Limits of Life History in Taxonomic Classification of Lampreys with Implications for

Conservation

A thesis presented to

the faculty of

the Voinovich School of Leadership & Public Affairs

In partial fulfillment

of the requirements for the degree

Master of Science

Aaron B. Cranford

August 2013

© 2013 Aaron B. Cranford. All Rights Reserved. 2

This thesis titled

Limits of Life History in Taxonomic Classification of Lampreys with Implications for

Conservation

by

AARON B. CRANFORD

has been approved for

the Program of Environmental Studies

and the Voinovich School of Leadership & Public Affairs by

Matthew M. White

Associate Professor of Biological Sciences

Mark Weinberg

Director, Voinovich School of Leadership & Public Affairs 3

ABSTRACT

CRANFORD, AARON B., M.S., August 2013, Environmental Studies

Limits of Life History in Taxonomic Classification of Lampreys with Implications for

Conservation

Director of thesis: Matthew M. White

In the genus there are closely related species that are morphologically and ecologically similar in their larval stage known as paired species

(Docker et al., 2012). The nonparasitic Mountain Brook (I. greeleyi) and the parasitic Lamprey (I. bdellium) is a species pair found in the Eastern .

The is endangered in Ohio (OhioDNR, 2013). These two species are genetically very similar. This raises questions about the appropriateness of using trophic behavior alone for lamprey classification, and a better understanding of this relationship may better inform any conservation efforts. The current study examined the relationship between the nonparasitic I. greeleyi and parasitic I. bdellium using the ND3 and Noncoding I genes in the lamprey mitochondrial genome.

My results support multiple origins with recent expansion of I. greeleyi and I. bdellium. These results are consistent with other studies of paired lamprey species.

However, alternative hypotheses such as ongoing gene flow with ecotypes of the same species, single origin, and mitochondrial introgression cannot be ruled out. Additional data are needed to clarify these hypotheses for paired lamprey species. The results will inform decisions on whether the two feeding modes can be managed as a single species. 4

DEDICATION

I dedicate this to my friends and family. Thank you for all of your support and guidance through the years. None of this would have been possible without you. I also dedicate this

to my advisor, committee members, and other collaborative colleges. You input and

support has been invaluable.

5

ACKNOWLEDGMENTS

I would like to acknowledge the following: Ohio EPA, Ohio State University

Museum of Biodiversity, Ohio University, United States National Museum (USNM),

North Carolina Museum of Natural History (NCMNH), and Margaret F. Docker for the samples they contributed; my advisor Dr. White for his patience, guidance, support, unwavering optimism, and willingness to work with me; my committee members, Dr.

Kuchta and Dr. Ballard for their patience and understanding; Dr. Kruse for helping with my locality map; Vijay Nadella and his technicians for the quick turnaround on sequences; OFMWA for allowing me to present my poster at their conference; Jeff

Thuma for printing my poster; The Voinovich School of Leadership and Public Affairs for accepting me into their program and giving me the opportunity to continue to peruse my education; and all the other MSES and EEB graduate students for stimulating conversations that kept me fascinated with science. 6

TABLE OF CONTENTS Page

Abstract ...... 3 Dedication ...... 4 Acknowledgments ...... 5 List of Tables ...... 7 List of Figures ...... 8 Introduction ...... 9 Materials and Methods ...... 14 and Localities ...... 14 DNA Sequencing ...... 14 Data Analysis ...... 15 Results ...... 17 ND3 ...... 17 NCI ...... 18 ND3/NCI Combined ...... 18 Discussion ...... 19 Genetics Results ...... 19 Relationship to Other Species Pairs ...... 20 Conservation Implications ...... 21 Future Directions for Research ...... 22 References ...... 38 7

LIST OF TABLES

Page

Table I: Dataset and GenBank Accession Numbers ...... 23 8

LIST OF FIGURES

Page

Figure 1. Mountain Brook lamprey Oral Disc ...... 26

Figure 2. Mountain Brook lamprey Profile ...... 26

Figure 3. Oral Disc ...... 27

Figure 4. Localities Map ...... 28

Figure 5. Mountain Brook lamprey distribution map ...... 29

Figure 6. Ohio lamprey distribution map ...... 30

Figure 7: ND3 Maximum Likelihood Tree ...... 31

Figure 8: ND3 Minimum Spanning Network ...... 32

Figure 9: ND3 Frequency Distribution Chart ...... 33

Figure 10: NCI Maximum Likelihood Tree ...... 34

Figure 11: NCI Minimum Spanning Network ...... 35

Figure 12: ND3/NCI Combined Maximum Likelihood Tree ...... 36 9

INTRODUCTION

The traditional convention for lamprey classification is based on life history characteristics, (Potter and Bailey, 1972; William et al., 1988; Kucheryavyi et al., 2007) morphometrics, and meristics (Potter, 1980; William et al., 1988; Lanteigne, 1988; Bird et al., 1994). The life history characteristic refers to lamprey’s feeding mode (parasitic and nonparasitic) and the timing of sexual maturation relative to metamorphosis.

Morphometric and meristic characteristics include dentition patterns, body size, myomere counts, fin patterns, and pigmentation. Some studies have used body proportions

(percentages of a particular body part relative to the total length) for species classification(Richards et al., 1982; Meeuwig et al., 2006; Renaud, 2011). Yet, considerable evidence suggests diagnostic characteristics are not the best tool for assessing the status of and relationships among lamprey species. This is because many lamprey species occur as species pairs, and there is support for considering some pairs a single species.

Many species in the family Petromyzontidae exist as closely related species pairs(Lang et al., 2009). Paired lamprey species are characterized by larvae that are morphologically and ecologically similar, but after metamorphosis they are morphologically distinct (Docker et al., 2012). These pairs are also characterized as being either a parasitic form or a nonparasitic form(Salewski, 2003; Docker et al., 2012).

Within a species pair, life history traits differ between the parasitic and nonparasitic forms. The primary difference in life history between two species that make up a pair is the feeding mode after metamorphosis. After metamorphosis, a parasitic species will 10 make its way to a larger water body (river, pond, lake, or ocean) and feed on the blood of other fish. A nonparasitic species does not feed and will stay in its resident stream to spawn (Potter, 1980; Beamish, 1987; Docker et al., 2012). Alternative hypotheses concerning the relationship between the parasitic and nonparasitic species include: single origin, multiple origin, ecotypes with ongoing gene flow, and mitochondrial introgression. Furthermore, much contemporary research suggests there is no single model to explain this relationship, and in many cases there is little evidence to consider species pairs two valid species (Brown, 2009). A heterochronic shift that results in an alteration in the timing of metamorphosis, relative to sexual maturity, is proposed as one of the modes of reproductive isolation in which a nonparasitic form would be produced.

However, little is known about the mechanism responsible for this change in relative timing of sexual maturation (Brown, 2009).

In addition, the difference in feeding modes, spatial and temporal separation, and ecological separation allows for more mechanisms of reproductive isolation. A combination of heterochrony and difference in feeding modes has been proposed for the origin of reproductive isolation through size-assortative mating. Hardisty and Potter

(1971) found that spawning required precise positioning of the genitals, and size- assortative mating was later demonstrated in stream tanks by (Malmqvist, 1983).

However, other attempts to demonstrate size-assortative mating showed successful fertilization even when size differences were greater than 30% (Beamish, 1992).

Furthermore, Kucheryavyi et al. (2007) were also unable to find evidence of size- assortative mating among three life history types of Arctic lamprey ( 11 camtschaticum). Beamish (1992) concluded that there might be additional forms of reproductive isolation via habitat differences. Although it is generally accepted that parasitic and nonparasitic species prefer different habitats, species have been found spawning at the same time and location in several cases(Morman, 1979; Kucheryavyi et al., 2007; Kucheryavyi et al., 2007).

The parasitic form has delayed metamorphosis and its gonads remain immature during the feeding phase, whereas the nonparasitic form has fully matured gonads after metamorphosis. Due to accelerated maturation the nonparasitic form has reduced fecundity because of its reduced size at metamorphosis, compared to the parasitic form

(Docker et al., 2012). Although these life history traits are useful in characterizing the lifecycle of a particular form (parasitic vs. nonparasitic), it does not necessarily mean that the paired species are two valid species. Therefore, methods using mitochondrial DNA can add resolution when assessing the limits of these species.

Where paired species occur sympatrically there is evidence of gene flow (Beamish,

1987; Docker et al., 2012). Kucheryavyi et al. (2007) found three forms of lamprey from the Utkholok River (anadromous parasitic, resident parasitic or forma praecox, and resident nonparasitic) of the same species. Espanhol et al. (2007) concluded there is evidence for ongoing gene flow and multiple origins in fluviatilis and

L.planeri. These examples question the appropriateness of using trophic behavior alone for lamprey classification.

Studies using mitochondrial DNA sequences have not revealed fixed differences in many paired species(Docker et al., 1999; Yamazaki et al., 2006; Espanhol et al., 2007; 12

Hubert et al., 2008). However, fixed differences have been found in mtDNA between L. camtschaticum and American brook (L. appendix) lampreys from Russia and the

Laurentian Great Lakes(Docker et al., 1999). Nevertheless, the lack of fixed differences in most paired species suggests that divergence occurred within the last 5-15KYA. Blank et al. (2008) estimated a rate of less than 0.03% and 0.01% sequence difference between

European river (L. fluviatilis) and European brook (L. planeri) respectively. This low rate of sequence difference has also been reported in silver (I. unicuspis) and northern brook

(I. fossor) lampreys (Docker, 2006). Reciprocal monophyly may not have been achieved due to large population sizes and incomplete linage sorting. The lack of fixation might not be seen even over much longer periods of time. Species may be isolated reproductively, but have yet to express fixed differences in the gene regions examined due to insufficient time (Brown, 2009).

In the genus Ichthyomyzon, paired species are morphologically indistinguishable in the larval stage, but after metamorphosis one species becomes parasitic and the other skips the adult feeding phase while rapidly becoming sexually mature(Docker et al.,

2012). The nonparasitic Mountain Brook lamprey (I. greeleyi) and the parasitic Ohio lamprey (I. bdellium) constitute a species pair found in the eastern United States (Fig. 5,

Fig. 6). The Mountain Brook Lamprey is endangered in Ohio (OhioDNR, 2013), vulnerable in (USFWS, 2013), and is rare in West (WVDNR,

2009).

There were two specific objectives of this study: (1) use two molecular markers in the mitochondrial DNA of I. greeleyi and I. bdellium to compare the divergence between 13 the species I. greeleyi and I. bdellium from the same drainage; (2) determine if there is an increase in divergence between the species when they are in different drainages. This analysis allowed me to assess the possible relationships between I. greeleyi and I. bdellium, as well as relate the results to other paired lamprey species. The results may also provide guidance for the management of Mountain brook lamprey, a regionally endangered species. 14

MATERIALS AND METHODS

Animals and Localities

A total of 33 samples from 15 localities were collected. The samples consisted of adult and juvenile Mountain Brook lamprey (I. greeleyi), Ohio lamprey (I. bdellium),

Silver lamprey (I. unicuspis), and Northern Brook lamprey (I. fossor) that were collected from several sources (Table I, Fig. 4). Thirty-three samples from 15 localities and four species were sequenced for the ND3 region of the mtDNA genome (Table I). Twenty-two specimens from 8 localities and three species were partially sequenced for the Noncoding

I (NCI) region of the mtDNA (Table I). In addition, four sequences from four localities were obtained from GenBank (Table I, GenBank accession numbers, DQ889811-

DQ889813). All samples were preserved in either 70% or 95% EtOH. Several samples were initially fixed in formalin. Samples of Northern Brook lamprey (I. fossor) and Silver lamprey (I. unicuspus) were used as outgroups.

DNA Sequencing

Total genomic DNA was extracted using a DNeasy Kit (Qiagen, Inc.) following the manufacturer’s protocol. A modification of the Chase et al. (1998) protocol was used to purify DNA from formalin preserved specimens. I amplified the mitochondrial ND3 gene and a portion of the control region using Polymerase Chain Reaction (PCR). Each 20µl

reaction contained 10X biotin NH4 reaction, 0.2mM each dNTP, 3.0mM MgCl2, 0.5µl of each primer, 1.0 U of Taq polymerase, and 2.0µl of template DNA. Amplification was performed in an MJ Research PTC 25 thermal cycler. The ND3 light-strand primer (5’- 15

ACG TGA ATT CTA TAG TTG GGT TCC AAC CA-3’) and heavy-strand primer (5’-

ATG CGG ATC CTT TTG AGC CGA AAT CA-3’) (Docker et al., 2012) were used for all amplification and sequencing reactions. The PCR conditions for ND3 were 25 cycles of 94°C for 30 seconds, 50°C for 30 seconds, and 72°C for 30 seconds.

The mitochondrial control region was amplified using three primer pairs designed for I. fossor (GenBank accession number DQ889855). The control region light-strand primer IGNCIF (5’-ACC CAA CTA CTC CCA ATA AA-3’), heavy-strand primer

IGNCIR (5’-CTT CAG TCG CTG GTT TAC AA-3’) and with IGNCIR2 (5’-AGA GTA

CCT TAG TCT GGG ATA AGT-3’). The last primer pair is IGNCIF2 (5’-GTA CCC

ACT CCC CAA AAC TA-3’) with IGNCIR (5’-CTT CAG TCG CTG GTT TAC AA-

3’). The PCR reaction consists of an initial denature at 94°C for 1 min, primer annealing for 1 min at 60°C (Docker et al., 2012), extension at 72°C for 1.5 min, and a final extension at 72°C for 5 min. All PCR products are verified on 0.8% agarose gels.

Between 10 and 40 ng of purified PCR product are used as template for the PCR sequencing reaction (ABI Big-DyeTM Terminator Cycler Sequencing v3.0). The sequences were determined on a 310 Genetic Analyzer or a 3730 DNA Analyzer (Applied

Biosystems, Inc.).

Data Analysis

Sequences were read using FinchTV v.1.3.0 and aligned for comparison using

CLUSTALX v1.81). Both genes were aligned with I. unicuspus. The ND3 and NCI data sets were analyzed individually then combined. I evaluated the evolutionary relationships 16 among haplotypes using maximum likelihood trees, that I generated in MEGA5 (Tamura et al., 2011) to identify haplotype groups. These trees were also used to evaluate relatedness between species and possibility for expansion.

The demographic history of I. greeleyi and I. bdellium was estimated at ND3 with a mismatch distribution of pair-wise differences between haplotypes (Rogers and

Harpending, 1992) using Arlequin version 3.5 (Excoffier et al., 2005). A unimodal distribution of differences is predicted under a model of population expansion. A multimodal distribution is expected under a scenario of a long-term stable population.

The raggedness index (Harpending et al. 1993) and sum of squared deviations (Schneider and Excoffier 1999) were calculated to test the goodness of fit between the observed distribution and a unimodal distribution.

The statistical parsimony method(Templeton et al., 1992)was used to identify connections among haplotypes with TCS version 1.21(Clement et al., 2000). A default

0.95-probability connection limit was used, and indels were treated as missing data.

Estimates of times since divergence between I. greeleyi and I. bdellium haplotypes were made based on divergence times suggested in(Blank et al., 2008). 17

RESULTS

ND3

The complete ND3 sequence is 351 nucleotides(Docker et al., 1999). Within I. greeleyi, there were 13 polymorphic sites. Among the polymorphic sites, four were at the first position, one was at the second position, and eight were at the third position. Three substitutions were non-synonymous. Within I. bdellium, there were six polymorphic sites.

Among the polymorphic sites, two were at the first position, one was at the second position, and three were at the third position. Three substitutions were non-synonymous.

Thirteen variable sites were observed between I. greeleyi and I. bdellium. Among the polymorphic sites, four were at the first position, 1 were at the second position, and eight were at the third position. Six of the substitutions were non-synonomous. The maximum liklihood tree was generated with the HKY+G model (AIC=1433.050) and 500 bootstrap iterations. It revealed six haplotypes in I. greeleyi and I. bdellium from the 33 individuals sampled (Fig. 7). The tree suggests multiple origins with expansion. The ND3 mismatch distribution revealed a bimodal distribution, but it is not significantly different from a unimodal distribution (Fig. 9). The raggedness index (0.14; P=0.12) and sum of squared deviations (0.05; P=0.15) were not significant. This is consistent with a recent population expansion. The parsimony network resolved relationships among haplotypes

(Fig. 8).

18

NCI

Noncoding region I is approximately 560 base pairs(Docker et al., 2012). Partial sequences of 229 base pairs of the control region were resolved. Some manual adjustment was necessary in NCI due to indels (1-10 bp). Within I. greeleyi there were 20 variable sites. Within I. bdellium there were four variable sites. The maximum likelihood tree was generated with the GRT+I model (AIC=1470.078) and 500 bootstrap iterations. It revealed seven haplotypes in I. greeleyi and I. bdellium from the 20 individuals sampled

(Fig. 10). The tree suggests a single origin with expansion. The parsimony network was able to resolve relationships among haplotypes (Fig. 11).

ND3/NCI Combined

The combined dataset had 580 base pairs. Within I. greeleyi there were six variable sites. Among the polymorphic sites, two were at the first position, one was at the second position, and three were at the third position. Within I. bdellium there were four variable sites, none were in the coding region. Thirty variable sites were observed between I. greeleyi and I. bdellium. Among the polymorphic sites in the coding region, two were at the first position, one was at the second position, and four were at the third position. There were six non-synonymous substitutions. The maximum likelihood tree was generated with the T93+G model (AIC=2909.230) and 500 bootstrap iterations. It revealed eight haplotypes in I. greeleyi and I. bdellium from the 15 individuals sampled

(Fig. 12). 19

DISCUSSION

Genetics Results

My objective was to determine if I. greeleyi and I. bdellium were genetically similar in and between the and River drainages. Analysis of the two mtDNA loci allowed for some resolution of the relationship between I. greeleyi and

I. bdellium. The most notable result was that the ND3 dataset supported a recent divergence between I. greeleyi and I. bdellium. This is consistent with many studies(Salewski, 2003; Kucheryavyi et al., 2007; Docker et al., 2012). Espanhol et al.

(2007) concluded ongoing gene flow was more prevalent. As more research is being conducted at increasing phylogenetic resolution, the paired relationships are being revealed as more complex than previously thought (Brown, 2009).

There are differences between the phylogenies of the two genes (Fig. 3 & 4), suggesting there may be different mechanisms, beyond the rate of evolution, acting on each locus. The results are consistent with NCI being highly conserved between species and among drainages, whereas the ND3 gene has more variability. The region of NCI that was resolved is known to harbor several regulatory sequences that are conserved (Lee and Kocher, 1995; White and Martin, 2009). The lack of diagnostic difference in the mtDNA is consistent with previous studies on paired lamprey species(Beamish, 1987;

Kucheryavyi et al., 2007; Espanhol et al., 2007; Lang et al., 2009) and more specifically paired lamprey species where ND3 and NCI were sequenced(Docker et al., 1999; Docker et al., 2012). 20

The increasing understanding of the complexity of the relationships between species pairs raises the question of the appropriateness of using trophic behavior alone for lamprey classification. While trophic behavior may be acceptable for characterizing a species, it alone may not be the best method for understanding the limits in species pairs.

Relationship to Other Species Pairs

The lack of divergence shown in the I. greeleyi/I. bdellium pair is consistent with other species pairs within the Petromyzontidae family(Espanhol et al., 2007; Lang et al.,

2009; Brown, 2009; Docker et al., 2012). Furthermore, my frequency distribution results are also consistent with other species pairs in the Petromyzontidae family having a recent population expansion(Salewski, 2003; Kucheryavyi et al., 2007; Docker et al., 2012)The

I. unicuspis/I. fossor pair was used to demonstrate the degree of divergence among multiple loci (Docker et al., 2012). They concluded that the I. unicuspis/I. fossor pair represents ecotypes of the same species and that there may be ongoing gene flow.

The possibility for ongoing gene flow cannot be ruled out for the I. greeleyi/I. bdellium pair. Esphanol et al., (2007) ruled out a single origin hypothesis in L. fluviatilis and L. planeri, but not the multiple origins or ecotypes hypothesis. Docker et al. (2012) also had support for the ecotype hypothesis. Esphanol et al. (2007) concluded that the multiple origins hypotheses and ecotypes hypothesis are not mutually exclusive.

Furthermore, mitochondrial introgression has been shown to act on lampreys that are in a species pair (Yokoyama et al., 2009). Therefore, mitochondrial introgression is another hypothesis that cannot be ruled out of paired lamprey species relationships. Given the 21 growing complexity of paired species relationships, it would be difficult to ascribe any one mechanism to an entire genus or even a species pair. Therefore, very few assumptions should be made when it comes to management.

Conservation Implications

The inability to reliably identify species during the ammocetes phase has considerable conservation implications. Many lamprey species are of conservation concern (Renaud, 2011), and the inability to distinguish species (morphologically or genetically) during their long larval stage presents a barrier to understanding the current distribution and population size of these species. I ran into this barrier in my study. I received specimens that were labeled as one species in a pair, but they sequenced as a species from another pair. Paired species are thought to have similar or identical habitat and risk factors during development, but the habitat and risk can change drastically following metamorphosis.

Barriers to migration have a particular impact on migratory and parasitic adults.

These barriers impact lamprey by reducing their prey base. While this may not impact nonparasitic species directly, there is an indirect effect. If future research reveals gene flow is extensive between paired species, it could not be assumed that nonparasitic species would not be impacted. Furthermore, if migratory and parasitic lampreys mediate gene flow between relatively isolated nonparasitic, resident lamprey, a reduction in parasitic lamprey populations could lead to a loss in nonparasitic lamprey species and 22 genetic diversity. It seems prudent to conserve phenotypic and genetic diversity in lamprey species pairs.

Future Directions for Research

My dataset was biased toward ND3 (n=34). The 18 sequences for NCI were less than half of the control region. Furthermore, my NCI sequences were from the 3’ end, which has been shown to be less variable than the 5’ end (White and Martin, 2009).

Therefore, it would be valuable to have the entire sequence.

Even with my reasonable sample size for ND3, there are areas for improvements.

Additional I. bdellium samples are needed throughout the Ohio distribution as well as in

North Carolina, especially in northeast Ohio. Just one or two more samples in some localities would make a considerable difference, and one or two more from every locality and two or three new localities would be ideal. Ideally these localities would still be in the Ohio River or Tennessee River drainage, and the species would be found in sympatry and syntopy.

Fresh samples from the field are needed. Using museum samples proved problematic when trying to extract and amplify DNA. Fresh samples would make extraction and amplification much easier. This is especially true for NCI, because it is a larger mtDNA fragment.

Finally, microsatellites are a logical next step in studying the relationship between 23

I. greeleyi and I. bdellium. Dr. White and I currently have primer pairs to run microsatellites. These microsatellites will give better resolution for inferences into evolutionary history between I. greeleyi and I. bdellium. 24

Table. I. Data set for all specimens sequenced; L&D= Lock & Dam, TW=Tail Waters, N,N= Samples sequenced for ND3 and NCI Species Locality N,N Sample ID Voucher ND3 NCI

I. unicuspis Unknown 0,1 unicuspis01 OUVC X X

I. fossor Ashtabula River OH 1,0 fossor01 OUVC X

I. bdellium Spring Creek, below RR trestle, SC 1,0 bdellium09 NCSM 26291 X

Paint Creek confluence, French Broad River,TN 1,0 bdellium10 NCSM 26307 X

French Broad River off of SR 1304, NC 1,0 bdellium08 NCSM 50198 X

Below London L&D, Kanawha River,WV 1,1 bdellium01 OUVC 11035 X X

Below London L&D, Kanawha River, WV 1,1 bdellium02 OUVC 11036 X X

Ohio River Marietta Island, OH 1,1 bdellium03 OUVC 11041 X X

Ohio River Marietta Island, OH 1,1 bdellium05 OUVC 11043 X X

Ohio River Marietta Island, OH 1,1 bdellium06 OUVC 11044 X X

I. greeleyi Slow Creek at SR 1531, NC 3,1 greeleyi14-16 NCSM 44783 X X

Persimmon Creek at SR 1127, NC 3,3 greeleyi17-19 NCSM 44825 X X

South Hominy Creek at NC 151 SR, NC 4,4 greeleyi23-26 NCSM 47401 X X

West Branch Mahoning River, OH 1,0 greeleyi11 OSUM 111860 X 25

Table. I. Continued

Kokosing River at Laymon Rd Bridge, OH 1,1 greeleyi09 OSUM 111763 X X

Kokosing River at Zion Rd Bridge, OH 1,1 greeleyi41 OSUM 111764 X X

Kokosing River at Staats Rd, OH 2,2 greeleyi38-39 OSUM 112704 X X

West Branch Mahoning River, OH 1,1 greeleyi42 OSUM 112742 X X

S.F. Eagle Creek, RM 2.7, OH 1,0 greeleyi06 OUVC X

Little Kanawha River, at Falls Mill, WV 1,0 greeleyi20 USNM 171992 (6) X

Little Kanawha River, at Falls Mill, WV 0,1 greeleyi21 USNM 171992 (13) X

Little Kanawha River, at Falls Mill, WV 0,1 greeleyi22 USNM 171992 (15) X

Little Kanawha River, at Falls Mill, WV 1,0 greeleyi02 USNM 171992 (18) X

Little Kanawha River, at Falls Mill, WV 1,0 greeleyi03 USNM 171992 (21) X

Little Kanawha River, at Falls Mill, WV 1,0 greeleyi04 USNM 117992 (22) X

Ischua Cr 1,0 greeleyi43 G10 DQ889811

French Cr 1,0 greeleyi44 G11 DQ889812

W Br Conewango R 1,0 greeleyi45 G12 DQ889813 26

Fig. 1. Profile photo of a Mountain Brook lamprey (I. greeleyi)

Fig. 2. Profile of a Mountain Brook lamprey (I. greeleyi) 27

Fig. 3. Oral Disc of Ohio lamprey (I. bdellium)

28

Fig. 4. Map showing all localities of collected specimens that were sequenced for either

ND3 or NCI genes. The localities for sequences acquired through GenBank are

not shown. 29

Fig. 5. Distribution of the Mountain Brook lamprey (I. greeleyi) (Page and Burr, 2011) 30

Fig. 6. Distribution map of the Ohio lamprey (I. bdellium) (Page and Burr, 2011)

31

Fig. 7. The maximum likelihood tree of the ND3 gene. Red squares denote samples from

the Ohio River drainage, and blue squares denote samples from the Tennessee

River drainage.

32

Fig. 8. Minimum spanning network of the six mitochondrial DNA ND3 haplotypes.

Numbers along the connections identify the number of substitutions between two

haplotypes (when the number of haplotypes is > 1). Ig and Ib stand for I. greeleyi

and I.bdellium respectively. The filled circle represents a hypothetical haplotype

that was not sampled in this study. 33

Fig.9. Frequency distribution of pairwise nucleotide differences between six ND3

haplotypes observed in I. greeleyi and I. bdellium (solid line = observed values;

dashed lines = 95% confidence interval). The raggedness index (0.14; P=0.12) and

sum of squared deviations (0.05; P=0.15) were not significant.

34

Fig.10. The maximum likelihood tree of the partially sequenced NCI gene. Red squares

denote samples from the Ohio River drainage, and blue squares denote samples

from the Tennessee River drainage.

35

Fig. 11. Minimum spanning network of the seven mitochondrial DNA NCI haplotypes.

Numbers along the connections identify the number of substitutions between two

haplotypes (when the number of haplotypes is > 1). Ig and Ib stand for I. greeleyi

and I.bdellium respectively. The filled circle represents a hypothetical haplotype

that was not sampled in this study. 36

Fig.12. The maximum likelihood tree for the ND3/NCI combined datasets. Red squares

denote samples from the Ohio River drainage, and blue squares denote samples

from the Tennessee River drainage. 37

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