Saybrook 3 -79.0559 1996/9/21 PSU PSU3094 17 17 W. Br. Tionesta Cr - 185 41.76023 79.08252199 PSU- warren W. Br. Tionesta Cr. Warren Co. Pa., near Weldbank on route 6 083370 640 8/27/69 Cooper 61a 10 10 W. Br. Tionesta Cr - 186 W. Branch Caldwell Cr., Warren Co., 1 st. bridge above Selkirk Staion at 41.69990 79.55217428 PSU- warren jet of W. Br. and Middle Br., 1/2 mi. N. of Selkirk 021300 410 5/29/66 Cooper 315d 1 1 W. Br. Caldwell Cr. 42.02533 187 Erie W. Branch French Creek, Erie Co., N. of Wattsburg on route #89 3 -79.8181 8/26/68 ELC 580 W. Br. French Creek W. Branch of French Creek, Erie Co., at Little Hope Highway, Rt. 89 42.10263 188 Erie crossing 3 -79.822 5/15/75 ELC 1529 W. Br. French Creek 42.07333 PSU1005 189 Erie Walnut Creek - .35 km upstream of mouth (Lake Erie) 3 -80.2341667 2008/5/24 RWC08013 0 1 Walnut Creek 41.46028 190 venango Warden Run - at Foster, 5 mi W of Cooperstown 33 -79.93205 1996/8/19 PSU PSU3082 10 10 Warden Run - 191 41.46018 79.93261095 PSU- venango Warden Run, Venango Co., 5 mi. SW of Cooperstown 189940 060 5/1/75 Cooper 1546 9 9 Warden Run - 192 41.46018 79.93261095 PSU- venango Warden Run, Venango Co., trib. to Sugar Run, near Utica of route 548 189940 060 8/5/68 Cooper 607 2 2 Warden Run 41.69416 193 warren West Branch Caldwell Creek 7 -79.571667 2003/7/1 PFBC West Branch Caldwell Creek 41.97406 194 Potter West Branch Genesee River - just above mouth at Hickox 67 -77.859 2005/6/7 RWC05044 PSU4035 6 4 W. Br. Genesee River 41.97583 195 Potter West Branch Genesee River - Rt 244 1 mi SW of Genesee 3 -77.883667 1996/10/1 PSU PSU3103 32 32 W. Br. Genesee River - 196 42.12149 79.77826760 PSU- Erie West Branch of French Creek, Erie Co., Pa. rte 430 crossing 943570 330 7/27/81 Cooper 2256 1 1 W. Br. of French Creek - 197 West Branch of Potato Creek, McKean Co., 0.5 mi. above junction of 41.67395 78.39419095 PSU- McKean East Branch 544510 690 8/9/78 Cooper 2136 12 12 W. Br. of Potato Creek MA 198 Shellgren, 41.72423 DG warren West Branch of Tionesta Cr. below bridge at Saybrook, Sheffield Quad 3 -79.0559 7/27/59 Badarick MAS 024 W. Br. of Tionesta Cr. - 199 41.74155 79.19156875 warren West Branch Tionesta Cr. at Wildcat Run, Sheffield Run 005810 290 7/24/59 Cornell MAS T19 1 1 W. Br. Tionesta Cr. - 200 41.71037 79.21396749 warren West Branch Tionesta Creek 0.25 mi. below Jones Run, Sheffield Quad. 285760 790 7/24/59 Cornell MAS T20 3 3 W. Br. Tionesta Cr.

201 McKean West Brnach Tunungant Creek - 2 mi SW of Bradford 41.9378 -78.677317 2005/5/17 RWC05032 PSU4025 25 3 W. Br. Tunungant Creek 41.63277 202 warren West Hickory Creek 8 -79.448056 2004/7/13 PFBC West Hickory Creek

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- 203 White Clay Creek, Chester Co., at Stroud Research Lab, near Avondale, 39.81916 75.78392793 PSU- chester Pa. 891510 160 4/15/69 Cooper 1534 6 6 White Clay Creek - 204 White Clay Creek, Chester Co., at Stroud Research Lab, near Avondale, 39.81916 75.78392793 PSU- chester Pa. 891510 160 5/12/78 Cooper 2050 5 5 White Clay Creek - 205 39.85370 75.82625449 PSU- chester White Clay Creek, Chester Co., near Chatham Pa., below bridge 851640 870 5/12/78 Cooper 2051 2 2 White Clay Creek PSU1007 206 Erie Whitney Run - upstream of Warren Co. line 41.86084 -79.61297 2009/9/25 RWC09038 0 1 Whitney Run Norman & 207 Herbert warren Wildcat Run, 8 mi SW of Clarendon; Ohio River drainage 41.7415 -79.1922 Hartweg MAS t19 Whitney Run 41.68948 208 Crawford Woocock creek rt 198, 1 mile east of Woodcock lake NW of Bloomsburg 33 -80.0518 8/3/1996 PSU PSU3083 9 Wood creek

209 Crawford Woodcock Creek 41.6891 -80.0525833 1990/11/2 PSU PSU2452 1 Woodcock Creek

210 Crawford Woodcock Creek - SR 198 bridge upstream of Woodcock lake 41.6891 -80.0525833 1991/4/2 PSU PSU2633 1 1 Woodcock Creek 41.68948 211 Crawford Woodcock Creek - SR 198 NW of Blooming Valley 33 -80.0518 1993/8/3 PSU PSU3083 8 8 Woodcock Creek

Appendix G. Petromyzon marinus historical sites

DEPOSI NO INDIVI NO CO LOCATION LAT LON DATE COLLECT T RET DUAL CREEK

1 Monroe Big Bushkill Creek at rt.209 bridge in Bushkill, PA 41.09136667000 -75.00303330000 1987/8/12 ELC 1906 1 Big Bushkill Creek

2 Monroe Brodhead Creek at Pinebrook Park North Enterance 41.03565 -75.20896667 8/20/2008 AMH 4605 19 Brodhead Creek Northamp 3 ton Delaware River at Porterland, PA 40.9240889 -75.09569444 1987/8/11 ELC 1916 2 Delaware River

4 Chester Hodgeson Run at Rt 841, 6 miles SE of Oxford 39.74636667000 -75.86663330000 9/29/1996 EAH, HDP 3104 1 Hodgeson Run

5 Wayne Main channel Delaware River at NPS HQ 41.627553 -75.046333 1985/12/9 ELC 1707 3 Delaware River Marshalls Creek - site 7, downstream rt. 209 bridge @ LTS 6 Monroe Builders 41.04206667 -75.12843333 6/10/2008 BDL 4672 1 Marshalls Creek

7 Wayne Nat. Park Service Hq Delaware River, NY 41.62755278 -75.0463333 1986/5/21 CEH 1771 3 Delaware River RWC0903 PSU100 8 Chester E.Br. Big Elk Creek - SR 896 bridge SE of Forestville 39.81308000000 -75.90066000000 2009/8/27 0 91 5 E.Br. Big Elk Creek RWC0902 9 Chester E.Br. White Clay Creek - Garden Sta. Rd. S of Avondale 39.80331 -75.78196 2009/8/27 8 2 E.Br. White Clay Creek

10 Chester Hodgson Run at SR 841 bridge 39.74636667 -75.8666333 10/2/2002 RWC 3753 1 Hodgson Run RWC0902 PSU100 Middle Br. White Clay 11 Chester Middle Br. White Clay Creek - SR 841 bridge SE of Wickerton 39.78615 -75.81759 2009/8/27 7 92 4 Creek RWC0902 PSU100 12 Chester W.Br. White Clay Creek - SR 3022 bridge near New London 39.78836 -75.85952 2009/8/27 6 89 7 W. Br. White Clay Creek

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13 Chester White Clay Creek, 1/4 mi. upstream of Delaware State line 39.73853333 -75.7709 10/2/2002 RWC 3750 4 White Clay Creek RWC0703 PSU100 14 Craw Conneaut Creek - Fish Rd. bridge (ammocoetes) 41.72916667 -80.35523333 2007/5/20 5 25 5 2 Conneaut Creek RWC0703 PSU100 15 Craw Conneaut Creek - Fisher Rd. bridge (photo- adult) 41.77335 -80.37895 2007/5/20 6 26 1 1 Conneaut Creek

16 Craw Conneuat Creek - 1 mi N of Springboro (2 adults) 41.81216667 -80.3845 1975/5/22 ELC ELC1536 2 2 Conneuat Creek RWC0900 PSU100 17 Erie Conneaut Creek - Griffey Rd. bridge 41.91773000000 -80.46910000000 2009/5/23 6 65 1 Conneaut Creek Raccoon Creek - 2.5 mi upstr of L. Erie; just dnstr of SR 5 18 Erie (adults) 41.96493333 -80.45998333 1975/5/28 ELC ELC1537 3 3 Raccoon Creek Harvey's Lake - 250mmTL found on large On. Mykiss taken by 19 Luzerne angler 41.367 -76.043167 1980/5/24 PFBC ELC2189 1 1 Harvey's Lake Cherry Creek - Cherry Valley Rd 1.2 mi SE of Delaware Water RWC0500 20 Monr Gap 40.97458333000 -75.16405000000 2005/4/13 7 7 4 Cherry Creek

21 Monr Delaware River - vic. Poxono island 41.05023333000 -75.02348330000 1995/9/15 RWC 1 1 Delaware River RWC0904 PSU100 22 Monroe Marshalls Creek - along SR 209 41.01880000000 -75.12949000000 2009/9/26 5 90 5 Marshalls Creek

23 Monroe Marshall's Creek, Broken bridge off Gap___Drive 41.00188333 -75.13641667 2007/5/8 BDL 4452 1 Marshall's Creek

24 Monroe Marshall's creek, PennDot site 7, rt. 209 bridge at LTS Builders 41.04206667 -75.12843333 2008/6/10 BDL 4672 Marshall's creek RWC0805 25 Pike Delaware River - SE of Indian Point 41.28281000000 -74.83425000000 2008/9/1 9 10 Delaware River PSU309 26 Wayn Delaware River - SR1019 - National Park HQ 41.6275 -75.046389 1996/9/30 PSU 5 54 54 Delaware River RWC0400 27 Wayn Delaware River - vic Narrowsburg, NY 41.61158333 -75.0601833 2004/5/18 4 3 1 Delaware River PSU310 28 Wayn Dyberry Creek - Rt 191 1 mi N of Honesdale 41.586033 -75.261283 1996/9/27 PSU 1 65 65 Dyberry Creek PSU309 29 Wayn Lackawaxen River - Rt 6 1 mi W of Rt 652 (near E Honesdale) 41.550167 -75.231 1996/9/30 PSU 6 77 77 Lackawaxen River

30 Wayne Delaware River 41.851397 -75.1455222 1990/7/11 ELC 2359 12 Delaware River

31 Wayne Delaware River 2.4 road miles S. of NY border in Wayne Co. 41.97322778 -75.3410833 1987/5/27 ELC 1865 1 Delaware River

32 Wayne Delaware River at NPS HQ 41.62755278000 -75.04633330000 1990/8/21 MJG 2537 5 Delaware River

33 Wayne Delaware River at NPS HQ 41.62755278 -75.0463333 1991/10/4 MJG 2450 1 Delaware River 1984/10/1 34 Wayne Delaware River at NPS HQ, 41.62755278000 -75.04633330000 6 ELC 1761 7 Delaware River Lackawaxen River - between Hawley and Lackawaxen on rt 590 AMH/TAW 35 Wayne across from Riverside Drive 41.48305167 -75.010245 8/20/2008 /DPF 4702 3 Lackawaxen River

36 Wayne Main branch of Delaware River at Dillontown, Pa. 41.868675 -75.26371389 2007/5/8 BDL 4455 2 Delaware River

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Appendix H. 2011 ammocoete sampling sites in PA with historical collection information.

ID CO LOCATION LAT LON EXPECTED SPECIES DATE_ COLLECT DEPOSIT CREEK GENUS SPECIES Raccoon Creek - 2.5 mi upstr of L. Erie; 1 Erie just dnstr of SR 5 (adults) 41.964933 -80.459983 Petromyzon marinus 1975/5/28 ELC ELC1537 Raccoon Creek Lampetra appendix 2003/5/22 RWC PSU3719 Raccoon Creek Conneaut Cr at PA Rte 198 bridge W of 2 Crawford Conneautville, PA 41.758112 -80.371587 Lampetra appendix PSU-Stauffer JMB 85.14 Conneaut Creek Petromyzon marinus 2007/5/20 RWC07036 PSU10026 Conneaut Creek Ichthyomyzon Fossor Conneaut Creek Oil Creek below Centerville Crawford 3 Crawford Co. 41.732333 -79.768833 Ichthyomyzon bdellium 1972/7/24 1449 Oil Creek Lampetra appendix 2003/8/18 RWC03066 PSU4006 Oil Creek 4 Warren Blue Eye Run 41.830833 -79.438333 Ichthyomyzon bdellium 1977/6/17 PFBC Blue Eye Run Lampetra appendix 1973/7/19 ELC1587 Blue Eye Run Woodcock Creek - SR 198 NW of 5 Crawford Blooming Valley 41.689483 -80.0518 Lampetra appendix 1993/8/3 PSU PSU3083 Woodcock Creek Ichthyomyzon bdellium 1991/5/8 2635 Woodcock Creek East Branch Oil Creek, Crawford Co., .5 6 Crawford mi. down from mouth of Brannon Run 41.764 -79.728167 Lampetra appendix ELC 1446 E. Br. Oil Creek Ichthyomyzon greeleyi 5/22/75 ELC 1524 E. Br. Oil Creek 7 Warren Spring Creek 41.8625 -79.517222 Lampetra appendix 1997/8/20 PFBC Spring Creek Ichthyomyzon greeleyi 2005/5/30 Lbush PSU4069 Spring Creek 8 Erie Crooked Creek 41.988833 -80.411 Ichthyomyzon unicuspis 1987/5/24 PFC PSU1951 Crooked Creek 9 Potter Elevenmile Creek 41.932222 -78.096667 Lampetra appendix 1978/7/25 PFBC Elevenmile Creek 10 Potter Mill Creek, Potter Co., at Sweden Valley 41.753437 -77.949887 Lampetra appendix PSU-Cooper 1540 Mill Creek E. Branch Tionesta route 133 at picnic 11 McKean area 41.688243 -78.92524 Lampetra appendix PSU-Cooper 3a E. Branch Tionesta Martin Run, Elk Co., trib to S. Branch of 12 Elk Tionesta, at end of F.S. route 195 41.604585 -78.899409 Lampetra appendix PSU-Cooper 42b Martin Run S. Br. of Kinzua Creek about 1/4 mile 13 McKean above mouth of Hubert Run 41.712042 -78.816086 Lampetra appendix PSU-Cooper 2b S. Br. Kinzua Creek Trib to West Branch White Clay Creek, W. Br. White Clay 14 Chester Chester Co., first trib. crossing Pa. rte 39.769033 -75.824286 Lampetra appendix PSU-Cooper 2052 Creek

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841, at Schoolhouse Road Hodgeson Run at Rt 841, 6 miles SE of 15 Chester Oxford 39.746367 -75.866633 Petromyzon marinus 9/29/1996 EAH, HDP 3104 Hodgeson Run Bull Creek - upstr. Fr. Upper edge of Alleghen 850' culvert below new concrete br bel. 16 y Golf course 40.61445 -79.75925 Lampetra aepyptera 0/0/1980 ELC CU7006 Bull Creek Crooked Creek - collected at 4 stations 17 Indiana in headwaters 40.693 -79.0825 Lampetra aepyptera 6/0/1980 ELC2230 Crooked Creek Westmor 18 eland Shannon Run (reported as I. greeleyi) 40.372398 -79.082901 Lampetra aepyptera 1977/8/29 PFBC Shannon Run 19 Beaver Raccoon Creek State Park 40.507116 -80.3556 Lampetra aepyptera 1956/4/28 CU64853 Raccoon Creek Mountain Creek, Fayette Co., trib. to Georges Creek, Monongahela R., 2 20 Fayette miles S. of Smithfield 39.777745 -79.824241 Lampetra aepyptera 7/15/68 PSU-Cooper 669 Mountain Creek Little Pucketa Creek, 2.5 mi up stream Westmor from the Allegheny River, New GO UMMZ Little Pucketa 21 eland Kensington 40.575433 -79.74225 Lampetra aepyptera 4/29/34 Hollibaugh 26.1 Creek

Appendix I. Sampling results by species

No. of Genus Species Coll. No. Date County Creek specimens Lampetra aepyptera SLL-18-11 6/2/2011 Indiana Crooked Creek 49 Lampetra aepyptera SLL-16-11 6/2/2011 Allegheny Bull Creek 2 Lampetra aepyptera SLL-14-11 6/1/2011 Westmoreland Shannon Creek 5 Lampetra aepyptera SLL-15-11 6/1/2011 Fayette Mountain Creek 31 Lampetra aepyptera SLL-19-11 6/9/2011 Chester Hodgcson Creek 24 Lampetra appendix SLL-4-11 5/10/2011 Warren Blue Eye Run 8 Lampetra appendix SLL-3-11 5/10/2011 Warren Spring Creek 2 Lampetra appendix SLL-5-11 5/11/2011 Erie Crooked Creek 4 Lampetra appendix SLL-2-11 5/9/2011 Crawford East Branch of Oil Creek 1

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Lampetra appendix SLL-11-11 5/25/2011 McKean South Br. Of Kinzua Creek 11 Lampetra appendix SLL-13-11 5/26/2011 Potter Elevenmile Creek 18 Lampetra appendix SLL-9-11 5/25/2011 Elk Martin Run 53 Lampetra appendix SLL-10-11 5/25/2012 McKean East Branch of Tionesta 16 Lampetra appendix RBT-006-11 5/10/2011 Erie Patrick Run 69 Lampetra/Petromyzon appendix/marinus SLL-7-11 5/11/2011 Crawford Conneaut Creek 1 Ichthyomyzon greeleyi SLL-2-11 5/9/2011 Crawford East Branch of Oil Creek 8 Ichthyomyzon greeleyi SLL-2-11 5/9/2011 Crawford East Branch of Oil Creek 13 Ichthyomyzon greeleyi SLL-3-11 5/10/2011 Warren Spring Creek 2 Ichthyomyzon greeleyi SLL-2-11 5/9/2011 Crawford East Branch of Oil Creek 5 Petromyzon marinus RBT-014-11 6/8/2011 Monroe Mashalls Creek 8 Petromyzon marinus SLL-19-11 6/9/2011 Chester Hodgcson Creek 27