CONSERVATION GENETICS AND POPULATION STATUS OF THE

FLAME CHUB, HEMITREMIA FLAMMEA

By:

Kathlina Frances Alford

Approved:

Anna L. George Joey Shaw Director of TNACI Professor of Biology (Co-Chair) (Co-Chair)

Mark S. Schorr Jeff S. Elwell Professor of Biology Dean of the College of Arts and Sciences (Committee Member)

A. Jerald Ainsworth Dean of the Graduate School

CONSERVATION GENETICS AND POPULATION STATUS OF THE

FLAME CHUB, HEMITREMIA FLAMMEA

By:

Kathlina Frances Alford

A Thesis Submitted to the Faculty of the University of at Chattanooga in Partial Fulfillment of the Requirements of the Degree of Master’s of Science of Environmental Science

The University of Tennessee at Chattanooga Chattanooga, Tennessee

August 2013

ii

ABSTRACT

The southeastern has a rich geologic history that contributed to the evolution of an extremely diverse aquatic fauna throughout the region. The Flame Chub,

Hemitremia flammea, is a brightly colored, spring endemic native to the

Cumberland, Tennessee, and Coosa river drainages. In this study, the cytochrome-b gene region was analyzed for 230 individuals from 29 populations across the three drainages. Results from maximum parsimony and Bayesian analyses recovered shallow divergence between the 31 haplotypes. AMOVA analyses indicated that most genetic variation distributed within and between populations, not between drainages. Based on these results, this species may not be restricted to spring as was originally presumed and can move within river systems and likely even between drainages. Further analyses using microsatellites and geospatial modeling would refine these results. Species like H. flammea are indicators of the health of groundwater resources that are under increasing anthropogenic pressure.

iii

DEDICATION

To my parents for instilling in me an inquiring mind and a desire for higher learning and for never letting me settle for less than my very best. To my husband for loving me through my

quirks everyday no matter what.

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ACKNOWLEDGEMENTS

This thesis would not have been possible without the guidance and the help of so many individuals who in one way or another contributed and extended their valuable assistance in the preparation and completion of this study. First and foremost, my utmost gratitude goes to my husband, Lee, without whose wavering support and encouragement would I have completed this study. Dr. Anna George has been so many things to me throughout the years: a boss, a mentor and a friend. Her passion for Southeastern aquatics is contagious and inspiring. Dr. Joey Shaw graciously allowed me to use his genetics laboratory for this project and also served as a co-chair on my committee. Dr. Mark Schorr also served on my committee and provided ecological insight on this study. Dr. Bernie Kuhajda provided perspective, revisions, and reassurance when I needed it most. Dr. David Neely was amazing with his awe-inspiring field expertise and never- ending collection energy. Evan “Bird Dog” Collins was priceless as a collector, GIS technician and friend. Steve Alford, Stephanie Brandt, Matt Hamilton, Ben Stenger, Dr. Matt Thomas and

Josh White all provided field assistance, and Greg Knothe and Bob Jett provided GIS mapping powers. The Tennessee Aquarium not only funded my education but also allowed me the time and flexibility to finish this study while working full-time. AZA, NANFA, and the UTC

Graduate Association all helped to fund my research and travel to make presentations at multiple conventions. Last but not the least, my extended family, friends, and fellow students endured this long process with me and provided so much encouragement and prayer. Thanks to you all.

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TABLE OF CONTENTS

DEDICATION v

ACKNOWLEDGEMENTS vi

LIST OF TABLES ix

LIST OF FIGURES x

LIST OF ABBREVIATIONS xi

CHAPTERS

1. INTRODUCTION 1

2. METHODS AND MATERIALS 15

Field Collections 15 Mitochondrial Analyses 16

3. RESULTS 19

Field Collections 19 Mitochondrial Analyses 20 Gene Analyses 20 Parsimony Analyses 20 Bayesian Analyses 21 Haplotype Analyses 21 Population Analyses 22

4. DISCUSSION 24

REFERENCES 31

APPENDICES

A. SEQUENCE DATA FOR ALL 31 HAPLOTYPES RECOVERED FOR HEMITREMIA FLAMMEA 54

vi

B. GENBANK ACCESSION NUMBERS AND SEQUENCE DATA FOR ALL OUTGROUPS INCLUDED IN ANALYSES 62

C. PROOF OF PROJECT APPROVAL BY THE UTC INSTITUTIONAL CARE AND USE COMMITTEE 70

VITA 72

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LIST OF TABLES

Table 1 All sites sampled for Hemitremia flammea from November 2009 to April 2012 39

Table 2 Sites where Hemitremia flammea were collected from November 2009 to April 2012 42

Table 3 Primers used in mitochondrial analyses of Hemitremia flammea 43

Table 4 Haplotype and nucleotide diversity for each population of Hemitremia flammea 44

Table 5 Φst values for all populations of Hemitremia flammea 45

Table 6 Haplotypes recovered for Hemitremia flammea , as represented at each site 46

viii

LIST OF FIGURES

Figure 1 Map of the Cumberland, Tennessee, and Coosa river drainages 47

Figure 2 Map of all sites sampled for Hemitremia flammea 48

Figure 3 Map of sites where Hemitremia flammea were collected 49

Figure 4 Map of sites included in population comparison analyses for Hemitremia flammea 50

Figure 5 Maximum parsimony concensus tree for Hemitremia flammea 51

Figure 6 Bayesian consensus tree using GTR+I+G model for Hemitremia flammea 52

Figure7 Haplotype network for Hemitremia flammea 53

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LIST OF ABBREVIATIONS

µL microliter / 0.001 liter

A adenine

AMOVA analysis of molecular variance

C cytosine

CI consistency index

CPUE catch per unit effort cyt-b cytochrome-b gene region

DNA deoxyribonucleic acid et al. and others

G guanine h haplotype diversity

HI homoplasy index

ICUN International Union for the Conservation of Nature m meter mM millimolar / a concentration of 0.001 mole per liter

MP maximum parsimony mya million years ago oC degrees Celsius p probability statistic

x

PCR polymerase chain reaction r Pearson product-moment correlation coefficient

RCI rescaled consistency index sp. species sp. cf. species confer / undescribed species

T thymine

Taq Thermus aquaticus polymerase

TVA Tennessee Valley Authority

UAIC University of Ichthyological Collection

UMMZ University of Michigan Museum of Zoology

UTEIC University of Tennessee Etnier Ichthyological

Collection ya years ago

Z Mantel’s test statistic

π nucleotide diversity

ΦCT phi-ct / genetic variation among metapopulations

relative to taxon

Φsc phi-sc / genetic variation within populations relative

to metapopulations

ΦST phi-st / genetic variation within populations relative

to the taxon

xi

CHAPTER 1

INTRODUCTION

Simply defined, biogeography is the geography of life. The field of biogeography provides the framework by which scientists study species distributional patterns, speciation events, and ancestor relationships within and between species through space and geologic time

(Lomolino et al. 2006). By identifying the processes that impact species distributions we are better able to protect imperiled species and habitats as well as develop strategies to control invasive species. Biogeographical research answers questions about spatial patterns of organisms through assessments, regional species lists, and even molecular studies. The science can be subdivided into more narrow concentrations such as: ecological, historic, terrestrial, aquatic, floral, faunal, and by specific taxonomic groups.

When researching within each subdivision, unique limitations must be considered. For example, freshwater organisms are restricted by the water pathways in which they live for dispersal opportunities. Freshwater in particular have a unique set of biogeographic constraints in comparison to terrestrial organisms in the same regions. The ability of freshwater fishes to move in response to climate change or geological events is limited to the patterns of connectivity of the water bodies where they exist. Opportunities for range expansion between isolated drainages are limited to rare events such as stream captures, geologic events (e.g., tectonic plate shifts) and major flooding events. is often more likely than migration due to their limited dispersal abilities. Climatic changes, disease, interspecific competition and

1 habitat destruction are all threats that freshwater fishes are often unable to escape (Swift et al.

1986, Warren et al. 2000).

The southeastern United States is a hotspot for biogeographical research on freshwater organisms. This region has the highest aquatic biodiversity of any temperate region in the world and the highest aquatic biodiversity in the country. This biodiverse region contains many endemic species that don’t exist anywhere else in the world. In part because of the many endemic and rare species, there are a high number of imperiled species in this region. Warren et al. (2000) stated that at least 28% of freshwater species are imperiled in the Southeast, including

6% endangered, 7% threatened and 15% vulnerable. These numbers represent a 125% increase in imperilment since 1980 (Warren et al. 2000), indicating an urgent need for aquatic conservation in this region. In general, decline of native populations can be attributed to habitat degradation and fragmentation, both in the Southeast and globally (Burkhead et al. 1997,

Warren et al. 2000). Allan & Flecker (1993) described six major causes of species loss: habitat loss, invasive species, overharvesting, pollution, climate change and secondary that occur when a keystone species is lost.

The source of the rich biological diversity in the southeastern United States can be traced back through geologic history. The Oligocene (33.9-23.03 mya), Miocene (23.03-5.332 mya) and Pliocene (5.332-2.588 mya) sea-level fluctuations and the Pleistocene (2.588 mya-11,700 ya) glacial cycles are major historical events that shaped current distributions of aquatic fauna in

North America (Bermingham & Avise 1986, Swift et al. 1986, Mayden 1988, Strange & Burr

1997, Cohen et al. 2012). Sea-level fluctuations during the Oligocene, Miocene and Pliocene epochs were up to 80 m higher than current levels. This fluctuation changed the physiographic structure and likewise impacted the relationship between freshwater, brackish and oceanic

2 habitats, pushing freshwater fish further inland and upland (Winker & Howard 1977,

Bermingham & Avise 1986, Swift et al. 1986). Advancing and retreating Pleistocene glaciers began to change the landscape of North America and its fauna. During each glacial cycle the topography of the continent was altered and northern species were pushed south to the warmer climate (Mayden 1988). In addition, much of the Earth’s water was captured in ice sheets, causing sea levels to be much lower than modern levels, again impacting the relationship between freshwater and saltwater habitats. Throughout geologic time, the upland provinces of the southeastern United States served as a stable refuge for freshwater organisms.

The rich geologic history of our region has resulted in a diversity of physiographic provinces, providing an abundance of different habitat types throughout the region. The upland physiographic provinces in the Southeast include the Highland Rim, Cumberland Plateau, Ridge and Valley, Blue Ridge and Piedmont. The Coastal Plain covers the western portion of the states of and Tennessee, northeastern and northwestern Alabama, surrounding the upland provinces and sloping towards the ocean (Etnier & Starnes 1993, Mettee et al. 1996,

Boschung & Mayden 2004).

The five upland provinces of the southeastern United States provide a diversity of aquatic habitats. The Highland Rim is a relatively low lying upland province with an average elevation of 200 m (Etnier & Starnes 1993). This province extends from northern Alabama up to southern

Ohio, Illinois and Indiana and is underlain with limestone, chert and shale. To the east of the

Highland Rim, a series of plateaus extends from Alabama north through New England. In the south, this province is called the Cumberland Plateau and is characterized by Pennsylvania sandstone, shale and coal deposits, capped by hard sandstone. Elevation in the Cumberland

Plateau province averages 800 m and forms a border between the Highland Rim province to the

3 west and the Ridge and Valley province to the east (Etnier & Starnes 1993). The Ridge and

Valley, as the name implies, is a series of higher and lower elevations that are reminiscent of a dramatic folding event caused by continental collision or seafloor spreading with elevations of

180-650 m (Mettee et al. 1996). These “ripples” form the foothills of the Appalachian

Mountains, or the Blue Ridge province. The Blue Ridge province has the highest elevations in this region with peaks of up to 1800 m (Etnier & Starnes 1993). These mountains were formed when Precambrian rock was thrust on top of younger rock, the same event that created the Ridge and Valley province. The foothills province to the east of the Blue Ridge province is called the

Piedmont or the “older Appalachians” (Fenneman 1938) where elevations have eroded to 150-

370 m (Mettee et al. 1996). The Coastal Plain province encircles the south and east of the entire upland region and is characterized by a dramatic drop in elevation that slopes to the ocean. The

Fall Line is used to define the transition from upland provinces to the coastal plain and because of the elevation change, there are many waterfalls along the Fall Line (Etnier & Starnes 1993,

Mettee et al. 1996, Boschung & Mayden 2004). Aquatic species composition is very different between upland provinces and the coastal plain, which is an ongoing research topic within the biogeography of the Southeast (Swift et al. 1986, Boschung & Mayden 2004).

In addition to the formation of a diverse arrangement of physiographic provinces, North

American river pathways, especially in the Southeast, have experienced dramatic changes throughout geologic time. River connection fluctuations contributed to the number of endemic species throughout the region as well as shared species between river drainages. The

Cumberland, Tennessee and Coosa rivers are the three drainages pertinent to this study. All three drainages are located in the upland provinces described above; however, the Coosa River is a smaller system within the Mobile Basin while the Tennessee and Cumberland rivers are both

4 larger systems that drain into the Ohio River near its confluence with the

(Etnier & Starnes 1993, Boschung & Mayden 2004).

The Tennessee and Cumberland rivers are currently part of the Ohio River Basin but historically may have had their own connection to the Gulf of Mexico and later to the Mississippi

River (Starnes & Etnier 1986). They may have also had an ancient connection to each other, with at least 152 species of fishes shared between these two systems (Etnier & Starnes 1993). This connection may have been through the Powell River, currently a tributary to the through the Cumberland Gap at the Tennessee/Kentucky border, or a Powell River tributary

(Hayes & Campbell 1894, McFarlan 1943, Ross 1972, Starnes & Etnier 1986).

The Cumberland and Tennessee rivers also share at least 48 species of fishes with the

Coosa River of the Mobile Basin, again indicating periods of historical connectivity (Etnier &

Starnes 1993, Boschung & Mayden 2004). At least 61 species of fish are shared between the

Tennessee and Mobile basins (Boschung & Mayden 2004). It is hypothesized that a large river known as the Appalachian River once ran through Tennessee and into Alabama, encompassing what is now the upper Tennessee River and the upper Mobile Basin. The Appalachian River was not a stream capture or a temporary system but was believed to be a stable river system up into the early Miocene (16-23 mya). At that time, the headwaters of the Tennessee River were located in modern Alabama in what is now considered the middle Tennessee River. Through headwater erosion the upper Appalachian River was ultimately captured by the Tennessee River, and the

Tennessee and Mobile systems were isolated (Mayden 1988, Boschung & Mayden 2004).

Swift et al. (1986) concluded that two or more connections between the Tennessee and

Mobile basins were necessary to explain modern fish distributions. In support, shared species between the Tennessee and Mobile river drainages show different patterns of isolation, some

5 being very recent and others being ancient. Layman & Mayden (2012) described nine total species of darters in the subgenus Doration that support the theory of ancient river connections and a common ancestor species. Other examples of ancient species divergence in the Southeast are suckers in the genus Hypentelium (Berendzen et al. 2003), darters in the subgenus Percina

(Williams et al. 2007, Near 2002 , Mayden 1989, Dimmick & Burr 1999), and studfishes in the subgenus Xenisma (Ghedotti et al. 2004). Fishes are not the only group of aquatic organisms that demonstrate this pattern, with freshwater mussels (Simpson 1900, van der Schalie 1939), turtles such as Sternotherus (Iverson 1977, Seidel & Lucchino 1981, Reynolds & Seidel 1983) and salamanders such as Hemidactylium (Herman 2009) also showing sister taxa patterns that support an ancient connection or multiple connections between the Tennessee and Mobile drainages (Swift et al. 1986, Mayden 1988).

In contrast to theories of ancient isolation, Near et al. (2011) analyzed approximately 250 darter species and estimated that the majority of extant species of darters experienced isolation in the last 15 million years, later than the Appalachian River capture, which indicates more recent connectivity patterns. Cumberland River darters exhibit extremely high levels of diversification over very fine geographic scales. Hollingsworth and Near (2009) estimated divergence time at 2-

8 million years for the five clades within the basilare (Corrugated Darter) species complex distributed throughout the tributaries of the Caney Fork River. While Keck and Near

(2010) found a similar pattern in Nothonotus darters, they explained the divergence as more recent with extensive microendemism, which they attributed to niche conservatism. Other species show patterns of very recent colonization between river basins as well. Distributions of an undescribed darter ( E. sp. cf. zonistium ) and two ( bellus () and baileyi ()) reveal evidence for stream capture events in the area at the

6

Fall Line where tributaries of the upper Tombigbee and Tennessee rivers are in very close proximity to the Sipsey Fork (Wall 1968, Boschung and Mayden 2004, Fluker 2011). These could be a result of small stream captures or large flood events, but no geologic evidence exists to support or refute specific theories (Starnes & Etnier 1986, Mayden 1988, Mettee et al. 1996,

Boschung & Mayden 2004).

Throughout the Cumberland, Tennessee and Coosa river drainages, many unique aquatic habitat types exist including large rivers, headwater mountain streams, urban streams, caves, ponds, and springs. Springs are located where groundwater exits to the surface of the earth and are generally characterized by cool, clear water (Mirarchi 2004). This formation occurs primarily along geologic fault lines and within rock formations (Mirarchi, 2004), which would explain why this habitat is so prominent throughout the southeastern United States. Spring habitats are relatively stable in terms of temperature, water chemistry and flow levels when compared to adjoining streams, and typically have a robust population (Hubbs 1995). Often the water levels in these microhabitats are too low for large predatory fish species so the waters become a sanctuary for many species of fishes and amphibians (Mirarchi 2004).

These unique spring habitats have been extensively altered and even destroyed by anthropogenic activities such as agricultural development, deforestation, aquaculture, installation of impervious surfaces, point and non-point source pollution, water withdrawal, and impoundments (Etnier 1997, Mirarchi 2004, Boschung & Mayden 2004, Timpe et al. 2009).

Addition of invasive species such as Gambusia affinis (Western Mosquitofish) (Sossomon 1990,

Hubbs 1995, Burkhead et al. 1997, Greenwald & Sandel 2009, Fluker et al. 2010) or Orconectes virilis (Northern Crayfish) (Fluker et al. 2009) can also destroy the habitat and limit resources for natives, especially in spring habitats. In the case of the federally endangered Etheostoma

7 phytophilum (Rush Darter ), even another can become invasive and competitive when introduced outside of its natural range (i.e., interactions with Etheostoma nuchale ( Darter)) (George et al. 2009). Loss of habitat, regardless of the cause, can be devastating to populations of spring endemics because they are not capable of migrating to a new location as they are dependent on the spring environment for survival. Spring-adapted fishes depend on a constant flow of clear cool water and a unique and diverse aquatic plant community for food, reproduction, and shelter. This dependency limits species mobility, naturally fragmenting their range and restricting gene flow, making these species more vulnerable to imperilment (Greenwald & Sandel 2009, Fluker et al. 2010).

The southeastern United States is not the only area of the world with springs containing endemic species. Spring habitats in the Great Artesian Basin of Australia are protected as

“endangered ecological community” by the Australian government and host endemic snails

(Ponder 1995, Wilmer and Wilcox 2007, Guzik et al. 2012). The warmwater springs of the western United States are home to several rare desert ( salinus, C. nevadensis, and C. diabolis ) (Duvernell and Turner 1998, 1999; Martin and Wilcox 2004) and killifish (Fundulus lima ) (Bernardi et al. 2007).

Probably the best known conservation story of a spring endemic is that of Cyprinodon radiosus (Owens ) in the western United States where approximately 800 remaining individuals were rescued from a rapidly drying spring fed pond and reestablished in stable habitat (Pister 1993). This story illustrates the vulnerability of one species to environmental and anthropogenic pressures. There are many other species of vulnerable isolated spring endemics including salamanders (Eurycea aquatica, E. pterophila, E. nana, and E. neotenes ) (Lucas et al.

2009, Timpe et al. 2009), killifish (Fundulus lima ) (Bernardi et al. 2007), darters (Etheostoma

8 nuchale, E. ditrema, E. swaini, and E. cinereum ) (Fluker et al. 2010, Mayden et al. 2005, Powers et al. 2004), sculpins ( Cottus paulus ), other pupfishes (Cyprinodon salinus, C. nevadensis, C. diabolis, C. pocosensis, and C. variegatus ) (Duvernell and Turner 1998, 1999, Martin and

Wilcox 2004, Echelle and Connor 1989) and ( Fonscochlea accepta ) (Ponder 1995,

Wilmer and Wilcox 2007).

Because springs are naturally fragmented systems, their inhabitants can be studied as surrogates for how other aquatic species will react to habitat alteration and fragmentation. The theory of island biogeography (MacArthur & Wilson 1967) outlines the basic principles of isolated populations in comparison to other populations based on their geographic proximity to each other and habitat size. Island populations have a much higher risk of extinction than mainland populations because of their isolation from other populations, which restricts colonization and gene flow (Diamond 1984, Vitousek 1988, Flesness 1989, Frankham 1997).

This theory can be applied to other areas of research outside of the idealized oceanic island setting, including springs. Spring endemics have been compared to island populations due to their isolated gene flow, low levels of genetic diversity, patchy distributions, levels of genetic structuring and susceptibility to stochastic events (Fluker et al. 2010).

Genetically distinct populations indicate long-term isolation of the population and would suggest that management on a population level would be appropriate. Optimally, the long-term viability of any species is linked to genetic variability within populations (Frankel 1974,

Frankham 2005, Spielman et al. 2004) though continued survival is possible even with reduced genetic diversity (Johnson et al. 2009). Widespread low genetic variability across populations may indicate historic, naturally low levels of diversity (Fluker et al. 2010). Similar genetic structure and variability across all populations, whether low or high, may indicate that recent

9 fragmentation is responsible for the isolation and management of all populations as a single genetic unit could be appropriate.

Genetics research involves the study of different portions of DNA to answer different types of questions about taxonomic relationships, population genetic health and species ranges.

These types of studies are especially useful when making decisions about imperiled species and how to spend conservation funds. Single-copy nuclear DNA evolves very slowly and is used to answer questions at the species level and higher (e.g., between taxonomic families). In fish, mitochondrial DNA mutates 5-10 times faster than single copy nuclear DNA so is appropriate for answering questions within a single species or about recent isolation events. Finally, microsatellite loci are repeating patterns within the nuclear DNA. These fragments evolve 100-

1000 times faster than entire genes within the single copy nuclear DNA so are useful for answering questions at the species level and below (Wan et al. 2004).

When studying spring endemic species, we expect to find a distinct genetic structure at the population level so mitochondrial DNA or microsatellite loci are the most commonly used genetics tools. This genetic structure in springs is due to long-term isolation of populations.

Sister species Etheostoma boschungi (Slackwater Darter) and E. tuscumbia (Tuscumbia Darter), exhibit very different genetic structures in the lower bend of the Tennessee River. Etheostoma boschungi is a seep-dependent species that exhibits broad differentiation to the point of being multiple undescribed species, based on mitochondrial and microsatellite analyses. In contrast, E. tuscumbia , though having a very similar geographical distribution, shows little population differentiation even at the microsatellite level (Fluker 2011). By using mitochondrial DNA and microsatellite loci to study these two species, Fluker (2011) was not only able to determine the

10 genetic structure of the species, but this data also answered questions about the life history of these species and their ability to disperse out of the spring habitat.

In addition to molecular studies, status surveys are necessary to assess the changing ranges and population sizes of a species, and the two areas of research complement each other.

Though the Southeast is home to one of the most diverse aquatic ecosystems in the world, the status of many freshwater fish species in this region is poorly documented (Warren et al. 2000,

Butler 2002, Stallsmith 2010). As threats to aquatic species increase it is likely that ranges, especially for already imperiled species, will shrink, which can negatively impact the overall genetic diversity of the species and the genetic diversity within populations. Limited genetic diversity makes a species susceptible to genetic bottlenecking. As the size of a breeding population decreases, haplotypes are lost and fewer genetic differences exist between individuals, creating a bottleneck. Once genetic diversity is lost, it can only be restored through natural mutations or the immigration of new breeding individuals into the population. In order to provide protection under state and federal legislation, survey work must be completed so that the most up to date information is available for decision making (Ceas & Page 1995).

The Flame Chub, Hemitremia flammea (Jordan & Gilbert 1878) , is a brightly colored member of the family , the minnows. H. flammea was originally described as

Hemitremia vittata (Cope 1870) and also recognized as Phoxinus flammea (Jordan & Gilbert

1878) and currently is the only species in the genus Hemitremia. This species earns its common name and specific epithet from the bright red coloration of nuptial males. Hemitremia is a conjugation of Greek prefixes meaning half (hemi) and aperture (trema) which together describe the incomplete lateral line of the species. A dark lateral stripe follows the incomplete lateral line and a dark caudal spot is present. Above the dark lateral stripe is a gold stripe and the dorsal

11 portion of the body is brown to olive green. The head is blunt and rounded as are the fin apexes.

The body is short, stout and covered in comparatively large scales. Maximum standard length is

64 mm (Etnier & Starnes 1993, Mettee et al. 1996, Boschung & Mayden 2004). This unique minnow is historically known from the Tennessee, Cumberland and Coosa river drainages in

Tennessee, , Alabama, and Kentucky (Etnier & Starnes 1993, Boschung & Mayden

2004).

Literature on this species is limited to one life history study from a spring-fed pond in

Loudon County, Tennessee (Sossomon 1990), two reproductive studies (Katula 1999, Maurakis et al. 2001) and a population survey of historic sites in the state of Alabama (Stallsmith 2010).

From these studies, we know that Hemitremia flammea is usually found in small groundwater- fed streams, ephemeral groundwater seeps or slackwater areas along the banks of larger streams where aquatic vegetation is abundant, specifically Watercress ( Nasturtium sp.) , Swamp

Smartweed ( Polygonum sp.), and Small Pondweed ( Potamogeton sp. ) (Sossomon 1990, Etnier &

Starnes 1993, Butler 2002). The species spawns during the winter-spring, December-May, with the peak of spawning season occurring in March (Sossomon 1990, Katula 1999, Maurakis et al.

2001). Males exhibit an orange to scarlet red lateral coloring throughout the year, but this color is exceptionally bright and extensive during the breeding season. Single rows of tubercles can also be observed on pectoral rays two through six of nuptial males as early as October and lasting through May (Sossomon 1990, Etnier & Starnes 1993). Breeding occurs on clean gravel in a brisk current area of the stream, and no nests are constructed. Instead, eggs are released into natural depressions in the substrate (Maurakis et al. 2001). Breeding aggregates have been observed at spring heads, in streams and in flooded pastures (Etnier and Starnes 1993, Mettee et al. 1996, Maurakis et al. 2001). Eggs hatch at 3-4 days and at 6-8 days from observed spawning

12 the larva are free-swimming. By 8 months, fry in lab studies were reported to be 25 mm

(Maurakis et al. 2001), and individuals reach sexual maturity at one year old and only 33-39 mm

(Sossomon 1990). No parental care has been documented for the species (Katula 1999, Maurakis et al. 2001).

The only published status survey for H. flammea was conducted at 53 historic sites throughout the Tennessee and Coosa river drainages in Alabama (Stallsmith 2010). Collections were made using a 3 x 1.3 m seine with a mesh size of 3 x 3 mm. Each site was visited once throughout the study from May 2005 through October 2007. Of the 53 sites, 40 were visited outside of breeding season in June, July or October, and H. flammea were captured at 32.5%, or

13 of these sites. Only 13 sites were visited during breeding season in February, April, or May and H. flammea were captured at 38.5%, or five of these sites. Relative abundance was assigned to each site according to the number of fish captured: single fish (1), uncommon (2-4) and common (>5). Of the 53 sample sites, H. flammea were considered common at 10, uncommon at

2, and a single fish was captured at six (Stallsmith 2010). Based on these results, Stallsmith

(2010) concluded that this species is in decline and in need of increased protection. Currently, H. flammea is listed as Vulnerable by the American Fisheries Society (Jelks et al. 2008) and the states of Alabama (S3) and Tennessee (S3) (Stallsmith 2010), and considered Endangered in the state of Georgia (Straight 2009). The International Union for the Conservation of Nature (IUCN) lists the species as Data Deficient (DD) (Stallsmith 2010), indicating limited research and a poor understanding of the species’ distribution and abundance. It has been suggested that the IUCN and the Alabama status in particular are too optimistic and should be changed due to limited catch in recent surveys (Stallsmith 2010).

13

Because H. flammea is a southeastern spring endemic that exhibits a naturally fragmented range within a specialized habitat, the species is highly susceptible to localized habitat degradation. As has been shown in groundwater-dependent endemic species, these naturally isolated populations may have limited genetic diversity and therefore could be susceptible to genetic bottlenecking which would reduce genetic diversity even further. These threats, compounded with recent anthropogenic fragmentation, could dramatically impact the future conservation needs of this species. Based on this knowledge, the objectives of this project were to: 1) Examine the genetic diversity and spatial distribution of genetic variation in the

Hemitremia flammea across its range using mitochondrial DNA, and 2) Evaluate the current of Hemitremia flammea and provide recommendations on delineation of management units.

I developed two hypotheses concerning my objectives at the beginning of this study.

First, I hypothesized that because of shared patterns of biogeography, I would recover divergence patterns in H. flammea between major river drainages (Cumberland, Tennessee and

Coosa rivers) indicating that individuals in these drainages had been isolated from one another for millions of years. Secondly, I hypothesized that by conducting my sampling during spawning season and across the entire range of the species I would capture H. flammea at a larger percentage of sites than Stallsmith (2010). This hypothesis was based on the assumption that H. flammea is concentrated at spring heads during spawning season and also on previous personal field experience with this species where I perceived the species to be abundant.

14

CHAPTER 2

METHODS AND MATERIALS

Field Collections:

Hemitremia flammea were collected from sites across the species’ range from November

2009 to April 2012. Collections were made under scientific collection permits from Tennessee

(1433), Alabama (5416) and Kentucky (SC1211103). Animal care and welfare protocols were approved under UTC IACUC #1025AG-01 (Appendix C) and Tennessee Aquarium Animal

Health and Management proposal 09-03. Historical collection data were obtained from

University of Alabama Ichthyological Collection (UAIC), University of Michigan Museum of

Zoology (UMMZ), University of Tennessee Etnier Ichthyology Collection (UTEIC), Tennessee

Valley Authority (TVA) and Fishnet2 (online database of museum collections). A total of 64 historic sites were sampled in addition to two new sites that were chosen based on visible habitat

(Table 1 & Figure 2). Specimens were collected with a 3 x 1.3 m seine with a mesh size of 3 x 3 mm mesh, dip nets and backpack electrofisher. Collection times, gear used and the number of individuals on the collection team were recorded as an indication of catch per unit effort (Table

1). The physiographic province in which each site is located is also included in Table 1.

Captured H. flammea were euthanized using a 100 ppm buffered solution of MS-222, and whole fish or fin tissue samples were preserved in 95% ethanol. If fin tissue samples were taken and preserved in ethanol, whole fish were preserved in 10% formaldehyde to be used as museum specimens. A target number of 10 specimens from each site was set to provide enough tissue for

15 comparative molecular analyses. Tissues and specimens will be sent to UAIC to be archived upon completion of this study.

Mitochondrial Analyses:

Total genomic DNA was isolated from fin tissue using QIAGEN DNeasy blood and tissue extraction kit. A 1% agar test gel was used to determine that DNA was successfully extracted. Once confirmed, genomic DNA was used as template for amplification of the ND2 and cytochrome-b (cyt-b) mitochondrial genes. The ND2 gene was amplified using universal primers ND2 E-H and ND2 B-L (Broughton & Gold 2000). The cyt-b mitochondrial gene was amplified using universal primers L14724 and H15915 (Schmidt & Gold, 1993). All primers used for mitochondrial analyses are listed in Table 3. Amplifications were performed using

Eppendorf (Westbury, New York, USA) Mastercycler gradient or Mastercycler personal thermal cyclers. All PCR amplifications were performed in 50-L reactions using the following reaction components: 25.8 L water, 10 L of 5× Taq reaction buffer (Applied Biosystems, Foster City,

California), 4 L of 25 mM MgCl2, 4 L of 10 mM solution of each deoxynucleotide triphosphate (dNTPs), 1 L of 10 M solution of each primer, 0.2 L of 5 U/ L Taq polymerase (Applied Biosystems) and 4 L of DNA solution. PCR parameters for both genes were as follows: 35 cycles, each consisting of 60 seconds denaturation at 94 oC, 60 seconds annealing at 48 oC, and 120 seconds extension at 72 oC. A 1% agar gel was used to confirm successful amplification.

Confirmed PCR products were cleaned of unincorporated primers and nucleotides using

QIAGEN Gel Extraction kits (QIAGEN, Valencia, CA), and sequenced on an ABI 3700

(Macrogen, Seoul, Korea). Both light and heavy strands were sequenced for all samples.

16

Sequences were verified by consensus between the two strands, edited and aligned by eye using

BioEdit version 7.0.9.0 software (Hall 1999). Veracity of all mutation was assessed via comparative alignment and examination of the electropherograms using BioEdit.

Haplotype networks were constructed using TCS 1.21 (Clement et al. 2000). Haplotype networks were created with TCS and then recreated using a 1010 base pair region of the gene to limit missing data. All haplotypes were labeled alphabetically according to the number of individuals represented in that haplotype. After A-Z, labeling continued using AA, AB, AC, etc.

Relationships between haplotypes were inferred under both parsimony and Bayesian criteria. Parsimony analysis (MP) utilized the heuristic search option in PAUP*4.0b10 (Swofford

2003) with ACCTRAN and tree-bisection-reconnection during 1000 replicates of random sequence addition. Calculation of decay indices (Bremer, 1994) was also conducted using

PAUP*4.0b10 and are illustrated in Figure 4. Parsimony analyses were conducted with molecular characters unweighted and unordered. All minimal-length trees were kept, and zero- length branches collapsed. Support for individual nodes was assessed by performing 1000 bootstrap replicates. Outgroup taxa for MP analyses included atromaculatus, Cousius plumbeus, Lepidomeda vittata, Meda fulgia, Snyderichthys copei, and Margariscus margarita as suggested by Mayden et al. (2008) and Simons et al. (2003) . All outgroup sequence data were taken from GenBank (Appendix B).

Bayesian analysis utilized MrBayes v3.2.1 (Huelsenbeck and Ronquist 2001; Ronquist and Huelsenbeck 2003) under a GTR+I+G model (Tavaré 1986), based on the Akaike

Information Criterion as implemented in MrModelTest v2.3 (Nylander 2004). Markov chain

Monte Carlo sampling was conducted for 1,000,000 generations, sampling trees every 1000 generations. Stationarity was assessed by plotting log-likelihood scores against number of

17 generations. A consensus tree of post-stationarity topologies was used to derive posterior probabilities. Bayesian analyses were also conducted by applying the best-fit model to each codon position independently (1: SYM+I, 2: HKY, 3: GTR+G) as well as analyses that excluded the third codon position. In addition to outgroups used in MP analyses, Micropterus salmoides

(Appendix B) was used in Bayesian analyses.

Arlequin 3.5.1.3 was used to examine nucleotide variation, substitution patterns, pairwise

Φst values, and AMOVAs under a distance model of sequence evolution (Excoffier & Lischer

2010). Haplotype diversity (Nei & Tajima 1981) and nucleotide diversity (Nei 1987) were calculated for all populations using DNAsp (Rozas et al. 2003). The correlation between pairwise Φst values and geographic distance was calculated using a Mantel test in Arlequin with

1000 permutation of the pairwise distance matrix to test for significance. Geographic distance between sites was calculated using aerial, not river, distance. A total of 23 populations were analyzed, each of which represented a site locality where 8 or more individuals were collected.

Three metapopulations were defined in Arlequin as the populations within each of the three major drainages: Cumberland, Tennessee, and Coosa rivers. The AMOVAs measured Φsc, the genetic variation within a population relative to the metapopulation; Φct, the genetic variation among metapopulations relative to taxon; and Φst, the genetic variation within populations relative to the taxon (Excoffier & Lischer 2010).

18

CHAPTER 3

RESULTS

Field Collections:

Sixty-six sites were sampled during this study. Table 1 lists all of the site localities, the time spent at each site, the number of collectors present, the collection gear used and the province in which each site exists. All sites are illustrated in Figure 2. Table 2 lists sites where Hemitremia flammea were collected, with sites used for population analyses shown in bold. These sites are illustrated in Figures 3 and 4 respectively. Only sites with eight or more specimens were included in analyses to compare populations, with the exception of three sites in the Choccolocco Creek drainage in Alabama. A total of six fish was collected from these three sites, and these were the only H. flammea found in the Coosa River drainage. For this reason, all six of these specimens were included in the population comparison analyses.

An overall capture rate of 43.9% (29 of 66 sites) was calculated from collections during this study. Within the Cumberland River drainage, a capture rate of 81.8% (nine of 11 sites) was achieved, compared to 46.5% (20 of 43 sites) in the Tennessee River drainage and 16.7% (two of

12 sites) in the Coosa River drainage. One site in the Cumberland River drainage was the Red

River in Simpson County, Kentucky. Hemitremia flammea was believed to be extirpated from the state of Kentucky prior to this study, based on the fact that the species had not been collected there since 1883 (Burr & Warren 1986).

19

The majority of sample sites were located in the Highland Rim physiographic province

(39 of 66). Of these 39 survey sites, H. flammea were captured at 21. Eight sample sites were located in the Cumberland Plateau physiographic province and H. flammea were captured at five.

Finally, 19 sample sites were located in the Ridge and Valley physiographic province and H. flammea were captured at five.

Mitochondrial Analyses:

Gene Analyses: Attempts to amplify the ND2 gene were unsuccessful. Universal primers

ND2 E-H and ND2 B-L were used. In addition, primers were developed based on Genbank sequence data and labeled ND2 Hhemi and ND2 Lhemi. Amplifications were considered weak based on the bands visualized in the 1% agar gel and sequence data generated by the ABI 3700

(Macrogen, Korea) provided less than 10 usable peaks per sample.

A total of 230 H. flammea were successfully amplified for cyt-b. The cyt-b gene region consists of 1140 base pairs. Of the 237 individuals amplified, 917-1140 base pairs were usable.

Seven individuals were eliminated from the dataset and the remaining 230 sequences were reduced to 1010 base pairs of complete data, starting at position 71 (second codon position) and ending at position 1081. G+C content ranged from 41.88-42.18% and A+T content ranged from

57.72-58.42%. Nucleotide ranges were as follows: A=254-263, C=264-267, G=155-163, and

T=326-329. Of the 1010 base pairs analyzed, 952 characters were constant, 28 were parsimony uninformative, and 30 were parsimony informative. Thirty-one unique haplotypes were found among the 230 individuals sequenced (Appendix A).

Parsimony Analyses: Parsimony analysis recovered 10 trees (length = 670, CI = 0.6746,

RCI = 0.4853, HI = 0.3254). Figure 5 illustrates a majority-rule consensus tree with bootstrap

20 values and decay indices for each node. A maximum decay value of 4 was recovered for the species. H. flammea was recovered as monophyletic with 100% posterior probability, and exhibited very shallow divergence across its range.

Bayesian Analyses: The GTR+I+G model was selected as the best-fit and applied to the dataset in MrBayes. Because MP and Bayesian trees were not congruent, analysis was also performed applying the best-fit model for each individual codon position and with the third codon position excluded from the data set. Applying the best-fit model to each codon position (1:

SYM+I, 2: HKY, 3: GTR+G) did not change the clade structure but did change the clade credibility values at many nodes (Figure 6). Analysis of the dataset without the third codon position was conducted due to the rapid rate of mutation at this position, which can cause homoplasy in the results. This analysis resulted in the loss of the nodes supporting a T+M clade,

K+X clade, and N+AB clade. Within the H+E+C+AE+G+Y+D+I+L+Q+R clade, three of the branches collapsed, dissolving the C+AE+G clade and the Q+R clade. Hemitremia flammea was recovered as monophyletic with 100% posterior probability, and exhibited very shallow divergence across the range.

Haplotype Analyses: Overall, the haplotype diversity ( h) for H. flammea was 0.894 and nucleotide diversity ( π) was 0.00873. Within the Cumberland River drainage, h of individual populations ranged from 0-0.844 and π from 0-0.01087. Within the Tennessee River drainage, h of individual populations ranged from 0-0.933 and π from 0-0.01067. Within the Coosa River drainage, h of individual populations ranged from 0.5-1.0 and π ranged from 0.0005-0.01782

(Table 4). Mutations within the cyt-b gene did not deviate from the neutral theory model according to the Tajima’s D, Fu and Li’s F*test, and Fu and Li’s D*test, which were all non- significant (P>0.05).

21

The haplotype networks created using the entire 1140 base pair dataset illustrated many unique haplotypes connected to several other haplotypes in a looping pattern. This type of pattern indicates extreme ambiguity, and the software is unable to determine maximum parsimony. These ambiguities were likely caused by missing data, so the sequence dataset was reduced from 1140 base pairs to 1010 base pairs to eliminate this missing data. The TCS algorithm was unable to connect two groups at the 95% connection limit of 20 steps (Figure 7).

The smaller of these networks contains three haplotypes and is primarily comprised of individuals from the Cumberland River (26 of 31 individuals), 25 from the Caney Fork drainage and one from the Barren Fork drainage. The remaining five individuals are from the Tennessee

River; two from the Elk River drainage, one from Little Cypress Creek drainage, one from

Cypress Creek drainage, and one from Pond Spring. There is very little structure in this group with 67.7% of individuals sharing one haplotype. The larger network is comprised of the other

28 haplotypes, and 46.7% of individuals share one of two haplotypes (A or B). Seventeen haplotypes are unique to one population, and eight individuals were recovered as having a unique haplotype. There is one loop in this network, indicating some ambiguity in the prediction of the evolutionary pathway. Two individuals from Swan Creek (Tennessee River) and two individuals from the Paint Rock River (Tennessee River) are located in this loop. Table 6 lists the number of individuals in each population represented in each haplotype.

Population Analyses: An AMOVA using a distance model of evolution was conducted using the Cumberland, Tennessee, and Coosa river drainages as groups. Of the original 230 individuals, 211 were included in this analysis, representing 23 populations where 8 or more individuals were analyzed. The Cumberland River group was composed of populations from the

Red River, Caney Fork (Pine Creek, Sink Creek, Morgan Branch, and Witty Creek), and Barren

22

Fork (Duke Creek and Bullpen Creek). The Tennessee River group was composed of populations from the Duck River (Wiley Spring), Cypress Creek (Little Cypress Creek and Buffler Spring),

Elk River (Richland Creek, Briar Fork, Beans Creek, and Bluespring Branch), Pond Spring,

Swan Creek, Little Cotaco Creek, Paint Rock River, Sequatchie River (Clear Spring), South

Chickamauga Creek, and Turkey Creek. The Coosa River group was composed of two populations in Choccolocco Creek. Significant genetic structure was resolved among populations but not among drainages; 20.1% of the variation was found among groups, 47.99% among populations, and 31.91% within populations ( ΦSC = 0.60059, ΦCT = 0.20104, ΦST = 0.68089,

P<0.05 for all). The Mantel test revealed a significant correlation between geographic distance and pairwise ΦST values (Z = 18982.84, r = 0.455, p = 0.001). Pairwise ΦST values are illustrated in Table 5.

23

CHAPTER 4

DISCUSSION

The Cumberland, Tennessee and Coosa river drainages are home to many endemic species that have experienced deep divergence along drainage divides. O’Neil & Shepard (2007) identified the Tennessee River as a separate ichthyofaunal region in Alabama from all other drainages above the Fall Line. Several other members of the family Cyprinidae show speciation between Cumberland and Tennessee rivers when compared to the Mobile Basin. For example,

Notropis boops (Bigeye Shiner) and N. xaenocephalus (Coosa Shiner) form a species pair within the N. texanus clade and are geographically divided by drainage. N. boops is found in the

Tennessee and Cumberland river drainages and N. xaenocephalus is found in the Coosa River.

Similarly, Notropis leuciodus (Tennessee Shiner) is found in the Tennessee and Cumberland river drainages while its sister species, N. chrosomus () is found in the Mobile

Basin (Boschung & Mayden 2004). Notropis volucellus (Mimic Shiner), though currently considered one species across its entire range has been described as a complex of species that needs further study (Mayden & Kuhajda 1989), especially to differentiate between potential species in the Mobile Basin and the Tennessee River in northern Alabama. Like these minnows,

I expected to recover significant divergence patterns between sites and individuals from each of the major drainages in Hemitremia flammea. In contrast, I recovered very little genetic variation 24 across the range of the species. Therefore, I have rejected my first hypothesis and propose that the results are an indication of the unique life history of this species. A similar unexpected genetic structure was recovered by Fluker (2011) for Etheostoma tuscumbia . Though E. tuscumbia is described as a spring endemic that does not seasonally migrate out of this habitat, they exhibit very shallow divergence across “isolated” populations indicating an extreme dispersal ability that was previously unknown. I hypothesize that within H. flammea there are periods of isolation and connectivity between populations and that maintain genetic diversity across metapopulations. The headwaters of the Caney Fork, Barren Fork, Elk, and Duck rivers lie in close proximity in central Tennessee providing the opportunity for temporary connectivity.

Seasonal flooding is common, allowing for gene flow between drainages. During periods of isolation, a population may become fixed for a unique haplotype. During periods of connectivity, gene flow across populations and even drainages may then spread these haplotypes. An example of this type of mobility was observed on Little Cotaco Creek at Eudy Cave. We collected no H. flammea during extensive sampling of the spring run but on the walk back to the vehicle we collected 15 in the middle of a flooded cow pasture, presumably spawning in the grasses.

Semotilus atromaculatus (Creek Chub) juveniles are often found in the same sites as H. flammea and though it is not considered a spring endemic species, they prefer small clear streams with undercut banks and/or vegetation for cover (Boschung & Mayden 2004). Semotilus thoreauianus (Dixie Chub) prefers small headwater streams throughout the coastal plain, south of the Fall Line in Alabama and upwards to the Flint River, the only Tennessee River population

(Boschung & Mayden 2004). These two species have been recovered as sister to H. flammea

(Mayden et al. 2008, Simons et al. 2003), and it is plausible that their life histories are more similar than previously known. Instead of being a truly spring endemic species that is localized

25 to this specialized habitat (Mettee et al. 1996, Etnier & Starnes 1993, Sossamon 1990), H. flammea could be capable of traversing through large creek systems where they have been collected close to the bank or in slackwater areas (Boschung & Mayden 2004) and even over flooded land (personal observation).

Life history research for this species is sparse. Sossamon (1990) conducted a life history study in a spring fed pond in Loudon County, Tennessee, but her thesis did not address the migration abilities of this species. Other life history studies focus solely on the reproduction of this species and are limited to laboratory observations and single field sites (Maurkaris 2001,

Katula 1992). Further survey work targeting this species and conducted throughout the year in addition to life history studies at various sites throughout the range of H. flammea are necessary to verify dispersal ability for this species.

All of my analyses indicate shallow divergence between haplotypes and within drainages.

Both Bayesian and parsimony analyses resolved a comb structure for all haplotypes where any clade structure was weakly supported. The starburst pattern that emerged in the haplotype network is also a good illustration of shallow divergence. Many haplotypes differ by only one or two base pairs, even between major drainage divides. Three haplotypes were recovered in the

Coosa River. One was unique to this drainage, but the other two were shared with individuals from the Tennessee and Cumberland rivers. One haplotype was represented in all three major drainages. The smaller of the two haplotype networks is primarily composed of Caney Fork of the Cumberland River individuals, but these haplotypes are also represented in the Barren Fork of the Cumberland River, the Elk River (Tennessee River) and in Little Cypress Creek of the

Tennessee River. All of these haplotypes shared between the three river drainages support the hypothesis that H. flammea readily migrate. AMOVA results also support the likelihood of

26 migration and indicate that populations are more diverse between populations than between drainages. An alternative explanation for these results is that the shared haplotypes could be explained as relict ancestral haplotypes that still exists in multiple drainages; however, no literature to support this hypothesis in Southeastern fishes was found.

The Mantel Test recovered a correlation between geographic distance and the haplotypes present. This may have been driven by geographically extreme populations such as the Red

River (Cumberland River) and Turkey Creek (upper Tennessee River), both of which were fixed for a single haplotype. In addition, a unique haplotype exists in the two sites located in the Coosa

River, which are also geographically isolated. When the Mantel Test was calculated without each of these sites individually the correlation between geographic distance and the haplotypes present was still significant. Likewise, when all four sites were removed the correlation was significant.

These analyses strengthen the argument that H. flammea is not restricted by modern river drainage divides.

Based on my collection results, I cannot entirely accept my second hypothesis that H. flammea are not as rare as previously published. The species is widespread in the Tennessee

River and Cumberland River and is still extant in the Coosa River. A comparison of my field results with previous work indicates that H. flammea are not as rare as they were recently reported to be (Stallsmith 2010) when sampled across their entire range; however, similar results were recovered for the state of Alabama. Of the 66 sites visited during this study, at least one individual was captured at 31 (47.0%), including 81.8% of Cumberland River sites, 46.5% of

Tennessee River sites and 16.7% of Coosa River sites. Stallsmith (2010) captured H. flammea at

18 of 53 sample sites (33.9%) exclusively in the state of Alabama. In comparison, I captured H. flammea at 10 out of 30 sites sampled in Alabama, yielding approximately the same percentage

27 of success within the state (33.3%). In the Tennessee River drainage, my capture rate was 46.5% and when calculated by state my capture rate was 47.8% in Tennessee and 44.4% in Alabama for this drainage. In comparison, 52 of 53 sites visited by Stallsmith were in the Tennessee River drainage in Alabama with a capture rate of 34.6%. Only one site from the Coosa River was included in the Stallsmith publication, and no H. flammea were captured at that site. In contrast, I visited 12 sites in the Coosa River drainage and captured H. flammea at two. When the Coosa

River sampling was removed from the dataset, a 53.7% capture rate was calculated for the

Cumberland and Tennessee river drainages combined. Based on these comparisons, I conclude that H. flammea are not as common in Alabama as they are in Tennessee. My overall capture rate as well as my Tennessee state capture rate, however, were driven by collections in the

Cumberland River where I captured specimens at nine of 11 sites visited (81.8%).

Hemitremia flammea may not be as rare in the Cumberland and Tennessee rivers as they are in the Coosa River due to conservation work and habitat restoration projects for other species. Fundulus julisia (Barrens Topminnow) and Etheostoma boschungi (Slackwater Darter) are imperiled species in these drainages that are currently benefiting from conservation initiatives throughout the Highland Rim and Cumberland Plateau provinces. Because H. flammea exist at many of the same sites as F. julisia and E. boschungi , the habitat improvements and protection are extended to this species as well. Conversely, many of the sites where we did not capture H. flammea were found to have extensively altered habitat such as ponds being built on the springs.

One site where we did not collect H. flammea was of particular interest. Kelly Creek in the Coosa River drainage historically supported a robust population of this species, where catches of over 30 were reported in the 1960s. This site currently maintains seemingly pristine habitat, but we have sampled this site three times since 2009 and no H. flammea have been

28 found. This type of absence may indicate a lack of understanding in the life history of this species and/or an unidentified water quality issue at this site to which H. flammea are particularly sensitive.

Based on this study, the delineation of management units is not merited because of the lack of divergence between drainages. Though there were two haplotype networks resolved, they both include individuals from the Cumberland and Tennessee rivers so they do not define units that are useful on a management scale. There are, however, a few areas of concern for H. flammea . The Coosa River and upper portions of the Cumberland River where H. flammea was believed to be extirpated (Stallsmith 2010) are the areas of highest conservation priority. In addition, we only collected specimens in Turkey Creek of the upper Tennessee River because all other sites visited were inaccessible or the habitat was completely destroyed, including

Sossamon’s life history site in Loudon County (1990). Applying computer modeling techniques in these drainages could prove to be highly beneficial in locating additional new populations based on land cover, geology, climate and other habitat parameters (Meixler & Bain 2012, Wall et al. 2004). A comprehensive sample across the entire range for this species is needed to determine population density and current species distribution.

In order to more fully understand migration rates within this species, additional genetics studies are needed. Because microsatellite loci mutate faster than mitochondrial gene regions, this type of analysis is able to recover finer scale and more recent range fragmentation (Fluker

2011). It is possible that certain populations or drainages are isolated from the rest of the range and that analysis of microsatellite loci could detect more clade structure for this species. For microsatellite analyses, at least 30 individuals from each population are needed to provide

29 significant results so further field survey work is required, especially in the Coosa River drainage where collection numbers are still very low.

Studies that analyze the health and abundance of spring endemic species are an important part of monitoring the health and persistence of Earth’s groundwater resource. H. flammea and other groundwater dependent species serve as environmental indicators. When these species are plentiful, we know that their habitats are healthy. The human population continues to grow and consequently is negatively impacting freshwater resources. This is a global problem that is only going to get worse over time, so we need to be concerned. A decline in the abundance of spring endemic populations is a warning sign that our freshwater resource is in danger.

30

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38

Table 1 All sites sampled for Hemitremia flammea from November 2009 to April 2012. All locations are mapped in Figure 2. States are abbreviated as follows: Alabama (AL), Georgia (GA), Kentucky (KY) and Tennessee (TN). Physiographic provinces are abbreviated as follows: Highland Rim (HR), Cumberland Plateau (CP) and Ridge and Valley (RV).

Site ID Major Physiographic Number Time Number Date Drainage System Site State County Latitude Longitude Province Collected Method (minutes) Collectors CPUE Jan- 1 12 Cumberland Red River Spring Creek KY Simpson 36.76188 -86.72092 HR 12 BPE 60 3 0.067 Feb- 2 11 Cumberland Caney Fork Pine Creek TN DeKalb 35.92939 -85.85574 HR 10 Seine 20 3 0.167 Feb- 3 11 Cumberland Caney Fork Pine Creek TN DeKalb 35.91082 -85.81885 HR 1 BPE 105 3 0.003 Feb- 4 10 Cumberland Caney Fork Sink Creek TN DeKalb 35.87107 -85.84688 HR 15 Seine ─ ─ ─ Sep- 5 09 Cumberland Barren Fork Hickory Creek TN Grundy 35.48304 -85.85232 HR 10 Seine 30 3 0.111 Nov- 6 09 Cumberland Barren Fork Duke Creek TN Cannon 35.67232 -86.08801 HR 10 Seine 30 3 0.111 Feb- 7 11 Cumberland Barren Fork Bullpen TN Cannon 35.75464 -85.97562 HR 10 Seine 20 3 0.167 Feb- 8 11 Cumberland Caney Fork Walker Spring TN VanBuren 35.77155 -85.40606 HR 0 Seine 60 3 0.000 Feb- 9 11 Cumberland Caney Fork Millstone Branch TN VanBuren 35.78310 -85.40560 HR 0 BPE 45 3 0.000 Feb- 10 10 Cumberland Caney Fork Morgan Branch TN DeKalb 35.57000 -85.49038 HR 10 Seine ─ ─ ─ Apr- 11 10 Cumberland Caney Fork Witty Creek TN Warren 35.40630 -85.58575 HR 10 Seine ─ ─ ─ Feb- 12 11 Tennessee Duck Parks Creek TN Coffee 35.55068 -86.08272 HR 0 BPE & Seine 30 3 0.000 Apr- 13 11 Tennessee Duck Wiley Spring TN Coffee 35.42620 -86.11420 HR 10 ─ ─ 3 ─ Mar- 14 11 Tennessee Middle Tennessee Cedar Creek AL Franklin 34.44075 -87.75909 HR 0 BPE 15 3 0.000 Mar- 15 11 Tennessee Middle Tennessee Cedar Creek AL Franklin 34.43856 -87.72334 HR 0 BPE 30 3 0.000 Mar- 16 11 Tennessee Cypress Creek Buffler Spring AL Lauderdale 34.85720 -87.65420 HR 10 BPE & Seine 30 3 0.111 Mar- 17 11 Tennessee Cypress Creek Little Cypress Creek TN Wayne 35.02680 -87.68083 HR 20 BPE & Seine 45 3 0.148 Mar- 18 11 Tennessee Cypress Creek Little Cypress Creek TN Wayne 35.05140 -87.68882 HR 0 BPE & Seine 10 3 0.000 Mar- 19 11 Tennessee Cypress Creek Little Cypress Creek TN Wayne 35.08016 -87.73842 HR 0 BPE & Seine 20 3 0.000 Mar- 20 11 Tennessee Elk Richland Creek TN Giles 35.23428 -87.08424 HR 0 BPE & Seine 30 3 0.000 Mar- 21 11 Tennessee Elk Richland Creek TN Giles 35.30027 -87.07944 HR 10 BPE & Seine 60 3 0.056 Mar- 22 11 Tennessee Elk Richland Creek TN Giles 35.29906 -87.08910 HR 0 BPE 20 3 0.000 Feb- BPE & Dip 23 11 Tennessee Elk Briar Fork TN Lincoln 34.99893 -86.67658 HR 10 Net 40 4 0.063 Mar- 24 11 Tennessee Elk Teel Hollow Spring TN Lincoln 35.11619 -86.47025 HR 0 BPE 10 3 0.000

39

Table 1 continued.

Mar- 25 11 Tennessee Elk Mulepen Creek TN Lincoln 35.05725 -86.40790 HR 0 BPE 10 3 0.000 Mar- 26 11 Tennessee Elk Beans Creek TN Franklin 35.07733 -86.24934 HR 18 BPE 25 3 0.240 Mar- 27 11 Tennessee Elk Bluespring Branch TN Franklin 35.23702 -86.03076 HR 10 BPE 20 3 0.167 Nov- 28 09 Tennessee Elk Spring Creek TN Franklin 35.30000 -86.12000 HR 6 Seine 60 3 0.033 Mar- 29 11 Tennessee Elk Betsy Willis Creek TN Coffee 35.30167 -85.93042 HR 1 BPE 50 3 0.007 Nov- 30 09 Tennessee Elk Henley Creek TN Grundy 35.27461 -85.85713 HR 0 Seine 15 3 0.000 Feb- 31 11 Tennessee Middle Tennessee Pond Spring AL Lawrence 34.65260 -87.25270 HR 14 BPE & Seine 30 4 0.117 Feb- 32 11 Tennessee Middle Tennessee Swan Creek AL Limestone 34.83166 -86.95190 HR 10 BPE 25 4 0.100 Mar- 33 11 Tennessee Middle Tennessee Crowabout Creek AL Morgan 34.35993 -87.10202 HR 0 BPE 5 3 0.000 Mar- 34 11 Tennessee Flint Creek Flint Creek AL Morgan 34.36008 -87.11148 HR 0 BPE 20 3 0.000 Mar- 35 11 Tennessee Flint Creek Dutton Creek AL Morgan 34.36026 -87.10602 HR 0 BPE 15 3 0.000 Mar- 36 11 Tennessee Middle Tennessee Little Cotaco Creek AL Marshall 34.39674 -86.54892 CP 1 BPE 20 1 0.050 Mar- 37 11 Tennessee Middle Tennessee Little Cotaco Creek AL Marshall 34.39415 -86.55099 CP 15 BPE & Seine 60 3 0.083 Mar- 38 11 Tennessee Middle Tennessee Little Cotaco Creek AL Marshall 34.39806 -86.54533 CP 0 BPE & Seine 30 3 0.000 Feb- 39 11 Tennessee Flint River Mint Spring AL Madison 34.93056 -86.45369 HR 0 BPE 30 4 0.000 Feb- 40 11 Tennessee Flint River Mint Spring AL Madison 34.93557 -86.45271 HR 0 BPE & Seine 60 4 0.000 Feb- 41 11 Tennessee Flint River Mountain Fork AL Madison 34.91340 -86.42790 HR 4 BPE & Seine 90 4 0.011 Mar- 42 11 Tennessee Paint Rock Paint Rock AL Jackson 34.74374 -86.31738 CP 10 BPE 20 3 0.167 Mar- 43 11 Tennessee Middle Tennessee Big Spring Creek AL Blount 34.15474 -86.47185 CP 0 BPE 10 3 0.000 Mar- 44 11 Tennessee Middle Tennessee Big Spring Creek AL Blount 34.14127 -86.49455 CP 0 BPE 30 3 0.000 Mar- 45 11 Tennessee Upper Tennessee Lookout Creek AL DeKalb 34.65614 -85.55750 CP 1 BPE & Seine 60 3 0.006 Mar- 46 11 Tennessee Middle Tennessee Crow Creek TN Franklin 35.10078 -85.92253 HR 0 BPE 45 3 0.000 Nov- 47 09 Tennessee Sequatchie Clear Spring TN Marion 35.10268 -85.60204 CP 9 Seine 30 3 0.100 Aug- ─ 48 08 Tennessee Upper Tennessee South Chickamauga GA Catoosa 34.92970 -85.00620 RV 10 ─ 1 ─ Nov- 49 09 Tennessee Upper Tennessee North Mouse Creek TN McMinn 35.52007 -84.60907 RV 0 Seine 30 2 0.000 Nov- 50 09 Tennessee Upper Tennessee Town Creek TN Loudon 35.80632 -84.27672 RV 0 Seine 30 2 0.000 Nov- 51 09 Tennessee Upper Tennessee Turkey Creek TN Knox 35.91242 -84.15363 RV 1 Seine 60 4 0.004 Apr- 52 10 Tennessee Upper Tennessee Turkey Creek TN Knox 35.89920 -84.14140 RV 10 ─ ─ 3 ─ 40

Table 1 continued.

Nov- 53 09 Tennessee Upper Tennessee Turkey Creek TN Knox 35.88477 -84.15403 RV 0 Seine 40 4 Nov- 54 09 Tennessee Upper Tennessee Second Creek TN Knox 36.00887 -83.97337 RV 0 Seine 60 4 Apr- BPE & Dip 55 12 Coosa Cheaha Creek Cheaha Creek AL Talladega 33.53175 -86.04205 RV 0 Net 60 2 Apr- BPE & Dip 56 12 Coosa Cheaha Creek Kelly Creek AL Talladega 33.48043 -86.02452 RV 0 Net 100 2 Apr- 57 12 Coosa Choccolocco Creek Choccolocco Creek AL Talladega 33.56107 -86.02065 RV 4 Seine 60 3 Apr- BPE & Dip 58 12 Coosa Choccolocco Creek Choccolocco Creek AL Calhoun 33.61674 -85.81367 RV 0 Net 30 2 Feb- 59 11 Coosa Choccolocco Creek Joseph Creek AL Calhoun 33.68333 -85.66758 RV 0 BPE & Seine 40 4 Feb- 60 11 Coosa Choccolocco Creek Joseph Creek AL Calhoun 33.68227 -85.66840 RV 0 BPE 20 4 Feb- 61 11 Coosa Choccolocco Creek Chosea Spring AL Calhoun 33.69365 -85.67541 RV 0 BPE & Seine 10 4 Apr- BPE & Dip 62 12 Coosa Choccolocco Creek Chosea Springs AL Calhoun 33.69270 -85.67850 RV 0 Net 90 2 Feb- 63 11 Coosa Choccolocco Creek Joseph Spring AL Calhoun 33.70931 -85.67005 RV 0 BPE 20 4 Feb- 64 11 Coosa Choccolocco Creek Murray Spring AL Calhoun 33.72501 -85.69030 RV 2 BPE 30 4 Feb- 65 11 Coosa Choccolocco Creek Murray Spring AL Calhoun 33.72467 -85.68989 RV 0 BPE & Seine 45 4 Apr- 66 12 Coosa Coosa Blue Eye Creek AL Talladega 33.64917 -86.02917 RV 0 BPE 5 1

41

Table 2 Sites where Hemitremia flammea were collected from November 2009 to April 2012. All sites are illustrated in Figure 3 and site numbers are correlated to that map. Sites shown in bold were used for population analyses and are illustrated separately in Figure 4. States are abbreviated as follows: Alabama (AL), Georgia (GA), Kentucky (KY) and Tennessee (TN).

Site ID Major Number Number Date Drainage System Site State County Latitude Longitude Analyzed 1 Jan-12 Cumberland Red River Spring Creek KY Simpson 36.76188 -86.72092 12 2 Feb-11 Cumberland Caney Fork Pine Creek TN DeKalb 35.92939 -85.85574 10 4 Feb-10 Cumberland Caney Fork Sink Creek TN DeKalb 35.87107 -85.84688 9 5 Sep-09 Cumberland Barren Fork Hickory Creek TN Grundy 35.48304 -85.85232 8 6 Nov-09 Cumberland Barren Fork Duke Creek TN Cannon 35.67232 -86.08801 9 7 Feb-11 Cumberland Barren Fork Bullpen TN Cannon 35.75464 -85.97562 10 10 Feb-10 Cumberland Caney Fork Morgan Branch TN DeKalb 35.57000 -85.49038 10 11 Apr-10 Cumberland Caney Fork Witty Creek TN Warren 35.40630 -85.58575 9 13 Apr-11 Tennessee Duck Wiley Spring TN Coffee 35.42620 -86.11420 10 Cypress 16 Mar-11 Tennessee Creek Buffler Spring AL Lauderdale 34.85720 -87.65420 10 Cypress Little Cypress 17 Mar-11 Tennessee Creek Creek TN Wayne 35.02680 -87.68083 10 21 Mar-11 Tennessee Elk Richland Creek TN Giles 35.30027 -87.07944 10 23 Feb-11 Tennessee Elk Briar Fork TN Lincoln 34.99893 -86.67658 10 26 Mar-11 Tennessee Elk Beans Creek TN Franklin 35.07733 -86.24934 10 Bluespring 27 Mar-11 Tennessee Elk Branch TN Franklin 35.23702 -86.03076 10 28 Nov-09 Tennessee Elk Spring Creek TN Franklin 35.30000 -86.12000 6 Betsy Willis 29 Mar-11 Tennessee Elk Creek TN Coffee 35.30167 -85.93042 1 Middle 31 Feb-11 Tennessee Tennessee Pond Spring AL Lawrence 34.65260 -87.25270 9 Middle 32 Feb-11 Tennessee Tennessee Swan Creek AL Limestone 34.83166 -86.95190 10 Middle Little Cotaco 37 Mar-11 Tennessee Tennessee Creek AL Marshall 34.39415 -86.55099 11 41 Feb-11 Tennessee Flint River Mountain Fork AL Madison 34.91340 -86.42790 4 42 Mar-11 Tennessee Paint Rock Paint Rock AL Jackson 34.74374 -86.31738 10 Upper 45 Mar-11 Tennessee Tennessee Lookout Creek AL DeKalb 34.65614 -85.55750 1 47 Nov-09 Tennessee Sequatchie Clear Spring TN Marion 35.10268 -85.60204 9 Upper South 48 Aug-08 Tennessee Tennessee Chickamauga GA Catoosa 34.92970 -85.00620 10 Upper 51 Nov-09 Tennessee Tennessee Turkey Creek TN Knox 35.91242 -84.15363 1 Upper 52 Apr-10 Tennessee Tennessee Turkey Creek TN Knox 35.89920 -84.14140 10 Choccolocco Choccolocco 57 Apr-12 Coosa Creek Creek AL Talladega 33.56107 -86.02065 4 Choccolocco 64 Feb-11 Coosa Creek Murray Spring AL Calhoun 33.72501 -85.69030 2

42

Table 3 Primers used in mitochondrial analyses.

Gene Primer Sequence (5'-3') ND2 ND2 E-H TTCTACTTAAAGCTTTGAAGCC ND2 ND2 B-L AAGCTTTCGGGCCCATACCC ND2 ND2 Hhemi TTCCCACTATCCCGGCTCAGGC ND2 ND2Lhemi TGCCAATTGCACTAGCAGGCC cyt-b H15915 CAACGATCTCCGGTTTACAAGAC cyt-b L12474 GTGACTTGAAAAACCACCGTTG

43

Table 4 Haplotype and nucleotide diversity for each population of Hemitremia flammea .

Site Number of Number of Haplotype Nucleotide ID Population Name Individuals Haplotypes Diversity Diversity 1 Red River 9 1 0.000 0.00000 2 Pine Creek 10 5 0.822 0.01087 4 Sink Creek 9 2 0.556 0.00055 6 Duke Creek 9 2 0.222 0.00044 7 Bullpen Creek 10 6 0.844 0.00636 10 Morgan Branch 10 2 0.200 0.00020 11 Witty Creek 8 1 0.000 0.00000 13 Wiley Spring 10 1 0.000 0.00000 16 Buffler Spring 10 5 0.822 0.00499 17 Little Cypress Creek 10 6 0.889 0.00477 21 Richland Creek 11 4 0.709 0.00234 23 Briar Fork 10 7 0.933 0.01067 26 Beans Creek 9 6 0.833 0.00330 27 Bluespring Branch 10 5 0.756 0.00273 31 Pond Spring 9 6 0.889 0.00831 32 Swan Creek 10 6 0.889 0.00169 37 Little Cotaco 11 4 0.709 0.00158 42 Paint Rock River 10 4 0.800 0.00158 47 Clear Spring 9 2 0.389 0.00655 South Chickamauga 48 Creek 10 2 0.356 0.00211 52 Turkey Creek 11 1 0.000 0.00000 57 Choccolocco Creek 4 2 0.500 0.00050 64 Murray Spring 2 2 1.000 0.01782

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Table 5 Φst values for all populations of Hemitremia flammea . Significant values are in bold (p<0.05).

Red Witty Sink Bullpen Duke Morgan Pine Beans Wiley Clear Turkey S. Chickamauga Swan Pond Rock Paint Cypress Little Cotaco Little Briar Buffler Richland Bluespring Murray Choccolocco

Red 0.000

Witty 1.000 0.000

Sink 0.984 0.983 0.000

Bullpen 0.334 0.002 0.788 0.000 - Duke 0.900 0.014 0.972 0.009 0.000

Morgan 0.994 0.993 0.432 0.803 0.982 0.000

Pine 0.534 0.491 0.570 0.285 0.496 0.274 0.000 - - Beans 0.516 0.014 0.895 0.057 0.000 0.907 0.425 0.000

Wiley 1.000 0.000 0.985 0.028 0.012 0.994 0.572 0.012 0.000

Clear 0.439 0.217 0.814 0.060 0.217 0.827 0.362 0.150 0.255 0.000

Turkey 1.000 1.000 0.989 0.809 0.986 0.996 0.723 0.900 1.000 0.769 0.000 S. Chickamauga 0.930 0.925 0.934 0.760 0.917 0.945 0.669 0.841 0.933 0.701 0.830 0.000

Swan 0.712 0.187 0.938 0.018 0.175 0.948 0.492 0.070 0.222 0.115 0.946 0.888 0.000 - Pond 0.314 0.104 0.740 0.079 0.112 0.755 0.232 0.007 0.138 0.042 0.766 0.717 0.076 0.000

Paint Rock 0.767 0.464 0.941 0.119 0.429 0.951 0.502 0.251 0.500 0.099 0.949 0.889 0.090 0.112 0.000 - - Little Cypress 0.422 0.009 0.832 0.058 0.015 0.845 0.339 0.036 0.036 0.111 0.852 0.799 0.056 0.014 0.177 0.000

Little Cotaco 0.750 0.299 0.942 0.052 0.274 0.952 0.497 0.151 0.334 0.134 0.952 0.896 0.050 0.095 0.136 0.091 0.000 - - Briar 0.264 0.112 0.615 0.042 0.117 0.629 0.113 0.060 0.145 0.018 0.661 0.613 0.121 0.062 0.148 0.010 0.126 0.000 - - - Buffler 0.422 0.038 0.826 0.051 0.043 0.840 0.339 0.018 0.066 0.121 0.847 0.795 0.067 0.027 0.190 0.091 0.112 0.012 0.000 - - - Richland 0.601 0.091 0.915 0.027 0.097 0.925 0.468 0.004 0.121 0.091 0.902 0.866 0.012 0.000 0.111 0.023 0.035 0.077 0.041 0.000 - - - - - Bluespring 0.553 0.009 0.907 0.059 0.005 0.917 0.445 0.095 0.016 0.146 0.911 0.855 0.037 0.007 0.233 0.002 0.128 0.070 0.010 0.019 0.000

Murray 0.775 0.751 0.878 0.326 0.736 0.883 0.258 0.493 0.795 0.256 0.786 0.584 0.672 0.174 0.684 0.453 0.705 0.057 0.447 0.596 0.547 0.000

Choccolocco 0.991 0.990 0.975 0.736 0.971 0.987 0.608 0.851 0.992 0.666 0.980 0.693 0.917 0.673 0.921 0.790 0.928 0.538 0.783 0.886 0.871 0.399 0.000

45

Table 6 Haplotypes recovered for Hemitremia flammea , as represented at each site.

Drainages Cumberland Tennessee Coosa

Sites Red Morgan Sink Pine Witty Hickory Duke Bullpen Wiley Briar Richland Beans Bluespring Spring Betsy Willis Paint Rock Swan Pond Little Cypress Buffler Clear Little Cottaco Fork Mountain Lookout S. Chickamauga Turkey Murray Choccolocco Number of Haplotypes 1 2 2 5 1 3 2 6 1 7 4 5 5 3 1 4 6 6 6 5 2 4 3 1 2 1 2 2 1 A 8 2 8 4 0 2 3 5 5 3 3 2 2 1

B 2 2 2 5 1 2 2 3 1 1 7 5 2

C 9 4 4 2 1 1 1 D 1

E 1 1 1 1 1 1 1 2 1 1

F 1 1 3 4

G 5 2 1 1

H 9

I 8

J 1 1 1 2 1 1

K 1 1 1 1 2 1

L 2 2

M 3 1

N 4

O 2 1 1

P 4 Haplotypes Haplotypes

Q 1 1 1

R 3

S 1 2

T 2

U 2

V 2

W 2

X 1

Y 1

Z 1

AA 1

AB 1

AC 1

AD 1

AE 1

46

Kentucky

Georgia Alabama

0 120 240 Kilometers

Figure 1 Map of the Cumberland, Tennessee and Coosa river drainages 47

Kentucky

Georgia

Alabama

0 120 240 Kilometers

Figure 2 Map of all sites sampled for Hemitremia flammea from November 2009 to April 2012 . Dots correspond to the locations listed in Table 1.

48

Kentucky

Georgia

Alabama

0 120 240 Kilometers

Figure 3 Map of sites where Hemitremia flammea were collected from November 2009 to April 2012. Dots correspond to the locations and site ID numbers listed in Table 2.

49

Kentucky

Georgia

Alabama

0 120 240 Kilometers

Figure 4 Map of sites included in population comparison analyses. Dots correspond to the locations and site ID numbers listed in bold in Table 2.

50

Figure 5 Maximum parsimony consensus tree for Hemitremia flammea. The tree illustrates bootstrap values for each node and decay indices are in parentheses. Letters represent the 31 haplotypes recovered in analyses and the colors correspond to the river drainages recovered in those haplotypes. 51

Figure 6 Combined Bayesian consensus trees using GTR+I+G model and codon specific models for Hemitremia flammea. Tree illustrates clade credibility values for the single model followed by the three model approach (i.e. 1/3). Letters represent the 31 haplotypes recovered in analyses and the colors correspond to the river drainages recovered in those haplotypes. 52

Figure 7 Haplotype network for Hemitremia flammea. Letters are unique to one haplotype. Numbers in parentheses correspond to the number of individuals recovered in each haplotype. Colors indicate the river drainages represented in each haplotype.

53

APPENDIX A

SEQUENCE DATA FOR ALL 31 HAPLOTYPES RECOVERED FOR HEMITREMIA FLAMMEA

54

Haplotype Sequence A CCTCTAACATTTCCGCGCTCTGAAATTTTGGGTCCCTTCTTGGATTATGTTTAATTACTCAAATTTTAACAGGATTATTTTTGGCAATACATT ACACCTCTGATATCTCAACCGCATTTTCATCCGTAGCCCATATCTGCCGGGACGTAAATTACGGCTGACTCATTCGAAACTTACACGCTAAC GGGGCATCCTTCTTCTTCATCTGCGTTTATATACACATCGCCCGAGGACTTTATTACGGGTCCTATCTTTATAAAGAAACCTGAAACATTGG TGTAGTCTTACTCCTATTAGTTATAATGACAGCCTTCGTGGGATATGTGCTTCCCTGAGGACAAATATCCTTTTGAGGCGCCACAGTAATC ACAAATTTATTATCAGCAGTCCCTTATATAGGAGACATGCTTGTACAATGAATTTGAGGGGGCTTTTCTGTAGATAACGCAACCCTGACAC GGTTCTTCGCATTCCATTTTCTACTCCCTTTCGTCATCGCCGGCGCGACCATCCTCCACTTACTGTTTTTACACGAAACAGGATCGAACAAC CCGGCCGGCCTAAACTCTGATGCAGATAAAATCTCTTTCCATCCATACTTTTCATACAAAGACCTTCTTGGTTTCATTGTAATATTATTAGCC CTGACATCATTAGCTTTATTCTCCCCAAACTTACTAGGTGACCCAGACAATTTTACACCCGCAAACCCTTTAGTTACTCCCCCTCATATTCAG CCTGAGTGATACTTCTTATTTGCCTACGCCATTCTTCGATCTATTCCAAATAAGCTAGGCGGGGTCCTTGCACTACTATTTAGCATCCTAGT GCTAATGGTTGTACCTATTCTACATACCTCTAAACAACGAGGACTCACTTTCCGTCCTATGACCCAATTTCTATTTTGAACCCTGGTGGCAG ATATACTTATTCTTACATGAATTGGTGGTATACCCGTAGAACACCCCTATGTCATAATTGGCCAAATCGCATCCGTCCTATACTTTGCC

B CCTCTAACATTTCCGCGCTCTGAAATTTTGGGTCCCTTCTTGGATTATGTTTAATTACTCAAATTTTAACAGGATTATTTTTGGCAATACATT ACACCTCTGATATCTCAACCGCATTTTCATCCGTAGCCCATATCTGCCGGGACGTAAATTACGGCTGACTCATTCGAAACTTACACGCTAAC GGGGCATCCTTCTTCTTCATCTGCGTTTATATACACATCGCCCGAGGACTTTATTACGGGTCCTATCTTTATAAAGAAACCTGAAACATTGG TGTAATCTTACTCCTATTAGTTATAATGACAGCCTTCGTGGGATATGTGCTTCCCTGAGGACAAATATCCTTTTGAGGCGCCACAGTAATC ACAAATTTATTATCAGCAGTCCCTTATATAGGAGACATGCTTGTACAATGAATTTGAGGGGGCTTTTCTGTAGATAACGCAACCCTGACAC GGTTCTTCGCATTCCATTTTCTACTCCCTTTCGTCATCGCCGGCGCGACCATCCTCCACTTACTGTTTTTACACGAAACAGGATCGAACAAC CCGGCCGGCCTAAACTCTGATGCAGATAAAATCTCTTTCCATCCATACTTTTCATACAAAGACCTTCTTGGTTTCATTGTAATATTATTAGCC CTGACATCATTAGCTTTATTCTCCCCAAACTTACTAGGTGACCCAGACAATTTTACACCCGCAAACCCTTTAGTTACTCCCCCTCATATTCAG CCTGAGTGATACTTCTTATTTGCCTACGCCATTCTTCGATCTATTCCAAATAAGCTAGGCGGGGTCCTTGCACTACTATTTAGCATCCTAGT GCTAATGGTTGTACCTATTCTACATACCTCTAAACAACGAGGACTCACTTTCCGTCCTATGACCCAATTTCTATTTTGAACCCTGGTGGCAG ATATACTTATTCTTACATGAATTGGTGGTATACCCGTAGAACACCCCTATGTCATAATTGGCCAAATCGCATCCGTCCTATACTTTGCC

C CCTCCAACAT TTCCGCGCTCTGAAATTTTGGGTCCCTTCTTGGATTATGTTTAATTACTCAAATTTTAACAGGATTATTTTTGGCAATACATT ACACCTCTGATATCTCAACCGCATTTTCATCCGTGGCCCATATCTGCCGGGACGTAAATTACGGCTGACTCATTCGAAACCTACACGCTAAT GGAGCATCCTTCTTCTTCATCTGCGTTTATATACACATCGCCCGAGGACTTTATTACGGGTCCTATCTTTATAAAGAAACCTGAAACATTGG TGTAGTCTTACTCCTATTAGTTATAATGACAGCCTTCGTAGGGTATGTACTTCCCTGAGGACAAATATCCTTTTGAGGCGCCACAGTAATC ACAAATTTATTATCAGCAGTCCCTTATATAGGAGACATGCTTGTACAGTGAATTTGAGGGGGCTTTTCTGTAGATAACGCAACCCTGACAC GGTTCTTCGCATTCCATTTTCTACTCCCTTTCGTCATCGCCGGCGCGACCATCCTCCACTTACTATTTTTACACGAAACAGGGTCGAACAAC CCGGCCGGCCTAAACTCTGATGCAGATAAAATCTCTTTCCATCCATACTTTTCATACAAAGACCTTCTTGGTTTCATTGTAATATTATTAGCC CTAACATCATTAGCTTTATTTTCCCCAAACTTACTAGGTGACCCAGACAATTTTACACCCGCAAACCCTTTAGTTACTCCCCCTCATATTCAG CCTGAATGATACTTCTTATTTGCCTACGCCATTCTTCGATCTATCCCAAATAAGCTAGGTGGGGTCCTTGCACTACTATTTAGCATCCTAGT GCTAATGGTTGTACCTATTCTACATACCTCTAAACAACGAGGACTCACTTTCCGTCCTATAACCCAATTTCTATTTTGAACCCTGGTGGCAG ATATACTTATTCTTACATGAATTGGTGGTATACCCGTAGAACACCCCTATGTCATAATTGGCCAAATCGCATCCGTCCTATACTTTGCC

D CCTCTAACATTTCCGCGCTC TGAAATTTTGGATCCCTTCTTGGATTATGTTTAATTACTCAAATTTTAACAGGATTATTTTTAGCAATACATT ACACCTCTGATATCTCAACCGCATTTTCATCCGTAGCCCATATCTGCCGGGACGTAAATTACGGCTGACTCATTCGAAACCTACACGCTAAT GGAGCATCCTTCTTCTTCATCTGCGTTTACATACACATCGCCCGAGGACTTTATTACGGGTCCTATCTTTATAAAGAAACCTGAAACATTGG TGTAGTCTTACTGCTATTAGTTATAATGACAGCCTTCGTGGGATATGTGCTTCCCTGAGGACAAATATCCTTTTGAGGCGCCACAGTAATT ACAAATTTATTATCAGCAGTCCCTTATATAGGAGACATACTTGTACAATGAATTTGAGGGGGCTTTTCTGTAGATAACGCAACCCTGACAC GGTTCTTCGCATTCCATTTTCTACTCCCCTTCGTCATCGCCGGCGCGACCATCCTCCACTTACTGTTTTTACACGAAACAGGATCGAACAAC CCGGCCGGCCTAAACTCTGATGCAGATAAAATCTCTTTCCATCCATACTTTTCATACAAAGACCTTCTTGGTTTCATTGTAATATTATTAGCC CTGACATCATTAGCTTTATTCTCCCCAAACTTACTAGGTGACCCAGACAATTTTACACCCGCAAACCCTTTAGTTACTCCCCCTCATATTCAG CCTGAATGATACTTCTTATTTGCCTACGCCATTCTTCGATCTATTCCAAATAAGCTAGGCGGGGTCCTTGCACTACTATTTAGCATCCTAGT GCTAATGATTGTACCTATTCTACATACCTCTAAACAACGAGGACTCACTTTCCGTCCTGTAACCCAATTTCTATTTTGAACCCTGGTGGCAG ATATACTTATTCTTACATGAATTGGTGGTATACCCGTAGAACACCCCTATGTCATAATTGGCCAAATCGCATCCGTCCTATACTTTGCC

55

E CCTCTAACATTTCCGCGCTCTGAAATTTTG GGTCCCTTCTTGGATTATGTTTAATTACTCAAATTTTAACAGGGTTATTTTTGGCAATACATT ACACCTCTGATATCTCAACCGCATTTTCATCCGTAGCCCATATCTGCCGGGACGTAAATTACGGCTGACTCATTCGAAACCTACACGCCAAT GGGGCATCCTTCTTCTTCATCTGCGTTTATATACATATCGCCCGAGGACTTTATTACGGGTCCTATCTTTATAAAGAAACCTGAAACATTGG TGTAGTCTTACTCCTATTAGTTATAATAACAGCCTTCGTGGGATATGTGCTTCCCTGAGGACAAATATCCTTTTGAGGCGCCACAGTAATC ACAAATTTATTATCAGCAGTCCCTTATATAGGAGACATGCTTGTACAATGAATTTGAGGGGGCTTTTCTGTAGATAACGCAACCCTCACAC GGTTCTTCGCATTCCATTTTCTACTCCCTTTCGTCATCGCCGGCGCGACCATCCTCCACTTACTGTTTTTACACGAAACAGGATCGAACAAC CCGGCCGGCCTAAACTCTGATGCAGATAAAATCTCTTTCCATCCATACTTTTCATACAAAGACCTTCTTGGTTTCATTGTAATATTATTAGCC CTGACATCATTAGCTTTATTCTCCCCAAACTTACTAGGTGACCCAGACAATTTTACACCCGCAAACCCTTTAGTTACTCCCCCTCATATTCAG CCTGAGTGATACTTCTTATTTGCCTACGCCATTCTTCGATCTATTCCAAATAAGCTAGGCGGGGTCCTTGCACTACTATTTAGCATCCTAGT GCTAATGGTTGTACCTATTCTACATACCTCTAAACAACGAGGACTCACTTTCCGTCCGATAACCCAATTTCTATTTTGAACCCTGGTGGCAG ATATACTTATTCTTACATGAATTGGTGGTATACCCGTAGAACACCCCTATGTCATAATTGGCCAAATCGCATCCGTCCTATACTTTGCC

F CCTCTAACATTTCCGCGCTCTGAAATTTTGGGTCCCTTCTTGGATTATGTTTAATTACTCAAATTTTAACAGGATTATTTTTGGCAATACATT ACACCTCTGATATCTCAACCGCATTTTCATCCGTAGCCCATATCTGCCGGGACGTAAATTACGGCTGACTCATTCGAAACTTACACGCTAAC GGGGCATCCTTCTTCTTCATCTGCGTTTATATACACATCGCCCGAGGACTTTATTACGGGTCCTATCTTTATAAAGAAACCTGAAACATTGG TGTAGTCTTACTCCTATTAGTTATAATGACAGCCTTCGTGGGATATGTGCTTCCCTGAGGACAAATATCCTTTTGAGGCGCCACAGTAATC ACAAATTTATTATCAGCAGTCCCTTATATAGGAGACATGCTTGTACAATGAATTTGAGGGGGCTTTTCTGTAGATAACGCAACCCTGACAC GGTTCTTCGCATTCCATTTTCTACTCCCTTTCGTCATCGCCGGCGCGACCATCCTCCACTTACTGTTTTTACACGAAACGGGATCGAACAAC CCGGCCGGCCTAAACTCTGATGCAGATAAAATCTCTTTCCATCCATACTTTTCATACAAAGACCTTCTTGGTTTCATTGTAATATTATTAGCC CTGACATCATTAGCTTTATTCTCCCCAAACTTACTAGGTGACCCAGACAATTTTACACCCGCAAACCCTTTAGTTACTCCCCCTCATATTCAG CCTGAGTGATACTTCTTATTTGCCTACGCCATTCTTCGATCTATTCCAAATAAGCTAGGCGGGGTCCTTGCACTACTATTTAGCATCCTAGT GCTAATGGTTGTACCTATTCTACATACCTCTAAACAACGAGGACTCACTTTCCGTCCTATGACCCAATTTCTATTTTGAACCCTGGTGGCAG ATATACTTATTCTTACATGAATTGGTGGTATACCCGTAGAACACCCCTATGTCATAATTGGCCAAATCGCATCCGTCCTATACTTTGCC

G CCTCCAACATTTCCGCGCTCTGAAATTTTGGGTCCCTTCTTGGATTATGTTTAATTACTCAAATTTTAACAGGATTATTTTTGGCAATACATT ACACCTCTGATATCTCAACCGCATTTTCATCCGTGGCCCATATCTGCCGGGACGTAAATTACGGCTGACTCATTCGAAACCTACACGCTAAT GGAGCATCCTTCTTCTTCATCTGCGTTTATATACACATCGCCCGAGGACTTTATTACGGGTCCTATCTTTATAAAGAAACCTGAAACATTGG TGTAGTCTTACTCCTATTAGTTATAATGACAGCCTTCGTAGGGTATGTACTTCCCTGAGGACAAATATCCTTTTGAGGCGCCACAGTAATC ACAAATTTATTATCAGCAGTCCCTTATATAGGAGACATGCTTGTACAGTGAATTTGAGGGGGCTTTTCTGTAGATAACGCAACCCTGACAC GGTTCTTCGCATTCCATTTTCTACTCCCTTTCGTCATCGCCGGCGCGACCATCCTCCACTTACTATTTTTACACGAAACAGGGTCGAACAAC CCGGCCGGCCTAAACTCTGATGCAGATAAAATCTCTTTCCATCCATACTTTTCATACAAAGACCTTCTTGGTTTCATTGTAATATTATTAGCC CTAACATCATTAGCTTTATTTTCCCCAAACTTATTAGGTGACCCAGACAATTTTACACCCGCAAACCCTTTAGTTACTCCCCCTCATATTCAG CCTGAATGATACTTCTTATTTGCCTACGCCATTCTTCGATCTATCCCAAATAAGCTAGGTGGGGTCCTTGCACTACTATTTAGCATCCTAGT GCTAATGGTTGTACCTATTCTACATACCTCTAAACAACGAGGACTCACTTTCCGTCCTATAACCCAATTTCTATTTTGAACCCTGGTGGCAG ATATACTTATTCTTACATGAATTGGTGGTATACCCGTAGAACACCCCTATGTCATAATTGGCCAAATCGCATCCGTCCTATACTTTGCC

H CCTCTAACAT TTCCGCGCTCTGAAATTTTGGGTCCCTTCTTGGATTATGTTTAATTACTCAAATTTTAACAGGATTATTTTTGGCAATACATT ACACCTCTGATATCTCAACCGCATTTTCATCCGTAGCCCATATCTGCCGGGACGTAAATTACGGCTGACTCATTCGAAACCTACACGCTAAC GGGGCATCCTTCTTCTTCATCTGCGTTTATATACACATCGCCCGAGGACTTTATTACGGGTCCTATCTTTATAAAGAAACCTGAAACATTGG TGTAGTCTTACTCCTATTAGTTATAATGACAGCCTTCGTGGGATATGTGCTTCCCTGAGGACAAATATCCTTTTGAGGCGCCACAGTAATC ACAAATTTATTATCAGCAGTCCCTTATATAGGAGACATGCTTGTACAATGAATTTGAGGGGGCTTTTCTGTAGATAACGCAACCCTGACAC GGTTCTTCGCATTCCATTTTCTACTCCCTTTCGTCATCGCCGGCGCGACCATCCTCCACTTACTGTTTTTACACGAAACAGGATCGAACAAC CCGGCCGGCCTAAACTCTGATGCAGATAAAATCTCTTTCCATCCATACTTTTCATACAAAGACCTTCTTGGTTTCATTGTAATATTATTAGCC CTGACATCATTAGCTTTATTCTCCCCAAACTTACTAGGTGACCCAGACAATTTTACACCCGCAAACCCTTTAGTTACTCCCCCTCATATTCAG CCTGAGTGATACTTCTTATTTGCCTACGCCATTCTTCGATCTATTCCAAATAAGCTAGGCGGGGTCCTTGCACTACTATTTAGCATCCTAGT GCTAATGGTTGTACCTATTCTACATACCTCTAAACAACGAGGACTCACTTTCCGTCCTATGACCCAATTTCTATTTTGAACCCTGGTGGCAG ATATACTTATTCTTACATGAATTGGTGGTATACCCGTAGAGCACCCCTATGTCATAATTGGCCAAATCGCATCCGTCCTATACTTTGCC

I CCTCTAACATTTCCGCGCTC TGAAATTTTGGATCCCTTCTTGGATTATGTTTAATTACTCAAATTTTAACAGGATTATTTTTGGCAATACATT ACACCTCTGATATCTCAACCGCATTTTCATCCGTAGCCCATATCTGCCGGGACGTAAATTACGGCTGACTCATTCGAAACCTACACGCTAAT GGAGCATCCTTCTTCTTCATCTGCGTTTACATACACATCGCCCGAGGACTTTATTACGGGTCCTATCTTTATAAAGAAACCTGAAACATTGG TGTAGTCTTACTGCTATTAGTTATAATGACAGCCTTCGTGGGATATGTACTTCCCTGAGGACAAATATCCTTTTGAGGCGCCACAGTAATT ACAAATTTATTATCAGCAGTCCCTTATATGGGGGACATACTTGTACAATGAATTTGAGGGGGCTTTTCTGTAGATAACGCAACCCTGACAC GGTTCTTCGCATTCCATTTTCTACTCCCCTTCGTCATCGCCGGCGCGACCATCCTCCACTTACTGTTTTTACACGAAACAGGATCGAACAAC CCGGCCGGCCTAAACTCTGATGCAGATAAAATCTCTTTCCATCCATACTTTTCATACAAAGACCTTCTTGGTTTCATTGTAATATTATTAGCC CTGACATCATTGGCTTTATTCTCCCCAAACTTACTAGGTGACCCAGACAATTTTACACCCGCAAACCCTTTAGTTACTCCCCCTCATATTCAG CCTGAATGATACTTCTTATTTGCCTACGCCATTCTTCGATCTATTCCAAATAAGCTAGGCGGGGTCCTTGCACTACTATTTAGCATCCTAGT GCTAATGGTTGTACCTATTCTACATACCTCTAAACAACGAGGACTCACTTTCCGTCCTGTAACCCAATTTCTATTTTGAACCCTGGTGGCAG ATATACTTATTCTTACATGAATTGGTGGTATACCCGTAGAACACCCCTATGTCATAATTGGCCAAATCGCATCCGTCCTATACTTTGCC

56

J CCTCTAACATTTCCGCGCTCTGAAATTTTG GGTCCCTTCTTGGATTATGTTTAATTACTCAAATTTTAACAGGATTATTTTTGGCAATACATT ACACCTCTGATATCTCAACCGCATTTTCATCCGTAGCCCATATCTGCCGGGACGTAAATTACGGCTGACTCATTCGAAACTTACACGCTAAC GGGGCATCCTTCTTCTTCATCTGCATTTATATACACATCGCCCGAGGACTTTATTACGGGTCCTATCTTTATAAAGAAACCTGAAACATTGG TGTAGTCTTACTCCTATTAGTTATAATGACAGCCTTCGTGGGATATGTGCTTCCCTGAGGACAAATATCCTTTTGAGGCGCCACAGTAATC ACAAATTTATTATCAGCAGTCCCTTATATAGGAGACATGCTTGTACAATGAATTTGAGGGGGCTTTTCTGTAGATAACGCAACCCTGACAC GGTTCTTCGCATTCCATTTTCTACTCCCTTTCGTCATCGCCGGCGCGACCATCCTCCACTTACTGTTTTTACACGAAACAGGATCGAACAAC CCGGCCGGCCTAAACTCTGATGCAGATAAAATCTCTTTCCATCCATACTTTTCATACAAAGACCTTCTTGGTTTCATTGTAATATTATTAGCC CTGACATCATTAGCTTTATTCTCCCCAAACTTACTAGGTGACCCAGACAATTTTACACCCGCAAACCCTTTAGTTACTCCCCCTCATATTCAG CCTGAGTGATACTTCTTATTTGCCTACGCCATTCTTCGATCTATTCCAAATAAGCTAGGCGGGGTCCTTGCACTACTATTTAGCATCCTAGT GCTAATGGTTGTACCTATTCTACATACCTCTAAACAACGAGGACTCACTTTCCGTCCTATGACCCAATTTCTATTTTGAACCCTGGTGGCAG ATATACTTATTCTTACATGAATTGGTGGTATACCCGTAGAACACCCCTATGTCATAATTGGCCAAATCGCATCCGTCCTATACTTTGCC

K CCTCTAACATTTCCGCGCTCTGAAATTTTGGGTCCCTTCTTGGATTATGTTTAATTACTCAAATTTTAACAGGATTATTTTTGGCAATACATT ACACCTCTGATATCTCAACCGCATTTTCATCCGTAGCCCATATCTGCCGGGACGTAAATTACGGCTGACTCATTCGAAACTTACACGCTAAC GGGGCGTCCTTCTTCTTCATCTGCGTTTATATACACATCGCCCGAGGACTTTATTACGGGTCCTATCTTTATAAAGAAACCTGAAACATTGG TGTAGTCTTACTCCTATTAGTTATAATGACGGCCTTCGTGGGATATGTGCTTCCCTGAGGACAAATATCCTTTTGAGGCGCCACAGTAATC ACAAATTTATTATCAGCAGTCCCTTATATAGGAGACATGCTTGTACAATGAATTTGAGGGGGCTTTTCTGTAGATAACGCAACCCTGACAC GGTTCTTCGCATTCCATTTTCTACTCCCTTTCGTCATCGCCGGCGCGACCATCCTCCACTTACTGTTTTTACACGAAACAGGATCGAACAAC CCGGCCGGCCTAAACTCTGATGCAGATAAAATCTCTTTCCATCCATACTTTTCATACAAAGACCTTCTTGGTTTCATTGTAATATTATTAGCC CTGACATCATTAGCTTTATTCTCCCCAAACTTACTAGGTGACCCAGACAATTTTACACCCGCAAACCCTTTAGTTACTCCCCCTCATATTCAG CCTGAGTGATACTTCTTATTTGCCTACGCCATTCTTCGATCTATTCCAAATAAGCTAGGCGGGGTCCTTGCACTACTATTTAGCATCCTAGT GCTAATGGTTGTACCTATTCTACATACCTCTAAACAACGAGGACTCACTTTCCGTCCTATGACCCAATTTCTATTTTGAACCCTGGTGGCAG ATATACTTATTCTTACATGAATTGGTGGTATACCCGTAGAACACCCCTATGTCATAATTGGCCAAATCGCATCCGTCCTATACTTTGCC

L CCTCTAACATTTCCGCGCTCTGAAATTTTGGATCCCTTCTTGGATTATGTTTAATTACTCAAATTTTAACAGGATTATTTTTGGCAATACATT ACACCTCTGATATCTCAACCGCATTTTCATCCGTAGCCCATATCTGCCGGGACGTAAATTACGGCTGACTCATTCGAAACCTACACGCTAAT GGAGCATCCTTCTTCTTCATCTGCGTTTACATACACATCGCCCGAGGACTTTATTACGGGTCCTATCTTTATAAAGAAACCTGAAACATTGG TGTAGTCTTACTGCTATTGGTTATAATGACAGCCTTCGTGGGATATGTACTTCCCTGAGGACAAATATCCTTTTGAGGCGCCACAGTAATT ACAAATTTATTATCAGCAGTCCCTTATATAGGAGACATACTTGTACAATGAATTTGAGGGGGCTTTTCTGTAGATAACGCAACCCTGACAC GGTTCTTCGCATTCCATTTTCTACTCCCCTTCGTCATCGCCGGCGCGACCATCCTCCACTTACTGTTTTTACACGAAACAGGATCGAACAAC CCGGCCGGCCTAAACTCTGATGCAGATAAAATCTCTTTCCATCCATACTTTTCATACAAAGACCTTCTTGGTTTCATTGTAATATTATTAGCC CTGACATCATTAGCTTTATTCTCCCCAAACTTACTAGGTGACCCAGACAATTTTACACCCGCAAACCCTTTAGTTACTCCCCCTCATATTCAG CCTGAATGATACTTCTTATTTGCCTACGCCATTCTTCGATCTATTCCAAATAAGCTAGGCGGGGTCCTTGCACTACTATTTAGCATCCTAGT GCTAATGGTTGTACCTATTCTACATACCTCTAAACAACGGGGACTCACTTTCCGTCCTGCAACCCAATTTCTATTTTGAACCCTGGTGGCAG ATATACTTATTCTTACATGAATTGGTGGTATACCCGTAGAACACCCCTATGTCATAATTGGCCAAATCGCATCCGTCCTATACTTTGCC

M CCTCTAACAT TTCCGCGCTCTGAAATTTTGGGTCCCTTCTTGGATTATGTTTAATTACTCAAATTTTAACAGGATTATTTTTGGCAATACATT ACACCTCTGATATCTCAACCGCATTTTCATCCGTAGCCCATATCTGCCGGGACGTAAATTACGGCTGACTCATTCGAAACTTACACGCTAAC GGGGCATCCTTCTTCTTCATCTGCGTTTATATACACATCGCCCGAGGACTTTATTACGGGTCCTATCTTTATAAAGAAACCTGAAACATTGG TGTAGTCTTACTCCTATTAGTTATAATGACAGCCTTCGTGGGATATGTGCTTCCCTGAGGACAAATATCCTTTTGAGGCGCCACAGTAATC ACAAATTTATTATCAGCAGTCCCTTATATAGGAGACATGCTTGTACAATGAATTTGAGGGGGCTTTTCTGTAGATAACGCAACCCTGACAC GGTTCTTCGCATTCCATTTTCTACTCCCTTTCGTCATCGCCGGCGCGACCATCCTCCACTTACTGTTTTTACACGAAACAGGATCGAACAAC CCGGCCGGCCTAAACTCTGATGCAGATAAAATCTCTTTCCATCCATACTTTTCATACAAAGACCTTCTTGGTTTCATTGTAATATTATTAGCC CTGACATCATTAGCTTTATTCTCCCCAAACTTACTAGGTGACCCAGACAATTTTACACCCGCAAACCCTTTAGTTACTCCCCCTCATATTCAG CCTGAATGATACTTCTTATTTGCCTACGCCATTCTTCGATCTATTCCAAATAAACTAGGCGGGGTCCTTGCACTACTATTTAGCATCCTAGT GCTAATGGTTGTACCTATTCTACATACCTCTAAACAACGAGGACTCACTTTCCGTCCTATGACCCAATTTCTATTTTGAACCCTGGTGGCAG ATATACTTATTCTTACATGAATTGGTGGTATACCCGTAGAACACCCCTATGTCATAATTGGCCAAATCGCATCCGTCCTATACTTTGCC

N CCTCTAACATTTCCGCGCTC TGAAATTTTGGGTCCCTTCTTGGATTATGTTTAATTACTCAAATTTTAACAGGATTATTTTTGGCAATACATT ACACCTCTGATATCTCAACCGCATTTTCATCCGTAGCCCATATCTGCCGGGACGTAAATTACGGCTGACTCATTCGAAACTTACACGCTAAC GGGGCATCCTTCTTCTTCATCTGCGTTTACATACACATCGCCCGAGGACTTTATTACGGGTCCTATCTTTATAAAGAAACCTGAAACATTGG TGTAATCTTACTCCTATTAGTTATAATGACAGCCTTCGTGGGATATGTGCTTCCCTGAGGACAAATATCCTTTTGAGGCGCCACAGTAATC ACAAATTTATTATCAGCAGTCCCTTATATAGGAGACATGCTTGTACAATGAATTTGAGGGGGCTTTTCTGTAGATAACGCAACCCTGACAC GGTTCTTCGCATTCCATTTTCTACTCCCTTTCGTCATCGCCGGCGCGACCATCCTCCACTTACTGTTTTTACACGAAACAGGATCGAACAAC CCGGCCGGCCTAAACTCTGATGCAGATAAAATCTCTTTCCATCCATACTTTTCATACAAAGACCTTCTTGGTTTCATTGTAATATTATTAGCC CTGACATCATTAGCTTTATTCTCCCCAAACTTACTAGGTGACCCAGACAATTTTACACCCGCAAACCCTTTAGTTACTCCCCCTCATATTCAG CCTGAGTGATACTTCTTATTTGCCTACGCCATTCTTCGATCTATTCCAAATAAGCTAGGCGGGGTCCTTGCACTACTATTTAGCATCCTAGT GCTAATGGTTGTACCTATTCTACATACCTCTAAACAACGAGGACTCACTTTCCGTCCTATGACCCAATTTCTATTTTGAACCCTGGTGGCAG ATATACTTATTCTTACATGAATTGGTGGTATACCCGTAGAACACCCCTATGTCATAATTGGCCAAATCGCATCCGTCCTATACTTTGCC

57

O CCTCTAACATTTCCGCGCTCTGAAATTTTG GGTCCCTTCTTGGATTATGTTTAATTACTCAAATTTTAACAGGATTATTTTTGGCAATACATT ACACCTCTGATATCTCAACCGCATTTTCATCCGTAGCCCATATCTGCCGGGACGTAAATTACGGCTGACTCATTCGAAACTTACACGCTAAC GGGGCATCCTTCTTCTTCATCTGCGTTTATATACACATCGCCCGAGGACTTTATTACGGGTCCTATCTTTATAAAGAAACCTGAAACATTGG TGTAGTCTTACTCCTATTAGTTATAATGACAGCCTTCGTGGGATATGTGCTTCCCTGAGGACAAATATCCTTTTGAGGCGCCACAGTAATC ACAAATTTATTATCAGCAGTCCCTTATATAGGAGACATGCTTGTACAATGAATTTGAGGGGGCTTTTCTGTAGATAACGCAACCCTGACAC GGTTCTTCGCATTCCATTTTCTACTCCCTTTCGTCATCGCCGGCGCGACCATCCTCCACTTACTGTTTTTACACGAAACAGGATCGAACAAC CCGGCCGGCCTAAACTCTGATGCAGATAAAATCTCTTTCCATCCATACTTTTCATACAAAGACCTTCTTGGTTTCATTGTAATATTATTAGCC CTGACATCATTAGCTTTATTCTCCCCAAACTTACTAGGTGACCCAGACAATTTTACACCCGCAAACCCTTTAGTTACTCCCCCTCATATTCAG CCTGAGTGATACTTCTTATTTGCCTACGCCATTCTTCGATCTATTCCAAATAAGCTAGGCGGGGTCCTTGCACTACTATTTAGCATCCTAGT GCTAATAGTTGTACCTATTCTACATACCTCTAAACAACGAGGACTCACTTTCCGTCCTATGACCCAATTTCTATTTTGAACCCTGGTGGCAG ATATACTTATTCTTACATGAATTGGTGGTATACCCGTAGAACACCCCTATGTCATAATTGGCCAAATCGCATCCGTCCTATACTTTGCC

P CCTCTAACATTTCCGCGCTCTGAAATTTTGGGTCCCTTCTTGGATTATGTTTAATTACTCAAATTTTAACAGGATTATTTTTGGCAATACATT ACACCTCTGATATCTCAACCGCATTTTCATCCGTAGCCCATATCTGCCGGGACGTAAATTACGGCTGACTCATTCGAAACTTACACGCTAAC GGGGCATCCTTCTTCTTCATCTGCGTTTATATACACATCGCCCGAGGACTTTATTACGGGTCCTATCTTTATAAAGAAACCTGAAACATTGG TGTAGTCTTACTCCTATTAGTTATAATGACAGCCTTCGTGGGATATGTGCTTCCCTGAGGACAAATATCCTTTTGAGGCGCCACAGTAATC ACAAATTTATTATCAGCAGTCCCTTATATAGGAGACATGCTTGTACAATGAATTTGAGGGGGCTTTTCTGTAGATAACGCAACCCTGACAC GGTTCTTCGCATTCCATTTTCTACTCCCTTTCGTCATCGCCGGCGCGACCATCCTCCACTTACTGTTTTTACACGAAACAGGATCGAACAAC CCGGCCGGCCTAAACTCTGATGCAGATAAAATCTCTTTCCATCCATACTTTTCATACAAAGACCTTCTTGGTTTCATTGTAATATTATTAGCC CTGACATCATTAGCTTTATTTTCCCCAAACTTACTAGGTGACCCAGACAATTTTACACCCGCAAACCCTTTAGTTACTCCCCCTCATATTCAG CCTGAGTGATACTTCTTATTTGCCTACGCCATTCTTCGATCTATTCCAAATAAGCTAGGCGGGGTCCTTGCACTACTATTTAGCATCCTAGT GCTAATGGTTGTACCTATTCTACATACCTCTAAACAACGAGGACTCACTTTCCGTCCTATGACCCAATTTCTATTTTGAACCCTGGTGGCAG ATATACTTATTCTTACATGAATTGGTGGTATACCCGTAGAACACCCCTATGTCATAATTGGCCAAATCGCATCCGTCCTATACTTTGCC

Q CCTCTAACATTTCCGCGCTCTGAAATTTTGGATCCCTTCTTGGATTATGTTTAATTACTCAAATTTTAACAGGATTATTTTTGGCAATACATT ACACCTCTGATATCTCAACCGCATTTTCATCCGTAGCCCATATCTGCCGGGACGTAAATTACGGCTGACTCATTCGAAACCTACACGCTAAT GGAGCATCCTTCTTCTTCATCTGCGTTTACATACACATCGCCCGAGGACTTTATTACGGGTCCTATCTTTATAAAGAAACCTGAAACATTGG CGTAGTCTTACTGCTATTAGTTATAATGACAGCCTTCGTGGGATATGTACTTCCCTGAGGACAAATATCCTTTTGAGGCGCCACAGTAATT ACAAATTTATTATCAGCAGTCCCTTATATAGGAGACATACTTGTACAATGAATTTGAGGGGGCTTTTCTGTAGATAACGCAACCCTGACAC GGTTCTTCGCATTCCATTTTCTACTCCCCTTCGTCATCGCCGGCGCGACCATCCTCCACTTACTGTTTTTACACGAAACAGGATCGAACAAC CCGGCCGGCCTAAACTCTGATGCAGATAAAATCTCTTTCCATCCATACTTTTCATACAAAGACCTTCTTGGTTTCATTGTAATATTATTAGCC CTGACATCATTAGCTTTATTCTCCCCAAACTTACTAGGTGACCCAGACAATTTTACACCCGCAAACCCTTTAGTTACTCCCCCTCATATTCAG CCTGAATGATACTTCTTATTTGCCTACGCCATTCTTCGATCTATTCCAAATAAGCTAGGCGGGGTCCTTGCACTACTATTTAGCATCCTAGT GCTAATGGTTGTACCTATTCTACATACCTCTAAACAACGAGGACTCACTTTCCGTCCTGCAACCCAATTTCTATTTTGAACCCTGGTGGCAG ATATACTTATTCTTACATGAATTGGTGGTATACCCGTAGAaCACCCCTATGTCATAATTGGCcAAATCGCATCCGTCCTATACTTTGCC

R CCTCTAACAT TTCCGCGCTCTGAAATTTTGGATCCCTTCTTGGATTATGTTTAATTACTCAAATTTTAACAGGATTATTTTTGGCAATACATT ACACCTCTGATATCTCAACCGCATTTTCATCCGTAGCCCATATCTGCCGGGACGTAAATTACGGCTGACTCATTCGAAACCTACACGCTAAT GGAGCATCCTTCTTCTTCATCTGCGTTTACATACACATCGCCCGAGGACTTTATTACGGGTCCTATCTTTATAAAGAAACCTGAAACATTGG CGTAGTCTTACTGCTATTAGTTATAATGACAGCCTTCGTGGGATATGTACTTCCCTGAGGACAAATATCCTTTTGAGGCGCCACAGTAATT ACAAATTTATTATCAGCAGTCCCTTATATAGGAGACATACTTGTACAATGAATTTGAGGGGGCTTTTCTGTAGATAACGCAACCCTGACAC GGTTCTTCGCATTCCATTTTCTACTCCCCTTCGTCATCGCCGGCGCGACCATCCTCCACTTACTGTTTTTACACGAAACAGGATCGAACAAC CCGGCCGGCCTAAACTCTGATGCAGATAAAATCTCTTTCCATCCATACTTTTCATACAAAGACCTTCTTGGTTTCATTGTAATATTATTAGCC CTGACATCATTAGCTTTATTCTCCCCAAACTTACTAGGTGACCCAGACAATTTTACACCCGCAAACCCTTTAGTTACTCCCCCTCATATTCAG CCTGAATGATACTTCTTATTTGCCTACGCCATTCTTCGATCCATTCCAAATAAGCTAGGCGGGGTCCTTGCACTACTATTTAGCATCCTAGT GCTAATGGTTGTACCTATTCTACATACCTCTAAACAACGAGGACTCACTTTCCGTCCTGCAACCCAATTTCTATTTTGAACCCTGGTGGCAG ATATACTTATTCTTACATGAATTGGTGGTATACCCGTAGAACACCCCTATGTCATAATTGGCCAAATCGCATCCGTCCTATACTTTGCC

S CCTCTAACATTTCCGCGCTC TGAAATTTTGGGTCCCTTCTTGGATTATGTTTAATTACTCAAATTTTAACAGGATTATTTTTGGCAATACATT ACACCTCTGATATCTCAACCGCATTTTCATCCGTAGCCCATATCTGCCGGGACGTAAATTACGGCTGACTCATTCGAAACTTACACGCTAAC GGGGCATCCTTCTTCTTCATCTGCGTTTATATACACATCGCCCGAGGACTTTATTACGGGTCCTATCTTTATAAAGAAACCTGAAACATTGG TGTAGTCTTACTCCTATTAGTTATAATGACAGCCTTCGTGGGATATGTGCTTCCCTGAGGACAAATATCCTTTTGAGGCGCCACAGTAATC ACAAATTTATTATCAGCAGTCCCTTATATAGGAGACATGCTTGTACAATGAATTTGAGGGGGCTTTTCTGTAGATAACGCAACCCTGACAC GGTTCTTCGCATTCCATTTTCTACTCCCTTTCGTCATCGCCGGCGCGACCATCCTCCACTTACTGTTTTTACACGAAACAGGATCGAACAAC CCGGCCGGCCTAAACTCTGATGCAGATAAAATCTCTTTCCATCCATACTTTTCATACAAAGACCTTCTTGGTTTCATTGTAATATTATTAGCC CTGACATCATTAGCTTTATTCTCCCCAAACTTACTAGGTGACCCAGACAATTTTACACCCGCAAACCCTTTAGTTACTCCCCCTCATATTCAG CCTGAGTGATACTTCTTATTTGCCTACGCCATTCTTCGATCTATTCCAAATAAGCTAGGCGGGGTCCTTGCACTACTATTTAGCATCCTAGT GCTAATGGTTGTACCTATTCTACATACCTCTAAACAACGAGGACTCACTTTCCGTCCTATGACCCAATTTCTATTTTGAACCCTGGTGGCAG ATATACTTATTCTTACATGAATTGGTGGTATACCCGTAGAACACCCTTATGTCATAATTGGCCAAATCGCATCCGTCCTATACTTTGCC

58

T CCTCTAACATTTCCGCGCTCTGAAATTTTG GGTCCCTTCTTGGATTATGTTTAATTACTCAAATTTTAACAGGATTATTTTTGGCAATACATT ACACCTCTGATATCTCAACCGCATTTTCATCCGTAGCCCATATCTGCCGGGACGTAAATTACGGCTGACTCATTCGAAACTTACACGCTAAC GGGGCATCCTTCTTCTTCATCTGCGTTTATATACACATCGCCCGAGGACTTTATTACGGGTCCTATCTTTATAAAGAAACCTGAAACATTGG TGTAGTCTTACTCCTATTAGTTATAATGACAGCCTTCGTGGGATATGTGCTTCCCTGAGGACAAATATCCTTTTGAGGCGCCACAGTAATC ACAAATTTATTATCAGCAGTCCCTTATATAGGAGACATGCTTGTACAATGAATTTGAGGGGGCTTTTCTGTAGATAACGCAACCCTGACAC GGTTCTTCGCATTCCATTTTCTACTCCCTTTCGTCATCGCCGGCGCGACCATCCTCCACTTACTGTTTTTACACGAAACAGGATCGAACAAC CCGGCCGGCCTAAACTCTGATGCAGATAAAATCTCTTTCCATCCATACTTTTCATACAAAGACCTTCTTGGTTTCATTGTAATATTATTAGCC CTGACATCATTAGCTTTATTCTCCCCAAACTTACTAGGTGACCCAGACAATTTTACACCCGCAAACCCTTTAGTTACTCCCCCTCATATTCAG CCTGAATGATACTTCTTATTTGCCTACGCCATTCTTCGATCTATTCCAAATAAGCTAGGCGGGGTCCTTGCACTACTATTTAGCATCCTAGT GCTAATGGTTGTACCTATTCTACATACCTCTAAACAACGAGGACTCACTTTCCGTCCTATGACCCAATTTCTATTTTGAACCCTGGTGGCAG ATATACTTATTCTTACATGAATTGGTGGTATACCCGTAGAACACCCCTATGTCATAATTGGCCAAATCGCATCCGTCCTATACTTTGCC

U CCTCTAACATTTCCGCGCTCTGAAATTTTGGGTCCCTTCTTGGATTATGTTTAATTACTCAAATTTTAACAGGATTATTTTTGGCAATACATT ACACCTCTGATATCTCAACCGCATTTTCATCCGTGGCCCATATCTGCCGGGACGTAAATTACGGCTGACTCATTCGAAACTTACACGCTAAC GGGGCATCCTTCTTCTTCATCTGCGTTTATATACACATCGCCCGAGGACTTTATTACGGGTCCTATCTTTATAAAGAAACCTGAAACATTGG TGTAATCTTACTCCTATTAGTTATAATGACAGCCTTCGTGGGATATGTGCTTCCCTGAGGACAAATATCCTTTTGAGGCGCCACAGTAATC ACAAATTTATTATCAGCAGTCCCTTATATAGGAGACATGCTTGTACAATGAATTTGAGGGGGCTTTTCTGTAGATAACGCAACCCTGACAC GGTTCTTCGCATTCCATTTTCTACTCCCTTTCGTCATCGCCGGCGCGACCATCCTCCACTTACTGTTTTTACACGAAACAGGATCGAACAAC CCGGCCGGCCTAAACTCTGATGCAGATAAAATCTCTTTCCATCCATACTTTTCATACAAAGACCTTCTTGGTTTCATTGTAATATTATTAGCC CTGACATCATTAGCTTTATTCTCCCCAAACTTACTAGGTGACCCAGACAATTTTACACCCGCAAACCCTTTAGTTACTCCCCCTCATATTCAG CCTGAGTGATACTTCTTATTTGCCTACGCCATTCTTCGATCTATTCCAAATAAGCTAGGCGGGGTCCTTGCACTACTATTTAGCATCCTAGT GCTAATGGTTGTACCTATTCTACATACCTCTAAACAACGAGGACTCACTTTCCGTCCTATGACCCAATTTCTATTTTGAACCCTGGTGGCAG ATATACTTATTCTTACATGAATTGGTGGTATACCCGTAGAACACCCCTATGTCATAATTGGCCAAATCGCATCCGTCCTATACTTTGCC

V CCTCTAACATTTCCGCGCTCTGAAATTTTGGGTCCCTTCTTGGATTATGTTTAATTACTCAAATTTTAACAGGATTATTTTTGGCAATACATT ACACCTCTGATATCTCAACCGCATTTTCATCCGTAGCCCATATCTGCCGGGACGTAAATTACGGCTGACTCATTCGAAACTTACACGCTAAC GGGGCATCCTTCTTCTTCATCTGCGTTTATATACACATCGCCCGAGGACTTTATTACGGGTCCTATCTTTATAAAGAAACCTGAAACATTGG TGTAGTCTTACTCCTATTAGTTATAATGACAGCCTTCGTGGGATATGTGCTTCCCTGAGGACAAATATCCTTTTGAGGCGCCACAGTAATC ACAAATTTATTATCAGCAGTCCCTTATATAGGAGACATGCTTGTACAATGAATTTGAGGGGGCTTTTCTGTAGATAACGCAACCCTGACAC GGTTCTTCGCATTCCATTTTCTACTCCCTTTCGTCATCGCCGGCGCGACCATCCTCCACTTACTGTTTTTACACGAAACAGGATCGAACAAC CCGGCCGGCCTAAACTCTGATGCAGATAAAATCTCTTTCCATCCATACTTTTCATACAAAGACCTTCTTGGTTTCATTGTAATATTATTAGCC CTGACATCATTAGCTTTATTCTCCCCAAACTTACTAGGTGACCCAGACAATTTTACACCCGCAAACCCTTTAGTTACTCCCCCTCATATTCAG CCTGAGTGATACTTCTTATTTGCCTACGCCATTCTTCGATCTATTCCAAATAAGCTAGGCGGGGTCCTTGCACTACTATTTAGCATCCTAGT GCTAATGGTTGTACCTATTCTACATACCTCTAAACAACGAGGACTCACTTTCCGTCCTATGACCCAATTTCTATTTTGAACCCTGGTGGCAG ATATACTTATTCTTACATGAATTGGCGGTATACCCGTAGAACACCCCTATGTCATAATTGGCCAAATCGCATCCGTCCTATACTTTGCC

W CCTCTAACAT TTCCGCGCTCTGAAATTTTGGGTCCCTTCTTGGATTATGTTTAATTACTCAAATTTTAACAGGATTATTTTTGGCAATACATT ACACCTCTGATATCTCAACCGCATTTTCATCCGTAGCCCATATCTGCCGGGACGTAAATTACGGCTGACTCATTCGAAACTTACACGCTAAC GGGGCATCCTTCTTCTTCATCTGCGTTTATATACACATCGCCCGAGGACTTTATTACGGGTCCTATCTTTATAAAGAAACCTGAAACATTGG TGTAATCTTACTCCTATTAGTTATAATGACAGCCTTCGTGGGATATGTGCTTCCCTGAGGACAAATATCCTTTTGAGGCGCCACAGTAATC ACAAATTTATTATCAGCAGTCCCTTATATAGGAGACATGCTTGTACAATGAATTTGAGGGGGCTTTTCTGTAGATAACGCAACCCTGACAC GGTTCTTCGCATTCCATTTTCTACTCCCTTTCGTCATCGCCGGCGCGACCATCCTCCACTTACTGTTTTTACACGAAACAGGATCGAACAAC CCGGCCGGCCTAAACTCTGATGCAGATAAAATCTCTTTCCATCCATACTTTTCATACAAAGACCTTCTTGGTTTCATTGTAATATTATTAGCC CTGACATCATTAGCTTTATTCTCCCCAAACTTACTAGGTGACCCAGACAATTTTACACCCGCAAACCCTTTAGTTACTCCCCCTCATATTCAG CCTGAGTGATACTTCTTATTTGCCTACGCCATTCTTCGATCTATTCCAAATAAGCTAGGCGGGGTCCTTGCACTACTATTTAGCATCCTAGT GCTAATGGTTGTACCTATTCTACATACCTCTAAACAACGAGGACTCACTTTCCGTCCTATGACCCAATTTCTATTTTGAACCCTGGTGGCAG ATATACTTATTCTTACATGAATTGGCGGTATACCCGTAGAACACCCCTATGTCATAATTGGCCAAATCGCATCCGTCCTATACTTTGCC

X CCTCTAACATTTCCGCGCTC TGAAATTTTGGGTCCCTTCTTGGATTATGTTTAATTACTCAAATTTTAACAGGATTATTTTTGGCAATACATT ACACCTCTGATATCTCAACCGCATTTTCATCCGTAGCCCATATCTGCCGGGACGTAAATTACGGCTGACTCATTCGAAACTTACACGCTAAC GGGGCGTCCTTCTTCTTCATCTGCGTTTATATACACATCGCCCGAGGACTTTATTACGGGTCCTATCTTTATAAAGAAACCTGAAACATTGG TGTAGTCTTACTCCTATTAGTTATAATGACGGCCTTCGTGGGATATGTGCTTCCCTGAGGACAAATATCCTTTTGAGGCGCCACAGTAATC ACAAATTTATTATCAGCAGTCCCTTATATAGGAGACATGCTTGTACAATGAATTTGAGGGGGCTTTTCTGTAGATAACGCAACCCTGACAC GGTTCTTCGCATTCCATTTTCTACTCCCTTTCGTCATCGCCGGCGCGACCATCCTCCACTTACTGTTTTTACACGAAACAGGATCGAACAAC CCGGCCGGCCTAAACTCTGATGCAGATAAAATCTCTTTCCATCCATACTTTTCATACAAAGACCTTCTTGGTTTCATTGTAATATTATTAGCC CTGACATCATTAGCTTTATTCTCCCCAAACTTACTAGGTGACCCAGACAATTTTACACCCGCAAACCCTTTAGTTACTCCCCCTCATATTCAG CCTGAGTGATACTTCTTATTTGCCTACGCCATTCTTCGGTCTATTCCAAATAAGCTAGGCGGGGTCCTTGCACTACTATTTAGCATCCTAGT GCTAATGGTTGTACCTATTCTACATACCTCTAAACAACGAGGACTCACTTTCCGTCCTATGACCCAATTTCTATTTTGAACCCTGGTGGCAG ATATACTTATTCTTACATGAATTGGTGGTATACCCGTAGAACACCCCTATGTCATAATTGGCCAAATCGCATCCGTCCTATACTTTGCC

59

Y CCTCTAACATTTCCGCGCTCTGAAATTTTG GATCCCTTCTTGGATTATGTTTAATTACTCAAATTTTAACAGGATTATTTTTGGCAATACATT ACACCTCTGATATCTCAACCGCATTTTCATCCGTAGCCCATATCTGCCGGGACGTAAATTACGGCTGACTCATTCGAAACCTACACGCTAAT GGAGCATCCTTCTTCTTCATCTGCGTTTACATACACATCGCCCGAGGACTTTATTACGGGTCCTATCTTTATAAAGAAACCTGAAACATTGG TGTAGTCTTACTGCTATTAGTTATAATGACAGCCTTCGTGGGATATGTACTTCCCTGAGGACAAATATCCTTTTGAGGCGCCACAGTAATT ACAAATTTATTATCAGCAGTCCCTTATATAGGAGACATACTTGTACAATGAATTTGAGGGGGCTTTTCTGTAGATAACGCAACCCTGACAC GGTTCTTCGCATTCCATTTTCTACTCCCCTTCGTCATCGCCGGCGCGACCATCCTCCACTTACTGTTTTTACACGAAACAGGATCGAACAAC CCGGCCGGCCTAAACTCTGATGCAGATAAAATCTCTTTCCATCCATACTTTTCATACAAAGACCTTCTTGGTTTCATTGTAATATTATTAGCC CTGACATCATTAGCTTTATTCTCCCCAAACTTACTAGGTGACCCAGACAATTTTACACCCGCAAACCCTTTAGTTACTCCCCCTCATATTCAG CCTGAATGATACTTCTTATTTGCCTACGCCATTCTTCGATCTATTCCAAATAAGCTAGGCGGGGTCCTTGCACTACTATTTAGCATCCTAGT GCTAATGGTTGTACCTATTCTACATACCTCTAAACAACGAGGACTCACTTTCCGTCCTGTAACCCAATTTCTATTTTGAACCCTGGTGGCAG ATATACTTATTCTTACATGAATTGGTGGTATACCCGTAGAACACCCCTATGTCATAATTGGCCAAATCGCATCCGTCCTATACTTTGCC

Z CCTCTAACATTTCCGCGCTCTGAAATTTTGGGTCCCTTCTTGGATTATGTTTAATTACTCAAATTTTAACAGGATTATTTTTGGCAATACATT ACACCTCTGATATCTCAACCGCATTTTCATCCGTAGCCCATATCTGCCGGGACGTAAATTACGGCTGACTCATTCGAAACTTACACGCTAAC GGGGCATCCTTCTTCTTCATCTGCGTTTATATACACATCGCCCGAGGACTTTATTACGGGTCCTATCTTTATAAAGAAACCTGAAACATTGG TGTAATCTTACTCCTATTAGTTATAATGACAGCCTTCGTGGGATATGTGCTTCCCTGAGGACAAATATCCTTTTGAGGCGCCACAGTAATC ACAAATTTATTATCAGCAGTCCCTTATATAGGAGACATGCTTGTACAATGAATTTGAGGGGGCTTTTCTGTAGATAACGCAACCCTGACAC GGTTCTTCGCATTCCATTTTCTACTCCCTTTCGTCATCGCCGGCGCGACCATCCTCCACTTACTGTTTTTACACGAAACAGGATCGAACAAC CCGGCCGGCCTAAACTCTGATGCAGATAAAATCTCTTTCCATCCATACTTTTCATACAAAGACCTTCTTGGTTTCATTGTAATATTATTAGCC CTGACATCATTAGCTTTATTCTCCCCAAACTTACTAGGTGACCCAGACAATTTTACACCCGCAAACCCTTTAGTTACTCCCCCTCATATTCAG CCTGAGTGATACTTCTTATTTGCCTACGCCATTCTTCGATCTATTCCAAATAAGCTAGGCGGGGTCCTTGCACTACTATTTAGCATCCTAGT GCTAATGGTTGTACCTATTCTACATACCTCTAAACAACGAGGACTCACTTTCCGTCCTATGACCCAATTTCTATTTTGAACCCTGGTGGCAG ATATACTTATTCTTACATGAATTGGTGGTATACCCGTAGAACACCCCTATATCATAATTGGCCAAATCGCATCCGTCCTATACTTTGCC

AA CCTCTAACATTTCCGCGCTCTGAAATTTTGGGTCCCTTCTTGGATTATGTTTAATTACTCAAATTTTAACAGGATTATTTTTGGCAATACATT ACACCTCTGATATCTCAACCGCATTTTCATCCGTAGCCCATATCTGCCGGGACGTAAATTACGGCTGACTCATTCGAAACTTACACGCTAAC GGGGCATCCTTCTTCTTCATCTGCGTTTATATACACATCGCCCGAGGACTTTATTACGGGTCCTATCTTTATAAAGAAACCTGAAACATTGG TGTAATCCTACTCCTATTAGTTATAATGACAGCCTTCGTGGGATATGTGCTTCCCTGAGGACAAATATCCTTTTGAGGCGCCACAGTAATC ACAAATTTATTATCAGCAGTCCCTTATATAGGAGACATGCTTGTACAATGAATTTGAGGGGGCTTTTCTGTAGATAACGCAACCCTGACAC GGTTCTTCGCATTCCATTTTCTACTCCCTTTCGTCATCGCCGGCGCGACCATCCTCCACTTACTGTTTTTACACGAAACAGGATCGAACAAC CCGGCCGGCCTAAACTCTGATGCAGATAAAATCTCTTTCCATCCATACTTTTCATACAAAGACCTTCTTGGTTTCATTGTAATATTATTAGCC CTGACATCATTAGCTTTATTCTCCCCAAACTTACTAGGTGACCCAGACAATTTTACACCCGCAAACCCTTTAGTTACTCCCCCTCATATTCAG CCTGAGTGATACTTCTTATTTGCCTACGCCATTCTTCGATCTATTCCAAATAAGCTAGGCGGGGTCCTTGCACTACTATTTAGCATCCTAGT GCTAATGGTTGTACCTGTTCTACATACCTCTAAACAACGAGGACTCACTTTCCGTCCTATGACCCAATTTCTATTTTGAACCCTGGTGGCAG ATATACTTATTCTTACATGAATTGGTGGTATACCCGTAGAACACCCCTATGTCATAATTGGCCAAATCGCATCCGTCCTATACTTTGCC

AB CCTCTAACA TTTCCGCGCTCTGAAATTTTGGGTCCCTTCTTGGATTATGTTTAATTACTCAAATTTTAACAGGACTATTTTTGGCAATACATT ACACCTCTGATATCTCAACCGCATTTTCATCCGTAGCCCATATCTGCCGGGACGTAAATTACGGCTGACTCATTCGAAACTTACACGCTAAC GGGGCATCCTTCTTCTTCATCTGCGTTTACATACACATCGCCCGAGGACTTTATTACGGGTCCTATCTTTATAAAGAAACCTGAAACATTGG TGTAATCTTACTCCTATTAGTTATAATGACAGCCTTCGTGGGATATGTGCTTCCCTGAGGACAAATATCCTTTTGAGGCGCCACAGTAATC ACAAATTTATTATCAGCAGTCCCTTATATAGGAGACATGCTTGTACAATGAATTTGAGGGGGCTTTTCTGTAGATAACGCAACCCTGACAC GGTTCTTCGCATTCCATTTTCTACTCCCTTTCGTCATCGCCGGCGCGACCATCCTCCACTTACTGTTTTTACACGAAACAGGATCGAACAAC CCGGCCGGCCTAAACTCTGATGCAGATAAAATCTCTTTCCATCCATACTTTTCATACAAAGACCTTCTTGGTTTCATTGTAATATTATTAGCC CTGACATCATTAGCTTTATTCTCCCCAAACTTACTAGGTGACCCAGACAATTTTACACCCGCAAACCCTTTAGTTACTCCCCCTCATATTCAG CCTGAGTGATACTTCTTATTTGCCTACGCCATTCTTCGATCTATTCCAAATAAGCTAGGCGGGGTCCTTGCACTACTATTTAGCATCCTAGT GCTAATGGTTGTACCTATTCTACATACCTCTAAACAACGAGGACTCACTTTCCGTCCTATGACCCAATTTCTATTTTGAACCCTGGTGGCAG ATATACTTATTCTTACATGAATTGGTGGTATACCCGTAGAACACCCCTATGTCATAATTGGCCAAATCGCATCCGTCCTATACTTTGCC

AC CCTCTAACATTTCCGCGC TCTGAAATTTTGGGTCCCTTCTTGGATTATGTTTAATTACTCAAATTTTAACAGGATTATTTTTGGCAATACATT ACACCTCTGATATCTCAACCGCATTTTCATCCGTAGCCCATATCTGCCGGGACGTAAATTACGGCTGACTCATTCGAAACTTACACGCTAAC GGGGCATCCTTCTTCTTCATCTGCGTTTATATACACATCGCCCGAGGACTTTATTACGGGTCCTATCTTTATAAAGAAACCTGAAACATTGG TGTAATCTTACTCCTATTAGTTATAATGACAGCCTTCGTGGGATATGTGCTTCCCTGAGGACAAATATCCTTTTGAGGCGCCACAGTAATC ACAAATTTATTATCAGCAGTCCCTTATATAGGAGACATGCTTGTACAATGAATTTGAGGGGGCTTTTCTGTAGATAACGCAACCCTGACAC GGTTCTTCGCATTCCATTTTCTACTCCCTTTCGTCATCGCCGGCGCGACCATCCTCCACTTACTGTTTTTACACGAAACAGGATCGAACAAC CCGGCCGGCCTAAACTCTGATGCAGATAAAATCTCTTTCCATCCATACTTTTCATACAAAGACCTTCTTGGTTTCATTGTAATATTATTAGCC CTGACATCATTAGCTTTATTCTCCCCAAACTTACTAGGTGACCCAGACAATTTTACACCCGCAAACCCTTTAGTTACTCCCCCTCATATTCAG CCTGAGTGATACTTCTTATTTGCCTACGCCATTCTTCGATCTATTCCAAATAAGCTAGGCGGGGTCCTTGCACTACTATTTAGCATCCTAGT GCTAATGGTTGTACCTATTCTACATACCTCTAAACAACGAGGACTCACTTTCCGTCCTATGACCCAATTTCTATTTTGAACCCTGGTGGCAG ATATACTTATTCTTACATGAATTGGTGGTATACCCGTAGAACACCCCTATGTCATAATTGGGCAAATCGCATCCGTCCTATACTTTGCC

60

AD CCTCTAACATTTCCGCGCTCTGAAATT TTGGGTCCCTTCTTGGATTATGTTTAATTACTCAAATTTTAACAGGATTATTTTTGGCAATACATT ACACCTCTGATATCTCAACCGCATTTTCATCCGTAGCCCATATCTGCCGGGACGTAAATTACGGCTGACTCATTCGAAACTTACACGCTAAC GGGGCATCCTTCTTCTTCATCTGCGTTTATATACACATCGCCCGAGGACTTTATTACGGGTCCTATCTTTATAAAGAAACCTGAAACATTGG TGTAGTCTTACTCCTATTAGTTATAATGACAGCCTTCGTGGGATATGTGCTTCCCTGAGGACAAATATCCTTTTGAGGCGCCACAGTAATC ACAAATTTATTATCAGCAGTCCCTTATATAGGAGACATGCTTGTACAATGAATTTGAGGGGGCTTTTCTGTAGATAACGCAACCCTGACAC GGTTCTTCGCATTCCATTTTCTACTCCCTTTCGTCATCGCCGGCGCGACCATCCTCCACTTACTGTTTTTACACGAAACAGGATCGAACAAC CCGGCCGGCCTAAACTCTGATGCAGATAAAATCTCTTTCCATCCATACTTTTCATACAAAGACCTTCTTGGTTTCATTGTAATATTATTAGCC CTAACATCATTAGCTTTATTCTCCCCAAACTTACTAGGTGACCCAGACAATTTTACACCCGCAAACCCTTTAGTTACTCCCCCTCATATTCAG CCTGAGTGATACTTCTTATTTGCCTACGCCATTCTTCGATCTATTCCAAATAAGCTAGGCGGGGTCCTTGCACTACTATTTAGCATCCTAGT GCTAATGGTTGTACCTATTCTACATACCTCTAAACAACGAGGACTCACTTTCCGTCCTATGACCCAATTTCTATTTTGAACCCTGGTGGCAG ATATACTTATTCTTACATGAATTGGTGGTATACCCGTAGAACACCCCTATGTCATAATTGGCCAAATCGCATCCGTCCTATACTTTGCC

AE CCTCCAACATTTCCGCGCTCTGAAATTTTGGGTCCCTTCTTGGATTATGTTTAATTACTCAAATTTTAACAGGATTATTTTTGGCAATACATT ACACCTCTGATATCTCAACCGCATTTTCATCCGTGGCCCATATCTGCCGGGACGTAAATTACGGCTGACTCATTCGAAACCTACACGCTAAT GGAGCATCCTTCTTCTTCATCTGCGTTTATATACACATCGCCCGAGGACTTTATTACGGGTCCTATCTTTATAAAGAAACCTGAAACATTGG TGTAGTCTTACTCCTATTAGTTATAATGACAGCCTTCGTAGGGTATGTACTTCCCTGAGGACAAATATCCTTTTGAGGCGCCACAGTAATC ACAAATTTATTATCAGCAGTCCCTTATATAGGAGACATGCTTGTACAGTGAATTTGAGGGGGCTTTTCTGTAGATAACGCAACCCTGACAC GGTTCTTCGCATTCCATTTTCTACTCCCTTTCGTCATCGCCGGCGCGACCATCCTCCACTTACTATTTTTACACGAAACAGGGTCGAACAAC CCGGCCGGCCTAAACTCTGATGCAGATAAAATCTCTTTCCATCCATACTTTTCATACAAAGACCTTCTTGGTTTCATTGTAATATTATTAGCC CTAACATCATTAGCTTTATTTTCCCCAAACTTACTAGGTGACCCAGACAATTTTACACCCGCAAACCCTTTAGTTACTCCCCCTCATATTCAG CCTGAATGATACTTCTTATTTGCCTACGCCATTCTTCGATCTATCCCAAATAAGCTAGGTGGGGTCCTTGCGCTACTATTTAGCATCCTAGT GCTAATGGTTGTACCTATTCTACATACCTCTAAACAACGAGGACTCACTTTCCGTCCTATAACCCAATTTCTATTTTGAACCCTGGTGGCAG ATATACTTATTCTTACATGAATTGGTGGTATACCCGTAGAACACCCCTATGTCATAATTGGCCAAATCGCATCCGTCCTATACTTTGCC

61

APPENDIX B

GENBANK ACCESSION NUMBERS AND SEQUENCE DATA FOR ALL OUTGROUPS INCLUDED IN ANALYSES

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GenBank Accession Species Number Sequence Semotilus HQ446761.1 ATGGCAAGCCTACGAAAAACTCACCCACTAATTAAAA atromaculatus TCGCTAATGACGCATTGGTTGATTTACCCACACCCTCT AATATCTCAGCACTCTGAAACTTCGGGTCCCTCCTAG GCCTATGTTTAATTACTCAAATCCTAACAGGACTGTTC CTAGCCATACACTACACCTCCGACATCTCAACCGCAT TTTCATCGGTAGCCCATATCTGCCGAGATGTTAATTAC GGCTGACTTATCCGAAACTTACACGCTAACGGCGCAT CCTTCTTCTTCATTTGTATTTATATACACATTGCCCGA GGCCTCTATTACGGATCTTACCTTTATAAAGAAACCT GAAACATCGGTGTAGTTTTACTTCTTTTAGTAATGATG ACCGCCTTCGTGGGCTACGTCCTTCCTTGAGGACAAA TATCCTTTTGAGGCGCCACAGTAATTACTAACTTATTA TCAGCAGTCCCTTACATAGGAGATACCCTTGTCCAGT GAATTTGAGGAGGCTTCTCTGTAGATAACGCGACCCT CACACGATTCTTCGCATTCCACTTCCTGCTGCCGTTCG TCATCGCCGGCGCAACCGTCCTACACCTTCTATTCCTA CACGAAACGGGATCGAACAACCCGGCCGGCCTGAAC TCTGATGCGGATAAAATTTCTTTCCATCCATACTTTTC ATACAAAGACCTTCTTGGCTTCATTCTAATATTGTTGG CCTTAACGTCACTGGCCCTATTCTCCCCAAATTTACTA GGTGACCCAGACAATTTTACCCCAGCAAACCCTCTGG TCACTCCTCCACATATTCAGCCAGAATGATACTTTCTA TTTGCCTACGCCATCTTACGTTCCATTCCTAATAAATT AGGAGGGGTCCTTGCACTACTATTCAGTATTTTAGTAC TAATAGTTGTACCTATTCTACATACCTCAAAACAACG AGGACTAACCTTCCGTCCTATGACCCAATTCTTATTCT GAACCCTCGTAGCAGATATACTTATCTTAACATGAAT TGGGGGTATACCCGTAGAACACCCCTACGTCATAATT GGCCAAATCGCATCCGTCTTATACTTTGCACTATTTCT CATTCTTATCCCTCTAACAGGATGAATTGAAAATAAA GCACTGAAATGA

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Couesius AY281053.1 ATGGCAAGCCTACGAAAAACCCACCCACTAATAAAA plumbeus ATCGCTAATGGTGCATTAGTTGATCTCCCAACACCAT CCAATATTTCAGCACTCTGAAACTTCGGGTCCCTCCTA GGACTATGTTTAATTACCCAAATTCTAACAGGATTGTT CCTAGCCATACACTATACCTCTGACATCTCAACTGCAT TTTCATCAGTGGCCCATATTTGCCGAGATGTAAATTAC GGCTGACTTATCCGAAACCTACATGCCAACGGAGCAT CATTCTTCTTTATCTGTATCTACATACACATTGCCCGC GGCCTATACTATGGGTCCTATCTTTATAAAGAAACCT GAAATATTGGCGTGGTGCTACTTCTTTTGGTCATGATA ACAGCCTTTGTTGGCTATGTCCTCCCATGGGGACAAA TATCTTTTTGAGGTGCCACAGTCATTACCAACCTGCTA TCAGCAGTCCCTTATATAGGAGACACCCTTGTTCAGT GAATTTGAGGGGGCTTCTCAGTAGACAACGCAACCCT AACGCGGTTCTTCGCATTCCACTTCCTCCTACCATTTG TCGTCGCCGGCGCAACCATCCTACATTTGTTGTTTTTA CACGAAACGGGATCAAACAACCCAGCCGGACTAAAC TCTGACGCGGATAAAATTTCTTTCCACCCATACTTCTC ATATAAAGATCTTCTCGGCTTCGTCGTAATACTACTAG CTCTCACATCATTAGCCCTATTTTCCCCAAATCTACTG GGCGACCCAGACAACTTCACTCCAGCAAATCCCCTGG TCACCCCGCCACACATCCAACCGGAATGATACTTTTT ATTTGCCTACGCCATCCTACGATCTATTCCTAACAAGC TGGGAGGGGTTCTTGCGCTATTGTTCAGTATTCTAGTG CTGATAGTTGTCCCCATTCTACACACCTCGAAACAGC GGGGACTCACTTTCCGCCCTATGACCCAGTTCCTATTC TGAACCTTAGTAGCAGATATGCTTATCCTTACATGAAT CGGAGGCATGCCTGTAGAACACCCATATGTCATGATT GGCCAAGTCGCATCAATCTTATACTTTGCACTCTTCCT CATCCTTATCCCACTAACAGGATGAGTTGAGAACAAA GCACTGAAATGAGCC

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Lepidomeda JX443056.1 ATGGCAAGCCTACGAAAGACTCACCCACTAATAAAA vittata ATCGCTAATGGTGCATTAGTCGATCTCCCCACACCAT CTAACATTTCAGCACTCTGAAACTTCGGATCCCTCCTA GGGCTCTGTTTAATTACCCAAATCCTAACAGGGTTATT TCTAGCCATACACTACACCTCTGATATTTCAACTGCGT TTTCATCAGTCACCCATATCTGCCGAGACGTTAATTAC GGCTGACTTATCCGAAACCTACATGCCAACGGCGCAT CCTTCTTCTTCATCTGTATTTATATACATATCGCCCGA GGCCTTTATTACGGGTCATACCTTTATAAGGAGACCT GAAACATCGGGGTGGTTCTACTCCTTCTAGTTATGATG ACAGCCTTTGTGGGCTATGTACTTCCATGGGGCCAAA TATCTTTTTGAGGGGCCACAGTAATCACGAACCTACT GTCGGCAGTCCCTTATATGGGCGATACCCTTGTTCAGT GGATTTGGGGCGGGTTCTCAGTAGATAACGCAACTCT TACACGGTTCTTCGCGTTCCACTTTCTCCTCCCATTCG TCATCGCCGGTGCAACCCTCCTGCATTTACTATTTTTA CACGAAACGGGGTCCAACAACCCAGCCGGACTAAAC TCTGACGCCGACAAGATTTCTTTCCACCCGTACTTCTC ATACAAAGACCTCCTTGGCTTTGTCGTAATATTATTAG CTCTTACGTCCCTGGCGCTGTTTTCCCCAAATCTCCTG GGTGACCCAGACAACTTTACCCCTGCAAACCCACTGG TTACCCCTCCGCACATCCAGCCCGAGTGATACTTTTTG TTTGCCTATGCTATTCTACGATCTATTCCAAACAAACT AGGAGGGGTCCTTGCACTACTCTTTAGTATTCTGGTAC TAATGGTAGTCCCGATTCTACACACCTCTAAACAACG AGGACTCACCTTCCGCCCCGTGACCCAATTCCTATTCT GAACCCTCGTAGCAGACATACTTATTCTGACATGAAT CGGAGGTATACCTGTAGAGCACCCCTATGTCATAATT GGCCAAGTCGCATCAATCTTGTACTTTACACTCTTTCT AGTTCTTATTCCACTAACGGGCTGGGTTGAAAATAAA GCACTGAAATGAGCC

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Meda fulgida JX443054.1 ATGGCAAGCCTACGAAAAACTCACCCACTAATAAAA ATCGCTAACAGTGCATTAGTAGACCTCCCCACACCGT CTAACATTTCAGCACTCTGAAACTTCGGGTCCCTCTTG GGGCTGTGCTTAATTGCCCAGCTCCTGACAGGACTAT TCCTTGCTATGCACTATGCCTCTGATATTTCAACTGCA TTTTCATCGGTCGCCCACATCTGCCGGGACGTTAATTA CGGCTGGCTCATTCGGAACCTTCATGCCAACGGCGCA TCCTTCTTCTTCATCTGTATCTACATACATATCGCCCG CGGCCTATACTACGGCTCTTACCTCTACAAGGAAACC TGGAATATTGGAGTAATCCTTCTCCTGCTGGTTATGAT GACTGCCTTTGTAGGCTATGTTCTTCCCTGAGGACAG ATATCTTTTTGGGGTGCCACGGTGATTACGAATTTACT ATCAGCAGTTCCTTACATAGGAGACACCCTTGTTCAG TGGATTTGGGGCGGCTTCTCGGTAGATAACGCCACTC TTACCCGGTTCTTCGCGTTCCACTTCCTCCTCCCGTTT GTCATCGTCGCCGCGACCCTTATGCACCTGTTGTTTCT TCACGAAACGGGGTCTAACAACCCAGCCGGACTAAA CTCCGACGCAGACAAAGTTTCTTTCCACCCGTACTTCT CGTACAAGGACCTCCTAGGCTTTATCGTGATGTTACT GGCCTTAACATCCCTTGCCCTGTTTTCCCCGAACCTGC TTGGCGATCCGGATAACTTCACCCCGGCAAACCCGCT AGTTACACCCCCACACATCCAGCCAGAATGATACTTC CTGTTTGCCTATGCTATCCTACGATCTATCCCGAACAA GCTAGGCGGGGTCCTCGCGCTACTCTTTAGTATTCTAG TGCTAATACTAGTCCCACTGATACACACCTCTAAGCA ACGTGGACTAACCTTCCGCCCAGTGACCCAATTCTTA TTCTGAGCCCTCGTAGCGGACGTACTTATTCTTACCTG AATTGGGGGCATACCCGTAGAACACCCATATGTCGCA ATTGGACAAATCGCATCCCTGTTATACTTTGCACTGTT TCTTGTACTTATCCCACTGGCAGGGTGAGTCGAGAAC AAAGCATTGAAATGAGCC

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Margariscus JX443011.1 ATGGCAAGCCTACGAAAGACTCATCCACTAATAAAAA margarita TTGCTAATGATGCATTAGTCGACCTCCCCACACCGTCC AATATTTCAGCACTCTGAAACTTCGGGTCCCTCCTAG GATTGTGTTTAATTACCCAAATCCTAACAGGGCTATTT CTAGCTATACACTATACATCTGATATCTCAACTGCATT TTCATCAGTGACACATATTTGTCGAGATGTTAACTACG GCTGACTTATCCGAAACCTTCATGCCAACGGAGCATC GTTCTTCTTCATCTGTATTTATATACACATTGCCCGCG GGCTATACTACGGGTCATACCTTTATAAAGAGACCTG AAATATTGGGGTAGTCCTTCTCCTTCTAGTCATGATGA CAGCCTTCGTCGGCTATGTACTTCCCTGAGGCCAAAT ATCCTTTTGAGGCGCCACTGTAATTACAAACCTTCTAT CAGCAGTCCCTTACATAGGCGACACCCTTGTTCAATG AATCTGAGGCGGCTTCTCAGTAGATAATGCAACGCTA ACACGATTCTTCGCGTTCCACTTCCTCCTGCCATTCGT CATCGCCGGCGCAACCGTCCTCCACTTGTTATTCTTAC ACGAAACGGGGTCGAACAACCCGGCCGGGCTAAACT CTGACGCGGATAAGATTTCTTTCCACCCATACTTCTCG TATAAGGACCTTCTTGGCTTCGTGGTAATATTATTAGC ACTCACATCACTGGCCCTATTTTCCCCTAATCTACTGG GCGACCCAGACAACTTCACCCCCGCCAACCCCTTAGT TACCCCGCCGCACATCCAGCCGGAATGATACTTCTTA TTTGCCTACGCCATCCTACGATCTATCCCTAACAAACT TGGGGGAGTTCTTGCATTATTATTTAGCATTCTAGTAC TAATGGTCGTGCCTATTCTACATACCTCGAAACAACG AGGACTCACCTTCCGTCCGATGACCCAATTTCTGTTCT GAACTTTAGTGGCAGATATACTCATTCTGACATGAAT TGGAGGCATACCCGTAGAACACCCATATGTCATAATT GGCCAAGTCGCCTCAATCTTATATTTCGCGCTCTTCCT CGTCCTTATCCCGCTAACAGGATGAGTTGAAAATAAA GCACTTAAATGAGCT

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Snyderichthys AF452086.1 ATGGCAAGCCTACGAAAGACTCACCCGCTAATAAAA copei ATCGCTAATAGTGCATTAGTCGATCTCCCTACACCATC TAACATTTCAGCACTCTGAAACTTCGGATCCCTCCTAG GGCTCTGTTTAATTACCCAAATCCTAACAGGGTTATTT CTAGCCATACACTACACCTCTGATATTTCAACCGCGTT TTCGTCAGTCACCCATATCTGCCGGGACGTTAATTAC GGATGACTTATCCGAAACCTACATGCCAACGCGGCAT CCTTCTTCTTCATCTGTATTTACATACATATCGCCCGG GGCCTCTATTACGGATCATACCTCTATAAGGAGACCT GAAGCATCGGGGTAGTCCTACTCCTTCTAGTTATGAT AACAGCCTTTGTGGGCTATGTGCTTCCATGGGGACAA ATATCTTTTTGAGGTGCCACAGTAATCACGAACCTGTT ATCAGCAGTCCCTTACATAGGCGATACCCTTGTTCAG TGAATTTGGGGCGGGTTCTCAGTAGATAACGCGACTC TTACACGGTTCTTCGCGTTCCACTTCCTCCTTCCATTC GTCATCGCCGGCGCAACTCTCCTCCATTTACTATTTTT ACACGAAACGGGGTCCAACAACCCAGCCGGACTGAA CTCTGACGCCGACAAAATCTCTTTCCACCCGTACTTCT CATATAAAGACCTCCTTGGCTTTGTCGTAATGTTGTTA GCTCTCACATCTCTGGCGCTGTTTTCCCCAAATCTGCT AGGCGATCCAGACAACTTTACCCCCGCAAACCCACTG GTTACCCCACCACACATCCAGCCCGAGTGATACTTTTT ATTTGCCTATGCTATTCTACGATCTATTCCAAACAAGC TAGGAGGAGTCCTAGCGCTACTATTTAGTATTCTGGT ACTAATAGTAGTCCCAATTCTGCACACCTCAAAACAA CGAGGACTTACCTTTCGCCCGATGACCCAATTCCTATT CTGAACCCTCGTAGCAGACATAATTATTCTGACGTGA ATCGGAGGTATACCCGTAGAACACCCGTATGTCATAA TTGGTCAAGTCGCATCAATCTTGTACTTTACGCTCTTT CTAGTTCTTATTCCACTAACGGGCTGGGTTGAAAATA AAGCACTGAAATGAGCC

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Micropterus ATGGCAAGCCTCCGAAAAACCCACCCCTTACTAAAAA salmoides TCGCAAACGACGCACTCGTCGACCTCCCCGCTCCATC AAACATCTCGGTCGGATGAAACTTCGGCTCCCTGCTG GGCCTCTGCCTAGCAACCCAAATCCTCACAGGCTTAT TCCTCGCTATACACTATACCTCTGATATCGCAACCGCC TTCTCATCCGTTGCCCACATTTTTTTTGATGTAAACTA CGGCTGACTAATTCGAAACATTCATGCTAATGGTGCA TCCTTCTTTTTCATCTGCATCTACCTGCATATCGGCCG GGGATTATATTATGGTTCATACCTTTACAAAGAGACA TGAAACATCGGAGTAATCCTCCTCCTCCTAGTCATAA TGACTGCCTTCGTAGGGTACGTCCTTCCCTGAGGACA AATATCCTTTTGAGGCGCAACAGTTATTACTAATCTCC TCTCGGCCGTCCCTTACATCGGTAACACCCTAGTTCAA TGAATCTGAGGCGGCTTCTCAGTCGACAATGCCACCC TCACCCGCTTCTTTGCCTTCCACTTCCTATTTCCATTTG TCATCGCTGCCGCCACAGTAATTCATCTACTCTTTCTT CACGAAACAGGATCTAACAACCCCCTAGGACTAAACT CTGACGCCGATAAAATTTCATTTCACCCCTACTTCTCC TATAAAGACTTGCTCGGGTTTGCAGCTCTCCTCATTGC CCTTACCTCATTAGCCTTATTTTCCCCCAACCTTCTAG GGGACCCAGACAACTTTACCCCCGCCAACCCCCTAGT CACACCCCCTCATATTAAACCAGAGTGATATTTCCTAT TTGCCTATGCCATCCTTCGCTCCATCCCCAACAAACTG GGCGGGGTCTTGGCCCTTCTCGCCTCAATCCTCGTACT AATAGTCGTCCCCATCCTCCACACCTCCAAACAACGA GGCCTAACGTTCCGCCCCCTCACGCAGCTCCTCTTCTG AGCCCTCGTCGCAGACGTCGCCATCTTAACCTGAATT GGAGGTATGCCCGTTGAACATCCTTTTATTATCATTGG CCAAGTTGCCTCTTTCTTGTACTTTTTCCTCTTCCTGTT CCTTTCTCCCCTAGCAGGCTGGCTGGAGAACAAGGCC CTCGAATGATCC

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APPENDIX C

PROOF OF PROJECT APPROVAL BY THE UTC INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE

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VITA

Kathlina Alford was born in Lawrenceburg, Tennessee, to the parents of Rev.

Russell and Dorothy Flatt. She is the youngest of three children, an older brother, Charles, and an older sister, Regina. She attended Livingston Academy in Livingston, Tennessee. After high school graduation, she attended Tennessee Technological University where she became interested in fishery sciences. She completed the Bachelors of Science degree in May 2004 in

Biology with a concentration in Zoology and a minor in Chemistry. Kathlina completed an internship her senior year of college at the Tennessee Aquarium and was subsequently offered a full-time position as an Aquarist. She worked for four years for the Tennessee Aquarium before entering the graduate school at the University of Tennessee at Chattanooga in the Environmental

Sciences Program. Kathlina graduated with a Masters of Science degree in Environmental

Science in August 2013. She continues to work at the Tennessee Aquarium as the Conservation

Associate doing propagation work with endangered species and conservation genetics research.

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