THE SPREAD OF INVASIVE VIRILE ( VIRILIS) IN THE CURRENT RIVER DRAINAGE OF MISSOURI, U.S.A.: HYBRIDIZATION AND POTENTIAL IMPACTS ON DETECTION AND MANAGEMENT

BY

ZACHARY ROZANSKY

THESIS

Submitted in partial fulfillment of the requirements for the degree of Master of Science in Natural Resources and Environmental Sciences in the Graduate College of the University of Illinois at Urbana-Champaign, 2019

Urbana, Illinois

Adviser:

Dr. Christopher A. Taylor

ABSTRACT

The introduction, establishment, and spread of invasive species has had many irreparable negative effects on ecosystems and causes significant economic harm. are a diverse group of freshwater which have proven to be harmful invasive species throughout the world. Additionally, invasive crayfishes are a leading cause of displacement of native crayfishes. Detecting invasive species and determining their distribution is critical to assessing risks to ecosystems and selecting effective management and control options. In this study, we wanted to determine the distribution of non-native in the Ozark National Scenic

Riverways, Missouri, U.S.A., where it has been spreading since the 1980s. Additionally, we wanted to develop an environmental DNA (eDNA) assay to aid in detection of F. virilis. While developing the F. virilis-specific eDNA assay, we noticed a discordance between the phenotype and mitochondrial DNA barcode of some native F. punctimanus specimens. As evidenced by a mismatch in mitochondrial, phenotypic, and microsatellite data, we have found that non-native

F. virilis have hybridized with native F. punctimanus in the Ozark National Scenic Riverways.

Hybridization has rarely been documented between crayfish species and before this study, the only genetically documented reports of North American crayfish species hybridizing have been between invasive F. rusticus and two congeners. Traditional sampling outperformed eDNA in our system and hybridization was not supported by modelling as a factor influencing eDNA detections. While it did not perform well for detecting F. virilis in the Current River watershed, eDNA remains an important tool. Overall, our results support previous researchers’ remarks that undocumented hybridization between native and non-native crayfish may be more common than previously thought.

ii ACKNOWLEDGEMENTS

My sincere thanks go to my advisor, Dr. Christopher Taylor, for his guidance over the past two years and for helping me develop skills that I will use in my career as a biologist. I would also like to extend thanks to Dr. Eric Larson, who was integral in the development and completion of this project. Thank you to Dr. Yong Cao, for being a member of my committee and helping improve my thesis. I would like to thank everyone who helped me in the field including George Balto, Danny Morrill, Amaryllis Adey, Kathleen Quebedeaux, and Caroline

Caton. I would also like to thank those who assisted and guided me with laboratory work including Amanda Curtis, Kurt Ash, and David Zaya. Finally, I would like to thank my family and friends for supporting me through my graduate education.

iii TABLE OF CONTENTS

CHAPTER 1: GENERAL INTRODUCTION ...... 1 CHAPTER 2: INVASIVE VIRILE CRAYFISH (FAXONIUS VIRILIS) HYBRIDIZES WITH NATIVE SPOTHANDED CRAYFISH (FAXONIUS PUNCTIMANUS) IN THE CURRENT RIVER WATERSHED OF MISSOURI, U.S.A ...... 12 CHAPTER 3: TRADITIONAL SAMPLING OUTPERFORMS ENVIRONMENTAL DNA FOR DETECTING INVASIVE VIRILE CRAYFISH (FAXONIUS VIRILIS) IN THE CURRENT RIVER WATERSHED OF MISSOURI, U.S.A ...... 48 SUMMARY ...... 82 APPENDIX A: MICROSATELLITE PRIMERS ...... 83 APPENDIX B: SUPPLEMENTAL TREES...... 84

iv CHAPTER 1: GENERAL INTRODUCTION

Species historically isolated by biogeographic barriers have spread beyond their native ranges due to human activities. The accidental or intentional transport, establishment, and spread of non-indigenous species has resulted in a decline in biodiversity and ecosystem function around the globe. While many species have been translocated, most fail to establish or have minimal impacts (Havel et al. 2015, Thomaz et al. 2015). However, invasive species are the few that successfully establish and cause considerable ecological disruption. Invasive species are recognized as a leading cause of biodiversity decline (Leung and Mandrak 2007) and the rate of introductions is growing (Thomaz et al. 2015). Freshwater ecosystems are particularly prone to harm by invasive species because of their high degree of isolation and endemism (Vander Zanden and Olden 2008). Aquatic invasive species have been shown to reduce biodiversity by altering habitats, restructuring food webs, competing for resources, and transferring pathogens (Dudgeon et al. 2006, Havel et al. 2015). Furthermore, economic costs associated with aquatic invasive species are tremendous and an estimated $7.7 billion is spent each year for control and mitigation of impacts (Pimental et al. 2005, Havel et al. 2015).

Occupying freshwaters on six continents, crayfish are a diverse group of organisms that include many narrow-ranging endemics and harmful aquatic invaders. There are currently 669 described species of crayfish (Crandall and De Grave 2017) and represents the richest region with nearly 400 species (Crandall and De Grave 2017; Richman et al. 2015).

Crayfish are important components of their native ecosystems as they process detritus, consume plant matter, and prey on fish and (Momot 1995, Usio 2000). Additionally, they are a source of prey for fish, amphibians, reptiles, mammals, and birds with some organisms relying heavily upon this food (Rabeni 1992; Beja 1996; DiStefano et al. 2009). Crayfish are considered

1 ecosystem engineers because they affect the habitat quality and resources for other organisms

(Reynolds et al. 2013). Through their burrowing and feeding activity, crayfish alter habitats and open interstitial spaces for other organisms (Reynolds et al. 2013). Because of their large influence on ecosystems, crayfish have proven to be detrimental invasive species throughout the world and invasive crayfish are a primary threat to native crayfish biodiversity (Lodge et al. 2000;

Lodge et al. 2012; Twardochleb et al. 2013).

Crayfish have been translocated by bait-bucket transfers, pet owners, aquaculture, and intentional introductions (Lodge et al. 2000; DiStefano et al. 2009; Chucholl and Wendler 2017).

Invasive crayfish have been shown to alter habitat and water chemistry by reducing macrophytes

(Olsen 1991, van der Wal et al. 2013) and increasing turbidity (Matsuzaki et al. 2009). Food webs are disrupted by invasive crayfish and populations of macroinvertebrates (Nilsson et al.

2011) and fish have been shown to decline (Olsen 1991, Mueller 2006). The spread of invasive crayfish has also resulted in economic losses (Keller et al. 2008) and has harmed agriculture and infrastructure such as levees (Correia and Ferreira 1995). One invasive crayfish species has even been shown to hybridize with a native species (Perry et al. 2001, Arcella et al. 2014). Invasive crayfish species are known to vector pathogens and have contributed to widespread declines in

European crayfish when the crayfish plague (Aphanomyces astaci) was introduced alongside

North American species (Lodge et al. 2000). Many species in the United States have narrow native ranges and the introduction and spread of invasive crayfish could gravely threaten those endemic species (Lodge et al. 2000).

The focal species for this study, the Virile Crayfish (Faxonius virilis), is the most widely distributed crayfish in North America (Taylor et al. 2015) and occurs in its native range from the

Hudson Bay watershed in south to across the midwestern United States. Faxonius virilis

2 has been introduced throughout North America and parts of Europe and is considered invasive in many locations. Faxonius virilis has spread through bait-bucket transfers by anglers (DiStefano et al. 2009; Kilian et al. 2012), deliberate stocking by state wildlife agencies as sport fish forage

(Sheldon 1989) and escaping from laboratory ponds (Riegel 1959). Faxonius virilis has been found on the Atlantic Slope, the Pacific Northwest, the Southwest deserts, and the Southeast.

Examples of introduced populations of F. virilis include the Potomac watershed of Maryland

(Schwartz et al. 1963), the Black Warrior and Coosa watersheds in Alabama (Smith et al. 2011), the Snake River in Idaho (Larson et al. 2018), the Columbia River in Montana (Sheldon 1988), the Colorado River in Arizona (Moody and Sabo 2013), and the Current River in Missouri

(DiStefano et al. 2015). Outside of the United States, F. virilis is now established in Mexico and has expanded its range in Canada (Philips et al. 2009; Williams 2012). In Europe, F. virilis has become established in England and the Netherlands (Ahern et al. 2008).

Faxonius virilis has been assessed by the United States Fish and Wildlife Service to be a high overall risk as an invasive species due in part to its history of successful invasions (USFWS

2015). Faxonius virilis has been shown to have measurable impacts on aquatic ecosystems. It can reduce macrophytes and macroinvertebrates (Hanson 1990, Moody and Sabo 2013) and lessen the reproductive success of fish by preying on eggs (Dorn and Mittelbach 2004). In

Arizona, where there are no native crayfish species, invasive F. virilis contributes to the decline in native fish by outcompeting them for food resources (Carpenter 2005). Although displacement mechanisms have not been determined, F. virilis has displaced native Pilose Crayfish

(Pacifastacus gambelii) from much of its native range in the western United States (Egly and

Larson 2018). Furthermore, F. virilis have been shown to increase turbidity and damage levees through burrowing activity (Ahern et al. 2008, Davidson et al. 2010). Attempts to control

3 invasive populations of F. virilis have utilized microbial agents (Davidson et al. 2010) and chemicals (Recsetar and Bonar 2015), but eradication remains a challenge. Trapping and electrofishing have been tested for removing invasive F. virilis but were found to be ineffective

(Rogowski et al. 2013). As is the case with other aquatic invasive species, the removal of established F. virilis populations has proven difficult (Davidson et al. 2010; Havel et al. 2015).

Detecting invasive species and determining their distribution is critical to assessing the risks that they pose and in deciding effective management and control options. In this project, we set out to determine the distribution of invasive F. virilis in the Ozark National Scenic Riverways,

Missouri, U.S.A, a protected area that is administered by the National Park Service. Ozark

National Scenic Riverways encompasses over 200 kilometers of the free-flowing Jacks Fork and

Current River and invasive F. virilis has been detected in the park since 1986 (Pflieger 1996).

Previously, F. virilis has been found in up to 67 km of the Current River, making this the largest contiguous crayfish invasion in Missouri (DiStefano et al. 2015). In addition to kick-seine sampling, we developed an environmental DNA (eDNA) assay and examined its utility as a tool to monitor the spread and detect previously unknown locations for F. virilis. While developing the eDNA assay, we noticed a discordance between phenotype and expected COI sequences and investigated suspected hybridization between F. virilis and the native Spothanded Crayfish, F. punctimanus. Here we discuss hybridization between F. virilis and F. punctimanus and the possible effects this previously unknown hybridization had on the performance of an eDNA assay. Determining the extent of the invasion of F. virilis and documenting potential genetic impacts in the Ozark National Scenic Riverways will provide the National Park Service and other relevant agencies with information to determine possible management options.

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11 CHAPTER 2: INVASIVE VIRILE CRAYFISH (FAXONIUS VIRILIS) HYBRIDIZES WITH NATIVE SPOTHANDED CRAYFISH (FAXONIUS PUNCTIMANUS) IN THE CURRENT RIVER WATERSHED OF MISSOURI, U.S.A.

INTRODUCTION

The introduction, establishment, and spread of invasive species has many irreparable negative effects on ecosystems and causes significant economic harm. Invasive species reduce biodiversity by altering habitats (Cuddington and Hastings 2004; Anderson et al. 2006; Byers et al. 2010), restructuring food webs (Vander Zanden et al. 1999; Zavaleta et al. 2001), competing for resources (Callaway and Aschehoug 2000; Bergstrom and Mensinger 2011), and transferring pathogens (Sepúlveda et al. 2014; Mrugała et al. 2015). Invasive species can also cause harm by hybridizing with native species. Many examples of hybridization between native and non-native species have been documented, including within mammals (Senn and Pemberton 2009), fish

(Muhlfeld et al. 2014), birds (van de Crommenacker et al. 2015), reptiles (Vuillaume et al. 2015), amphibians (Riley et al. 2003), and plants (Meyerson et al. 2009). Hybridization between closely-related species occurs naturally, and this gene flow can provide benefits for populations.

Natural hybridization can maintain or increase genetic diversity through the formation of stable hybrid zones, rescue of inbred populations, development of new adaptations, or enforcement of reproductive isolation (Todesco et al. 2016). More often, hybridization between native and non- native species often results in negative consequences for populations.

Hybridization can occur when non-native species are introduced into the range of a closely-related native species and they retain the ability to mate and reproduce (Harrison and

Larson 2014). Hybridization where sterile offspring are produced may have little effect on populations and displacement of species (Arnold et al. 1999; Huxel 1999), although the wasted reproductive effort and decline in fitness can cause populations to decline (Todesco et al. 2016).

12 However, fertile offspring and resulting introgression, or the incorporation of genes from one species into another with subsequent hybridization and backcrossing (Harrison and Larson 2014), can facilitate the displacement of a native species (Huxel 1999; Wolf et al. 2001) and decreases diversity. Rare species can be particularly impacted through hybridization and introgression with non-native species (Rhymer and Simberloff 1996) and extirpation or extinction can occur rapidly

(Wolf et al. 2001). Additionally, hybridization can significantly affect the establishment success and subsequent impact of invasive species by increasing their fitness and adaptation to new environments (Hänfling 2007).

Crayfishes are a diverse group of organisms that is composed of widespread, rare, and narrowly-endemic taxa, as well as some harmful invasive species (Taylor et al. 2019). Invasive crayfish damage ecosystems by degrading water quality, altering habitat, and decreasing biodiversity. They consume macrophytes (Olsen 1991, van der Wal et al. 2013) and reduce populations of macroinvertebrates (Nilsson et al. 2011) and fish (Olsen 1991, Mueller 2006).

Invasive crayfish have been identified as a primary threat to narrow-ranging endemic crayfish species (Lodge et al. 2000). While invasive crayfish and their effects on ecosystems are well studied, evolutionary effects of these invasions are understudied, and known cases of hybridization between native and non-native crayfish are infrequent. Initially, literature on crayfish hybridization pertained strictly to morphological evidence and identified specimens that exhibited morphologically intermediate characters (Crocker 1957; Boyd and Page 1978; Capelli and Capelli 1980; Smith 1981). An early account of hybridization utilizing genetic techniques documented natural hybridization between two native cave-dwelling Procambarus spp. in Mexico

(Cesaroni et al. 1992). Induced hybridization between crayfish has also been documented (Berrill

13 1985; Lawrence et al. 2000). In an Australian aquaculture system, directional hybridization between two spp. resulted in the production of all-male progeny (Lawrence et al. 2000).

Hybridization detected using genetic methods has been documented between the highly invasive (Faxonius rusticus) and two other species, the Northern Clearwater

Crayfish (F. propinquus) and Sanborn’s Crayfish (F. sanbornii). In the most in-depth investigation of crayfish hybridization, Perry et al. (2001a) reported that F. rusticus hybridized with F. propinquus in upper Midwestern lakes of the U.S.A. Morphologically intermediate individuals were recognized, and hybridization was confirmed through allozyme analysis.

Hybridization between F. rusticus and F. propinquus was shown to hasten the demise of pure F. propinquus genotypes, as the hybrids were fertile and backcrossing with F. rusticus (Perry et al.

2001b, Arcella et al. 2014). Zuber et al. (2012) documented another case of invasive F. rusticus hybridizing with the native species F. sanbornii in Ohio, by examining nuclear and mitochondrial

DNA and allozymes. We are unaware of any other cases of documented hybridization between non-native and native crayfish, although this particular impact of invasive species may be common, but little investigated.

While examining the distribution of the invasive Virile Crayfish (F. virilis) in the Current

River of Missouri, U.S.A., we noticed a mismatch between phenotypic life coloration and mitochondrial DNA (mtDNA) between two species. Our initial observations indicated that native

Spothanded Crayfish (F. punctimanus) had mtDNA sequences matching invasive F. virilis.

Noting this mismatch, we hypothesized that F. punctimanus and F. virilis were hybridizing.

Faxonius virilis is the widest ranging crayfish in North America (Taylor et al. 2015) and is a successful invader in many parts of the United States (Kilian et al. 2010; Moody and Sabo 2013) and abroad in Europe (Ahern et al. 2008; Filipová et al. 2010). In the Current River, Missouri,

14 U.S.A, the species has expanded its range since it was first documented by Pflieger (1992) in

1986. To confirm that hybridization was occurring, we compared phenotype, mtDNA, and microsatellite fragment data.

METHODS

Study Location

The Ozark National Scenic Riverways is administered by the National Park Service and encompasses over 200 kilometers of the free-flowing Jacks Fork and Current River. Situated in the Ozark Mountains in south-central Missouri, the park is dominated by karst topography and contains more than 425 springs which supply much of the baseflow to the Jacks Fork and Current

River (Bowles and Dodd 2015). The watershed is primarily rural and contains hills covered in mixed pine (Pinus spp.) and oak (Quercus spp.) woodlands. The upper Current River and Jacks

Fork are home to five native crayfish species: Golden Crayfish (Faxonius luteus), Ozark Crayfish

(F. ozarkae), Spothanded Crayfish (F. punctimanus), Hubbs’ Crayfish ( hubbsi), and

Salem Cave Crayfish (C. hubrichti; Pflieger 1996). The Faxonius species and C. hubbsi are stream dwellers, while C. hubrichti is a cave-dweller and is not typically found in surface waters.

In addition to the native species, F. virilis has been introduced to the Current River and has been detected since the 1986 (Pflieger 1996).

Faxonius virilis occurs natively throughout much of Missouri, but it has been introduced to new watersheds in the southcentral portion of the state (DiStefano et al. 2015). The presence of

F. virilis in the southern Ozark subregion, including the Current River watershed, is thought to be the result of bait bucket introductions (Pflieger 1996). In the Current River watershed, Pflieger

(1996) documented two F. virilis site records during extensive surveys in the 1980s and early

15 1990s (154 collections at 55 sites in the watershed) in the upper reaches of the River near Pulltite access and at Akers (DiStefano et al. 2015). National Park Service staff, Illinois Natural History

Survey researchers, and volunteers have found additional F. virilis specimens since the initial surveys throughout the upper portion of the Current River.

Collection Sites

Between 2018 and 2019, crayfish were collected from the Current River and tributaries within the Ozark National Scenic Riverways in southern Missouri by kick-seining. A 3.3 m seine net was placed in the stream and rocks and substrate immediately upstream were disturbed, allowing the current to carry dislodged crayfish into the net. We collected additional F. punctimanus from one allopatric site >50 kilometers from the nearest known F. virilis population

(Jacks Fork at Buck Hollow Access; Figure 2.1).

Faxonius virilis and sympatric species were originally collected to aid in the development of an environmental DNA assay for F. virilis. Because it was a previously known location for the non-native species, we collected F. virilis and F. punctimanus from Spring Valley Creek on 7

March 2018 and 23 May 2018. Faxonius virilis and F. punctimanus were collected from Pulltite

Access on the Current River on 7 March 2018 and F. punctimanus was collected from Sinking

Creek on 7 March 2018. Mitochondrial DNA sequencing of these Spring Valley Creek specimens for eDNA assay development identified the potential hybridization that motivated our subsequent research. Additional F. punctimanus were then collected from Spring Valley Creek on 13 June 2019. Grassy Creek was chosen as a site to collect F. virilis specimens after it was discovered that the species dominated the crayfish fauna and F. virilis were collected on 10 July

2019.

16 Phenotype Identification

Faxonius virilis and F. punctimanus are nearly indistinguishable morphologically with only slight differences in Form I male gonopod characteristics (Pflieger 1996), so we relied on life coloration to separate these species. Faxonius virilis and F. punctimanus in the Current River watershed have key phenotypic differences that distinguish them (Figure 2.2). The common name of F. punctimanus, the Spothanded Crayfish, indicates a distinguishing feature: a distinct black spot at the base of the moveable finger on the chelae. While not every population of F. punctimanus possesses this chelae spot, the Current River population reliably does (Pflieger

1996). Faxonius virilis may possess a chelae spot, however, these spots exhibit irregular edges and are not as pronounced. We observed that F. punctimanus in the Current River watershed also have a spot on the underside of the chelae, while F. virilis do not. Faxonius virilis may have multiple spots or mottling on the chelae and always have paired dark blotches on the dorsal surface of each abdominal segment while F. punctimanus lack both characters (Fig. 2). Finally,

Faxonius punctimanus have a pronounced black saddle across the posterior of their cephalothorax, while F. virilis lack the saddle (Figure 2.2).

Cytochrome c Oxidase Subunit I (COI) Sequencing

We amplified a portion of the cytochrome c oxidase subunit I (COI) gene for each crayfish specimen. A 15 mg muscle tissue sample was extracted from the abdomen of each crayfish specimen and preserved in 99% ethanol. Crayfish specimens were individually tagged, preserved in 70% ethanol, and accessioned into the Illinois Natural History Survey’s Crustacean Collection

(Champaign, IL, U.S.A). DNA was isolated from the muscle samples using a DNeasy Blood and

Tissue Kit (Qiagen, Hilden, Germany) following the manufacturer’s spin column protocol for tissues. We amplified COI on a PTC-100 thermal cycler (MJ Research, Inc., Watertown,

17 Massachusetts, U.S.A.) using 1 µl of LCO1490 primer (5'-

GGTCAACAAATCATAAAGATATTGG-3'), 1 µl of HCO2198 primer (5'-

TAAACTTCAGGGTGACCAAAAAATCA-3'; Folmer et al. 1994), 21 µl of water, 2 µl of template DNA, and PuReTaq Ready-To-Go PCR beads (GE Healthcare, Buckinghamshire, UK).

The thermal profile for the PCR reactions was 94°C for 3 min, 45 cycles of 94°C for 30 seconds,

55°C for 30 seconds, and 72°C for 90 seconds, followed by a final 72°C extension. PCR products were cleaned using a QIAquick PCR Purification Kit (Qiagen). Sanger sequencing was conducted by the University of Illinois at Urbana-Champaign Core Sequencing Facility (Urbana,

IL, U.S.A.). Forward and reverse sequence reads were analyzed and edited using Sequencher 5.4

(Gene Codes Corporation, Ann Arbor, Michigan, U.S.A.) to obtain the final COI consensus sequences.

Microsatellite Sequencing

We examined nine microsatellite loci primers developed for F. virilis populations in

Canada by Williams et al. (2010) and found that eight were informative for both F. virilis and F. punctimanus in the Current River watershed (Table A.1). To ensure that these primers worked with the Current River population of F. virilis and F. punctimanus, we first ordered and tested unlabeled forward and reverse primers (Integrated DNA Technologies, Coralville, Iowa, U.S.A).

Primers were tested using the thermal profile of 94°C for 1 minute, 3 cycles of 94°C for 30 seconds, 48°C or 52°C for 20 seconds, and 72°C for 1 second, followed by 33 cycles of 94°C for

15 seconds, 52°C for 20 seconds, and 72°C for 1 second, and a final 72°C extension. PCR amplifications were performed in 20 µl reactions consisting of 1 µl each of forward and reverse primers, 10 µl of GoTaq® G2 Colorless Master Mix (Promega Corporation, Madison, Wisconsin,

18 U.S.A.), 5 µl of water, and 3 µl of template DNA. Products were run on a 2% agarose gel for confirmation of amplification.

Final PCR amplifications were performed in 10 µl reactions consisting of 5 µl of GoTaq®

G2 Colorless Master Mix, 0.25 µl each of fluorescently-labelled forward primers (Applied

Biosystems, Foster City, California, U.S.A.) used in Williams et al. (2010) and unlabeled-reverse primers, and 3 µl water, and 1.5 µl of DNA template. The thermal profile for PCR was the same as during the microsatellite primers test. PCR reactions were performed individually, and products were combined for fragment analysis. Combined products were coloaded with

GS600LIZ size standard (Applied Biosystems) and analyzed on ABI 3730 DNA Analyzer

(Applied Biosystems) at the University of Illinois at Urbana-Champaign Core Sequencing Facility

(Urbana, IL). Fragments were genotyped using Genemapper® version 4.0 (Applied Biosystems).

Analyses

To visualize the differences and mismatches between phenotype identification, COI sequences, and microsatellite fragment data, we constructed a neighbor joining tree for COI sequences, UPGMA dendrogram for microsatellite fragment data, and a principal coordinate analysis (PCoA) displaying microsatellite fragment data, COI, and phenotype identifications. To visualize differentiation between F. virilis and F. punctimanus COI groups, we constructed a bootstrapped (2000 iterations) neighbor joining tree in Mega X (Kumar et al. 2018). Outgroups on the neighbor joining tree were F. ozarkae and F. luteus that we collected from Sinking Creek and Spring Valley Creek of the Current River watershed respectively and C. hubbsi that we acquired from GenBank (GenBank accession #MG872957.1). A maximum likelihood tree

(Figure B.1) and maximum parsimony tree with 5000 bootstraps (Figure B.2) were also made and we noted the same separation between the two main groups. Microsatellite Analyzer version 4.05

19 (Dieringer and Schlötterer 2003) was used to calculate Nei’s distance for the microsatellite fragment data and Mega X (Kumar et al. 2018) was used to generate a UPGMA dendrogram based on the Nei’s genetic distance matrix. Outgroups were not added to the UPGMA dendrogram as it is unlikely that the microsatellite primers would amplify more distantly related species. Based on the microsatellite data, a PCoA based on pairwise FST values was generated in

Microsoft Excel using the add-on GenAlEx 6.5 (Peakall and Smouse 2012) to further visualize differences between the F. virilis and F. punctimanus populations.

RESULTS The COI neighbor-joining tree displayed separate clades for F. virilis and F. punctimanus with 100% bootstrap support (Figure 2.3). However, nine specimens are mismatched with their phenotype assignments not aligning with the COI groupings. Seven specimens identified as phenotypic F. virilis group instead with F. punctimanus and two specimens identified as phenotypic F. punctimanus group instead with F. virilis. Table 2.1 displays all specimens used in the analyses and shows the phenotype and COI assignments. The UPGMA dendrogram generated from microsatellite data recover two main groups that separate at the first node and align with phenotype identifications in all but one instance (Figure 2.4). The only specimen that did not align was SVC003 which exhibited a COI sequence and phenotype consistent with F. punctimanus, but it is grouping with F. virilis in the UPGMA dendrogram.

The PCoA (Figure 2.5) visualizes major variance in the microsatellite distance data. The first two axes explained 27.9% of the total variance, with 17.4% explained by Axis 1 and 10.5% explained by Axis 2. Faxonius virilis phenotypes load positively on the dominant axis one, whereas F. punctimanus phenotypes load negatively on the dominant axis one. Convex hulls

20 bound the monophyletic F. virilis or F. punctimanus as identified by the UPGMA dendrogram

(Figure 2.4).

DISCUSSION

Hybridization has rarely been documented between crayfish species. The only genetically documented examples within North American crayfishes have been between invasive F. rusticus and two other species, F. propinquus (Perry et al. 2001a) and F. sanbornii (Zuber et al. 2012).

Our findings indicate that introduced F. virilis are hybridizing with native F. punctimanus in the

Current River watershed. To make this discovery, crayfish were identified by phenotypic coloration and genotyped using both microsatellite fragment analysis data and COI sequences.

Phenotype assignments were concordant with microsatellite data in all but one specimen.

Phenotype/microsatellite data compared to COI partial subunit I sequence data revealed nine mismatched specimens. Between 43 total crayfishes collected from Grassy Creek and Spring

Valley Creek, we identified two crayfish exhibiting F. punctimanus phenotypes and F. virilis COI sequences and seven crayfish exhibiting F. virilis phenotypes and F. punctimanus COI sequences.

The incorporation of mitochondrial DNA (mtDNA) from F. punctimanus to F. virilis and vice versa indicates that hybridization has occurred. The occurrence of hybridization between these crayfishes shows that reliance on mtDNA barcoding to supplement phenotype-based identifications can lead to species misidentifications. While the ecological and evolutionary implications of our findings are unknown, we have shown that the introduction of F. virilis has had a genetic impact on a native crayfish species.

The mitochondrial COI gene is a commonly used genetic barcode because it generally shows high levels of divergence across closely related species and can be amplified from many

21 different taxa (Hebert et al. 2003; Toews and Brelsford 2012). For many species, mtDNA aligns with other genetic markers and phenotypes (Toews and Brelsford 2012). However, many cases of discordance between mtDNA and nuclear DNA have been reported and problems can arise when relying solely on mtDNA for species assignment and phylogeny reconstruction (Funk and

Omland 2003; Cong et al. 2017). Often, a discordance between mtDNA and nuclear DNA is due to hybridization and introgression or incomplete lineage sorting (Funk and Omland 2003; Toews and Brelsford 2012). Our data highlights the potential pitfalls of relying on a single genetic marker for species assignment. If only interpreting the COI data for crayfish specimens from

Grassy Creek, the conclusion would be that these were F. punctimanus. If examining the phenotype or the microsatellite data, one would conclude that these specimens were F. virilis.

Many studies on crayfish genetics have utilized only a single marker type (Trontelj et al. 2005;

Schrimpf et al. 2011; Larson et al. 2012). To avoid taxonomic misclassifications, a combination of mtDNA and other genetic markers should be utilized. Utilizing a combination of independent genetic markers is more informative for inferring relationships between species than using a single marker (Edwards and Bensch 2009; Toews and Brelsford 2012). However, the lack of informative nuclear genetic markers for crayfishes has impeded phylogenetic and species identification research efforts.

Many nuclear genetic markers to date have been shown to be uninformative for distinguishing between crayfish at a species level (Sinclair et al. 2004; Zuber et al. 2012; Larson et al. 2016) and development of more informative nuclear genetic markers for crayfish has been advancing slowly. Microsatellites have been slow to develop for crayfish, especially for use in the family Cambaridae, but are increasingly being applied (Froufe et al. 2015; Huang et al. 2017;

Panicz et al. 2019). Demonstrating potential for improved understanding of crayfish evolution

22 and population genetics, nuclear approaches including single nucleotide polymorphisms (SNPs) and whole genome sequencing have recently been applied to the parthenogenetic Marble Crayfish

(Procambarus virginalis), a highly invasive species globally (Gutekunst et al. 2018). Single nucleotide polymorphisms (SNPs) have proven useful for other non-crayfish taxa (Pearse and

Garza 2015). Advances in next-generation sequencing have allowed for the recovery of large amounts of mitochondrial and nuclear genetic data from crayfish in museum collections through a cost-effective technique called genome skimming (Grandjean et al. 2016). Future work in crayfish genetics and phylogenetics will need to utilize more nuclear genome data to compliment past reliance on strictly mtDNA information.

A discordance between nuclear and mtDNA genetic markers has been reported for a variety of different organisms due to introgression (Toews and Brelsford 2012). The introgression of mtDNA occurs naturally between populations of organisms (Mallet 2005), but also occurs after secondary contact mediated by anthropogenic translocations (Senn and

Pemberton 2009; Guildea 2015). Mitochondrial DNA, including COI, is maternally inherited and haploid, therefore it does not undergo recombination, and variation is generally low within a population (Funk and Omland 2003; Hebert et al. 2003; Toews and Brelsford 2012). The low intraspecific variation and ability to distinguish between species makes mtDNA popular as a genetic marker, but because of the mode of inheritance, mtDNA is more likely to be introgressed than nuclear DNA and the effects are long-lasting (Funk and Omland 2003; Manstrantonio et al.

2016). In some studies, mtDNA has been introgressed without any evidence of nuclear DNA introgression (Zieliński et al. 2013; Pons et al. 2014). In cases of secondary contact, research has shown that mtDNA introgression is often asymmetric between populations and mostly occurs from the native to the introduced species (Manstrantonio et al. 2016). In contrast, microsatellites

23 are a popular genetic marker that are codominant and mutate rapidly, allowing them to exhibit high polymorphism within and between populations (Schlötterer 2004). With the information at hand, we cannot conclude whether the specimens we identified as hybrids are sterile or if they are fertile and backcrossing has occurred. Determining whether introgression is occurring, as opposed to hybridization with sterile offspring, would require further analysis involving identification of a source allopatric population of the parental F. virilis and tests to infer individual ancestry (Kothera et al. 2009; do Prado et al. 2017).

Overall, the frequency and consequences of hybridization and introgression between crayfishes, including native and non-native species, are poorly known and require further investigation. While we report here that hybridization has occurred between F. virilis and F. punctimanus, we have not examined the hybrid structure of this system. Future research could examine the prevalence of F1 hybrids, F2 hybrids and backcrosses by examining allopatric populations of both parental species, or populations free of hybridization, and the sympatric population. Perry et al. (2001a) investigated hybridization and assigned putative generations by comparing allozymes of allopatric parental species of F. rusticus and F. propinquus to sympatric populations. Outside of crayfishes, hybridization studies utilizing microsatellites have also compared allopatric parental species to sympatric populations (Muñoz-Fuentes et al. 2007; do

Prado et al. 2017). Determining individual ancestry and association to hybrid groups could determine whether the introduction of F. virilis poses a potential evolutionary risk to pure genotype native F. punctimanus in the watershed.

To further study the hybridization in this system, it would be important to identify an allopatric population of F. virilis with genetics like the introduced population in the Current River watershed to have the pure parental genotypes for comparison to the sympatric population and

24 hybrids. We did not attempt to identify a source population or try to perform microsatellite fragment analysis on any non-sympatric populations because of monetary and time constraints.

Additionally, F. virilis has an expansive native range resulting in a high amount of genetic diversity within the species (Williams 2012). In Missouri, multiple different genetic populations have been identified (Williams 2012). To identify a source population for examining the hybrid structure of this system, microsatellite fragment analysis could be conducted for F. virilis from different populations throughout Missouri.

Another future direction to examine hybridization in this system is to perform morphometric analyses of F. punctimanus, F. virilis, and hybrids from the Current River watershed. Identifying characteristics to distinguish hybrids in the field or by sight in the laboratory could be helpful. Anecdotally, the hybrid specimens from this system did not seem to exhibit any intermediate coloration or mixed pattern features; they either appeared to be typical F. punctimanus or F. virilis. Morphology between these two species is very close, if not identical, and relies on the examination of Form I males (Pflieger 1996; Taylor et al. 2015). Male crayfishes in the family Cambaridae exhibit a cyclic dimorphism in which they molt between a sexually reproductive Form I and a non-reproductive Form II. Previous research suggests that there is a difference in the ratio of gonopod length in Form I males where the primary process of the gonopod is more than half of the total length in F. punctimanus and less than half in F. virilis

(Taylor et al. 2015). We did not examine gonopod ratios because F. virilis males were non- reproductive Form II during our summer sampling period. Further, male F. virilis were less abundant than females in our collections and the analysis of gonopods was not applicable to female and juvenile individuals. Differences may exist in the shape of the sperm receptacle, known as the annulus ventralis, between females of F. virilis and F. punctimanus (Pflieger 1996).

25 In F. punctimanus, the posterior margin of the annulus ventralis is sharply angular and produces a triangular extension, which is absent in F. virilis where the posterior margin is rounded (Pflieger

1996). Morphological characteristics including differences in the annulus ventralis and gonopod lengths and morphometric ratios between the specimens could be examined. In the examination of hybrid F. rusticus and F. propinquus, Perry et al. (2001a.) examined 12 different characters from the exoskeletons of male crayfishes and found that many specimens from sympatric sites exhibited morphologies intermediate between allopatric parental species. In an attempt to visually distinguish hybrid crayfish, Zuber et al. (2012) examined morphological characteristics between allopatric and sympatric crayfish populations but were unable to determine traits that were fully indicative of hybrid or non-hybrid specimens due to overlapping morphometric ratios.

Being able to distinguish hybrids morphologically would provide a method to identify hybrid specimens without performing time consuming and costly genetic analyses.

Faxonius virilis and F. punctimanus have been previously identified as closely related by both morphological (Pflieger 1996; Taylor et al. 2015) and genetic analyses (Crandall and

Fitzpatrick 1996; Taylor and Knouft 2006). Accordingly, it may not be surprisingly that these species lack reproductive isolation. Many of the narrow-ranging endemic crayfishes of the

United States are likely not reproductively isolated from their closely-related congeners, with gene flow prevented by geographic rather than reproductive isolation. While F. punctimanus is common and abundant (McAllister and Robison 2012; NatureServe 2019), the introduction of F. virilis or other crayfishes into watersheds containing narrow-ranging endemics could pose negative genetic consequences which could result in extirpations or extinctions. Therefore, preventing the spread of crayfish outside of their native range should remain a priority.

26 Policies regarding the possession and translocation of crayfishes have been implemented to prevent impacts of invasive crayfishes (DiStefano et al. 2009; Dresser and Swanson 2013;

Patoka et al. 2018). Faxonius virilis is the widest ranging crayfish in North America and its range has been expanded by anthropogenic translocations (Kilian et al. 2010; Moody and Sabo 2013).

In Missouri, this species is native and wide-ranging, but has not historically occurred in several watersheds. Pflieger (1996) noted that occurrences of F. virilis in the southern Missouri Ozarks were likely the result of introductions. Further, DiStefano et al. (2015) recognized that F. virilis had two confirmed invasions within the state including the largest contiguous invasion in the

Current River. A full ban on the use of crayfish as bait in Missouri was attempted in 2011

(DiStefano et al. 2016). However, after debate, F. virilis was retained for use as bait as a consolatory measure because of its widespread distribution in Missouri compared to other common bait species (DiStefano et al. 2009). However, the species is still not native to many watersheds in the state, and the potential exists for further bait bucket introductions in new locations. Introductions of F. virilis are now known to pose a genetic impact through hybridization with native species. Although the ecological and evolutionary implications of this impact are unknown, our findings add to the long list of reasons to avoid the introduction of any crayfish species and could potential impact future policies regarding crayfishes.

Hybridization has rarely been documented between crayfish species and until this study, genetically-documented hybridization involving F. virilis has been unreported. We have shown that non-native F. virilis has hybridized with native F. punctimanus as evidenced by a mismatch in mitochondrial, microsatellite, and phenotypic data. The occurrence of hybridization between these crayfishes shows that reliance on mtDNA barcoding can lead to errors when conducting genetic analyses or inferring phylogenies of crayfishes. While the ecological and evolutionary

27 implications of our findings are unknown, we have shown that the introduction of F. virilis has had a genetic impact on a native crayfish species. Hybridization after human-mediated secondary contact has been reported for a variety of taxonomic groups and it can be assumed that where recently diverged crayfishes meet through secondary contact, hybridization and introgression could occur and rare genotypes may be lost. Perry et al. (2001a.) remarked that although undocumented, hybridization between crayfishes may be common. Our results add to the rarely documented cases of hybridization between crayfishes.

28 TABLE AND FIGURES Table 2.1: All crayfish specimens that were used in the analyses. Specimens with the species highlighted in red have COI sequences that do not match the indicated phenotype. The asterisk next to the specimen code indicates that these specimens (Cambarus hubbsi, Faxonius ozarkae, and F. luteus) were only used for outgroups in the COI tree. All specimens were collected from the Ozark National Scenic Riverways except for C. hubbsi. We obtained the COI data for C. hubbsi from GenBank (National Center for Biotechnology Information).

29

Figure 2.1: Collection sites of crayfishes in the Current River watershed, Missouri, U.S.A. Red squares represent sites where only F. punctimanus was collected. Green circles represent locations where F. virilis were collected with sympatric F. punctimanus. Red squares represent locations where no F. virilis were located, but F. punctimanus were collected. The black triangle represents a location where only F. virilis were located and collected. Inset maps display location of Missouri within the United States and the location of the Current River watershed within Missouri.

30 A. B.

Figure 2.2: Two 5 cm crayfish collected in the same seine haul from an unnamed tributary (common location “Huckleberry Hollow”) of the Current River in Shannon County, Missouri. Crayfish (A.) exhibits a color pattern consistent with F. virilis and crayfish (B.) exhibits a color pattern of F. punctimanus. Both crayfish (A.) and (B.) possessed the F. punctimanus COI sequence genotype.

31

Figure 2.3: A neighbor-joining tree generated using the COI sequences of F. virilis and F. punctimanus collected from the Current River watershed in Missouri, U.S.A. Bootstrap values (2000 iterations) are displayed at major nodes. Species assignment is based on the phenotype and the specimen number is displayed (Table 2.1). Faxonius virilis is displayed in red and F. punctimanus is displayed in blue.

32

Figure 2.4: A UPGMA dendrogram displaying microsatellite data for Faxonius virilis and F. punctimanus specimens from the Current River watershed in Missouri, U.S.A. Species assignment is based on the phenotype and the specimen number is displayed (Table 2.1). Faxonius virilis is displayed in red and F. punctimanus is displayed in blue.

33

Figure 2.5: A principal coordinate analysis (PCoA) visualizing variance in genetic distance data from the microsatellite fragment analysis for crayfishes collected at sites in Shannon and Texas Counties, Missouri, U.S.A (Figure 2.4). Convex hulls bound the monophyletic F. virilis or F. punctimanus as identified by the UPGMA dendrogram (Figure 2.4). Circles denote F. punctimanus phenotypes and squares denote F. virilis phenotypes (Figure 2.2). White color denotes F. punctimanus COI barcodes while black denotes F. virilis COI barcodes (Figure 2.3).

34 A. B.

Figure 2.6: Two crayfish collected from Grassy Creek, a tributary to the Current River in Shannon County, Missouri (Figure 2.1). Both crayfish exhibit a color pattern coinciding with F. virilis, but crayfish (A.) (GC001) exhibits a COI matching F. punctimanus and crayfish (B.) (GC006) exhibits a COI matching F. virilis.

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47 CHAPTER 3: TRADITIONAL SAMPLING OUTPERFORMS ENVIRONMENTAL DNA FOR DETECTING INVASIVE VIRILE CRAYFISH (FAXONIUS VIRILIS) IN THE CURRENT RIVER WATERSHED OF MISSOURI, U.S.A.

INTRODUCTION Improved detection has the potential to lessen ecological and economic damage of invasive species. Invasive aquatic species have been shown to reduce biodiversity by altering habitats, restructuring food webs, competing for resources, and transferring pathogens (Dudgeon et al. 2006, Havel et al. 2015). Economic costs associated with aquatic invasive species have been estimated at $7.7 billion each year for control and mitigation of impacts (Pimental et al.

2005, Havel et al. 2015). Because of the great expenses involved, decisions are often made by natural resource managers to prioritize funding and optimize management actions for preventing the introduction, slowing the spread, or attempting the eradication of these destructive species.

Detecting invasive species and determining their distribution is critical in assessing the risks that they pose to ecosystems and in deciding effective management and control options (Mehta et al.

2007). Early detection of invasion species increases the chance of success for control and elimination and decreases associated costs (Vander Zanden et al. 2010; Goldberg et al. 2013).

Determining the extent of an invasion also establishes baseline data for monitoring their future spread. Detecting aquatic species, including invasive species, traditionally involved direct collection or visual verification of presence. Increasingly, environmental DNA (eDNA) techniques have been employed for the detection of invasive species.

Environmental DNA is a surveillance technique which can detect the presence of a species by collecting and analyzing DNA extracted from environmental samples. As organisms interact with their environment, they emit DNA through the shedding of epidermal tissues and secretions, feces and urine, and reproductive cells (Turner et al. 2014). The presence of a species can be

48 inferred by collecting environmental samples and utilizing molecular techniques to determine whether the DNA of that species is present. Environmental DNA was initially utilized to detect microorganisms in the early 2000s (Taberlet et al. 2012) and has since proven useful for detecting macrobiota in terrestrial and aquatic environments. Environmental DNA can detect the presence of species with potentially greater sensitivity than traditional sampling methods making it ideal for detecting rare species including threatened, endangered, and recently introduced non-natives

(Jerde et al. 2011; Wilcox et al. 2016). Traditional sampling methods can be harmful and may disturb endangered and threatened species, while eDNA is non-disruptive (Beja-Pereira et al.

2009). Aquatic eDNA requires the collection of water samples and does not involve rock flipping, electroshocking, seining, or other traditional aquatic species sampling techniques. In

2008, eDNA was successfully used to detect an aquatic species, invasive American Bullfrogs, in

French wetlands (Ficetola et al. 2008). Since then, aquatic eDNA has been used for detecting amphibians (Spear et al. 2015), birds (Thomsen et al. 2012b), fish (Thomsen et al. 2012b; Wilcox et al. 2016; Nevers 2018), insects (Mächler et al. 2014), mammals (Ushio et al. 2017), mollusks

(Goldberg et al. 2013; Stoeckle et al. 2015), and reptiles (Davy et al. 2014; Piaggio et al. 2014).

Additionally, eDNA has been successfully used to detect threatened (Ikeda et al. 2016; Rice et al.

2018) and invasive crayfishes (Tréguier et al. 2014; Dougherty et al. 2016; Harper et al. 2018).

The goals of this project were to develop and evaluate the performance of an F. virilis eDNA assay for aiding in determining the distribution of the invasive species in the Ozark

National Scenic Riverways and the Current River watershed of Missouri, U.S.A. Developing a working eDNA assay for F. virilis could be useful for natural resource managers to identify and monitor invasions in other systems. Faxonius virilis is the most widely distributed crayfish in

North America (Taylor et al. 2015) and its range has been expanded by human activities. A

49 native species in Missouri, F. virilis has been transferred by humans to previously unoccupied watersheds including the Current River watershed where F. virilis was first detected in 1986

(Pflieger 1996). In Chapter 2, we presented evidence that F. virilis is hybridizing with a native congener, F. punctimanus, in the Current River watershed. We investigated whether hybridization between the target species, F. virilis, and a non-target species, F. punctimanus, limited the performance of our eDNA assay. Determining the distribution of F. virilis in the

Current River watershed and can aid in determining appropriate management actions and assessing the utility of an F. virilis eDNA assay could be useful to natural resource managers looking to monitor invasions.

METHODS Study Location

The Ozark National Scenic Riverways is administered by the National Park Service and encompasses over 200 kilometers of the free-flowing Jacks Fork and Current River. Situated in the Ozark Mountains in south-central Missouri, the park is dominated by karst topography and contains more than 425 springs which supply much of the baseflow to the Jacks Fork and Current

River (Bowles and Dodd 2015). The watershed is primarily rural and contains hills covered in mixed pine (Pinus spp.) and oak (Quercus spp.) woodlands. The upper Current River and Jacks

Fork are home to five native crayfish species: Golden Crayfish (Faxonius luteus), Ozark Crayfish

(F. ozarkae), Spothanded Crayfish (F. punctimanus), Hubbs’ Crayfish (Cambarus hubbsi), and

Salem Cave Crayfish (C. hubrichti; Pflieger 1996). The Faxonius species and C. hubbsi are stream dwellers, while C. hubrichti is a cave-dweller and is not typically found in surface waters.

In addition to the native species, F. virilis has been introduced to the Current River and has been detected since 1986 (Pflieger 1996).

50 Faxonius virilis occurs natively throughout much of Missouri, but it has been introduced to new watersheds in the southcentral portion of the state (DiStefano et al. 2015). The presence of

F. virilis in the southern Ozark subregion, including the Current River watershed, is thought to be the result of bait bucket introductions (Pflieger 1996). In the Current River watershed, Pflieger

(1996) documented two F. virilis site records during extensive surveys in the 1980s and early

1990s (154 collections at 55 sites in the watershed) in the upper reaches of the River near Pulltite access and at Akers (DiStefano et al. 2015). National Park Service staff, Illinois Natural History

Survey researchers, and volunteers have found additional F. virilis specimens since the initial surveys throughout the upper portion of the Current River.

Site Selection

We utilized ArcGIS Pro (Esri, Redlands, California, U.S.A.) to identify mainstem Current

River and tributary sites from the National Hydrography Dataset Plus Version 2 (NHDPlusV2) data (McKay et al. 2012) in and adjacent to the Ozark National Scenic Riverways. Tributaries with a Strahler (1952) stream order of 1 were eliminated as their low discharge would likely lead to complete drying during our summer field season and F. virilis has shown an intolerance to drying (Bovbjerg 1970). Additionally, second order and greater tributary streams were eliminated if they had a length less than 0.5 kilometers. The resulting 40 tributary sites were selected for sampling, and 30 with suitable access and wadeable water depths were sampled (Table 3.1). At the local site scale, sampling reaches were chosen approximately 100 meters up each tributary from its mouth or until a riffle/run/pool sequence was located. Tributary sites were accessed via car when possible and remaining sites were accessed via canoe.

To choose sites on the mainstem of the Current River, the sample function in R v3.4.3 (R

Core Team 2018) was used to randomly select sampling sites from a group of 207 stream

51 segments from NHDPlusV2 (i.e., between tributary confluences; Sowa et al. 2007; McKay et al.

2012) between the headwaters of the Current River and Van Buren, Missouri. We imposed a three river kilometers buffer between selected segments resulting in 13 segments being selected.

Subsequently, one segment was eliminated due to the potential presence of a federally endangered species (communication with the National Park Service), leaving sampling 12 segments (Table

3.1). An approximate middle point of each randomly selected segment was chosen for mainstem sites on the Current River but was adjusted downstream until the conditions permitted wading and were shallow enough for us to sample via seining. Mainstem Current River sites were accessed via car at one site and remaining sites were accessed via canoe.

Because there are no historical occurrences of F. virilis, the Jacks Fork was not as extensively sampled as the upper Current River. On the Jacks Fork, a 5th order tributary to the

Current River, three mainstem sites at access points were chosen that were accessible by car and were sampled using eDNA and kick-seine sampling (Table 3.1).

Environmental DNA Primer Development

To design primers for an environmental DNA (eDNA) assay, F. virilis and sympatric surface-dwelling crayfish species, F. punctimanus, F. luteus, and F, ozarkae, were collected from the Current River and sequenced for the cytochrome c oxidase subunit I (COI) gene utilizing the protocol outlined below. COI data for C. hubbsi was obtained from GenBank (National Center for Biotechnology Information; Table 3.2). An alignment file of COI sequences was visualized with Sequencher 5.4 (Gene Codes Corporation, Ann Arbor, Michigan, U.S.A.) and potential primer sequence locations unique to F. virilis were noted. Possible primers were analyzed using

IDT OligoAnalyzer (Integrated Data Technologies, Coralville, Iowa, U.S.A.) to check for hairpins and dimers and estimate the melting temperatures. After identifying candidate primers, the

52 sequences were entered on BLAST (National Center for Biotechnology Information) to check for specificity to F. virilis. Selected primers were ordered from Integrated Data Technologies and were hydrated to 10μm.

All tissue-derived DNA samples of F. virilis versus the non-target species were diluted

1:10 before testing and amplifications were performed using a QuantStudio3 Real-Time PCR

System (Applied Biosystems; Foster City, California, U.S.A.). Each 20 µl reaction consisted of 6

µl H2O, 10 µl iTaq Universal SYBR Green Supermix (Bio-Rad Laboratories, Inc., Hercules,

California, U.S.A.), 0.5 µl of 10 µM forward primer, 0.5 µl of 10 µM reverse primer, and 3 µl of

DNA template. The best-performing primer assay, 1Fvir (5'- GTA GGT ACG GGA TGA ACA

GT -3') and 1Rvir (5'- TGA CCA AAC AAA TAA TGG CAT A -3') repeatedly amplified a 198 bp length fragment from tissue-derived DNA of F. virilis, but also amplified non-target congeners at the starting 60°C annealing temperature (Table 3.2). Faxonius virilis and F. punctimanus both amplified at the initial PCR temperatures and could not be distinguished by melt curve analysis

(Tm=79-80°C). Raising the annealing temperature to 64°C eliminated amplifications of F. punctimanus and still allowed for F. virilis amplifications. Using the following thermal profile, we were able to exclude the non-target species and still amplify F. virilis consistently: 50°C for 2 minutes, 95°C for 10 minutes, then 40 cycles of 95°C for 15 seconds and 64°C for 1 minute, followed by melt curve of 95°C for 15 seconds, 60°C for 1 minute, and 95°C for 1 second. Each qPCR run had a heated lid temperature of 105°C.

Environmental DNA Sampling and Processing

Collection of eDNA samples occurred in tributaries and the mainstem of the Current

River, mostly within the Ozark National Scenic Riverways, Missouri, U.S.A between 21 May

2018 and 14 June 2018. Sites on the Jacks Fork were sampled on 15 October 2018. Separated

53 from eDNA sampling by a year, traditional sampling on the Current River occurred between 10

June 2019 and 19 July 2019. On the Jacks Fork, traditional sampling was conducted on 16

October 2018.

Four 250 mL samples were collected at each site: one from near the left side of the channel, one from near the right side of the channel, and two from the center. After rinsing the sample bottles three times in the stream and dumping downstream, water was collected from the surface. A fresh pair of nitrile gloves was used at each site. A field blank containing DI water was included with each site to act as a negative control for detecting contamination during field procedures and samples were stored on ice for filtering during the evening of the collection day.

To avoid contamination and prevent false positive results (Goldberg et al. 2016), 250 mL field water collection bottles were soaked overnight in a 50% sodium hypochlorite (bleach) solution, rinsed with DI water, dried, and sealed in gallon freezer bags prior to sampling.

A surface was prepared by wiping with a 50% bleach solution and the water samples were filtered. The filter setup consisted of a Rocker 300 vacuum pump (Sterlitech, Kent, Washington,

U.S.A.), three vacuum flasks and vacuum hoses, and 0.45-micron Whatman cellulose nitrate filters (GE Healthcare) in funnels. After filtering, sterile tweezers were used to transfer the folded filters to 2 mL centrifuge tubes containing 0.8 mL of cetrimonium bromide (CTAB) extraction solution. After a month in storage at room temperature, DNA was extracted from filters using a modified phenol-chloroform extraction (Renshaw et al. 2015) in a dedicated eDNA cleanroom.

The extracted DNA was stored in 2 mL centrifuge tubes at -80°C until amplification for detection using qPCR. Extraction controls were included to assess evidence of laboratory contamination with one control included for every round of extracted samples.

54 Environmental DNA Amplification

All samples were amplified using a QuantStudio3 Real-Time PCR System. Each 20 µl reaction consisted of 6 µl H2O, 10 µl iTaq Universal SYBR Green Supermix, 0.5 µl of 10 µM forward primer, 0.5 µl of 10 µM reverse primer, and 3 µl of DNA template. The thermal profile for the reactions was 50°C for 2 minutes, 95°C for 10 minutes, then 40 cycles of 95°C for 15 seconds and 64°C for 1 minute, followed by melt curve of 95°C for 15 seconds, 60°C for 1 minute, and 95°C for 1 second. Each qPCR run had a heated lid temperature of 105°C. For each sample and blank, four replicate reactions were performed. PCR tube strips with attached caps were used instead of plates to minimize the potential for contamination that could occur during the loading of 96 well plates. Three non-template controls, using 3 µl of water instead of template, were also included with each qPCR run to identify possible contamination. A gBlock synthetic COI fragment (Integrated Data Technologies) that matched F. virilis collected from

Spring Valley Creek acted as the positive control and each run contained a 1:10 serial dilution of gBlock F. virilis from 1 ag 10-10 to 100 fg 10-5. Any samples that amplified during qPCR and had a melt temperature of approximately 79°C were further amplified using traditional PCR, purified, and sent to the University of Illinois at Urbana-Champaign Core Sequencing Facility (Urbana, IL,

U.S.A.) to be Sanger sequenced for confirmation that F. virilis was amplified. A positive detection was counted for a sample if any replicate amplified and sequence matched to F. virilis.

We noted an R2 value of 0.99 for runs and a low efficiency of 54.8% was tolerated for this presence/absence assay as we were not trying to quantify our results. A limit of detection (LOD) was 10-9 ng µl-1 and was set as the lowest concentration of DNA that amplified on qPCR runs using the 1:10 serial dilution of F. virilis gBlock.

55 Traditional Crayfish Sampling and Habitat Variable Measurement

Sampling for crayfish using traditional methods was conducted between 10 June 2019 and

19 July 2019 on the mainstem Current River and tributaries, except on the Jacks Fork where we sampled on 15 and 16 October 2018. After arriving at a site access point, we determined whether to sample 100 meters upstream or downstream based on whether it contained more favorable F. virilis habitat (presence of woody debris or macrophytes). Faxonius virilis has shown a preference for abundant cover in the form of organic and woody debris (Pflieger 1996). At each site, the wetted width was measured in meters and was multiplied by ten to identify the length of the sampling site. The total site length was capped at 100 meters to reduce the amount of time spent at each site and the minimum site length was 50 meters. Each ten-meter subsection was marked with flags within each sampling reach and sampling proceeded from downstream to upstream. Within each 10 m subsection, sampling was conducted on both sides of the stream and each sample was taken 0.5-1 meters away from the bank in the center of the subsection. As was the case on some of the main channel sites, if crayfish habitat at a site was sparse, we would sample every other subsection. On the mainstem, one bank was often inaccessible due to deep water or high flow and we were limited to sampling one bank. On some smaller stream sites, the stream was not wide enough to accommodate two samples and one sample was taken per ten- meter reach. This sampling methodology lead to a minimum of five samples per site (on the mainstem where the site was consistent gravel with suboptimal habitat) to a maximum of 20 samples per site (on sites >ten-meter wetted width with wadeable access on both sides and varied habitat). After the randomly assigned sampling was completed, a 15-minute qualitative sample targeting preferred crayfish habitat (e.g., woody debris, macrophytes, boulders) was conducted for

56 sites with a wetted width less than ten meters and 30 minutes qualitative sample for sites with a wetted width greater than ten meters.

At most sampling sites, two field technicians were employed. One person would hold a

3.3-meter seine net downstream of a sample point and the other person would vigorously disturb an approximate 1.5 square meters of substrate so that the flow carried crayfishes into the net.

Alternatively, if the flow was low, substrate was disturbed, and debris was kicked into the net or the net was quickly dragged through and lifted. All collected crayfishes were placed in a bucket and the number of each species was noted for each sample. Native crayfishes were dumped downstream of the site after sampling in the ten-meter section was completed. Any F. virilis that were collected were stored in 70% ethyl alcohol and later accessioned into the Crustacean

Collection at the Illinois Natural History Survey. Up to ten of the vouchered specimens per site were COI sequenced to document the occurrence and frequency of phenotypic F. virilis that had

F. punctimanus COI and would not amplify using our eDNA assay. Specimens less than five centimeters in overall length were excluded from sequencing.

We collected data on canopy cover, discharge, and temperature at each site to act as covariates for relating eDNA detection in logistic regression modelling. More open canopies allow higher UV exposure which can degrade eDNA (Pilliod et al. 2014; Kessler et al. 2019), higher discharge causes eDNA to be transported quickly downstream (Deiner and Altermatt

2014), and higher temperatures can degrade eDNA more quickly (Strickler et al. 2015). At each sample point, we collected closed overstory density (%) using a Spherical Crown Densiometer

(Forestry Suppliers, Inc., Jackson, Mississippi, U.S.A) and averaged the values to get the site total. Discharge was calculated by measuring the wetted width at a subsection of the stream, dividing by ten, and taking a depth and flow sample at every “wetted width/10” meters. Flow was

57 collected using an FP111 digital flow probe (Global Water Instrumentation, College Station,

Texas, U.S.A.) and depth was taken using the markings on the flow probe staff. One temperature measurement was taken at each site using a floating aquarium thermometer (Rolf C. Hagen Corp.,

Mansfield, Massachusetts, U.S.A).

Cytochrome c Oxidase Subunit I (COI) Sequencing

We amplified a portion of the cytochrome c oxidase subunit I (COI) gene for vouchered phenotypic F. virilis crayfish specimens for determining potential hybridization. If phenotypic F. virilis had COI barcodes matching F. punctimanus, then these individuals could not be detected by the eDNA assay. Up to ten specimens >5 cm in length were sequenced per site. A 15 mg muscle tissue sample was extracted from the abdomen of each crayfish specimen and preserved in

99% ethanol. Crayfish specimens were individually tagged, preserved in 70% ethanol, and accessioned into the Illinois Natural History Survey’s Crustacean Collection (Champaign, IL,

U.S.A). DNA was isolated from the muscle samples using a DNeasy Blood and Tissue Kit

(Qiagen, Hilden, Germany) following the manufacturer’s spin column protocol for animal tissues.

We amplified COI on a PTC-100 thermal cycler (MJ Research, Inc., Watertown, Massachusetts,

U.S.A.) using 1 µl of LCO1490 primer (5'-GGT CAA ATC ATA AAG ATA TTG G-3'), 1 µl of

HCO2198 primer (5'-TAA ACT TCA GGG TGA CCA AAA AAT CA-3'; Folmer et al. 1994), 21

µl of water, 2 µl of template DNA, and PuReTaq Ready-To-Go PCR beads (GE Healthcare,

Buckinghamshire, UK). The thermal profile for the PCR reactions was 94°C for 3 min, 45 cycles of 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 90 seconds, followed by a final 72°C extension. PCR products were cleaned using a QIAquick PCR Purification Kit (Qiagen). Sanger sequencing was conducted by the University of Illinois at Urbana-Champaign Core Sequencing

Facility Forward and reverse sequence reads were analyzed and edited using Sequencher 5.4 to

58 obtain the final COI consensus sequences. If a specimen identified as F. virilis using live coloration pattern had a COI sequence matching F. punctimanus, we identified these specimens as presumed hybrids. We included the percent of specimens with concordant F. virilis COI as a predictor of eDNA performance in our model.

Statistical Analysis

We fitted logistic regression models relating our binary presence/absence F. virilis eDNA detections at sites to four site covariates in R v3.6.1 (R Core Team 2018). The covariates that we used were F. virilis abundance (virabundance), discharge (m3/s), temperature (°C), and the percentage of sequenced F. virilis specimens with concordant COI sequences (virilisCOI).

Overstory density was eliminated as a variable because it was correlated with discharge (r = 0.69), as wider and higher discharge sites also had more open canopies. Models were ranked according to Akaike Information Criterion with correction for small sample sizes (AICc; Akaike 1974) and best performing models were identified as those that had a ΔAICc <2. We calculated model- averaged parameter estimates from the best performing models (ΔAICc <2) using the package

MuMln (Barton 2014). McFadden’s pseudo-R2 values were calculated to assess model fit. We split our modeling into two analyses: one including all sites and a second including only sites where we collected F. virilis phenotype individuals. We conducted our modeling in two analyses to more accurately reflect the virilisCOI covariate, which was only relevant at sites where we collected F. virilis phenotype individuals. The first analysis containing all sites included the covariates of F. virilis abundance (virabundance), discharge, and temperature. For the second analysis at only sites where we collected F. virilis, we modelled discharge, temperature, and F. virilis COI (virilisCOI). Alternative approaches for modeling the data, such as occupancy

59 estimation modelling to account for detection probabilities (Fiske and Chandler 2011), were considered but we had too few detections to support these analyses.

RESULTS

Environmental DNA Sampling and Amplification

Our assay detected eDNA for F. virilis at only six of the 45 sampled sites in the Current

River and tributaries (Table 3.1; Figure 3.1). Out of the 12 mainstem Current River sites, F. virilis was detected at two sites. Of the 33 tributary sites sampled, F. virilis was detected at four sites. All sample amplifications matched the COI sequence of F. virilis. We detected F. virilis eDNA in two out of four replicates at two sites and in one out of four replicates in the remaining four sites. No contamination was detected in field, extraction, or plate controls.

Traditional Sampling

Of the 45 sites that were sampled for eDNA, we sampled 41 using our crayfish kick- seining method and phenotypic F. virilis was caught and vouchered at 26 sites (Table 3.1; Figure

3.2). We had no false positive eDNA detections and F. virilis was collected at all six sites where eDNA detected the species. DiStefano et al. (2015) reported that F. virilis had invaded at least 67 km of the Current River and some tributaries within that reach. Faxonius virilis is now present in at least 117 km, from Pigeon Creek in Montauk State Park, Dent County, to unnamed tributary

“Huckleberry Hollow,” Shannon County.

60 COI Hybrid Sequencing

We COI sequenced F. virilis vouchered specimens from the 26 sites where they were collected. Of the 74 vouchered and sequenced F. virilis phenotype specimens, 29 (39%) contained COI sequences matching F. virilis and 45 (61%) sequence matched F. punctimanus.

The number of phenotypic specimens collected per site, number of specimens COI sequenced, and percentage of sequenced specimens with F. virilis COI are summarized in Table 3.1 and shown in Figure 3.3.

Statistical Analysis

According to AICc ranking, our best model in the first round of analysis related eDNA detection to the number of F. virilis that were caught at each site (virabundance). No models were within ΔAICc <2. This model had a weak positive relationship and a low McFadden’s

2 pseudo R indicating a poor fit to the data. For the second round of analysis looking at sites where we collected phenotypic F. virilis, the best performing model related detections to the percentage of F. virilis with concordant COI sequences (virilisCOI). Models relating eDNA detection to temperature and eDNA temperature to discharge had a ΔAICc <2. We performed model-averaging because the cumulative AICc weight for these three models was 0.73. These models had very poor fit to the data as shown by low McFadden’s pseudo-R2 values. All three models included zero in their 95% confidence intervals. The modeling results are summarized in

Table 3.3 and best performing models in Table 3.4. Predicted detection probabilities are shown for virabundance and virilisCOI in Figure 3.4.

61 DISCUSSION

Environmental DNA is increasingly being utilized for the detection of invasive species

(Dougherty et al. 2016; Rose et al. 2019) and allows researchers to determine distributions and implement more informed management options. Our goal was to determine the range of F. virilis within the Ozark National Scenic Riverways and develop an eDNA assay for detecting the species. We evaluated the performance of the F. virilis eDNA assay compared to traditional kick- seine sampling. Using eDNA, we detected F. virilis at 6/45 sites while traditional sampling detected the species at those same six sites and an additional 20 sites. Environmental DNA is often touted as being more sensitive than traditional sampling for detecting species, but this method of detection did not perform well for detecting F. virilis in the Current River watershed.

We hypothesized that our poor eDNA performance was due in part to hybridization resulting in F. virilis individuals with F. punctimanus COI barcodes. Without the correct F. virilis barcode, a crayfish could not be detected with the eDNA assay. Sequencing of F. virilis vouchered specimens revealed that 45/74 (61%) sequence matched F. punctimanus. However, percentage of hybrids was not supported by the logistic regression modelling as a predictor of eDNA detection.

Abundance of F. virilis was the only covariate that was weakly supported by the logistic regression modelling and showed a positive relationship with eDNA detection. At sites where F. virilis was not abundant, the limit of detection of our assay may not have been low enough to successfully detect the species.

Our eDNA assay performed poorly for detecting F. virilis in the Current River watershed.

Developing a species-specific primer for F. virilis proved difficult because we were trying to exclude closely-related non-target congeners, especially F. punctimanus, from amplifying.

Finding candidate primer pairs that excluded F. punctimanus was the most difficult task, as these

62 species differ only by 2.6% at the COI region that we examined. We were successful in developing an assay which did exclude non-target species after altering the PCR conditions. The assay’s limit of detection was 10-9 ng µl-1, which is comparable to other crayfish eDNA assays including those developed for Procambarus clarkii (Tréguier et al. 2014; Cai et al. 2017), and better than assays developed for Austropotamobius pallipes (Dunn et al. 2017) and P. virginalis

(Mauvisseau et al. 2019). We hypothesized that hybridization was causing the low detectability of F. virilis, as our assay would fail to detect hybrid individuals with F. punctimanus COI barcodes. COI sequencing of vouchered specimens revealed that 61% of our 74 specimens COI sequenced matched F. punctimanus (Table 3.2). However, our modelling results did not show any support for our hypothesis and hybridization does not explain why we had so many false- negative detections.

The abundance of F. virilis at most sites may have been too low for detections with our assay and detection probability could be improved by increasing replication and accounting for the seasonal biology of F. virilis. Modelling revealed that abundance of F. virilis was the only covariate that had a weak relationship with eDNA detection. We used four 250 mL field samples, and we detected F. virilis eDNA in two out of four field replicates at two sites and in one out of four field replicates in the remaining four sites. To improve our chances of detection and decrease the likelihood of false-negative detections, more field samples could be collected (de

Souza et al. 2016; Dougherty et al. 2016). Collecting more field replicates would increase the chance that the limited amount of F. virilis eDNA present in the environment would be collected.

While we used four qPCR replicates, performing more plate replicates would also be a way to increase the probability of detection (Jerde and Mahon 2015). An additional way to improve the chances of detecting rare species is by timing eDNA collection with seasonal breeding activities

63 as this has been shown to increase detectability (Laramie et al. 2015; Spear et al. 2015). When crayfish are carrying eggs, increased eDNA detectability has been observed (Dunn et al. 2017).

Increasing the number of replicates and timing the collection of eDNA field samples with the breeding season of F. virilis could increase eDNA detections.

Environmental DNA has been proposed as a more sensitive tool for detecting and monitoring rare organisms (Jerde et al. 2011; Dejean et al. 2012), however, recent studies have reported alternative results. We add to case studies where eDNA doesn't perform as well as conventional sampling. Well-performing assays have been shown to underperform compared to conventional sampling. For detection in a neighboring river system, Rice et al. (2018) detected the Coldwater Crayfish (Faxonius eupunctus) at more sites using traditional sampling than they did using eDNA (~90% agreement) with a well performing eDNA assay. Outside of crayfishes,

Rose et al. (2019) utilized eDNA to detect two species of invasive watersnake (Nerodia spp.) but found that traditional trapping surveys had a higher probability of detections. Perez et al. (2017) and Ulibarri et al. (2017) both reported that traditional sampling techniques outperformed eDNA when sampling for fishes. Despite our poor eDNA performance, eDNA remains an important tool and successfully outperforms traditional sampling in many systems.

Other than evaluating the performance of eDNA for detecting F. virilis, we were able to document the distribution of F. virilis and identified additional evidence of hybridization between

F. virilis and F. punctimanus. DiStefano et al. (2015) reported that F. virilis had invaded at least

67 km of the Current River and some tributaries within that reach. Our traditional sampling was successful at locating F. virilis and based on vouchered specimens of phenotypic F. virilis, we report an increase to at least 117 km, from Pigeon Creek in Montauk State Park, Dent County, to unnamed tributary “Huckleberry Hollow,” Shannon County. Using the vouchered specimens, we

64 performed COI sequencing to identify potential hybrid individuals. We used COI as a hybridization indicator and phenotypic F. virilis individuals without concordant COI sequences were labelled as hybrids. Sequencing of F. virilis vouchered specimens revealed that 45/74

(61%) sequence matched F. punctimanus. These hybrid individuals are distributed throughout the range of phenotypic F. virilis in the Current River watershed (Figure 3.3). Faxonius virilis has spread in the Current River since it was first documented by Pflieger in 1986 and hybridization with native F. punctimanus has resulted in discordant COI barcodes.

Environmental DNA has proven to be a useful tool for detecting rare species, including recently introduced non-native species (Jerde et al. 2011), but did not perform well for detecting

F. virilis in the Current River watershed. The poor performance of our eDNA assay compared to traditional sampling shows that eDNA is not always the best method for monitoring biological invasions. The abundance of F. virilis was the most supported covariate in our modelling and had a weak positive effect on eDNA detection. Our assay may not have been sensitive enough to detect low level populations of F. virilis and increasing replication and timing sampling around seasonal breeding activity could increase detection probability. Traditional kick-seining was the best tool for monitoring this invasion and greatly outperformed eDNA. Environmental DNA has been promoted as a more sensitive tool for detecting and monitoring rare organisms, but this has not proven true for every species or system.

65 TABLES AND FIGURES Table 3.1: Locations for environmental DNA collection and crayfish sampling within the Current River watershed of Missouri, U.S.A.

66 Table 3.2: A comparison between the primer assay and the corresponding COI sequence segments of target F. virilis and non- target F. punctimanus, F. luteus, F. ozarkae, and C. hubbsi. Mismatched base-pairs are darkened and underlined. Accession numbers are for GenBank (National Center for Biotechnology Information).

67

Table 3.3: Logistic regression models for relating eDNA detections to covariates. The first models (A.) relate eDNA detections to all sites and the second group of models (B.) relate eDNA detections to covariates only at sites where we collected phenotypic F. virilis individuals.

A.

B.

68

Table 3.4: Coefficient estimates and model-averaged coefficient estimates from the best performing logistic regression models (ΔAICc <2) including upper and lower 95% confidence intervals.

A.

B.

69 Figure 3.1: Faxonius virilis eDNA detections and non-detections in the Current River watershed, Missouri, U.S.A. Detections are symbolized by green circles and non-detections are red circles. Inset maps display location of Missouri within the United States and the location of the Current River watershed within Missouri.

70

Figure 3.2: Faxonius virilis traditional kick-seining detections and non-detections in the Current River watershed, Missouri, U.S.A. Sites where we collected and vouchered F. virilis are symbolized by green circles and sites where we didn’t locate F. virilis are red circles.

71

Figure 3.3: Faxonius virilis traditional kick-seining detections and COI sequencing hybrid percentages in the Current River watershed, Missouri, U.S.A. Circle size indicates the number of phenotypic F. virilis that were collected from each site. The color represents the percentage of vouchered individuals with concordant F. virilis COI sequences.

72

Figure 3.4: Predicted detection probabilities with 95% confidence intervals for number of phenotypic F. virilis collected per site (virabundance) and percentage of phenotypic F. virilis with concordant COI barcodes (virilisCOI).

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81 SUMMARY

Crayfishes are a diverse group of freshwater crustaceans, which have proven to be harmful invasive species throughout the world. In Chapter 2, we assessed hybridization between invasive Virile Crayfish (Faxonius virilis) and native Spothanded Crayfish (Faxonius punctimanus) in the Current River watershed of Missouri. We compared phenotype, COI, and microsatellite fragment data to determine that hybridization was occurring. Phenotype and microsatellite data aligned, but COI was mismatched for certain individuals as a result of hybridization. In the future, examining the hybridization structure of this system could aid in determining potential impacts. Hybridization has rarely been documented between crayfish species and until this study, genetically-documented hybridization involving F. virilis has been unreported.

Improving detection of aquatic invasive species has been the goal of researchers to lessen potentially severe ecological and economic damage. Further, determining the distribution of invasive species is an important part in assessing management and control options. In Chapter 3, we determined the distribution of non-native Faxonius virilis in the Current River watershed and compared eDNA to traditional kick-seine sampling. Our traditional detection method of kick- seining outperformed eDNA. With eDNA, we detected F. virilis at 6/45 sites and with traditional kick-seining, we collected and vouchered F. virilis at 26/41 sites. Sequencing of vouchered F. virilis specimens revealed that the majority (61%) had COI barcodes matching F. punctimanus. Hybridization was not supported as a covariate affecting eDNA detection by logistic regression modelling and only the abundance of F. virilis collected per site had a weak positive relationship with eDNA detection. Despite our poor eDNA performance, eDNA remains an important tool and successfully outperforms traditional sampling in many systems.

82 APPENDIX A: MICROSATELLITE PRIMERS

Table A.1: Microsatellite primers from Williams et al. 2010 that amplified for both F. virilis and F. punctimanus from the Current River watershed in Missouri, U.S.A. The locus name and primer sequences are shown. NED, FAM, VIC, and PET are fluorescent dyes (Applied Biosystems, Foster City, California, U.S.A.).

83 APPENDIX B: SUPPLEMENTAL TREES

Figure B.1: A maximum-likelihood tree with 5000 bootstrap iterations for COI data from crayfishes collected in the Current River watershed of Missouri, U.S.A. Species identification for specimens is based on phenotype by coloration.

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Figure B.2: A maximum parsimony tree with 5000 bootstrap iterations for COI data from crayfishes collected in the Current River watershed of Missouri, U.S.A. Species identification for specimens is based on phenotype by coloration.

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