Anatomical and Genetic Variation of Western Oxyloma 

(Pulmonata: Succineidae) Concerning the Endangered 

Kanab Ambersnail (Oxyloma haydeni kanabense) in 

Arizona and Utah

U.S. Department of the Interior

U.S. Geological Survey

Scientific Investigations Report 2013–5164
Cover:

Foreground photograph: Kanab ambersnail (Oxyloma haydeni kanabense).

Background photograph: Vasey’s Paradise waterfalls, Colorado River, Arizona. 

Photograph taken by Jeff Sorensen, Arizona Game and Fish Department.
Anatomical and Genetic Variation of 

Western Oxyloma (Pulmonata: Succineidae) 

Concerning the Endangered Kanab 

Ambersnail (Oxyloma haydeni kanabense) in 

Arizona and Utah

By Melanie Culver, Hans-Werner Herrmann, Mark Miller, Barry Roth, and 

Jeff Sorenson

Scientific Investigations Report 2013–5164

U.S. Department of the Interior

U.S. Geological Survey
U.S. Department of the Interior

SALLY JEWELL, Secretary

U.S. Geological Survey

Suzette M. Kimball, Acting Director 

U.S. Geological Survey, Reston, Virginia: 2013

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Suggested citation:

Culver, Melanie, Herrmann, Hans-Werner, Miller, Mark, Roth, Barry, and Sorenson, Jeff, 2013, Anatomical and genetic 

variation of western Oxyloma (Pulmonata: Succineidae) concerning the endangered Kanab ambersnail (Oxyloma

haydeni kanabense) in Arizona and Utah: U.S. Geological Survey Scientific Investigations Report, 2013–5164, 66 p., 

http://pubs.usgs.gov/sir/2013/5164/.
iii

Contents

Abstract...........................................................................................................................................................1

Introduction.....................................................................................................................................................2

Background Information on Subspecies Trinomial.........................................................................4

Molecular Genetic Markers Used .....................................................................................................4

Study Objectives ...................................................................................................................................5

Purpose and Scope ..............................................................................................................................5

Methods ..........................................................................................................................................................5

Specimen Collection ............................................................................................................................5

Shell Morphology .................................................................................................................................7

Soft Tissue Anatomy.............................................................................................................................8

DNA Isolation.........................................................................................................................................8

Genetic Analyses..................................................................................................................................8

Mitochondrial DNA......................................................................................................................8

Data Generation..................................................................................................................8

Nuclear DNA.................................................................................................................................9

Single Copy ..........................................................................................................................9

Markers from Other Studies ....................................................................................9

Genomic Library.........................................................................................................9

Data Analyses—Mitochondrial DNA and Nuclear Single Copy DNA............10

Microsatellite (Single Locus)..........................................................................................10

Genomic Library.......................................................................................................11

PCR Conditions.........................................................................................................11

AFLP (Multilocus)..............................................................................................................11

Data Generation.......................................................................................................11

Data Analyses ..........................................................................................................11

Results ...........................................................................................................................................................12

Specimen Collection ..........................................................................................................................12

Shell Morphology................................................................................................................................12

Principal Components Analysis...............................................................................................12

Discriminant Analysis................................................................................................................18

Soft Tissue Anatomy...........................................................................................................................18

DNA Isolation.......................................................................................................................................18

Genetic Analyses ...............................................................................................................................18

Mitochondrial DNA....................................................................................................................26

Nuclear DNA...............................................................................................................................30

Single Copy ........................................................................................................................38

Microsatellite (Single Locus)..........................................................................................42

AFLP (Multilocus)..............................................................................................................50
iv

Contents—Continued

Discussion ....................................................................................................................................................52

Shell Morphology................................................................................................................................52

Soft Tissue Anatomy...........................................................................................................................52

Morphology and Anatomy—Taxonomic-Nomenclatural Considerations.................................53

Genetics................................................................................................................................................53

Each Marker Separate..............................................................................................................54

Markers Combined ....................................................................................................................56

Conservation Implications.................................................................................................................56

Future Research ..........................................................................................................................................57

Summary........................................................................................................................................................57

Acknowledgments ......................................................................................................................................57

References Cited..........................................................................................................................................58

Glossary of Molecular Genetic Terms......................................................................................................63

Appendix A. Genbank Accession Numbers ...........................................................................................65

Appendix B. DNA Sequences for Random Ambersnail Nuclear Clone 356......................................65

Appendix C. DNA Sequences for Ribosomal First and Second Internal Transcribed Spacer.......65

Figures

1. Map showing sampling locations for Oxyloma populations used in this study from

Arizona and Utah ..........................................................................................................................3

2. Graph showing scores of Oxyloma specimens from all study samples in Arizona

and Utah on principal components 2 and 3 (“factors”) from principal components

analysis.........................................................................................................................................15

3. Graphs showing scores of Oxyloma specimens from individual sampling locations

in Arizona and Utah on principal components 2 and 3 (“factors”) from principal

components analysis .................................................................................................................16

4. Graphs showing scores of Oxyloma specimens from sampling locations in Arizona

and Utah individually on principal components 3 and 4 (“factors”) from principal

components analysis .................................................................................................................17

5. Canonical scores plot of discriminant analysis using shell dimension variables SL

(shell length), SD (shell maximum diameter), SAL (shell aperture length), SAW

(shell aperture width), and SMD (height of maximum diameter)........................................19

6. Diagram showing anatomical features for Oxyloma specimens analyzed for this

study..............................................................................................................................................20

7. Maximum likelihood tree diagram showing the concatenated mitochondrial DNA

dataset (mtconcat) including the cytochrome oxidase subunit I (COI) and

cytochrome b (cyt b) genes for Oxyloma populations, used to show position of the

outgroups .....................................................................................................................................27
v

Figures—Continued

8. Bayesian (MB) 50-percent majority rule consensus tree diagram showing

mitochondrial cytochrome oxidase subunit I gene (COI) dataset analyzed for

Oxyloma populations sampled..................................................................................................31

9. Median-joining haplotype network diagram showing mitochondrial cytochrome

oxidase subunit I gene (COI) dataset analyzed for Oxyloma populations sampled.........32

10. Bayesian 50-percent majority rule consensus tree diagram showing mitochondrial

cytochrome b gene (cyt b) dataset analyzed for Oxyloma populations sampled............34

11. Median-joining haplotype network diagram showing mitochondrial cytochrome b

gene (cyt b) dataset analyzed for Oxyloma populations sampled......................................35

12. Bayesian 50-percent majority rule consensus tree diagram showing concatenated

mitochondrial DNA dataset (mtconcat) including the cytochrome oxidase subunit I

(COI) and cytochrome b (cyt b) genes analyzed for Oxyloma populations sampled.......37

13. Median-joining haplotype network drawing showing concatenated mitochondrial

DNA dataset (mtconcat) including the cytochrome oxidase subunit I (COI) and

cytochrome b (cyt b) genes analyzed for Oxyloma populations sampled.........................38

14. Bayesian 50-percent majority rule consensus tree diagram showing ribosomal

gene introns S7-1 and S7-2 (S7) dataset analyzed for Oxyloma populations sampled...39

15. Median joining haplotype network diagram showing ribosomal gene introns S7-1

and S7-2 (S7) dataset analyzed for Oxyloma populations sampled. S7 partial 1 and 2

(below the main network) represent magnified parts of the complete network .............40

16. Bayesian 50-percent majority rule consensus tree diagram of the ribosomal DNA

first and second internal transcribed spacer (ITS) dataset analyzed for Oxyloma

populations sampled ..................................................................................................................43

17. Bayesian 50-percent majority rule consensus tree diagram showing random

ambersnail nuclear clone 356 (356) dataset analyzed for Oxyloma populations

sampled ........................................................................................................................................44

18. Median-joining haplotype network diagram showing random ambersnail nuclear

clone 356 (356) dataset analyzed for Oxyloma populations sampled.................................45

19. Bayesian 50-percent majority rule consensus tree diagram showing random

ambersnail nuclear clone 458 (458) dataset analyzed for Oxyloma populations

sampled ........................................................................................................................................46

20. Median-joining haplotype network diagram showing random ambersnail nuclear

clone 458 (458) dataset analyzed for Oxyloma populations sampled.................................47

21. Diagram showing nuclear microsatellite alleles present in each individual,

indicated by cells with allele sizes listed across columns and individuals listed

down rows ...................................................................................................................................48

22. Unweighted Pair Group Method with Arithmetic mean (UPGMA) tree diagram

showing Amplified Fragment Length Polymorphism (AFLP) data analyzed for

Oxyloma populations sampled..................................................................................................50

23. Diagram showing results of a Bayesian STRUCTURE analysis of the Amplified

Fragment Length Polymorphism (AFLP) data analyzed for Oxyloma populations

sampled ........................................................................................................................................51
vi

Tables

1. Site name and locality description of ambersnail populations in Arizona and Utah

sampled for the anatomical and genetic analyses described in this study........................6

2. Number of ambersnail samples per population in Arizona and Utah, sequenced 

for two mitochondrial DNA (mtDNA) loci, the concatenated mtDNA loci, the 

four nuclear DNA loci, and the Amplified Fragment Length Polymorphism (AFLP) 

analyses .........................................................................................................................................9

3. Polymerase Chain Reaction (PCR) primer sequences used for PCR amplification

and the appropriate PCR annealing temperatures for each of the five nuclear loci

used in this study of Oxyloma populations .............................................................................10

4. Summary statistics of ambersnail shell morphology variables used in principal

components analysis and discriminant analysis, by sample localities in Arizona 

and Utah .......................................................................................................................................13

5. Pearson correlation pairwise matrix for ambersnail shell characters submitted to

principal components analysis and discriminant analysis used to analyze Oxyloma

populations ..................................................................................................................................14

6. Component loadings of variables measured in Oxyloma populations, eigenvalues,

and percent of total variance explained by the first four components in principal

components analysis of shells of Oxyloma ............................................................................15

7. Summary of Oxyloma anatomical data collected for this study, by sample localities

in Arizona and Utah ....................................................................................................................21

8. Analysis of molecular variance used to estimate genetic variation within versus

among populations; and Mantel test to assess correlation between genetic

distance and geographic distance for six different mitochondrial and nuclear DNA

loci and one concatenated mtDNA dataset used to analyze genetic variation in

Oxyloma populations..................................................................................................................26

9. Tajima’s D (a test for demographic expansion or contraction) values for 6

mitochondrial and nuclear DNA loci and 1 concatenated mitochondrial DNA 

region in 12 Oxyloma populations from Arizona and Utah...................................................28

10. Fu’s F statistic (a test for population expansion) for 6 mitochondrial and nuclear

DNA loci and 1 concatenated mitochondrial DNA region in 12 Oxyloma populations

from Arizona and Utah ...............................................................................................................28

11. Roza’s R2 value (a test for population growth) for 6 mitochondrial and nuclear DNA

loci, 1 concatenated mitochondrial DNA region, and 1 concatenated nuclear DNA

region in 12 Oxyloma populations from Arizona and Utah...................................................29

12. Pairwise distance matrices for mitochondrial DNA datasets for Oxyloma

populations from Arizona and Utah .........................................................................................36
vii

Conversion Factors

Inch/Pound to SI

Multiply By To obtain

Flow rate

cubic foot per second (ft3/s) 0.02832 cubic meter per second (m3/s)

SI to inch/pound

Multiply By To obtain

Length

millimeter (mm) 0.03937 inch (in.)

kilometer (km) 0.6214 mile (mi)

Volume

microliter (μL) 0.000034 ounce, fluid (fl. oz)

Mass

gram (g) 0.03527 ounce, avoirdupois (oz)

Temperature in degrees Celsius (°C) may be converted to degrees Fahrenheit (°F) as follows:

°F=(1.8×°C)+32.
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Anatomical and Genetic Variation of Western Oxyloma

(Pulmonata: Succineidae) Concerning the Endangered 

Kanab Ambersnail (Oxyloma haydeni kanabense) in 

Arizona and Utah

By Melanie Culver1, Hans-Werner Herrmann2, Mark Miller3, Barry Roth4, and Jeff Sorenson5

Abstract

The land snail genus Oxyloma (Pulmonata: Succineidae) 

includes the Federally endangered Kanab ambersnail 

(Oxyloma haydeni kanabense Pilsbry), which is known at 

the time of this study from only two locations in the United 

States: Three Lakes, Utah, and Vaseys Paradise, Arizona, on 

the Colorado River in Grand Canyon National Park. Since 

1994, the Kanab ambersnail has received much attention 

because its presence at Vaseys Paradise has implications for 

the ecosystem-wide management of the Colorado River. This 

attention is primarily because an experimental high-flow 

release of water from Glen Canyon Dam in 1996 destroyed 

or degraded Kanab ambersnail habitat at Vaseys Paradise. 

This experimental high flow was designed to replicate natural 

flow regimes throughout the Grand Canyon river corridor. 

However, as a result of the habitat destruction at Vaseys 

Paradise, in 1996, the U.S. Fish and Wildlife Service ruled 

that no further experimental high-discharge floods could be 

carried out until additional Kanab ambersnail populations 

were discovered or established. This mandate created a 

situation where the management of a single endangered 

species conflicted directly with the management of an entire 

ecosystem. Although since 1996, the U.S. Fish and Wildlife 

Service has permitted the use of flows as high as stage 

heights equivalent to 44,000 cubic feet per second, higher 

flows were requested by various Grand Canyon stakeholders 

and scientists but were not possible owing to low storage of 

Lake Powell.

1

U.S. Geological Survey, Arizona Cooperative Fish and Wildlife Research 

Unit and University of Arizona, School of Natural Resources and the 

Environment, Tucson.

2

University of Arizona, Department of Plant Sciences, Tucson.

3

U.S. Geological Survey, Forest and Rangeland Ecosystem Science Center, 

Corvallis, Oregon.

4

Consultant, San Francisco.

5

Arizona Game and Fish Department, Phoenix.

Adding to the controversy about Oxyloma and the Kanab 

ambersnail were previous anatomical and genetic analyses 

of the genus, which showed that genetic characteristics of 

specimens did not correspond with their identifications based 

on traditional taxonomic criteria, raising questions about the 

validity of the taxonomy of Oxyloma and the protected status 

of Kanab ambersnails. Specifically, a previous study suggested 

that the endangered Kanab ambersnail population at Three 

Lakes was more closely related to other, non-endangered 

ambersnail populations across the Southwest. In contrast, 

the Kanab ambersnail population at Vaseys Paradise 

appeared to be genetically distinct from all other ambersnail 

populations studied.

Management options for the ambersnail population 

at Vaseys Paradise, at the time of this study, conflict with 

ecosystem-wide measures proposed to benefit other natural 

resources in the Grand Canyon. The U.S. Fish and Wildlife 

Service will not revise the 1995 Kanab Ambersnail Recovery 

Plan until further genetic and anatomical analyses provide 

more fine-scale taxonomic resolution of the identity of 

Oxyloma populations on the Colorado Plateau and elsewhere 

in the American Southwest. Likewise, interagency cooperators 

cannot revise down-listing criteria for the Kanab ambersnail 

until substantial evidence is provided identifying distinct 

Oxyloma taxa or a larger group of conspecifics that reasonably 

could be managed as one species. Therefore, given the 

current controversy about the taxonomy of Oxyloma and the 

endangered Kanab ambersnail, new detailed analyses were 

completed of morphological and genetic variation from many 

Oxyloma specimens collected at 12 western North American 

locations. These new data have allowed us to evaluate many 

issues related to Kanab ambersnail taxonomy. Using this 

dataset, the study of shells and anatomy indicates that the 

holotype of Oxyloma haydeni kanabense plausibly can be 

regarded as a member of the same species as the populations 

of Oxyloma analyzed in this study. Additionally, the presence 

of gene flow among all populations is evidence that they 

are members of the same species. Almost all the observed 

genetic diversity can be accounted for by short-distance or 
2 Anatomical and Genetic Variation of Western Oxyloma Concerning the Endangered Kanab Ambersnail in Arizona and Utah

long-distance dispersal events between populations in this 

study. Our major taxonomic conclusion is that all samples 

collected for this study were drawn from populations of the 

same species.

Introduction

The land snail genus Oxyloma (Pulmonata: Succineidae) 

contains about 16 described taxa throughout the United States 

and Canada (Pilsbry and Ferris, 1911; Pilsbry, 1948; Turgeon 

and others, 1998) and includes the federally endangered 

Kanab ambersnail (Oxyloma haydeni kanabense Pilsbry). 

(Note: Although it is spelled “kanabensis” in most of the 

literature, this taxon name should be “kanabense” to agree in 

gender with the neuter noun Oxyloma [Turgeon and others, 

1998]. Similarly, Oxyloma nuttalliana and O. retusa should be 

O. nuttallianum and O. retusum, respectively.)

The taxon Oxyloma haydeni kanabense Pilsbry 

previously has been recognized at only two locations: 

Three Lakes, Utah, and Vaseys Paradise, Arizona, on the 

Colorado River in Grand Canyon National Park (fig. 1). It is 

protected under the Endangered Species Act as an endangered 

subspecies (U.S. Fish and Wildlife Service, 1992a and 

1992b), although some consider it deserves full species status 

(Oxyloma kanabense: Harris and Hubricht, 1982; Spamer and 

Bogan, 1993). The Kanab ambersnail purportedly differs from 

other Oxyloma in its shell morphology, whose “spire is more 

slender and drawn out than O. haydeni” (Pilsbry, 1948), and 

reproductive anatomy (genitalia). According to Pilsbry (1948), 

“the genitalia show some resemblance to O. nuttalliana, but 

this species differs by the short penis, more coiled epiphallus, 

the twinned seminal vesicles and (in the specimen dissected), 

the massive hermaphroditic duct.” There are many cases 

where the U.S. Fish and Wildlife Service (USFWS) is 

uncertain whether a group of populations should be classified 

as a subspecies or a species, and this issue is controversial 

(Scott and others, 2006). The term “evolutionarily significant 

unit (ESU)” is sometimes used to describe the same level of 

population divergence as a typical subspecies, so subspecies 

and ESU are often used synonymously (Moritz, 1994).

Since 1994, the Kanab ambersnail has received 

much attention because its presence at Vaseys Paradise 

has implications for the ecosystem-wide management of 

the Colorado River. In 1996, an experimental high-water 

discharge from Glen Canyon Dam destroyed or degraded 

about 17 percent of Kanab ambersnail habitat at Vaseys 

Paradise (Stevens and others, 2001). This planned flood 

release was designed to let resource managers evaluate an 

alternate strategy for Glen Canyon Dam operations and to 

allow the restoration of more natural flow regimes throughout 

the Grand Canyon river corridor. The ultimate goal was to 

mimic natural floods that occurred in the canyon prior to 

the completion of the dam in 1963. As a result of the habitat 

destruction at Vaseys Paradise in 1996, the USFWS ruled 

that no further experimental high-flows could be done until 

additional Kanab ambersnail populations were discovered or 

established (U.S. Fish and Wildlife Service, 1996, 1997). This 

mandate created a situation where the management of a single 

endangered species conflicted directly with the management 

of an entire ecosystem (Meretsky and others, 2000). Although 

since 1966, the USFWS permitted the use of flows as high 

as stage heights equivalent to 44,000 cubic feet per second 

(U.S. Fish and Wildlife Service, 1996, 1997), various Grand 

Canyon stakeholders and scientists requested higher flows. 

These higher flows, although potentially beneficial to the 

Grand Canyon ecosystem, would result in habitat destruction 

at Vaseys Paradise. Until the USFWS declares a new Finding 

of No Significant Impact for the High Flow Equivalent 

protocol Environmental Assessment, it is not clear what they 

find acceptable, although the USFWS has allowed three tests 

of that magnitude since the Environmental Impact Statement 

was completed.

In previous work on the genus Oxyloma (Miller and 

others, 2000), Amplified Fragment Length Polymorphism 

(AFLP) markers and deoxyribonucleic acid (DNA) sequences 

were used to characterize genetic diversity in southwestern 

Oxyloma populations. This analysis was performed 

on individuals from the two known Kanab ambersnail 

populations and individuals from the only two known 

southwestern populations of the widespread, non-endangered, 

Niobrara ambersnail (Oxyloma haydeni haydeni) from Indian 

Garden, in Grand Canyon National Park, and the Minus 

9-Mile Spring, upstream of Lees Ferry on the Colorado River. 

A controversial result of this analysis was the indication that 

the Kanab ambersnail population at Three Lakes is closely 

related to the non-endangered Niobrara ambersnail population 

at Indian Garden. Additionally, the Vaseys Paradise population 

appeared to be distinct from the other three populations. These 

findings raised multiple questions about the taxonomic status 

of the Kanab ambersnail and its protected status under the 

Endangered Species Act.

Because of the conflicting morphological and molecular 

genetic information from Miller and others (1996) and Stevens 

and others (2000) on the evolution of the genus Oxyloma, a 

discrepancy existed, as the actual taxonomic and conservation 

status of the Kanab ambersnail was unclear. As a result of 

this discrepancy, efforts were made to locate additional 

southwestern populations of Oxyloma, and field surveys 

identified many new populations of the genus throughout the 

Southwest (Meretsky and others, 2002).
Introduction 3

tac11-0685_fig 01

Grand Canyon 

National Park 

MOHAVE COCONINO APACHE

NAVAJO

GILA

LINCOLN

YAVAPAI

MARICOPA

IRON

CLARK

SAN JUAN

KANE

GARFIELD

LA PAZ

MILLARD

EMERY GRAND

BEAVER

WAYNE

SEVIER

WASHINGTON

WHITE PINE

PIUTE

SANPETE

Verde River

Escalante River

Dirty Devil R

Green River San Juan River

Colorado River

Colorado River

Little Colorado River

Sevier River

Virgin River

Colorado River

Sevier

Lake

Salt River

Dolores River

Kanab Creek

East Fork Sevier River

Fremont River

Panguitch

Creek

E Fk Virgin R

Puerco River

Phoenix

St.

George

Laughlin

Prescott

Page

Flagstaff

COLORADO

CALIFORNIA

ARIZONA

NEVADA

NEW MEXICO

UTAH

PasCrk

PanCrk

SR

CCrk

ER

LF

ECh IG

VP

3L

Greens

GHwy

EXPLANATION

Sample location

CCrk, Center Creek

ER, Escalante River

GHwy, Glendale Highway

IG, Indian Garden

LF, Lees Ferry

PasCrk, Pass Creek

PanCrk, Panguitch Creek

SR, Sevier River

Greens, The Greens

3L, Three Lakes

ECh, Elves Chasm

VP, Vaseys Paradise

114° 112°

38°

36°

34°

Base map modified from USGS and other digital data sources, 

variable sources. Projection: Albers Equal Area Conic, CM = -96°, 

SP1 = 29.5°, SP2 = 45.5°, LO = 23°.

0 20

20

40

40

60

60

80

80

100 Miles

0 100 Kilometers

110°

CA

AZ

NV

WY

OR ID

UT

NM

MN

TX

NE

MT

WA

CO

SD

ND

OK

KS

IA

MEXICO

CANADA

Map

Area

Figure 1. Sampling locations for Oxyloma populations used in this study from Arizona and Utah. Each location is coded with a 

different color. These colors are consistent for those same populations in the phylogenetic trees figures.
4 Anatomical and Genetic Variation of Western Oxyloma Concerning the Endangered Kanab Ambersnail in Arizona and Utah

Given the discovery of new Oxyloma populations, 

Stevens and others (2000) conducted new analyses of 

anatomical and mitochondrial genetic variation (cytochrome b, 

cyt b) in succineids from across western North America. 

These analyses corroborated findings of the previous study 

by Stevens and others (2000) in that taxonomic designations 

of Oxyloma specimens did not correspond with their 

mitochondrial haplotypes. For example, the Kanab ambersnail 

population at Three Lakes and the Niobrara ambersnail 

population at Indian Garden (Haplotype BB), when included 

with a sample of a more diverse set of populations, continued 

to form part of the same evolutionary clade. Similarly, the 

Vaseys Paradise population, which seemed distinct from the 

other three populations previously studied, appeared in its own 

basal group outside the other Oxyloma specimens included in 

analyses. This suggested that the Vaseys Paradise population 

diverged from other Oxyloma populations early in the 

evolutionary history of the genus in western North America. 

Interestingly, whereas the Kanab ambersnail population at 

Three Lakes seemed closely related to other newly discovered 

southwestern Oxyloma populations, the anatomical studies 

indicated that none of the new populations contained Kanab 

ambersnails. Instead, individuals identified as O. haydeni 

haydeni and O. retusum seemed closely related to specimens 

from the Three Lakes population.

Background Information on 

Subspecies Trinomial

Because the Kanab ambersnail was listed under the 

Endangered Species Act as a subspecies, it is relevant to give 

some background information on the issue of subspecies, 

particularly with respect to conservation. The term 

“subspecies,” as a Latin trinomial, was introduced by Esper to 

designate geographical varieties (Mayr, 1982). In 1942, Mayr 

defined a subspecies as “a geographically defined aggregate 

of local populations which (sic) differ taxonomically from 

other subdivisions of the species.” However, Wilson and 

Brown (1953) dismissed the subspecies trinomial as arbitrary, 

too subjective to be useful, a taxonomic designation that is 

often based on insignificant groupings or clines, and widely 

exploited. In 1981, with the emergence of conservation 

biology as a science and the legal incorporation of subspecies 

into the Endangered Species Act, debate over subspecies 

was revived at an ornithological forum (Barrowclough, 

1982; Gill, 1982; Mayr, 1982). Later, Avise and Ball (1990) 

suggested that subspecies designations should be made 

based on “concordant distribution of multiple independent 

genetic traits,” and that this would reduce the subjective 

and arbitrary nature of trinomials. Subsequent to Avise and 

Ball’s definition, O’Brien and Mayr (1991) proposed that 

members of a subspecies would share (1) a unique geographic 

range, (2) a set of “phylogenetically concordant phenotypic 

characters,” (3) a suite of molecular genetic similarities, 

and (4) derived adaptations relative to other subspecies. 

Furthermore, a subspecies could (1) go extinct, (2) become 

a new subspecies by hybridization with another subspecies, 

(3) become a new subspecies by changing its genetic character 

through stochastic processes like genetic drift, (4) become 

a new species by becoming reproductively isolated such 

as by developing intrinsic barriers, or (5) remain the same. 

Subspecies should be on their own “evolutionary trajectory,” 

not yet reproductively isolated but with the future potential to 

be isolated, as suggested by Moritz (1994) in his development 

of the Evolutionarily Significant Unit (ESU) concept; many 

systematists use the term ESU synonymously with subspecies. 

Molecular Genetic Markers Used

With the advance of molecular markers, in the 1990s, 

as a high-resolution tool to determine phylogenetically 

distinguishable intraspecific populations, molecular markers 

have revealed that subspecific taxonomy does not always 

represent evolutionary relationships (Avise and Ball, 1990; 

O’Brien and Mayr 1991). Additionally, the results of 

phylogenetic reconstruction with one gene do not always 

agree with the results of phylogenetic reconstruction with 

another gene, and results with one gene can differ among 

species (Avise, 1991; Graur and Li, 2000). Therefore, several 

different genes and several independent markers were selected 

for this study.

The first class of markers used here is mitochondrial 

DNA (mtDNA) genes. The mtDNA genome does not 

recombine and is inherited maternally (Brown, 1983; Harrison, 

1989; Gyllensten and others, 1991). Additionally, the mtDNA 

genome is rapidly evolving, making it useful in examining 

recently diverged populations (Brown, 1985; Wilson and 

others, 1985; Moritz and others, 1987; Avise, 1991). The 

second class of markers used is nuclear microsatellite DNA. 

These markers, composed of tandem repeats of 1–6 base 

pair (bp) units, are highly polymorphic nuclear markers that 

are randomly distributed throughout the genome (Weber, 

1993; Tautz and others, 1986; Tautz, 1989; Edwards and 

others, 1992). Because microsatellites have high levels of 

polymorphism, they are ideal for examining populations 

within a species. The next class of molecular markers is 

nuclear DNA sequence for genes and random regions. The 

nuclear DNA sequences complement the mtDNA because 

they are inherited bi-parentally instead of maternally. The 

final marker used in this study is Amplified Fragment Length 

Polymorphisms (AFLP), which is a multilocus marker that 

simultaneously can type hundreds of loci across the genome. 

The advantage of AFLPs is their ability to sample broadly 

across the nuclear genome of the organism. Each marker 

is characterized by distinct rates of evolution and thereby 

possesses different levels of resolution from resulting data and 

analyses, and was selected to analyze recent divergences.
Methods 5

Study Objectives

The main objective of this study was to obtain additional 

information to help resolve the controversial Oxyloma haydeni

taxa at the species and subspecies-population level. The 

first step in resolving differences among datasets required 

a thorough understanding of the anatomical variation used 

at the time of this study to distinguish taxa and to designate 

conservation units. This step is essential, given that previous 

anatomical analyses of southwestern Oxyloma were performed 

on very small sample sizes (1–3 individuals) from each 

location (Stevens and others, 2000). Furthermore, in the course 

of performing the new anatomical work, tissue samples were 

preserved separately and used to obtain DNA for genetic and 

molecular evolutionary analyses of nuclear and mitochondrial 

genetic variation. This provided an unprecedented opportunity 

to generate molecular and morphologic datasets from an 

identical array of specimens. We sampled a relatively 

large number of individuals (about 20) within each of 

12 populations. Generation of these new genetic data, along 

with the new anatomical information, allowed us to address 

the following questions:

1. What are the extent and nature of anatomical variation 

among individuals within populations?

2. Does the anatomical variation within populations 

appear to reflect the presence of one or more species at 

a location? 

3. Do anatomical investigations tend to confirm the 

taxonomic significance of structures originally regarded 

as significant by Pilsbry (1948), or do other anatomical 

characters better reflect the evolutionary history of 

Oxyloma in the western United States?

4. Does the previous pattern observed in Stevens and others 

(2000) hold when large numbers of individuals from each 

location are used for analyses?

5. Do the genetic data suggest the presence of distinct 

evolutionary lineages within and among populations? 

6. Do the genetic data, when interpreted as a whole along 

with data from analyses of anatomical variation, suggest 

that one of the three hypotheses described in questions 2, 

3, or 4 is likely correct?

Question 1 is observational; questions 2, 3, and 4 are 

interpretive. Interpretations of explicit criteria have been made 

for species definitions (Roth, 2004).

Purpose and Scope

This report documents all known land snail populations 

of the genus Oxyloma in Arizona and Utah and examines 

taxonomic distinctions among populations. Additional 

taxonomic considerations at the subspecies level are 

discussed as relevant to the Kanab ambersnail. The Kanab 

ambersnail was listed as federally endangered in 1992, and the 

endangered subspecies is thought to occur at Vaseys Paradise, 

Arizona. The management of ambersnails at Vaseys Paradise, 

Arizona, is balanced with ecosystem-wide management 

of other natural resources in the Grand Canyon. Data used 

to resolve the taxonomic status of ambersnails include 

anatomical variation, morphological variation, and genetic 

variation of mitochondrial and nuclear DNA. Much data were 

collected for anatomical, morphological, and genetic markers 

for ambersnails from 12 populations across Arizona and Utah.

Methods 

A combination of morphological and molecular methods 

was used in this study, starting with careful sampling of all 

12 known ambersnail populations in Arizona and Utah. The 

same samples were used for all data collection methods; a 

small part of each sample was used for mitochondrial and 

nuclear genetic analyses and the remainder was used for 

anatomical and morphological analyses.

Specimen Collection

We restricted the sample collections to 12 southwestern 

ambersnail populations that were selected based on their 

historical or conservation importance, previous taxonomic 

designations applied to specimens obtained from these 

locations, or because the population in question recently has 

been discovered and has never been the subject of genetic 

or morphological analyses. Samples were collected from 

12 locations in Arizona and Utah (table 1). Sample collections 

were also attempted at one location in Nevada (Maggie 

Creek), but several trips there did not reveal any snails. Each 

of the 12 sampling locations generated a “batch” of samples: 

Batches 1–12, corresponding to each of 12 sampling locations. 

Between 20 and 25 samples were collected from each 

location except Batch 1 (with 14 samples) and Batch 12 (with 

16 samples). Because samples from Batch 11 were not mature 

individuals, Batch 11 sample specimens were not included in 

the anatomical analyses. Four additional batches were sampled 

(test batches A, B, C, and D), which were used exclusively 
6 Anatomical and Genetic Variation of Western Oxyloma Concerning the Endangered Kanab Ambersnail in Arizona and Utah

for genetic testing. Samples from Batches 1–10 and 12 are 

considered voucher specimens and are curated by Barry Roth 

for placement into a museum. DNA sequences linked to 

these voucher specimens have been deposited in GenBank®

(appendix A) with the exceptions of nuclear 356 sequences, 

which were too short to deposit in GenBank®, and nuclear 

internal transcribed spacer (ITS) sequences, which had a high 

likelihood of homoplasy (convergent evolution). Because 

multiple copies of the ITS sequence were located throughout 

the genome of each individual, we were uncertain if the 

comparable versions of the ITS gene among individuals were 

being analyzed. Instead, the sequences for 356 and ITS are 

included in appendixes B and C, respectively.

Sterile sample vials and fresh preservative solutions were 

used for all collections to prevent possible contamination. 

All snails were handled with clean forceps. Live specimens 

were drowned in a closed container of water and transferred 

by stages to 70 percent ethanol (EtOH). Following the 

initial drowning, a small piece of foot tissue from each 

animal was preserved separately in 95 percent EtOH for 

use in genetic analyses. Each specimen was labeled with a 

unique identification number that was linked to a database 

of information indicating the specimen’s population 

affiliation, collection date, and other relevant information. 

This information was duplicated in a field journal as backup. 

When satellite coverage was available, a GarminTM GPS 

unit was used to record locality information: a minimum of 

180 waypoints were averaged and saved to improve accuracy 

of site location. Representative digital photographs were 

made of each locality for later reference. Localities and their 

descriptions are provided in figure 1 and table 1, respectively, 

with the figure including only Oxyloma localities and the table 

including descriptions of Oxyloma and Catinella localities.

Table 1. Site name and locality description of ambersnail populations in Arizona and Utah sampled for the anatomical and genetic 

analyses described in this study.

[Information is provided about the taxonomic designations of the limited numbers of snails previously identified from the 12 sampling locations. Abbreviations: 

km, kilometer; mi, mile]

Batch 

No. Site name State Locality Previous taxonomic designation

1 Sonoita Creek Arizona Near Fresno Canyon Catinella

2 Lees Ferry Arizona Minus 9-Mile Spring upstream of Lees Ferry on the 

Colorado River

Oxyloma haydeni haydeni

3 Indian Garden Arizona Indian Garden Campground in Grand Canyon 

National Park

Oxyloma haydeni haydeni

4 Vaseys Paradise Arizona Vaseys Paradise, River Mile 31.5R along 

Colorado River1

Oxyloma haydeni kanabense

5 The Greens2 Utah Best Friends Ranch along Highway 89 north of 

Kanab, Utah

Oxyloma retusum, O. haydeni haydeni

6 Glendale Highway Utah KOA Campground 3 km north of Glendale on 

Highway 89

Oxyloma retusum 

7 Center Creek Utah Tributary to East Fork Sevier River, south of Antimony Oxyloma? (undescribed species)

8 Sevier River Utah North of The Nature Conservancy, along Highway 89 Oxyloma?

9 Three Lakes Utah 6 km north of Kanab, Utah, along Kanab Creek Oxyloma haydeni kanabense

10 Pass Creek Utah Along Highway 143, 0.5 mi north of Pass Creek Road, 

north of Panguitch Lake

Oxyloma? (undescribed species)

11 Panguitch Creek Utah Pole Hollow Bridge Oxyloma retusum 

12 Escalante River Utah Wet meadow upstream Oxyloma?

1

River Mile (RM) is used to describe distances along the Colorado River in the study area. The use of the river mile has historical precedent and provides a 

reproducible method for describing location. Lees Ferry is considered the reference point, RM 0, with mileage measured for both upstream and downstream 

locations. Locations upstream of Lees Ferry in the Glen Canyon National Recreation Area are assigned negative river-mile designations. “R” in “31.5R” refers 

to the right bank of the river viewed in the downstream direction. 

2

Although considered to be the Kanab ambersnail (Oxyloma haydeni kanabense) type locality, no Kanab ambersnails have been observed there since 1991 

(Meretsky and others, 2002).
Methods 7

Shell Morphology 

The counts, dimensions, and calculated values recorded 

from shells in the samples were selected to provide a dataset 

that effectively expresses size and shape variability in the 

system of shell characters. The succineid shell is simple 

for a gastropod shell; there is little surface sculpture (none 

considered significant during this investigation) and there is 

no departure from the initial coiling trajectory as an individual 

nears adulthood, unlike in some other land-snail families. 

For this reason, relatively few parameters were adequate to 

characterize the shells in the samples at an acceptable level 

of generality. More measurements would introduce the risk 

of tracking individual, secular variation that would amount to 

noise in the phylogenetic system.

Shells were released from the soft parts (held with 

fine forceps) by gentle, twisting pressure. Where necessary, 

algal film on the surface was removed by brief immersion 

in an ultrasonic pen cleaner (Koh-i-noor®). Shells were 

photographed digitally in standard apertural view with both 

shell axis and maximum width of the aperture in the plane of 

the page. The images were printed at a standard enlargement 

(8.5 times). On the resulting prints, two lines were drawn 

tangent to the right and left sides of the first whorl and the 

body whorl, respectively, delineating the apical angle of the 

shell. The coiling axis was delineated by a line from the vertex 

of this angle through the columellar side of the aperture.

The following measurements and counts were recorded 

for each shell image (SI):

Apical angle (APA)—angle between two lines tangent 

to the right and left sides of the first whorl and the body 

whorl, respectively, measured with a protractor.

SI (shell image) length (IL)—distance between the 

apex of the shell and the lowest extent of the aperture, 

measured parallel to the axis.

SI maximum diameter (ID)—greatest width of the shell 

measured normal to the axis.

SI height of maximum diameter (IMD)—distance from 

the lowest extent of the aperture to the point of greatest 

diameter, measured parallel to the axis.

SI aperture length (IAL)—distance from the junction of 

the posterior lip of the aperture with the preceding whorl 

to the lowest extent of the aperture, measured parallel to 

the axis.

SI aperture width (IAW)—maximum width of the 

aperture measured perpendicular to the axis.

Number of whorls (WN)—counted by the method of 

Verduin (1982).

Width of whorl 1 (W1)—distance from axis to insertion 

of first whorl upon suture of second whorl, on apertural 

(anatomically, right) side of shell, measured perpendicular 

to the axis.

Width of whorl 2 (W2)—distance from insertion of first 

whorl to insertion of second whorl upon suture of third 

whorl, on apertural side of shell, measured perpendicular 

to the axis.

Width of whorl 3 (W3)—distance from insertion of second 

whorl to insertion of third whorl upon suture of fourth 

whorl, on apertural side of shell, measured perpendicular 

to the axis.

Width of whorl 4 (W4)—distance from insertion of third 

whorl to maximum extent of fourth whorl, on apertural 

side of shell, measured perpendicular to the axis.

These SI measurements were divided by 8.5 to convert 

them to the following shell dimensions: Shell length (SL), 

shell maximum diameter (SD), height of maximum diameter

(SMD), shell aperture length (SAL), and shell aperture width

(SAW). Shell dimensions, rather than SI measurements, were 

used in statistical analysis.

From these dimensions, the following sum and ratios 

were calculated, generating some of the traditional characters 

used in the description of gastropod shells and used in this 

study as characters for phylogenetic analysis:

Shell size (SS)—shell length plus shell maximum 

diameter (SL + SD); for use of this sum as a measure 

of overall size, see Gould (1969) and Galler and Gould 

(1979).

Relative height (SRH)—shell length/shell maximum 

diameter (SL/SD).

Relative aperture height (SRA)—shell aperture length/

shell length (SAL/SL).

Aperture asymmetry (APY)—height of maximum 

diameter/shell aperture length (SMD/SAL). This character 

expresses the departure of the shape of the aperture from, 

approximately, an ellipse. A value of 0.50 represents a 

figure with its widest extent at its midpoint, like an ellipse. 

A low value produces what is traditionally described as an 

“effuse” aperture, which is wide near the anterior end.
8 Anatomical and Genetic Variation of Western Oxyloma Concerning the Endangered Kanab Ambersnail in Arizona and Utah

Shell coiling parameters (Raup, 1966; also see Kohn 

and Riggs, 1975; Lindberg, 1985) were calculated from 

unconverted image measurements:

S (“apertural shape”) = SI aperture length/SI aperture 

width.

W (“whorl expansion rate”) = (W1/W2 + W2/W3 + 

W3/W4) / (WN-1)

T (“translation”) = Y/R, where Y is the distance of the 

center of the generating curve from the apex of the shell, 

measured parallel to the axis (calculated herein as shell 

image length minus one-half of SI aperture length), 

and R is the distance of the center of the generating 

curve from the axis (calculated herein as one-half the SI 

aperture width).

A fourth coiling parameter, D (Raup, 1966), representing 

the distance of the generating curve from the axis, was 

observed, from inspection of the shells, to equal zero in all 

cases. No specimens had an umbilicus, the void created down 

the center of a shell when the generating curve migrates away 

from the coiling axis. This parameter was not considered 

further in the study.

The data on samples identified as Oxyloma (that is, all but 

the sample from Sonoita Creek, Arizona, which is a sample 

of Catinella included in the study to serve as an outgroup 

in phylyogenetic analysis) were examined by principal 

components analysis and discriminant analysis with the 

software SYSTAT 12® for Windows. Principal components 

analysis was used to examine the sources of variation among 

the entered variables and to generate a reduced set of factors 

(“components”) to express that variation in coherent terms. 

Discriminant analysis was used to assess the performance 

of individual variables for discriminating among samples. 

Three sets of variables were subjected to discriminant 

analysis, representing three different approaches of traditional 

taxonomy toward recognizing discrete taxonomic units.

The holotype of Oxyloma haydeni kanabensis Pilsbry, 

1948 (Academy of Natural Sciences of Philadelphia specimen 

103166) was submitted to discriminant analysis as an 

unclassified case, to determine to which of the established 

groups it would be assigned. Previous studies using 

discriminant analysis to assign type specimens to subsequently 

defined or recognized categories include those of Shaklee and 

Tamaru (1981) and Lindberg (1985).

Soft Tissue Anatomy

Specimens were dissected on paraffin in 70 percent 

EtOH following standard methods for this type of anatomical 

examination (for example, Patterson, 1973; Hoagland and 

Davis, 1987). Soft parts were held with fine forceps and 

released from the shells by gentle, twisting pressure. Color 

and patterning of the mantle and head-foot were recorded. The 

body cavity was opened by dorsal or right-lateral incision and 

by paring back the mantle over the lung. Organs of the body 

cavity were observed in place. Anatomical features examined 

included the character and size of the hermaphrodite duct, 

albumen gland, prostate gland, spermatheca and its duct, vas 

deferens, epiphallus, penis, penial appendix (where present), 

vagina, and free oviduct.

DNA Isolation

We extracted DNA from 292 snail tissue samples (foot 

pieces and whole soft bodies) from 12 populations and 

4 batches of samples used only for testing marker variability. 

We used phenol-chloroform and standard silica-based methods 

of DNA extraction and purification, and DNA was quantified 

using PicoGreen® and measured in a fluorometer.

Genetic Analyses

Several types of molecular genetic markers were used 

to analyze the ambersnail specimens. In total these markers 

represented a mixture of coding regions (or genes) and noncoding regions (including repetitive and non-repetitive DNA). 

Mitochondrial DNA

Mitochondrial DNA evolves more rapidly than nuclear 

DNA and is maternally inherited. Two genes were selected 

within the mitochondrial genome based on their rate of 

evolution and widespread use in other mollusk studies. 

Data Generation

Two mitochondrial genes were sequenced for use 

in the analyses. First, we used the universal molluscan 

primers HCO12198 and LCO1490 described in Folmer and 

others (1994) to amplify about 600 bp of the mitochondrial 

cytochrome oxidase subunit I gene (COI). Likewise, we 

used the universal primers of Merritt and others (1998) to 

amplify about 410 bp of the mitochondrial cytochrome b gene 

(cyt b). Fragments were sequenced bi-directionally on an 

ABI3730 DNA sequencer using standard methods. Sequence 

chromatograms were aligned and edited using the Seqman 

and Megalign software packages (Lasergene®, Inc.). After 

editing, sequences were trimmed to 510 bp for the COI gene 

and to 366 bp for the cyt b gene. In total, 176 individuals 

were sequenced for the COI gene and 183 individuals were 

sequenced for the cyt b gene. Specific information on sample 

sizes from each location is provided in table 2. Five members 

of the Catinella outgroup population amplified and we also 

downloaded three mtDNA COI sequences from GenBank®

to use as outgroups for this study, from Catinella baldwini 

(GenBank® accession number AY148571.1), and Neosuccinea 

kofui (GenBank® accession number AY150085.1). 

Additionally, we sequenced the partial COI gene for 

Succinea concordialis.
Methods 9

Nuclear DNA

Nuclear DNA has a broad variety of marker types from 

coding, non-coding, single copy, multilocus, and repetitive 

regions—each with different evolutionary rates. Our selection 

of nuclear markers included all these categories and spanned a 

sampling of the entire nuclear genome.

Single Copy

To identify nuclear loci for Polymerase Chain Reaction 

(PCR) amplification, we applied two strategies. First, we 

tested published primers, which had previously been used in 

other studies, and, second, we created a genomic library.

Markers from Other Studies

We designed primers and optimized PCR amplification 

conditions for seven different nuclear loci: an intron of the 

aminopeptidase N gene (AP54; Sokolova and Boulding, 

2004), ribosomal DNA first and second internal transcribed 

spacer (ITS-1 and ITS-2; Armbruster and others, 2000), 

large subunit 28S rDNA (LSU; Teshima and others, 2003), 

6-phosphogluconate dehydrogenase (PGD; Lee and Foighil, 

2002), and S7 ribosomal gene introns (S7-1 and S7-2; Chow 

and Hazama, 1998). These loci had been used in genetic 

studies in different animal groups ranging from gastropods 

to fish. We designed new primers for some loci (AP54, ITS 

and S7). Next, we sequenced 10 different DNA fragments 

from 6 of the initial 7 published loci for one snail from Vaseys 

Paradise (“Kanab ambersnail”), and one snail from Indian 

Garden (“Niobrara ambersnail”). We were able to align 

sequences in 8 of the 10 fragments. An 800-bp fragment of 

S7-1 (S7) and a 400-bp fragment of ITS-1 (ITS) showed much 

variation between the Vaseys Paradise and the Indian Garden 

specimens. The other eight fragments showed no variation 

between the specimens of the two populations and, therefore, 

were determined to be uninformative for this study.

Genomic Library

We constructed a non-enriched genomic library to 

develop a set of random single nucleotide polymorphism 

(SNP) markers for Oxyloma. We amplified 250 positive 

clones and sequenced 150 clones for a total of about 65 kilo 

base pairs (kbp). We designed primers using OLIGO 6.6 

(Molecular Biology Insights, Inc.) and PRIMER3 (Rozen and 

Skaletzky, 2000), and optimized PCR reactions to amplify and 

sequence snail test panels from different populations to assess 

marker variability and informativeness. Two cloned sequence 

fragments, 458 and 356, showed much variation and were 

suitable for this study. However, there were many variable 

sites per clone, instead of single sites typical for SNPs. We, 

therefore, performed genotyping by sequencing the complete 

fragment for all individuals. For primer sequences and 

annealing temperatures, see table 3.

Table 2. Number of ambersnail samples per population in Arizona and Utah, sequenced for two mitochondrial DNA (mtDNA) loci, the 

concatenated mtDNA loci, the four nuclear DNA loci, and the Amplified Fragment Length Polymorphism (AFLP) analyses.

[Samples from the introduced Elves Chasm population were not sequenced for mtDNA loci nor included in the AFLP analyses. COI: cytochrome oxidase 

subunit I gene. cyt b: cytochrome b gene. mtconcat: mitochondrial genes concatenated. S7: ribosomal gene introns S7-1 and S7-2. ITS: ribosomal DNA first 

and second internal transcribed spacer. 356: random ambersnail nuclear clone 356. 458: random ambersnail nuclear clone 458. AFLP: Amplified Fragment 

Length Polymorphism. –, no data]

Locality

DNA genes or regions

COI cyt b mtconcat S7 ITS 356 458 AFLP

Minus 9-Mile Spring-Lees Ferry (LF) 19 20 16 22 20 12 10 23

Indian Garden (IG) 23 20 19 21 7 0 9 25

Vaseys Paradise (VP) 13 16 13 17 17 13 16 18

The Greens (Greens) 19 21 16 24 1 24 21 24

Glendale Highway (GHwy) 17 21 15 22 20 14 9 25

Center Creek (CCrk) 16 20 16 18 3 18 4 19

Sevier River (SR) 5 11 4 17 0 10 9 19

Three Lakes (3L) 13 4 2 20 16 16 0 20

Pass Creek (PasCrk) 9 14 8 15 6 3 2 17

Panguitch Creek (PanCrk) 0 2 0 20 10 5 12 0

Escalante River (ER) 7 14 7 16 16 16 9 16

Elves Chasm (ECh) – – – 10 10 7 8 0

Total 141 163 116 222 126 138 109 206
10 Anatomical and Genetic Variation of Western Oxyloma Concerning the Endangered Kanab Ambersnail in Arizona and Utah

DNA fragments were sequenced bidirectionally on an 

ABI3730XL DNA Analyzer. Sequences were assembled and 

aligned with SEQUENCHER 4.2.2. (Gene Codes Corporation, 

2010) and edited using MacClade version 4.03 (Maddison 

and Maddison, 2001). As analysis software treats indels as 

missing data, we recoded indels of the same length and at 

the same position as an additional binary nucleotide site. 

If we encountered indels of different lengths at the same 

position, we treated each indel defined by a specific number 

of nucleotides as a separate binary character. If polymorphism 

was present within one individual, this could be detected from 

direct sequencing using our DNA sequencing methodology, 

allowing both alleles to be scored for an individual. After 

sequence alignment, the program PHASE 2.1 (Stephens 

and others, 2001; Stephens and Scheet, 2005), which uses a 

Bayesian algorithm, was used for haplotype reconstruction of 

autosomal loci (to set phase for each allele and to determine 

haplotypes in polymorphic individuals).

Data Analyses—Mitochondrial DNA and Nuclear Single 

Copy DNA

We performed an analysis of molecular variance 

(AMOVA) (Excoffier and others, 1992), using the program 

package ARLEQUIN 3.1 (Excoffier and others, 2006), and 

likewise calculated pairwise Fst values between all analyzed 

populations. We graphically visualized pairwise Fst values 

between populations by applying multidimensional scaling 

(MDS) using NTSSpc 2.0 (Applied Biostatistics Inc.).

To test for isolation by distance, we used NTSSpc 

2.0 (Applied Biostatistics Inc.) to perform Mantel tests (to 

estimate the correlation between geographical distances 

and genetic distances between populations) (Slatkin and 

Maddison, 1990). The analysis was performed separately for 

each gene.

To test for neutrality and demographic stability, we 

computed Tajima’s D (Tajima, 1989), Fu’s Fs

 (Fu, 1997), 

and mismatch distribution (Rogers and Harpending, 1992) 

using ARLEQUIN 3.1. Values for R2 (Ramos-Onsins and 

Rozas, 2002) were calculated with DNASP 4.10 (Rozas and 

others, 2003).

Median joining haplotype networks were constructed 

with the program NETWORK 4.2.0.1 of Fluxus Technology 

Ltd. (Bandelt and others, 1999). For phylogenetic analyses, 

we used the software package PAUP 4.0b10 (Swofford, 

2002). We performed neighbor joining (NJ) analyses as 

well as maximum parsimony (MP) analyses with heuristic 

searches, tree bisection and reconnection (TBR), and branch 

swapping and bootstrapping (100 replicates). Additionally, 

we performed Bayesian analyses (MB) using the program 

MrBayes version 3.1 (Ronquist and Huelsenbeck, 2003). We 

calculated the best fitting model of nucleotide substitution 

by using MODELTEST 3.7 (Posada and Crandall, 1998). 

The following models were found: COI TVM+I+G, cyt b

HKY+I+G, concatenated mitochondrial loci TVM+I+G, 

356 K81uf+I, 458 TVM+I, ITS GTR+G, S7 HKY+I, and 

concatenated autosomal loci TVM+I+G. MrBayes ran with 

10,000,000 generations in four Markov Chain Monte Carlo 

chains. An appropriate burnin was selected by assessing the 

MrBayes trace files with the program TRACER 1.3 (Rambaut 

and Drummond, 2005). The number of generations at burnin 

was 4,000 in all analyses. Because no outgroup data were 

available for many of the analyses, some of the phylogenetic 

analyses were performed without an outgroup. Those 

phylogenetic trees, therefore, are unrooted. However, a single 

outgroup rooted tree was performed using COI sequences 

from GenBank® from the outgroups Catinella, Succinea, and 

Neosuccinea. These sequences were aligned with mtDNA 

concatenated sequences from this study, and a maximum 

likelihood phylogenetic analysis was performed with RAxML 

7.0.3 (Stamatakis, 2006). RAxML parameters were set to 

GTRGAMMA substitution model, 1,000 bootstrap iterations, 

and Catinella was designated as the outgroup.

Microsatellite (Single Locus)

Because of the repetitive sequences at microsatellite 

loci, these markers have rapid evolutionary rates. As a result, 

microsatellites are a high-resolution molecular genetic tool 

suitable for subspecies-level taxonomic analyses. 

Table 3. Polymerase Chain Reaction (PCR) primer sequences 

used for PCR amplification and the appropriate PCR annealing 

temperatures for each of the five nuclear loci used in this study of 

Oxyloma popul ations.

[Samples from the introduced Elves Chasm population were not sequenced for 

mtDNA loci nor included in the AFLP analyses. Primer sequence (5’→3’):

5 prime to 3 prime. Abbreviations: Ta, annealing temperature; °C, degrees 

Celsius; F, forward; R, reverse; S7, ribosomal gene introns S7-1 and S7-2; 

ITS, ribosomal DNA first and second internal transcribed spacer; 356, random 

ambersnail nuclear clone 356; 458, random ambersnail nuclear clone 458; 

KS6511, simple tandem repeat (STR) locus KS6511]

Locus Primer sequence (5’→ 3’) Ta (°C)

S7 F: CTGACTTTGTTCTGTTTCTGT 50

R: TCTATCTCCTGGTTCATTGAT

ITS F: TGCGGAAGGATCATTATCGT 56

R: CGCTTAAAGTTGTCAGATGA

356 F: TGATCGGCTAATGCTCTTGG 58

R: AAAACAAACATTAAACACCCAGTAGA

458 F: TAAAATATGGTTTGGTTTCGTT 51

R: CGTTGGTGTTGGTCAGCA

KS6511 F: TATTGGCACGCTGCTTAATG 53

R: CGGTATTGTGTTGACGTGTTTT
Methods 11

Genomic Library

For microsatellite analyses, we constructed a Kanab 

ambersnail genomic library, enriched for microsatellite motifs 

following the protocol by Hamilton and others (1999) using 

the Invitrogen TOPO® TA Cloning® Kit. We developed 

multiple different primer pairs that were designed to amplify 

nuclear genomic regions containing short tandem repeats 

within the genus Oxyloma. These microsatellite primers 

were designed following analyses of about 200 microsatellite 

enriched clones of restriction fragments from the Oxyloma

genome. Unfortunately, almost all subsequent attempts at 

implementing these microsatlellite primers in the full dataset 

were uninformative. Microsatellite primers frequently could 

not be amplified across all populations from the dataset, and 

often also produced multiple peaks that were inconsistent 

with amplification of a single Mendelian genetic locus. Only 

one microsatellite locus, KS6511, successfully amplified 

across all populations using forward and reverse primers, 

which are listed in table 3. Although we have included data 

from microsatellite KS6511 in this study, given our goal of 

characterizing nuclear variation within the genus Oxyloma, we 

subsequently implemented the AFLP procedure to generate 

“highly variable” multilocus nuclear genetic data for our full 

dataset, as described in section AFLP (Multilocus) of the 

Methods section.

PCR Conditions

PCR conditions for the one microsatellite locus that 

amplified, KS6511, were 10 ng DNA in 10 millimolar (mM) 

Tris pH 8.3, 50 mM potassium chloride (KCl), 2.5 mM 

magnesium chloride (MgCl2), 1 mM dNTPs, 0.5 Units 

Platinum Taq (Invitrogen), 2 picomolar (pM) forward primer 

(with M13 5’ extension TGTAAAACGACGGCCAGT), 4 pM 

reverse primer (with PIGtail 5’ extension GTTTCTT), 4 pM 

M13 primer with fluorescent label (FAM6) in a final volume 

of 10 μL. The cycling conditions were: initial denaturation 

at 94°C for 3 minutes, followed by 20 cycles of denaturation 

at 94°C for 30 seconds, annealing at 55°C for 30 seconds, 

and extension at 72°C for 30 seconds; then 15 cycles of 

denaturation at 94°C for 30 seconds, annealing at 53°C for 

30 seconds, and extension at 72°C for 30 seconds; followed by 

a final extension at 72°C for 10 minutes.

AFLP (Multilocus)

AFLPs have a moderate mutation rate, but tend to 

be informative because many loci are typed at one time. 

Multilocus markers, such as AFLPs, have the advantage of 

being useful markers when not much information is available 

about the study organism’s genome.

Data Generation

We used the AFLP procedure of Vos and others (1995) 

to generate nuclear genetic markers for 206 individuals 

(see table 2 for information about sample sizes from each 

population). AFLP reactions were performed using the 

methods described in Miller and others (2006). In total, the 

second selective PCR reaction (the stage at which multilocus 

AFLP fingerprints were generated) was performed using two 

different sets of primers: Eco-ACG/Mse-ACG and Eco-ACG/

Mse-ATC. AFLP fragments produced in the second selective 

PCR reaction were resolved with an ABI 3100 automated 

DNA sequencer (Applied Biosystems, Inc.) using the 

manufacturer’s protocols and analyzed using the GeneScan®

Analysis software package (Life Technologies Corp., 2013). 

After analysis, we viewed sample files and scored the presence 

or absence of individual AFLP markers using Genographer 1.6 

(Benham, 2001). In total, data from 69 polymorphic loci were 

recorded from the two primer combinations examined.

Data Analyses

AFLP data initially were analyzed using the Unweighted 

Pair Group Method with Arithmetic mean (UPGMA) 

clustering algorithm implemented in the program NT-SYSpc 

v2.10 (Exeter Software, Inc.). The Dice coefficient option 

was used when calculating pairwise similarity coefficients 

among all analyzed individuals. To examine more thoroughly 

the population structure among southwestern Oxyloma

populations, we used the Bayesian clustering algorithm of 

Pritchard and others (2000) as implemented in the software 

package STRUCTURE 2.2 (Pritchard and others, 2000). This 

analysis was used to (1) identify the most likely number of 

genetic clusters or populations (K) that exist, and (2) estimate 

the proportion of each analyzed individual’s genome that was 

derived from each of the K clusters. Analyses were performed 

using 5 replicates for each value of K, where K ranged from 

4 to 13. For each analysis replicate, 5,000 burnin steps were 

used followed by an additional 20,000 Markov chain Monte 

Carlo sweeps. The “admixture model” and “correlated allele 

frequencies” options were used when performing each 

analysis. When analyses were completed, likelihood scores 

from each replicate value of K were evaluated using the 

guidelines in Pritchard and others (2000) to infer the true 

number of distinct genetic clusters. A graphical summary of 

STRUCTURE analyses was subsequently generated using the 

program Distruct (Rosenberg, 2004).
12 Anatomical and Genetic Variation of Western Oxyloma Concerning the Endangered Kanab Ambersnail in Arizona and Utah

Results

Results are summarized below on all aspects of this 

study. The organismal aspects are presented with specimen 

collection, anatomical analyses, and morphological analyses, 

followed by the molecular aspects with the DNA analyses.

Specimen Collection

Based on observations from our field collections team 

prior to and during this study, the number of species present at 

each of the sampling locations was 

• Vaseys Paradise—in addition to one species of 

Oxyloma, there also were three species of Catinella

at the location, albeit much less common than the 

Oxyloma that dominated that location; 

• Three Lakes—a single Succineid snail shell was found 

at the location during a visit in 1996, but Oxyloma was 

the dominant land snail at the location; 

• Elves Chasm—during 2008–10, only a few 

Catinella were detected in the area where Oxyloma

were released; 

• Lees Ferry—Catinella occurred at the location, but 

prefers slightly drier habitat than Oxyloma (similar to 

Vaseys Paradise with these Succineids); 

• Indian Garden—no Catinella reported in the areas 

where we found Oxyloma; and 

• other locations in Utah—no Catinella observed at 

the specimen collection locations for this study, but 

that was expected because our focus areas were those 

believed to have the best Oxyloma habitat.

At the time of our collections for this study, our lead 

field biologist had about 9–10 years of experience in sampling 

for and conducting field identifications of Succineid snails in 

the Grand Canyon region. He has the expertise and ability to 

distinguish the difference in shells and appearance of Oxyloma

and Catinella. Other members of the field team likewise had 

the same level of experience in distinguishing between the 

two genera while in the field. Although the differences in 

shells and appearance between the two genera are subtle, and 

difficult for novices to recognize, we are highly confident 

that we properly distinguished the difference between the two 

genera at locations that were visited and used in this study.

Shell Morphology

Sample ranges, means, and standard deviations of the 

entered variables are presented in table 4. The correlation 

matrix for the ungrouped data indicated that a factor-analytic 

model was marginally applicable. Fifty-two (66 percent) of 

78 pairs of variables have correlation coefficients of less than 

0.3 (table 5). The strongest correlations were among the raw 

measurements of size (SL, SD, SAL, SAW, and SMD).

Principal Components Analysis

Thirteen principal components were computed. The 

first four components account for 89.572 percent (44.570, 

26.226, 10.278, and 8.498 percent, respectively) of the total 

variance. Table 6 shows loadings of the entered variables. 

The first principal component, shell size (SS), primarily 

expressed size. A high score for this component indicated a 

large shell with a large aperture (although not necessarily an 

aperture large in relation to overall shell size). Apical angle 

(APA), apertural asymmetry (APY), and whorl expansion 

rate (W) load negatively on component 1, although not with 

high values. Translation (T, negatively), APA, and relative 

height (SRH, negatively) load strongly on component 2. A 

high score on this component indicated a shell with an obtuse 

apex and a relatively broad shell, in which the center of the 

generating curve translated slowly along the axis of coiling. 

Relative aperture height also loads strongly on component 

2, indicating a shell with a relatively short spire posterior 

to the aperture. Component 3 largely was an expression 

of the effuseness of the aperture, with APY and height of 

maximum diameter (SMD) loading strongly; a high score on 

this component indicated a shell with an effuse, asymmetric 

aperture. Apertural shape (S) and, to a lesser extent, relative 

aperture height (SRA), and APY (negatively) load strongly on 

component 4. A high score on this component indicated a shell 

with a proportionally narrow aperture that was symmetrical 

and long relative to shell length.

A plot of individual Oxyloma specimen scores on 

principal components 2 (the “narrow shell” component) 

and 3 (the “effuse aperture” component) (fig. 2) showed 

extensive overlap among all samples (“batches”). As shown 

by individual sample plots (fig. 3), shells from Three Lakes 

and The Greens tended to have asymmetrical, effuse apertures 

(scoring high on principal component 3). Indian Garden shells 

tended to be relatively broad, with obtuse apices (generally 

scoring high on the principal component 2). Shells from 

Vaseys Paradise tended to be “middle of the pack” with 

respect to effuseness, and moderate to obtuse and broad with 

respect to shell shape.

Plots of individual Oxyloma specimen scores on principal 

components 3 and 4 (the “narrow, symmetrical aperture” 

component) (fig. 4) showed less difference among the point 

scatters. Some samples, such as Glendale Highway and Lees 

Ferry, varied widely in their scores on principal component 4.
Results 13

Table 4. Summary statistics of ambersnail shell morphology variables used in principal components analysis and discriminant analysis, by sample localities in Arizona and Utah.[For each population, range, mean, and standard deviation are included for each variable. Sample localities: n, number of samples; SonCr, Sonoita Creek; LF, Lees Ferry; IG, Indian Garden; VP, Vaseys 

Paradise; Greens, The Greens; GHwy, Glendale Highway; CCrk, Center Creek; SR, Sevier River; 3L, Three Lakes; PassCrk, Pass Creek; PanCrk, Panguitch Creek; ER, Escalante River. Abbreviations: APA, apical angle; S, apertural shape; T, translation; W, whorl expansion rate; SL, shell length; SD, shell maximum diameter; SAL, shell aperture length; SAW, shell aperture width; SMD, height of maximum diameter; SS, shell size; SRH, relative height; SRA, relative aperture height; APY, apertural asymmetry; mm, millimeter]

Sample 

localities

(n)

APA

(degrees)

S

(dimensionless)

T

(dimensionless)

W

(dimensionless) SL (mm) SD (mm) SAL (mm) SAW (mm) SMD (mm) SS (mm) SRH (dimension- less) SRA (dimension- less) APY (dimension- less)

SonCr (12) 54.50–70.00

62.83

5.069

127–1.63

1.390.092

2.93–3.63

3.240.269

0.47–0.72

0.57

0.085

7.53–10.24

8.840.769

4.76–6.12

5.670.453

4.53–5.88

5.310.442

3.29–4.24

3.830.329

1.59–2.71

2.330.333

12.29–16.35

14.52

1.148

1.43–1.72

1.560.090

0.55–0.65

0.600.031

0.33–0.49

0.440.051

LF (24) 54.00–68.00

62.60

4.061

1.38–1.66

1.570.070

2.74–3.75

3.190.262

0.37–0.67

0.470.072

6.65–10.35

8.560.975

4.35–6.47

5.290.485

4.59–7.12

5.630.555

2.94–4.53

3.600.350

0.88–2.53

1.940.376

11.00–16.59

13.85

1.402

1.48–1.88

1.620.106

0.57–0.71

0.660.037

0.19–0.47

0.340.059

IG (25) 64.00–78.00

71.29

4.072

1.35–1.84

1.520.117

2.39–3.67

2.810.264

0.31–0.62

0.430.076

8.47–11.71

10.04

1.092

5.47–8.00

6.870.758

5.65–8.38

7.060.856

3.76–5.49

4.660.572

1.26–3.44

2.410.505

14.18–19.41

16.92

1.771

1.29–1.59

1.460.091

0.65–0.75

0.700.026

0.21–0.46

0.340.055

VP (19) 47.00–71.50

59.09

7.867

1.39–1.76

1.600.100

2.77–4.05

3.340.368

0.34–0.57

0.460.064

9.71–15.88

12.70

1.738

6.76–9.41

7.730.684

6.71–10.94

8.201.008

4.35–6.35

5.140.551

2.59–3.79

3.010.307

16.71–25.29

20.43

2.263

1.39–1.97

1.640.168

0.59-–0.71

0.650.038

0.32–0.42

0.370.031

Greens (22) 60.00–77.50

66.17

4.795

1.49–1.79

1.640.087

2.88–3.57

3.200.179

0.35–0.60

0.460.071

7.29–11.44

9.361.100

4.88–6.82

5.900.560

4.82–7.53

6.340.678

3.24–4.59

3.860.400

2.18–3.06

2.570.265

12.18–18.26

15.26

1.635

1.42–1.71

1.580.070

0.62–0.72

0.680.026

0.37–0.45

0.410.023

GHwy (23) 64.00–86.00

71.88

5.182

1.40–1.79

1.550.095

2.59–3.46

2.960.233

0.30–0.65

0.420.085

7.18–13.59

10.65

2.037

4.47–9.29

7.071.352

4.82–9.82

7.38

1.597

2.82–6.35

4.761.034

1.94–3.85

2.840.537

11.88–22.88

17.72

3.365

1.37–1.66

1.510.071

0.61–0.74

0.690.032

0.32–0.48

0.390.041

CCrk (15) 65.00–78.50

71.72

4.049

1.33–1.70

1.570.109

2.60–3.49

3.090.250

0.36–0.83

0.510.119

6.58–12.67

10.22

1.595

4.71–8.20

6.780.915

4.47–8.94

6.891.118

2.94–5.35

4.390.601

1.82–3.26

2.600.425

11.2820.87

17.00

2.458

1.29–1.67

1.500.100

0.63–0.71

0.670.023

0.33–0.45

0.380.031

SR (18) 61.50–81.50

69.18

5.010

1.45–1.66

1.520.056

2.70–3.30

3.060.179

0.40–0.87

0.520.101

6.12–9.12

6.940.785

4.12–5.88

4.700.411

4.06–6.24

4.610.550

2.58–3.82

3.030.315

1.28–2.35

1.860.262

10.24–15.00

11.64

1.185

1.37–1.56

1.470.054

0.62–0.72

0.660.029

0.28–0.45

0.400.040

3L (17) 60.00–74.50

66.96

3.694

1.47–1.68

1.570.054

2.74–3.56

3.160.206

0.39–0.66

0.500.066

6.71–11.41

8.591.225

4.47–6.94

5.530.586

4.65–7.12

5.690.679

2.80–4.53

3.620.412

1.96–3.47

2.400.395

11.18–18.35

14.12

1.784

1.43–1.78

1.550.086

0.61–0.71

0.660.027

0.36–0.49

0.420.039

PassCrk (18) 61.20–77.00

66.20

4.432

1.31–1.72

1.590.100

2.75–3.47

3.130.200

0.36–0.64

0.450.087

8.94–12.35

10.81

0.929

5.75–7.53

6.820.434

6.12–8.24

7.280.692

4.02–5.04

4.580.313

2.12–3.36

2.640.332

15.14–19.76

17.63

1.335

1.44–1.67

1.580.063

0.63–0.72

0.670.024

0.31–0.44

0.360.036

PanCrk (1) 73.5 1.62 2.97 0.48 10.53 6.94 7.41 4.59 2.67 17.47 1.52 0.70 0.36

ER (16) 66.00–79.00

73.05

3.348

1.46–1.72

1.570.077

2.62–3.03

2.82

0.113

0.33–0.56

0.460.073

7.08–15.18

9.431.838

4.94–9.52

6.251.041

5.35–11.02

6.741.450

3.32–6.61

4.290.765

2.11–3.73

2.590.420

12.02–24.69

15.67

2.876

1.42–1.59

1.500.045

0.67–0.79

0.710.033

0.34–0.44

0.390.025
14 Anatomical and Genetic Variation of Western Oxyloma Concerning the Endangered Kanab Ambersnail in Arizona and Utah Table 5. Pearson correlation pairwise matrix for ambersnail shell characters submitted to principal components analysis and discriminant analysis used to analyze Oxyloma populations.

[Abbreviations: APA, apical angle; S, apertural shape; T, translation; W, whorl expansion rate; SL, shell length; SD, shell maximum diameter; SAL, shell aperture length; SAW, shell aperture width; SMD, height of maximum diameter; SS, shell size; SRH, relative height; SRA, relative aperture height; APY, apertural asymmetry]

APA S T W SL SD SAL SAW SMD SS SRH SRA APY

APA 1.000S -0.235 1.000T -0.753 0.528 1.000

W -0.101 0.018 0.131 1.000SL -0.234 0.252 0.221 -0.168 1.000

SD 0.056 0.064 -0.087 -0.216 0.931 1.000

SAL -0.033 0.280 0.009 -0.203 0.963 0.957 1.000

SAW 0.041 -0.022 -0.162 -0.223 0.921 0.977 0.952 1.000

SMD 0.017 0.311 0.097 -0.188 0.813 0.800 0.834 0.770 1.000

SS -0.129 0.185 0.109 -0.189 0.991 0.973 0.976 0.957 0.822 1.000

SRH -0.775 0.532 0.821 0.054 0.430 0.079 0.269 0.110 0.245 0.305 1.000

SRA 0.691 0.146 -0.760 -0.139 -0.044 0.169 0.223 0.193 0.141 0.036 -0.538 1.000

APY 0.091 0.054 0.162 0.037 -0.222 -0.233 -0.253 -0.280 0.311 -0.230 -0.044 -0.154 1.000
Results 15

Table 6. Component loadings of variables measured in Oxyloma 

populations, eigenvalues, and percent of total variance explained 

by the first four components in principal components analysis of 

shells of Oxyloma.

[Abbreviations: SS, shell size; SL, shell length; SAL, shell aperture length; 

SD, shell maximum diameter; SAW, shell aperture width; SMD, height of 

maximum diameter; T, translation; APA, apical angle; SRH, relative height; 

SRA, relative aperture height; APY, apertural asymmetry; S, apertural shape; 

W, whorl expansion rate]

Variable Component

1

Component

2

Component

3

Component

4

SS 0.992 0.016 -0.081 -0.057

SL 0.989 -0.109 -0.076 -0.031

SAL 0.988 0.111 0.002 0.095

SD 0.954 0.226 -0.086 -0.099

SAW 0.943 0.254 -0.163 -0.107

SMD 0.857 0.036 0.457 -0.203

T 0.121 -0.958 0.105 0.002

APA -0.131 0.865 0.275 0.028

SRH 0.349 -0.859 -0.004 0.165

SRA 0.087 0.788 0.276 0.502

APY -0.195 -0.130 0.797 -0.545

S 0.271 -0.427 0.530 0.665

W -0.228 -0.207 -0.049 0.094

Eigenvalue 5.794 3.409 1.336 1.105

Percent of variance 

explained

44.570 26.226 10.278 8.498

EXPLANATION

3

2

1

0

-1

-2

-3

-4

-4 -3 -2 -1 0 1 2 3

Factor(2)

Factor(3)

Sample location

CCrk, Center Creek

ER, Escalante River

GHwy, Glendale Highway

IG, Indian Garden

LF, Lees Ferry

PasCrk, Pass Creek

PanCrk, Panguitch Creek

SR, Sevier River

Greens, The Greens

3L, Three Lakes

VP, Vaseys Paradise

Figure 2. Scores of Oxyloma specimens from all study samples in Arizona and Utah on 

principal components 2 and 3 (“factors”) from principal components analysis.
16 Anatomical and Genetic Variation of Western Oxyloma Concerning the Endangered Kanab Ambersnail in Arizona and Utah

tac11-0685_fig 03

-4

-3

-2

-1

0

1

2

3

Factor(3)

Three Lakes Center Creek Escalante River Glendale Highway

-4

-3

-2

-1

0

1

2

3

Factor(3)

The Greens Indian Garden Lees Ferry Panguitch Creek

-4 -3 -2 -1 0 1 2 3

Factor(2)

-4

-3

-2

-1

0

1

2

3

Factor(3)

Pass Creek

-4 -3 -2 -1 0 1 2 3

Factor(2)

Sevier River

-4 -3 -2 -1 0 1 2 3

Factor(2)

-4 -3 -2 -1 0 1 2 3

Vaseys Paradise Factor(2)

Figure 3. Scores of Oxyloma specimens from individual sampling locations in Arizona and Utah on principal components 2 and 3 

(“factors”) from principal components analysis.
Results 17

tac11-0685_fig 04

-4

-3

-2

-1

0

1

2

3

Factor(4)

Three Lakes Center Creek Escalante River Glendale Highway

-4

-3

-2

-1

0

1

2

3

Factor(4)

The Greens Indian Garden Lees Ferry Panguitch Creek

-4 -3 -2 -1 0 1 2 3

Factor(3)

-4

-3

-2

-1

0

1

2

3

Factor(4)

Pass Creek

-4 -3 -2 -1 0 1 2 3

Factor(3)

Sevier River

-4 -3 -2 -1 0 1 2 3

Factor(3)

-4 -3 -2 -1 0 1 2 3

Vaseys Paradise Factor(3)

Figure 4. Scores of Oxyloma specimens from sampling locations in Arizona and Utah individually on principal components 3 and 4 

(“factors”) from principal components analysis.
18 Anatomical and Genetic Variation of Western Oxyloma Concerning the Endangered Kanab Ambersnail in Arizona and Utah

Discriminant Analysis

Discriminant analysis showed that no linear combination 

of submitted variables reliably classified or discriminated 

among samples. (The sample from Panguitch Creek, 

consisting of only one specimen, was excluded from 

this analysis.)

The first run of discriminant analysis included the coiling 

parameters of Raup (1966): S, T, and W. These performed very 

poorly, classifying only 31 percent of specimens into their 

samples of origin. The best-classified sample was that from 

Sevier River, with 51 percent of specimens correctly classified; 

the second best sample was that from Vaseys Paradise, with 

47 percent of specimens correctly classified. Adding apical 

angle (APA) to the analysis raised the total percentage of 

specimens correctly classified to 34 percent, with 53 percent 

of specimens from Vaseys Paradise correctly classified.

A second run used the raw dimensions SL, SD, SAL, 

SAW, and SMD. This set of variables classified 55 percent 

of all specimens correctly as to their samples of origin. 

The best-classified samples were those from Sevier River 

(78 percent), Escalante River (75 percent), Minus 9-Mile 

Spring (67 percent), and Vaseys Paradise (63 percent).

A third run used the overall size (SS) and shape (SRH, 

SRA, APY) variables, the kinds of shell morphological 

features a traditional taxonomist of the school of Pilsbry 

(1948) probably would have emphasized in preparing his or 

her descriptions. This set of variables classified 51 percent 

of all specimens correctly as to their samples of origin. 

The best‐classified samples were those from Sevier River 

(78 percent), Lees Ferry (67 percent), Escalante River 

(63 percent), and Vaseys Paradise (63 percent). Jackknifed 

classification matrices (which used functions from all cases 

except the one being classified) reduced the total percentages 

of the first, second, and third runs to 26 percent (29 percent 

with APA added), 51 percent, and 48 percent, respectively.

Plots of canonical variable scores (for example, fig. 5, 

based on the raw dimension dataset, the best-discriminating 

run of the three) showed substantial overlap among the 

samples. In this run, five canonical variables were calculated. 

The first canonical variable is the linear combination of the 

submitted variables that best discriminated among the groups, 

the second canonical variable is orthogonal to the first and is 

the next-best combination of submitted variables, and so on. 

The first canonical variable accounted for 53.2 percent of the 

total dispersion of the groups; the first two together accounted 

for 76.4 percent of the total dispersion. These figures, and 

canonical correlations for the first three canonical variables 

of 0.766, 0.618, and 0.529, respectively, represented the 

moderately good ability of these variables to discriminate 

among the groups.

The holotype of O. h. kanabense is 19.15 mm long, 

larger than any specimens collected in the present study. When 

submitted as an unclassified case to discriminant analysis, 

it was assigned to Vaseys Paradise in runs using each of 

the three sets of variables cited earlier in this Discriminant 

Analysis section.6 The same result was achieved with datasets 

emphasizing and de-emphasizing size ([SL, SD, SAL, SAW, 

SMD] and [S, T, W, APA], respectively), indicating that the 

similarity was not owing to shell size alone. When Vaseys 

Paradise was removed as an option, the analysis classified the 

holotype in the Pass Creek sample based on variable sets (SL, 

SD, SAL, SAW, SMD) and (SS, SRH, SRA, APY), and in the 

Lees Ferry sample based on variable set (S, T, W, APA).

Soft Tissue Anatomy

It proved difficult to achieve standard anatomical 

measurements with repeatability because of the flexibility 

and elasticity of soft tissues. Additionally, the reproductive 

organs lay within the body cavity in varied orientations, 

variously compressed within the available space (fig. 6). This 

“packing problem” made it inadvisable to rely on micrometer 

or ocular reticule measurements. Additionally, an unknown 

but considerable amount of ontogenetic variation was present. 

Half of the specimens in some lots proved, on dissection, to be 

reproductively immature, even though collected on the same 

date as other, mature specimens in the population (table 7).

DNA Isolation

A DNA yield of 1 ng to 19 μg of DNA was obtained from 

each of the 259 samples. This yield was sufficient for all PCR 

amplifications; however, some individuals did not amplify 

for all genes or regions attempted (discussed in more detail in 

Mitochondrial DNA and Nuclear DNA sections below).

Genetic Analyses 

Genetic analyses includes analyses of mitochondrial 

and nuclear DNA. The mitochondrial genome is composed 

almost entirely of genes, two of which were examined. The 

nuclear DNA has a wide variety of DNA types relative to the 

mitochondria, and many of these types were examined.

6

Indian Garden was removed as an option in order to keep the number of 

groups at 10, the maximum allowed by the SYSTAT program for this type 

of analysis.
Results 19

tac11-0685_fig 05

EXPLANATION

Center Creek

Sample location

Escalante River

Glendale Highway

Indian Garden

Lees Ferry

Pass Creek

Sevier River

The Greens

Three Lakes

Vaseys Paradise

Factor (1) Factor (2) Factor (3) Factor (4) Factor (5)

Factor (1) Factor (2) Factor (3) Factor (4) Factor (5)

Factor (1) Factor (5) Factor (4) Factor (3) Factor (2)Factor (1) Factor (5) Factor (4) Factor (3) Factor (2)

Canonical scores plot

Figure 5. Canonical scores plot of discriminant analysis using shell dimension variables SL (shell length), SD (shell 

maximum diameter), SAL (shell aperture length), SAW (shell aperture width), and SMD (height of maximum diameter). 

The axes are the first through fifth canonical variables (factor), and the points are the canonical variable scores of 

individual ambersnail specimens analyzed for this study. The confidence ellipses are centered on the centroid of each 

sample.
20 Anatomical and Genetic Variation of Western Oxyloma Concerning the Endangered Kanab Ambersnail in Arizona and Utah

tac11-0685_fig 06

AG

HD

PG

SP

VD

FO

PR

EP

PA

PE

PS

VA

AG Albumen gland

EP Epiphallus

FO Free oviduct

HD Hermaphrodite duct

PA Penial appendix

PG Prostate gland

PR Penial retractor muscle

PS Penial sheath (drawn in transparency to show contained organs)

SP Spermatheca

VA Vagina

VD Vas deferens

EXPLANATION

Figure 6. Anatomical features for Oxyloma specimens analyzed for 

this study.
Results 21

Table 7. Summary of Oxyloma anatomical data collected for this study, by sample localities in Arizona and Utah.

[Sample locality: LF, Lees Ferry; IG, Indian Garden; VP, Vaseys Paradise; Greens, The Greens; GHwy, Glendale Highway; CCrk, Center Creek; SR, Sevier 

River; 3L, Three Lakes; PassCrk, Pass Creek; PanCrk, Panguitch Creek; ER, Escalante River. Mantle over lung: ~, about]

Sample

locality Head-foot color Mantle color Mantle over lung(percent pigmented) Foot sole Penial sheath Penis

LF White, with distinct

charcoal-gray

patches of pigment,

tending toward 

longitudinal stripes

Lightly dotted with

black and blackish

brown; spots tending

to form branching 

network

~60 Tripartite; broad

white central zone 

flecked with black,

white lateral zones

Entire, thin, with a

few black flecks of

pigment; summit

acute, with penial

retractor muscle

inserted on it

Cylindrical, simplewalled, with conical

appendage lateral

to insertion of

epiphallus

IG White to pale buff,

with distinct

charcoal-gray

patches of pigment,

tending toward

longitudinal stripes

Anterior margin

with lobate pigment

blotches; rightanterior 60 percent

lightly black dotted;

diagonal black

streak; left-posterior 

part with scattered 

black patches

~70 Uniformly white

to pale buff

Entire, thin, with a

few black flecks of

pigment; summit

acute, with penial

retractor muscle

inserted on it

Cylindrical, simple

walled, bearing

small, elongateconical appendage

VP Pale buff, with

charcoal gray

pigment shading

toward head;

striped; nape dark

Closely, uniformly, 

black dotted

~100 Uniformly pale

buff

Entire, thin, with a

few black flecks of

pigment; summit

acute, with penial

retractor muscle 

inserted on it

Cylindrical, simplewalled, bearing 

small, elongateconical appendage

Greens Pale buff, with

small amount of

gray pigment

shading toward

head

Right-anterior 60

percent lightly black

dotted; diagonal

black streak; leftposterior part with

minor, scattered

black patches

~60 Uniformly pale

buff

Thin, with a few

black flecks of

pigment; summit 

acute, with penial

retractor muscle

inserted on it 

and apical gap

admitting vas

deferens

Cylindrical, simplewalled, bearing

small, corkscrewcoiled, conical

appendage

GHwy White, with gray

pigment shading

toward head;

spotted; nape dark

Entirely blackish

brown with some

darker streaks

100 Tripartite; broad

white central zone,

gray pigmented

lateral zones

Thin, with a few

black flecks of

pigment; summit

acute, with penial

retractor muscle

inserted on it

and apical gap

admitting vas

deferens

Cylindrical, simplewalled, upper

20 percent recurved,

bearing minute

conical appendage
22 Anatomical and Genetic Variation of Western Oxyloma Concerning the Endangered Kanab Ambersnail in Arizona and Utah

Table 7. Summary of Oxyloma anatomical data collected for this study, by sample localities in Arizona and Utah.—Continued

[Sample locality: LF, Lees Ferry; IG, Indian Garden; VP, Vaseys Paradise; Greens, The Greens; GHwy, Glendale Highway; CCrk, Center Creek; SR, Sevier 

River; 3L, Three Lakes; PassCrk, Pass Creek; PanCrk, Panguitch Creek; ER, Escalante River. Mantle over lung: ~, about]

Sample

locality Head-foot color Mantle color Mantle over lung(percent pigmented) Foot sole Penial sheath Penis

CCrk Gray, slightly darker

toward head

Closely, uniformly,

black dotted

100 Tripartite; broad

pale buff central

zone faintly mottled

with gray, gray

pigmented lateral

zones

Entire, thin, with

numerous black

flecks of pigment;

summit acute, with

penial retractor

muscle inserted on it

Cylindrical, simplewalled, upper

20 percent recurved,

bearing minute,

reflexed appendage

SR White, with small

amount of gray

pigment shading

toward head

Closely dotted with

black and dark

brown pigment,

somewhat clumped

~70 Tripartite; broad

white central zone,

gray pigmented

lateral zones

Thin, with a few

 black flecks of

 pigment; summit

 acute, with penial

 retractor muscle

 inserted on it and

apical gap admitting

vas deferens

Club-shaped, broader

distally; simplewalled, upper

20 percent recurved,

bearing small, 

reflexed appendage

3L White to pale buff,

with small amount

of gray pigment

shading toward 

head

Anterior margin

with lobate pigment

blotches; right half

lightly black dotted;

antero-posterior

 medial black streak;

left half with

scattered black

patches

~40 Uniformly pale

buff

Entire, thin,

unpigmented;

summit acute, with

penial retractor

muscle inserted

on it

Cylindrical,

simple-walled,

bearing hammershaped, reflexed

appendage

PassCrk Gray, slightly

darker toward head

Closely, uniformly,

black dotted

100 Tripartite; flecked

with charcoal-gray

pigmernt, flecks

slightly larger in

lateral zones than in

central zone

Entire, thin, with a

few black flecks of

pigment; summit

rounded, admitting

vas deferens

Cylindrical,

simple-walled,

with prominent,

conical reflexed

appendage lateral

to insertion of 

epiphallus

PanCrk Gray, slightly

darker toward head

Closely, uniformly,

blackish-brown

dotted

100 Tripartite; broad

white central zone,

lightly gray flecked

lateral zones 

Entire, thin, with a

few black flecks of

pigment; summit

rounded, admitting

 vas deferens

Cylindrical, simplewalled, upper

20 percent recurved,

bearing reflexed

appendage

ER White, with gray

pigment shading

toward head;

vertically striped;

nape dark

Closely, uniformly,

black dotted

100 Tripartite; broad

white central zone,

gray pigmented

lateral zones

Entire, thin, with a

few black flecks of

pigment; summit

acute, with penial

retractor muscle

inserted on it

Cylindrical, simplewalled, upper

20 percent recurved,

bearing minute,

reflexed appendage
Results 23

Table 7. Summary of Oxyloma anatomical data collected for this study, by sample localities in Arizona and Utah.—Continued

[Sample locality: LF, Lees Ferry; IG, Indian Garden; VP, Vaseys Paradise; Greens, The Greens; GHwy, Glendale Highway; CCrk, Center Creek; SR, Sevier 

River; 3L, Three Lakes; PassCrk, Pass Creek; PanCrk, Panguitch Creek; ER, Escalante River. Mantle over lung: ~, about]

Sample

locality Vas deferens Epiphallus Spermatheca Spermathecal duct Vagina Free oviduct

LF Simple, tubular,

lightly flecked with

black pigment;

entering epiphallus

terminally

Stout, borne in

2.5–3.0 coils,

simply confluent

with summit of

penis

Ovoid Rather broad at

base, narrowin

upward; confluent

with summit of

vagina

Cylindrical,

internally plicate,

about as long as

penis plusepiphallus

Originating laterally

on vagina and

broader than it,

contorted

IG Simple, tubular,

unpigmented;

entering epiphallus

terminally

Moderately stout,

coiled, simply

confluent with

summit of penis

Globose Slender, simple,

confluent with

summit of vagina

Fusiform, broad

medially, internally

plicate, about as

long as penis plus

epiphallus

Originating laterally

on vagina; of about

same width,

contorted

VP Simple, tubular, 

lightly flecked with

black pigment;

entering epiphallus

terminally

Contorted, simply

confluent with

summit of penis

Inflated, globose Slender, simple,

branching laterally

off summit of vagina

Cylindrical,

internally plicate,

about as long as

penis plus 

epiphallus

Confluent with

summit of vagina

and about the same

diameter as it

Greens Simple, tubular,

lightly flecked with

black pigment;

looping above

penial sheath,

entering epiphallus

terminally

Stout, contorted,

simply confluent

with summit of

penis

Inflated, globose Slender, simple,

confluent with

summit of vagina

Cylindrical,

internally plicate,

about same length

as penis plus

epiphallus or a 

little shorter

Originating laterally

on vagina; of about

same width,

contorted

GHwy Simple, tubular,

lightly flecked with

black pigment;

looping above 

penial sheath,

entering epiphallus

terminally

Stout, contorted,

simply confluent

with summit

of penis

Ovoid Very long,

moderately slender,

simple, confluent

with summit of

vagina

Cylindrical,

internally plicate,

slightly shorter

than penis plus

epiphallus

Originating laterally

on vagina; of about

same width,

contorted

CCrk Simple, tubular,

prominently flecked

 with black pigment;

looping above 

penial sheath,

entering epiphallus

terminally

Stout, contorted,

simply confluent

with summit

of penis

Globose Moderately slender,

simple, confluent

with summit of

vagina; narrowing

just below

spermatheca; fleck

with dark pigment

Cylindrical,

internally plicate,

about as long

as penis plus

epiphallus

Originating laterally

on vagina; of

about same width,

contorted
24 Anatomical and Genetic Variation of Western Oxyloma Concerning the Endangered Kanab Ambersnail in Arizona and Utah

Table 7. Summary of Oxyloma anatomical data collected for this study, by sample localities in Arizona and Utah.—Continued

[Sample locality: LF, Lees Ferry; IG, Indian Garden; VP, Vaseys Paradise; Greens, The Greens; GHwy, Glendale Highway; CCrk, Center Creek; SR, Sevier 

River; 3L, Three Lakes; PassCrk, Pass Creek; PanCrk, Panguitch Creek; ER, Escalante River. Mantle over lung: ~, about]

Sample

locality Vas deferens Epiphallus Spermatheca Spermathecal duct Vagina Free oviduct

SR Simple, tubular,

prominently flecked

with black pigment;

looping above

penial sheath,

entering epiphallus

terminally

Stout, contorted,

simply confluent

with summit

of penis

Ovoid Long, moderately

slender, simple,

confluent with

summit of vagina;

narrowing just

below spermatheca

Fusiform, internally

plicate, about as

long as penis plus

epiphallus

Originating laterally

on vagina; of about

same width,

contorted

3L Simple, tubular,

lightly flecked with

black pigment;

entering epiphallus

terminally

Stout, contorted,

simply confluent

with summit

of penis

Inflated, globose Moderately slender,

simple, confluent

with summit of

vagina; narrowing

just below

spermatheca

Cylindrical, about as

long as penis plus

epiphallus

Of about same

diameter as vagina,

contorted

PassCrk Simple, tubular,

lightly flecked with

black pigment;

entering epiphallus

terminally

Stout, borne in 3

coils, simply

confluent with

summit of penis

Inflated, globose Slender, simple,

confluent with

 summit of vagina;

 longer than penis

 plus epiphallus

Cylindrical,

internally plicate,

about as long

as penis plus

epiphallus

Originating laterally

 on vagina, of about

 same diameter,

 contorted

PanCrk Simple, tubular,

lightly flecked with

black pigment;

entering epiphallus

terminally

Stout, contorted,

simply confluent

with summit

of penis

Globose Thick (about as thick

as main body of 

penis), confluent

with summit of

vagina; flecked

with black

Fusiform, broad

medially, internally

plicate, about as 

long as penis plus 

epiphallus

Originating

laterally on

vagina, of about

same diameter,

contorted

ER Simple, tubular,

lightly flecked with

black pigment;

entering epiphallus

terminally

Stout, contorted,

simply confluent

with summit

of penis

Inflated, globose Moderately slender,

simple, confluent 

with summit of 

vagina; narrowing 

just below 

spermatheca

Cylindrical, 

internally plicate, 

about as long as

penis plus

epiphallus

Broader than vagina,

contorted
Results 25

Table 7. Summary of Oxyloma anatomical data collected for this study, by sample localities in Arizona and Utah.—Continued

[Sample locality: LF, Lees Ferry; IG, Indian Garden; VP, Vaseys Paradise; Greens, The Greens; GHwy, Glendale Highway; CCrk, Center Creek; SR, Sevier 

River; 3L, Three Lakes; PassCrk, Pass Creek; PanCrk, Panguitch Creek; ER, Escalante River. Mantle over lung: ~, about]

Sample

locality Prostate gland Albumen gland Hermaphrodite duct

LF Pinkish creamcolored, enclosing

sheath with

prominent black

flecks

Pale cream

colored,

long-triangular, 

enclosing sheath

unpigmented

Very large, stout,

darkly pigmented

throughout

IG Pinkish creamcolored, enclosing

sheath with

prominent black

flecks

Pale cream

colored,

long-triangular, 

enclosing sheath

unpigmented

Stout, darkly

pigmented

throughout

VP Pinkish creamcolored, enclosing

sheath

unpigmented

or with sparse

black flecks

Pale cream

colored,

long-triangular, 

enclosing sheath

unpigmented 

Not especially

large, darkly

pigmented

throughout

Greens Pinkish creamcolored, enclosing

sheath

unpigmented

or with sparse

black flecks

Pale cream

colored,

long-triangular, 

enclosing sheath

unpigmented

Not especially

large, darkly

pigmented

throughout

GHwy Pinkish creamcolored, enclosing

sheath

unpigmented

or with sparse

black flecks

Pale cream

colored,

long-triangular, 

enclosing sheath

unpigmented

Stout, mottled

with dark pigment

CCrk Pale cream

colored,

enclosing sheath

pigmented with

black flecks

Pale cream

colored,

long-triangular, 

enclosing sheath

with sparse black

flecks

Stout, darkly

pigmented 

throughout

Sample

locality Prostate gland Albumen gland Hermaphrodite duct

SR White, enclosing

sheath pigmented

with black flecks

White, triangular,

enclosing sheath

unpigmented or

with sparse black

flecks

Stout, darkly

pigmented 

throughout

3L Pale cream

colored,

enclosing sheath

unpigmented

Pale cream

colored,

long-triangular, 

enclosing sheath 

unpigmented 

Very large, stout,

darkly pigmented

throughout

PassCrk Pale cream

colored, 

enclosing sheath

with sparse black

flecks

Pale cream

colored,

triangular, 

enclosing sheath

unpigmented 

Stout, darkly

pigmented 

throughout

PanCrk Pinkish creamcolored, enclosing

sheath with sparse

black flecks

Pale cream

colored,

long-triangular, 

enclosing sheath

unpigmented 

Stout, darkly

pigmented

throughout

ER Pale creamcolored, lobate,

enclosing sheath 

unpigmented or

with sparse black

flecks

Pale cream

colored,

long-triangular, 

enclosing sheath

unpigmented 

Stout, darkly

pigmented

throughoutt
26 Anatomical and Genetic Variation of Western Oxyloma Concerning the Endangered Kanab Ambersnail in Arizona and Utah

Mitochondrial DNA

This study examined two mitochondrial regions: the 

cytochrome oxidase subunit I (COI) and cytochrome b (cyt b) 

genes. Catinella, Succinea, and Neosuccinea sequences from 

GenBank® were included in a single mtDNA analysis to 

generate a maximum likelihood phylogenetic tree indicating 

the location of the outgroup root (fig. 7). In this tree, the 

Catinella sequence from GenBank® is the first to branch 

off (most ancestral), followed by a clade of five Catinella

sequences from Batch 1 (Sonoita Creek), and then a group 

containing the other two outgroup sequences from GenBank®, 

Succinea and Neosuccinea. One individual from Batch 12 

(Escalante River), 12-6, clustered with the Batch 1 clade in 

this tree.

The COI fragment had a total length of 510 bp with 

131 variable sites of which 57 were parsimony informative. 

From Sevier River, Pass Creek, and Escalante River, we 

obtained only five, nine, and seven sequences, respectively. 

Samples from Panguitch Creek failed to amplify completely 

(table 2). The analyzed COI dataset represented 141 

specimens that successfully amplified for a total of 48 

different haplotypes.

The cyt b fragment was 366 bp long and had 99 

variable sites of which 30 were parsimony informative. 

From Three Lakes, we obtained only four sequences, and 

from Panguitch Creek, we obtained only 2 sequences. There 

are 163 specimens that successfully amplified for a total of 

32 haplotypes present in the cyt b dataset. Three haplotypes 

were shared by specimens from different locations: H2-1 

includes 21 specimens from Lees Ferry, one specimen from 

Vaseys Paradise, and one specimen from Sevier River; H10-1 

includes six specimens from Pass Creek, two from Panguitch 

Creek, and one from Sevier River; H9-5 includes four 

specimens from Three Lakes and one from Sevier River. 

AMOVA results for the concatenated mitochondrial 

loci indicated that greater than or equal to 71 percent of the 

observed variation existed among populations with less than 

or equal to 29 percent within populations (table 8). Pairwise 

genetic distance (FST) MDS plots (not shown) revealed no 

obvious groupings. However, the position of the Vaseys 

Paradise population is most distant from all other populations.

Pairwise genetic distance values (estimated using 

FST) compared to linear geographic distance (kilometers) 

between populations in a Mantel test showed a slight but 

statistically significant positive correlation between genetic 

and geographic distance for cyt b (average coefficient of 

determination [r2] less than or equal to 0.08; table 8). Thus, 

only 8 percent of the genetic distance variance observed 

could be explained by geographic distance for cyt b data. The 

analysis was performed separately on each gene.

Signals for population expansions with (significant) 

negative values for Tajima’s D (table 9), Fu’s Fs (table 10), 

and (significant) small positive values for Roza’s R2 (table 11) 

were found in Lees Ferry, Vaseys Paradise, The Greens, 

Glendale Highway, Center Creek, Three Lakes, Pass Creek 

and Escalante River. The only population that did not show 

such values was Indian Garden. The more conservative 

mismatch distribution (not shown) indicated a deviation from 

the multimodal distribution of a stable population at the COI 

locus of the Glendale Highway population only.

Table 8. Analysis of molecular variance used to estimate genetic 

variation within versus among populations; and Mantel test to 

assess correlation between genetic distance and geographic 

distance for six different mitochondrial and nuclear DNA loci 

and one concatenated mtDNA dataset used to analyze genetic 

variation in Oxyloma populations.

[Locus: COI, cytochrome oxidase subunit I gene; cyt b, cytochrome b gene; 

mtconcat, mitochondrial genes concatenated; 356, random ambersnail nuclear 

clone 356; 458, random ambersnail nuclear clone 458; ITS, ribosomal DNA 

first and second internal transcribed spacer; S7, ribosomal gene introns S7-1 

and S7-2. AMOVA: Analysis of molecular variance; FST, F statistic indicating 

genetic distance of a subpopulation relative to the total of all populations; km, 

kilometer; <, less than]

Locus

AMOVA

among populations

Mantel test 

pairwise FST/log km

Estimated using FST r

2

COI 75.4 10.06

cyt b 66.2 20.08

mtconcat 71.7 30.05

356 67.9 30.01

458 83.0 20.19

ITS 97.2 30.02

S7 71.8 20.07

1

 0.05˂p˂0.10

2

 p˂0.05

3

 Not significant
Results 27

tac11-0685-fig 07

'Vaseys Paradise' 

Succinea

Oxyloma haydeni 'Alberta'

'The Greens' 3 specimens

Oxyloma retusa

 'Alberta' Neosuccinea

'Indian Garden'

'Indian Garden'

'Escalante River'

Oxyloma retusa 'Utah'

Catinella

Catinella Catinella Catinella

Catinella

Catinella

Catinella

Oxyloma haydeni 'Alberta'

'The Greens' 3 specimens

Oxyloma retusa

'Alberta' Neosuccinea

'Indian Garden'

'Indian Garden'

'Escalante River'

Oxyloma retusa 'Utah'

Catinella

All Oxyloma

ingroup

NJoutgroup Inset

Inset

0.04 Nucleotide substitutions per site

Figure 7. Maximum likelihood tree diagram showing the concatenated mitochondrial DNA dataset (mtconcat) including the 

cytochrome oxidase subunit I (COI) and cytochrome b (cyt b) genes for Oxyloma populations, used to show position of the outgroups. 

All 12 ambersnail populations (12 Batches) are included in the tree along with several outgroup species. One Catinella “outgroup” 

sequence (the first branch of the tree and most ancestral group; far left) is shown in red, and two other “outgroup” sequences from 

GenBank®, Succinea and Neosuccinea, also are shown in red. The triangle represents all ingroup populations of Kanab ambersnail, 

with the width of the triangle indicating the total evolutionary divergence of that ambersnail group.
28 Anatomical and Genetic Variation of Western Oxyloma Concerning the Endangered Kanab Ambersnail in Arizona and Utah Table 9. Tajima’s D (a test for demographic expansion or contraction) values for 6 mitochondrial and nuclear DNA loci and 1 concatenated mitochondrial DNA region in 12 Oxyloma populations from Arizona and Utah.

[Locus: COI, cytochrome oxidase subunit I gene; cyt b, cytochrome b gene; mtconcat, mitochondrial genes concatenated; 356, random ambersnail nuclear clone 356; 458, random ambersnail nuclear clone 458; ITS, ribosomal DNA first and second internal transcribed spacer; S7, ribosomal gene introns S7-1 and S7-2. Abbreviations: LF, Lees Ferry; IG, Indian Garden; VP, Vaseys Paradise; Greens, The Greens; GHwy, Glendale Highway; CCrk, Center Creek; SR, Sevier River; 3L, Three Lakes; PasCrk, Pass Creek; PanCrk, Panguitch Creek; ER, Escalante River; ECh, Elves Chasm; n, number of samples; na, not applicable; –, no data; ˂, less than; ≤, less than or equal to]

Locus Sample localities

LF IG VP Greens GHwy CCrk SR 3L PasCrk PanCrk ER ECh

COI -1.16 1.31 -1.16 -0.69 1 -1.76 2 -1.40 3 -0.79 1 -2.35 1 -1.65 – 2 -1.43 –

cyt b 1 -1.73 2.43 1 -2.26 -0.50 2 -1.29 0.00 0.76 1 -1.52 -0.29 na -1.16 –

mtconcat 1 -1.87 2.38 1 -1.70 -0.47 2 -1.46 2 -1.40 -0.65 1 -1.59 0.14 – 2 -1.49 –

356 na – 1.77 3.06 -1.22 2.67 1.70 0.61 3 1.22 3 0.02 -1.19 3 1.00

458 0.72 na na na na 3 1.17 3 0.18 – 3 2.21 2.11 3 0.97 3 1.06

ITS 2.58 3 0.08 na 3 0.0 na 3 0.34 – na 3 0.70 -0.33 na na

S7 na 1.10 1.52 2.55 -0.49 na -0.74 0.15 2 -1.44 na -1.22 3 0.42

1 p˂0.052 0.05˂p˂0.103 n≤10

Table 10. Fu’s F statistic (a test for population expansion) for 6 mitochondrial and nuclear DNA loci and 1 concatenated mitochondrial DNA region in 12 Oxyloma populations 

from Arizona and Utah.

[Locus: COI, cytochrome oxidase subunit I gene; cyt b, cytochrome b gene; mtconcat, mitochondrial genes concatenated; 356, random ambersnail nuclear clone 356; 458, random ambersnail nuclear clone 458; ITS, ribosomal DNA first and second internal transcribed spacer; S7, ribosomal gene introns S7-1 and S7-2. Abbreviations: LF, Lees Ferry; IG, Indian Garden; VP, Vaseys Paradise; Greens, The Greens; GHwy, Glendale Highway; CCrk, Center Creek; SR, Sevier River; 3L, Three Lakes; PasCrk, Pass Creek; PanCrk, Panguitch Creek; ER, Escalante River; ECh, Elves Chasm; n, number of samples; na, not applicable; –, no data; ˂, less than; ≤, less than or equal to]

Locus Sample localities

LF IG VP Greens GHwy CCrk SR 3L PasCrk PanCrk ER ECh

COI -0.9 1.9 1 -11.2 9.4 1 -2.0 1 -2.0 2 -1.1 -0.7 1 -3.9 – 3 -2.0 –

cyt b 1 -4.0 6.4 1.3 21.8 1.2 n.a. -0.1 2 1.4 -0.6 na 1 -1.6 –mtconcat 1 -3.7 6.0 1 -2.4 15.5 -0.5 -0.6 2 -0.1 2 0.7 -1.2 – -1.8 –

356 na – 10.3 8.3 3.6 12.5 7.6 2.3 2 4.2 2 4.7 1.6 2 7.5

458 0.7 na na na na 2 0.9 2 3.7 – 2 6.0 7.7 2 11.3 1,2 -2.6

ITS 5.4 2 3.4 na 2 1.1 na 2,3 -1.2 – na 2 24.8 1 -3.5 na na

S7 na 2.0 1.1 6.7 1.1 na 2.7 -0.1 0.1 na 1.3 2 -0.1

1 p˂0.052 n≤103 0.05˂p˂0.10
Results 29

Table 11. Roza’s R2 value (a test for population growth) for 6 mitochondrial and nuclear DNA loci, 1 concatenated mitochondrial DNA region, and 1 concatenated nuclear DNA 

region in 12 Oxyloma populations from Arizona and Utah.

[Locus: COI, cytochrome oxidase subunit I gene; cyt b, cytochrome b gene; mtconcat, mitochondrial genes concatenated; 356, random ambersnail nuclear clone 356; 458, random ambersnail nuclear clone 458; ITS, ribosomal DNA first and second internal transcribed spacer; S7, ribosomal gene introns S7-1 and S7-2; ncon, all four nuclear genes and regions concatenated. Abbreviations: LF, Lees Ferry; IG, Indian Garden; VP, Vaseys Paradise; Greens, The Greens; GHwy, Glendale Highway; CCrk, Center Creek; SR, Sevier River; 3L, Three Lakes; PasCrk, Pass Creek; PanCrk, Panguitch Creek; ER, Escalante River; ECh, Elves Chasm; n, number of samples; na, not applicable; –, no data ; ˂, less than; ≤, less than or equal to]

Locus Sample localities

LF IG VP Greens GHwy CCrk SR 3L PasCrk PanCrk ER ECh

COI 0.22 0.21 0.24 0.11 1 0.09 0.11 1,2 0.13 0.15 3 0.14 – 0.16 –

cyt b 0.11 0.25 0.20 0.12 0.18 na 0.17 2 0.26 0.15 na 0.24 –

mtconcat 1 0.10 0.24 3 0.11 1 0.09 0.12 0.11 1,2 0.13 2 0.30 0.16 – 0.17 –356 na – 0.21 0.25 3 0.07 0.23 0.20 1 0.06 2 0.27 2 0.18 1 0.06 2 0.20

458 0.18 na na na na 2 0.27 2 0.16 – 2 0.33 0.21 2 0.18 2 0.21

ITS 0.26 na na 2 0.50 na 2 0.22 – na 2 0.22 0.13 na na

S7 na 0.16 0.19 0.20 0.08 na 0.09 0.16 0.12 na 1 0.07 2 0.16

ncon 2 0.19 – 0.21 0.20 2 0.28 2 0.27 2 0.18 – – 2 0.27 2 0.16 2 0.21

1 0.05˂p˂0.102 n≤103 p˂0.05
30 Anatomical and Genetic Variation of Western Oxyloma Concerning the Endangered Kanab Ambersnail in Arizona and Utah

phylogenetic analyses for this concatenated dataset supported 

predominately shallow structure for each sampling location. 

A distance matrix for this dataset is shown in table 12. The 

Vaseys Paradise population was monophyletic and The Greens 

population was polyphyletic. In the NJ and MP trees (not 

shown), three specimens from The Greens (haplotype H5-4) 

were monophyletic with the Vaseys Paradise population. 

In the Bayesian analysis, this relationship was unresolved 

(fig. 12). The position of one Three Lakes specimen (9-12) in 

all three mtDNA analyses showed it as unrelated to the rest 

of the Three Lakes haplotypes and more closely related to the 

three highly divergent The Greens specimens. Additionally, 

two specimens from Sevier River (8-9 and 8-10) were 

monophyletic with the rest of the Three Lakes specimens.

The haplotype network for the concatenated dataset 

(fig. 13) mainly showed the same pattern as the single locus 

networks. Geographic units (populations) corresponded 

largely with the haplotype network. Three specimens from 

The Greens (haplotype H5-4) were positioned at the end of 

an extraordinarily long branch, indicating many mutational 

steps between them and the other samples. All specimens from 

Vaseys Paradise and one specimen from Three Lakes showed 

the next-largest number of mutational steps.

Overall, the mitochondrial data showed shallow 

structure corresponding with populations. Exceptions were 

three specimens from The Greens (haplotype H5-4) and 

one specimen from Three Lakes. Taken together, these 

exceptions showed positions in the haplotype network that 

were supported by a considerably larger number of mutational 

steps than between other groups in this study. Interesting 

“outliers” were one specimen from Vaseys Paradise sharing its 

cyt b haplotype with specimens of the Lees Ferry population. 

Some specimens from the Sevier River population also 

were interesting, as they seemed to be monophyletic with 

other populations. The Sevier River population was the only 

population that was widely distributed over the phylogenetic 

trees and haplotype networks.

Nuclear DNA

The nuclear DNA regions examined in this study, and 

presented here, are markers that span genes (coding regions), 

non-coding regions, repetitive regions, single-copy regions, 

and multilocus markers. Taken together, these markers provide 

a more complete and scientifically rigorous analysis than any 

of these markers alone.

Figure 8 shows the Bayesian tree for our COI dataset. 

NJ and MP trees also were generated but are not shown. 

All trees show structure between different populations, 

mostly at a shallow level. All analyses agreed in that Vaseys 

Paradise specimens formed a monophyletic group but were 

not monophyletic with Three Lakes and all specimens from 

The Greens. The Greens were divided into two separate 

groups: one haplotype (H5-4) of three specimens that were 

monophyletic with the Vaseys Paradise population, and 

one monophyletic group that consists of seven different 

haplotypes. The Three Lakes population was not monophyletic 

in the Bayesian analysis. One specimen (9-12) from Three 

Lakes showed no close relationship with the other specimens 

of the group, as seen by the Three Lakes individual (yellow) 

that is placed on a branch far away from all other Three Lakes 

(yellow) individuals in the tree.

A haplotype network (fig. 9) resulted in a similar pattern 

with specimens from Vaseys Paradise showing the second 

largest distance from the network center. The largest distance 

shown connected a haplotype from The Greens (H5-4) to the 

network. The third largest distance was shown by a branch 

leading to one Three Lakes specimen (9-12).

Phylogenetic trees generated for cyt b used the same 

three algorithms. The Bayesian tree is shown in fig. 10. 

Phylogenetic analyses supported the monophyly of the Vaseys 

Paradise specimens with the exception of one specimen that 

grouped with Lees Ferry individuals (haplotype H2-1) and 

one Sevier River individual. Otherwise, the results showed 

a similar pattern of shallow structure as observed in the 

COI fragment.

The cyt b haplotype network (fig. 11) reflected the same 

structure seen in the phylogenetic analyses. Haplotypes 

corresponded largely with geographic populations. The long 

branches (many mutational steps) leading to Vaseys Paradise, 

one Three Lakes specimen (9-12), and to a group of three 

specimens from The Greens (haplotypes H5-4), indicate these 

to be the most divergent haplotypes in this study area.

As mitochondrial loci are linked, we also concatenated 

the COI and cyt b sequences for each individual. We excluded 

samples with missing sequences from this dataset. This 

resulted in only four concatenated sequences for Sevier River, 

two for Three Lakes, eight for Pass Creek and seven for 

Escalante River. As no COI sequences for Panguitch Creek 

samples were generated, no concatenated sequences for this 

location were analyzed.

The concatenated mitochondrial loci sequence represents 

876 bp with 85 informative nucleotide polymorphisms. A 

total of 116 specimens and 53 haplotypes are observed. The 
Results 31

tac11-0685-fig 08

Center Creek

Escalante River

Vaseys Paradise

Glendale Highway

Indian Garden

Pass Creek

Lees Ferry

The Greens

The Greens

Three Lakes

Three Lakes

Three Lakes

Three Lakes

Sevier River

Sevier River

Sevier River

*

*

*

*

*

*

COI

0.04 Nucleotide substitutions per site

Three Lakes

Indian Garden

Vaseys Paradise

The Greens

Sevier River

Pass Creek

Escalante River

Population

EXPLANATION

Glendale Highway

Lees Ferry

Center Creek

Figure 8. Bayesian (MB) 50-percent majority rule consensus tree diagram showing mitochondrial cytochrome oxidase subunit I gene 

(COI) dataset analyzed for Oxyloma populations sampled. The Panguitch Creek, Utah, population is not included in the tree. Asterisks 

indicate well-supported nodes (Bayesian posterior probability greater than 90 percent). Rectangles and triangles represent individuals 

and populations, respectively. The height of a rectangle or triangle indicates the relative number of individuals represented by the 

sample (larger height is more individuals), and the width of a rectangle or triangle indicates the relative evolutionary divergence (larger 

width is more divergence).
32 Anatomical and Genetic Variation of Western Oxyloma Concerning the Endangered Kanab Ambersnail in Arizona and Utah

Figure 9. Median-joining haplotype network diagram showing mitochondrial cytochrome oxidase subunit I gene (COI) dataset 

analyzed for Oxyloma populations sampled. COI partial (in the box) represents an enlargement of the lower part of the complete 

network. Color codes represent the different populations and are the same as in the COI Bayesian tree (fig. 8). The size of each circle is 

proportional to the number of individuals containing each haplotype, respectively. Branch lengths are proportional to mutational steps, 

and the shortest branch is one step.

tac11-0685-fig 09

COI

COI Partial

Population

EXPLANATION

Three Lakes

Indian Garden

Vaseys Paradise

The Greens

Sevier River

Pass Creek

Escalante River

Glendale Highway

Lees Ferry

Center Creek
Results 33

Figure 9.—Continued

tac11-0685-fig 09_inset

Population

EXPLANATION

COI

COI Partial

Three Lakes

Indian Garden

Vaseys Paradise

The Greens

Sevier River

Pass Creek

Escalante River

Glendale Highway

Lees Ferry

Center Creek
34 Anatomical and Genetic Variation of Western Oxyloma Concerning the Endangered Kanab Ambersnail in Arizona and Utah

tac11-0685-fig 10

Center Creek

Escalante River

Glendale Highway

The Greens

Indian Gardens

Indian Gardens

Sevier River

Vaseys Paradise

Pass Creek

Lees Ferry

Vaseys Paradise

Sevier River

Lees Ferry

Pass Creek

Pass Creek

Pass Creek

Sevier River

Sevier River

Sevier River

Panguitch Creek

The Greens Sevier River

Three Lakes

Escalante River

Three Lakes

Sevier River

Three Lakes

*

0.07 Nucleotide substitutions per site

cyt b

Panguitch Creek

Three Lakes

Indian Garden

Vaseys Paradise

The Greens

Sevier River

Pass Creek

Escalante River

Population

EXPLANATION

Glendale Highway

Lees Ferry

Center Creek

Figure 10. Bayesian 50-percent majority rule consensus tree diagram showing mitochondrial cytochrome b gene (cyt b) dataset 

analyzed for Oxyloma populations sampled. The Panguitch Creek population is not included in the tree. Asterisk indicates wellsupported nodes (Bayesian posterior probability greater than 90 percent). The height of a rectangle or triangle indicates the relative 

number of individuals represented by the sample (larger height indicates more individuals), and the width of a rectangle or triangle 

indicates the relative evolutionary divergence (larger width indicates more divergence).
Results 35

tac11-0685-fig 11

4-18

4-12

4-17

5-16

5-1

mv7

mv9

mv10

mv11

mv8

mv5

mv1 mv4

mv2 mv3

mv5

10-10

10-12

9-12

10-2

8-16 8-4 2-16 2-21

2-11 2-19

9-10

9-20 8-10

8-1

6-3

6-1

6-17

3-2 3-10

7-1

12-1

12-2

12-7

9-19

8-5 8-11

4-20

cyt b

cyt b Partial

cyt b Partial

5-4

Panguitch Creek

Three Lakes

Indian Garden

Vaseys Paradise

The Greens

Sevier River

Pass Creek

Escalante River

Population

EXPLANATION

Glendale Highway

Lees Ferry

Center Creek

Figure 11. Median-joining haplotype network diagram showing mitochondrial cytochrome b gene (cyt b) dataset analyzed for 

Oxyloma populations sampled. Cyt b partial (in the box) represents an enlargement of the lower part of the complete network. 

Color codes represent the different populations and are the same as in the cyt b Bayesian tree (fig. 10). The size of each circle is 

proportional to the number of individuals containing each haplotype, respectively. Branch lengths are proportional to mutational steps, 

and the shortest branch is one step.
36 Anatomical and Genetic Variation of Western Oxyloma Concerning the Endangered Kanab Ambersnail in Arizona and Utah

Table 12. Pairwise distance matrices for mitochondrial DNA datasets for Oxyloma populations from Arizona and Utah.

[Upper matrix is the complete concatenated dataset of the cytochrome b gene (cyt b) and cytochrome oxidase subunit I gene (COI) for a total of 876 base 

pairs. Two genetic distance measures are included; FST (Fixation Index for subpopulation relative to the total combined population) is below the diagonal, Dxy 

(corrected average genetic distance between two populations, x and y) is above the diagonal. Lower matrix is the same as the upper matrix, except without 

sample outliers 5_04, 5_19, and 5_23 from The Greens population. Abbreviations: n, number of individuals per population; PasCrk, Pass Creek; ER, Escalante 

River; LF, Lees Ferry; IG, Indian Garden; VP, Vaseys Paradise; Greens, The Greens; GHwy, Glendale Highway; CCrk, Center Creek; SR, Sevier River; 3L, 

Three Lakes; –, no data]

n PasCrk ER LF IG VP Greens GHwy CCrk SR 3L

PasCrk 11 – 0.0220 0.0051 0.0208 0.0492 0.0380 0.0215 0.0190 0.0210 0.0261

ER 12 0.8965 – 0.0202 0.0139 0.0520 0.0510 0.0092 0.0067 0.0102 0.0167

LF 20 0.7968 0.9164 – 0.0210 0.0472 0.0366 0.0221 0.0188 0.0209 0.0262

IG 19 0.8545 0.7353 0.8834 – 0.0533 0.0484 0.0132 0.0112 0.0114 0.0202

VP 16 0.9792 0.9677 0.9906 0.9544 – 0.0678 0.0539 0.0495 0.0507 0.0533

Greens 22 0.3722 0.5201 0.3655 0.4789 0.6573 – 0.0509 0.0500 0.0496 0.0539

GHwy 19 0.9127 0.7253 0.9412 0.7515 0.9762 0.5272 – 0.0075 0.0112 0.0171

CCrk 18 0.9442 0.7431 0.9747 0.7799 0.9907 0.5348 0.8239 – 0.0067 0.0128

SR 9 0.8317 0.5877 0.8586 0.5672 0.9420 0.4810 0.6594 0.5540 – 0.0148

3L 7 0.6713 0.4453 0.6941 0.5045 0.8500 0.4291 0.4831 0.3740 0.2907 –

PasCrk 11 – 0.0220 0.0051 0.0208 0.0492 0.0153 0.0215 0.0190 0.0210 0.0261

ER 12 0.8965 – 0.0202 0.0139 0.0520 0.0310 0.0092 0.0067 0.0102 0.0167

LF 20 0.7968 0.9164 – 0.0210 0.0472 0.0137 0.0221 0.0188 0.0209 0.0262

IG 19 0.8545 0.7353 0.8834 – 0.0533 0.0283 0.0132 0.0112 0.0114 0.0202

VP 16 0.9792 0.9677 0.9906 0.9544 – 0.0501 0.0539 0.0495 0.0507 0.0533

Greens 22 0.9127 0.9257 0.9219 0.8920 0.9789 – 0.0307 0.0297 0.0293 0.0342

GHwy 19 0.9127 0.7253 0.9412 0.7515 0.9762 0.9378 – 0.0075 0.0112 0.0171

CCrk 18 0.9442 0.7431 0.9747 0.7799 0.9907 0.9634 0.8239 – 0.0067 0.0128

SR 9 0.8317 0.5877 0.8586 0.5672 0.9420 0.8780 0.6594 0.5540 – 0.0148

3L 7 0.6713 0.4453 0.6941 0.5045 0.8500 0.7479 0.4831 0.3740 0.2907 –
Results 37

tac11-0685-fig 12

The Greens

Vaseys Paradise

Pass Creek

Lees Ferry

Escalante River

Center Creek

Glendale Highway

Sevier River

Indian Garden

The Greens

Three Lakes

Sevier River

Three Lakes

*

*

*

*

*

*

mtconcat

0.05 Nucleotide substitutions per site

Three Lakes

Indian Garden

Vaseys Paradise

The Greens

Sevier River

Pass Creek

Escalante River

Population

EXPLANATION

Glendale Highway

Lees Ferry

Center Creek

Figure 12. Bayesian 50-percent majority rule consensus tree diagram showing concatenated mitochondrial DNA dataset (mtconcat) 

including the cytochrome oxidase subunit I (COI) and cytochrome b (cyt b) genes analyzed for Oxyloma populations sampled. The 

Panguitch Creek population is not included in the tree. Asterisks indicate well-supported nodes (Bayesian posterior probability greater 

than 90 percent). The height of a rectangle or triangle indicates the relative number of individuals represented by the sample (larger 

height means more individuals), and the width of a rectangle or triangle indicates the relative evolutionary divergence (larger width 

means more divergence).
38 Anatomical and Genetic Variation of Western Oxyloma Concerning the Endangered Kanab Ambersnail in Arizona and Utah

Single Copy

AMOVA results for the 4 nuclear loci showed that most of 

the observed variation exists among populations (greater than 

67 to 97 percent, table 8). Pairwise FST MDS plots (not shown) 

revealed no consistent population groups. The Lees Ferry, 

Glendale Highway and Escalante River populations showed 

slight deviations from the distribution of the other populations at 

most loci. 

For the nuclear, single-copy loci, a Mantel test comparing 

pairwise genetic distance values (estimated using FST) with 

geographic distance (kilometers) between populations revealed 

a slight positive correlation between genetic and geographic 

distance for S7 and 458 (r2 greater than or equal to 0.07 and r2

greater than or equal to 0.19, respectively; table 8). Therefore, 

only 7 percent or less (and 19 percent or less for region 458) of 

the genetic distance variance could be explained by geographic 

distance. The S7 fragment was 485 bp long and included 48 

variable sites, 33 parsimony informative sites, and two indels 

(1 and 9 bp in length). The S7 dataset included 43 haplotypes 

detected among 222 individuals that successfully amplified from 

among all populations. This made S7 the locus with the largest 

and most representative dataset.

Signals for population expansion with statistically significant 

negative values for Tajima’s D (table 9), Fu’s Fss (table 10) and 

statistically significant small positive values for the Roza’s R2

(table 11) were seen in Glendale Highway, Center Creek, Three 

Lakes, Panguitch Creek and Escalante River populations. The 

Vaseys Paradise population deviated from the multimodal curve 

of a stable population at the S7 locus.

Phylogenetic trees were generated using Neighbor Joining 

(NJ), Maximum Parsimony (MP) and Bayesian algorithms. The 

trees had similar features so only the Bayesian tree was shown 

(fig. 14). Ten samples from Vaseys Paradise and 3 samples from 

Elves Chasm shared 1 haplotype with Panguitch Creek, Sevier 

River, Pass Creek, Three Lakes and The Greens. This haplotype 

was the most commonly found haplotype in the dataset. Lees 

Ferry samples shared their haplotype with Escalante River 

samples, and Center Creek samples shared their haplotype with 

Three Lakes, Sevier River and Pass Creek. Samples from The 

Greens formed three major groups, which were distributed in 

different areas of the trees. The only monophyletic population 

was Glendale Highway. It is noteworthy that one shared 

haplotype from The Greens (including 24 single haplotypes) is 

equally distant from all other groups in the trees, with a branch 

length that is the largest observed in the S7 analyses.

The haplotype network (fig. 15) showed some geographic 

mixing and some discrete geographic units. The network structure 

corresponded less to populations or geographic units than was 

observed for the mitochondrial loci networks. 

tac11-0685-fig13

mtconcat

Three Lakes

Indian Garden

Vaseys Paradise

The Greens

Sevier River

Pass Creek

Escalante River

Population

EXPLANATION

Glendale Highway

Lees Ferry

Center Creek

Figure 13. Median-joining haplotype network drawing showing concatenated mitochondrial DNA dataset (mtconcat) including the 

cytochrome oxidase subunit I (COI) and cytochrome b (cyt b) genes analyzed for Oxyloma populations sampled. Color codes represent 

the different populations and are the same as in the mtconcat Bayesian tree (fig. 12). The size of each circle is proportional to the 

number of individuals containing each haplotype, respectively. Branch lengths are proportional to mutational steps, and the shortest 

branch is one step.
Results 39

tac11-0685-fig 14

Indian Garden

The Greens

Escalante River

Lees Ferry

Vaseys Paradise

Glendale Highway

Pass Creek

Panguitch Creek

The Greens

Sevier River

Three Lakes

Center Creek

Sevier River

The Greens

Indian Garden

Indian Garden

Vaseys Paradise

Three Lakes

Vaseys Paradise

Sevier River

0.009 Nucleotide substitutions per site

S7

Panguitch Creek

Three Lakes

Indian Garden

Vaseys Paradise

The Greens

Sevier River

Pass Creek

Escalante River

Population

EXPLANATION

Glendale Highway

Lees Ferry

Center Creek

Figure 14. Bayesian 50-percent majority rule consensus tree diagram showing ribosomal gene introns S7-1 and S7-2 (S7) dataset 

analyzed for Oxyloma populations sampled. The height of a rectangle or triangle indicates the relative number of individuals represented 

by the sample (larger height indicates more individuals), and the width of a rectangle or triangle indicates the relative evolutionary 

divergence (larger width indicates more divergence).
40 Anatomical and Genetic Variation of Western Oxyloma Concerning the Endangered Kanab Ambersnail in Arizona and Utah

tac11-0685-fig 15

S7

S7 Partial 1

S7 Partial 2

Panguitch Creek

Three Lakes

Indian Garden

Vaseys Paradise

The Greens

Sevier River

Pass Creek

Escalante River

Population

EXPLANATION

Glendale Highway

Lees Ferry

Center Creek

Figure 15. Median joining haplotype network diagram showing ribosomal gene introns S7-1 and S7-2 (S7) dataset analyzed 

for Oxyloma populations sampled. S7 partial 1 and 2 (below the main network) represent magnified parts of the complete 

network. Color codes represent the different populations and are the same as in the S7 Bayesian tree (fig. 14). The size of each 

circle is proportional to the number of individuals containing each haplotype, respectively. Branch lengths are proportional to 

mutational steps, and the shortest branch is one step.
Results 41

tac11-0685-fig 15 insets

S7 Partial 1

S7 Partial 2

D09BA

O4_05B

O4_05B

O4_07A

O8_18A

O1006A

O1010B

O5_17B

O8_01B

O9_09A O6_08B

O6_01A

mvi

O1_1002A

mv2

O5_12B

mv16 mv3

mv15

mv10

mv4

O1206A

O1004B

O5_11A

O5_16B

O5_01A

O1011B

O5_10B

O5_11B

Panguitch Creek

Three Lakes

Indian Garden

Vaseys Paradise

The Greens

Sevier River

Pass Creek

Escalante River

Population

EXPLANATION

Glendale Highway

Lees Ferry

Center Creek

Figure 15.—Continued
42 Anatomical and Genetic Variation of Western Oxyloma Concerning the Endangered Kanab Ambersnail in Arizona and Utah

ITS had a length of 616 bp including 51 variable sites, 

33 parsimony informative sites, and one indel. The indel was 

extensive in size, with a minimum size of 142 bp, and had 

variable sizes within and among individuals, occurring in 

11 different size states ranging from 142 to 387 bp. This and 

the discovery of paralogous amplicons for ITS in other studies 

made this marker questionable, unless techniques such as 

cloning all alleles prior to sequencing are applied to ensure 

that compared sequences are orthologous (Rosselló and others, 

2007). Therefore, ITS was excluded from the concatenated 

dataset. ITS sequences represented 126 samples with some 

populations such as Vaseys Paradise, Lees Ferry and Glendale 

Highway, producing many sequences, and other populations 

producing none (Sevier River), or very few sequences 

(The Greens, Center Creek, Pass Creek and Indian Garden) 

(table 2). The 126 samples represented 22 different haplotypes.

The different phylogenetic analysis algorithms used 

for ITS sequences (NJ, MP, and Bayesian) largely agree for 

this sample size and character number reduced dataset. The 

Bayesian tree is shown in figure 16. Population-based structure 

was apparent. The only population that was not grouped by 

geography, but instead was scattered throughout the trees, was 

Pass Creek. Because of limited variability in the dataset, a 

haplotype network was not calculated for ITS sequences.

The 356 locus sequence was 192 bp long and included 

25 variable and 15 parsimony informative sites. Two indels 

were observed, one of which was between 1 and11 nucleotides 

long (occurring in 7 different length states). A total of 

138 sequences were obtained for the 356 locus. These 

covered all populations with the exception of Indian Garden. 

Only three and five sequences were obtained from Pass and 

Panguitch Creeks, respectively.

The Bayesian phylogenetic tree for the 356 locus 

(fig. 17) showed many unresolved relationships, with only 

one group supported by bootstrap values and Bayesian 

posterior probabilities. This group contained 18 haplotypes 

from Vaseys Paradise, all 28 haplotypes from Three Lakes, 

and 4 haplotypes from Sevier River. Haplotypes from the 

same populations mostly appeared in different parts of the 

tree. Only Lees Ferry samples showed one haplotype that was 

not shared with other populations. NJ and MP trees showed 

similar topology to the Bayesian tree and are not shown. The 

haplotype network shows the same pattern as the phylogenetic 

trees (fig. 18).

The 458 locus produced a sequence of 525 bp with 

36 variable sites, 32 parsimony informative sites, and five 

indels (one with two length states). A total of 109 samples 

that successfully amplified were sequenced and produced 

26 different haplotypes. None of the samples from the Three 

Lakes population produced sequences. From Pass Creek, only 

two samples amplified, and from Center Creek, only four 

samples could be sequenced.

NJ, MP and Bayesian phylogenetic trees were generated 

and showed similar topology, so only the Bayesian tree is 

shown (fig. 19). Lees Ferry haplotypes formed the only 

monophyletic group. All samples from Vaseys Paradise, The 

Greens, Indian Garden, and Glendale Highway had only 

one haplotype. Of these, Vaseys Paradise and The Greens 

shared the same haplotype, and Indian Garden and Glendale 

Highway shared the same haplotype. Other populations, such 

as Panguitch Creek and Sevier River, exhibited a number of 

different haplotypes, which were distributed over larger areas 

of the trees. The haplotype network for the 458 locus was 

consistent with the phylogenetic trees (fig. 20).

We concatenated the sequences of S7, 356 and 458 

for all individuals, which produced sequences for all three 

loci. The fourth locus ITS was not included for reasons of 

DNA sequence quality mentioned in Single Copy of the 

section Results. The concatenated dataset resulted in only 

74 individuals; Indian Garden, Three Lakes, and Pass Creek 

samples were not included. The dataset included 92 variable 

and 86 parsimony informative sites in 52 haplotypes.

Phylogenetic analysis, using NJ, MP, and Bayesian 

algorithms for the concatenated nuclear DNA dataset, showed 

population structure with a number of exceptions, such as the 

Panguitch Creek haplotypes, one Escalante haplotype, or two 

Sevier River haplotypes (tree not shown).

The haplotype network for the concatenated nuclear DNA 

dataset agreed largely with the phylogenetic tree, with the 

exception that the Glendale Highway population haplotypes 

were positioned within the Vaseys Paradise-Elves Chasm 

haplotypes. Furthermore, all Escalante River haplotypes 

clustered together with a monophyletic origin (network 

not shown).

Overall, phylogenetic patterns observed in the nuclear 

gene trees were not consistent with each other or with 

the patterns observed for the two mitochondrial loci. No 

population was monophyletic in all analyses and all loci. Two 

populations, Lees Ferry and Glendale Highway, showed the 

lowest amount of within-population variability. The Sevier 

River population showed the widest phylogenetic diversity 

across the phylogenetic analyses and haplotype networks.

Microsatellite (Single Locus)

One microsatellite locus, KS6511, was amplified across 

all sampled populations. KS6511 is a GA dinucleotide repeat 

with a complex structure of (GA)8....(GA)7 in the sequenced 

copy. The alleles ranged from 10 to 53 repeats, but because 

of the complex structure, allele numbering did not strictly 

correspond to the number of repeats. The Greens, Three Lakes, 

and to some degree Panguitch Creek populations showed 

the complete allele diversity possible with the marker. Four 

populations were monomorphic (homozygous): Lees Ferry, 

Indian Garden, Center Creek, and Escanante River. All other 

populations exhibited fewer alleles relative to The Greens and 

Three Lakes populations (fig. 21). Figure 21 also included 

samples from the northern United States and Canada, which 

may have represented source populations for long-distance 

migrants. The alleles observed in the Arizona and Utah study 

area appeared to be a subset of the alleles present in Oxyloma

from the northern areas.
Results 43

tac11-0685-fig 16

The Greens

Center Creek

Lees Ferry

Lees Ferry

Glendale Highway

Indian Garden

Escalante River

Vaseys Paradise

Panguitch Creek

Pass Creek

Three Lakes

Pass Creek

*

*

*

*

*

* ITS

0.0030 Nucleotide substitutions per site

Panguitch Creek

Three Lakes

Indian Garden

Vaseys Paradise

The Greens

Pass Creek

Escalante River

Population

EXPLANATION

Glendale Highway

Lees Ferry

Center Creek

Figure 16. Bayesian 50-percent majority rule consensus tree diagram of the ribosomal DNA first and second internal 

transcribed spacer (ITS) dataset analyzed for Oxyloma populations sampled. The Sevier River population is not included in the 

tree. Asterisks indicate well-supported nodes (Bayesian posterior probability greater than 90 percent). The height of a rectangle 

or triangle indicates the relative number of individuals represented by the sample (larger height means more individuals), and 

the width of a rectangle or triangle indicates the relative evolutionary divergence (larger width means more divergence).
44 Anatomical and Genetic Variation of Western Oxyloma Concerning the Endangered Kanab Ambersnail in Arizona and Utah

tac11-0685-fig 17

*

Vaseys Paradise

Panguitch Creek

Center Creek

The Greens

Glendale Highway

Escalante River

Three Lakes

Lees Ferry

Sevier River

Pass Creek

Glendale Highway

0.02 Nucleotide substitutions per site

356

Panguitch Creek

Three Lakes

Vaseys Paradise

The Greens

Sevier River

Pass Creek

Escalante River

Population

EXPLANATION

Glendale Highway

Lees Ferry

Center Creek

Figure 17. Bayesian 50-percent majority rule consensus tree diagram showing random ambersnail nuclear clone 356 (356) dataset 

analyzed for Oxyloma populations sampled. The Indian Garden population is not included in the tree. Asterisks indicate well-supported 

nodes (Bayesian posterior probability greater than 90 percent). The height of a rectangle or triangle indicates the relative number of 

individuals represented by the sample (larger height means more individuals), and the width of a rectangle or triangle indicates the 

relative evolutionary divergence (larger width means more divergence).
Results 45

tac11-0685-fig 18

356

Panguitch Creek

Three Lakes

Vaseys Paradise

The Greens

Sevier River

Pass Creek

Escalante River

Population

EXPLANATION

Glendale Highway

Lees Ferry

Center Creek

Figure 18. Median-joining haplotype network diagram showing random ambersnail nuclear clone 356 (356) dataset 

analyzed for Oxyloma populations sampled. Color codes represent the different populations and are the same as in 

the 356 Bayesian tree (fig. 17). The size of each circle is proportional to the number of individuals containing each 

haplotype, respectively. Branch lengths are proportional to mutational steps, and the shortest branch is one step.
46 Anatomical and Genetic Variation of Western Oxyloma Concerning the Endangered Kanab Ambersnail in Arizona and Utah

tac11-0685-fig 19

The Greens

Vaseys Paradise

Lees Ferry

Sevier River

Escalante River

Panguitch Creek

Center Creek

Glendale Highway

Indian Garden

Pass Creek

Panguitch Creek

Panguitch Creek

Vaseys Paradise

Vaseys Paradise

*

*

*

0.008 Nucleotide substitutions per site

458

Panguitch Creek

Indian Garden

Vaseys Paradise

The Greens

Sevier River

Pass Creek

Escalante River

Population

EXPLANATION

Glendale Highway

Lees Ferry

Center Creek

Figure 19. Bayesian 50-percent majority rule consensus tree diagram showing random ambersnail nuclear clone 458 (458) 

dataset analyzed for Oxyloma populations sampled. The Three Lakes population is not included in the tree. Asterisks indicate 

well-supported nodes (Bayesian posterior probability greater than 90 percent). The height of a rectangle or triangle indicates the 

relative number of individuals represented by the sample (larger height means more individuals), and the width of a rectangle or 

triangle indicates the relative evolutionary divergence (larger width means more divergence).
Results 47

tac11-0685-fig 20

458

Panguitch Creek

Indian Garden

Vaseys Paradise

The Greens

Sevier River

Pass Creek

Escalante River

Population

EXPLANATION

Glendale Highway

Lees Ferry

Center Creek

Figure 20. Median-joining haplotype network diagram showing random ambersnail nuclear clone 458 (458) dataset 

analyzed for Oxyloma populations sampled. Color codes represent the different populations and are the same as in the 

458 Bayesian tree (fig. 19). The size of each circle is proportional to the number of individuals containing each haplotype, 

respectively. Branch lengths are proportional to mutational steps, and the shortest branch is one step.
48 Anatomical and Genetic Variation of Western Oxyloma Concerning the Endangered Kanab Ambersnail in Arizona and Utah

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48

53

Figure 21. Nuclear microsatellite alleles present in each individual, indicated by cells with allele sizes listed across columns and 

individuals listed down rows. Oxyloma populations are labeled and color coded with the same colors used in the phylogenetic trees and 

map for ease of viewing population differences. Alleles for an individual are indicated as white stippled cells at the appropriate allele 

size. A group of samples from north of the study area also is shown.
Results 49

tac11-0685-fig 21 page2 7-3 7-4 7-5 7-6 7-7 7-8 7-9 7-10 7-11 7-12 7-13

7-147-157-167-177-187-197-208-98-108-128-189-19-29-39-49-59-69-79-89-99-109-119-129-139-149-159-169-179-189-199-2010-1811-211-412-412-812-1012-1112-1212-1412-1512-16965102103162178224229285315326351405

Pass Creek

Panguitch Creek

Center Creek Escalante River Northern Samples Sevier

River Three Lakes

Figure 21.—Continued
50 Anatomical and Genetic Variation of Western Oxyloma Concerning the Endangered Kanab Ambersnail in Arizona and Utah

AFLP (Multilocus)

The UPGMA tree generated from the AFLP data generally indicated 

that most sampling locations were distinct from one another (fig. 22). 

Lees Ferry

The Greens

Escalante River

Three Lakes

Sevier River

Pass Creek (two individuals)

Indian Garden

Pass Creek

Sevier River

Three Lakes (two individuals)

Sevier R (one individual)

Center Crk (one individual)

Center Creek

Sevier River (two individuals)

Vaseys Paradise

Glendale Highway

Dice Coefficient

0.64 0.73 0.82 0.91 1.00

Figure 22. Unweighted Pair Group Method with Arithmetic mean (UPGMA) tree diagram showing Amplified Fragment Length 

Polymorphism (AFLP) data analyzed for Oxyloma populations sampled. The Panguitch Creek, Utah, population is not included in the tree.
Results 51

However, two lines of evidence suggest that it may not be 

appropriate to consider each population as “genetically 

distinct.” First, although uncommon, multiple analyzed 

individuals did not directly cluster with other individuals 

from their shared sampling location. Second, there was 

often relatively large variation in branch lengths among 

individuals forming the distinct clusters. Together, these 

data suggested that in some instances, the genetic ancestry 

of some individuals may be, in part, derived from other 

source populations.

Analyses using STRUCTURE confirmed the general 

pattern observed in the UPGMA tree. Rather than identifying 

K=10 genetic clusters (the number of sampled locations due 

to the removal of the Panguitch population from the analysis), 

STRUCTURE suggested that K=9 was a more likely value 

(fig. 23). This was mainly because STRUCTURE tended to 

define the loosely-differentiated Pass Creek and Indian Garden 

populations as being from the same cluster, as indicated by 

the prevalence of shared pink coloration in the STRUCTURE 

output (fig. 23). The result of nine genetic clusters was 

consistent with the relatively weak differentiation of these two 

populations (Pass Creek and Indian Gardens) in the UPGMA 

tree (fig. 22). More importantly, the STRUCTURE analysis 

suggested the presence of diverse ancestry among analyzed 

individuals from several sampling locations (particularly 

Glendale Highway, Center Creek, Sevier River, Pass Creek, 

and Escalante River).

tac11-0685-fig 23

Three Lakes

Three Lakes

Indian Garden

Indian Garden

Vaseys Paradise

Vaseys Paradise

The Greens

The Greens

Center Creek

Center Creek

Sevier River

Sevier River

Pass Creek

Pass Creek

Escalante River

Escalante River

Population

EXPLANATION

Glendale Highway

Glendale Highway

Lees Ferry

Lees Ferry

Note: This population color 

scheme does not match the 

other graphics

Figure 23. Results of a Bayesian STRUCTURE analysis of the 

Amplified Fragment Length Polymorphism (AFLP) data analyzed 

for Oxyloma populations sampled. The analysis suggested the 

presence of K=9 genetic clusters of individuals, or 9 populations, 

from 11 sampled locations. Each individual from each sampling 

location is represented in columns along the horizontal axis. Each 

of the K=9 detected clusters is indicated by a different color. The 

proportion of each individual’s genome that is derived from each 

cluster is represented as a function of the height (vertical) of each 

colored sub-bar.
52 Anatomical and Genetic Variation of Western Oxyloma Concerning the Endangered Kanab Ambersnail in Arizona and Utah

Discussion 

The data obtained for this study dramatically increased 

our understanding of Oxyloma, and more specifically, 

the taxonomic status of the federally endangered Kanab 

ambersnail. All analyses from this study, morphologic and 

genetic, supported the recognition of a single species of 

Oxyloma occurring in this study area. Some of the more 

high-resolution genetic data from the nuclear DNA (AFLP 

and microsatellite) indicated that population samples for this 

study were differentiated into distinct genetic groups. The 

significance of those groups was less obvious and will be 

discussed in the section Conservation Implications.

Shell Morphology

From the principal components analysis and the 

discriminant analysis, it was apparent that variation in 

shell morphology in the examined samples showed little 

geographic coherence. No meaningful combination of 

variables was effective at classifying more than about one-half 

of the analyzed specimens with respect to their samples of 

origin. Shell characters were poor predictors of population 

membership among these samples. The best characters were 

size-related. Size in molluscan populations and samples is well 

known to be affected by ontogeny, nutrition, local climate, 

and other secular factors (Goodfriend, 1986). The conclusion 

was that most of the variation within the system studied 

likely came from non-taxonomic, non-phylogenetic factors 

and represented an expected level of variability within one 

succineid species.

Discriminant analysis failed to assign the holotype of 

O. h. kanabense to its type population (The Greens) over three 

other options (Vaseys Paradise, Pass Creek, and Lees Ferry). 

Pilsbry (1948) may have selected the holotype for its large 

size; the rest of the type lot is in the 13–15 mm length range. 

In the past it was common for taxonomists to select the largest 

and “finest” specimen of a type lot rather than the most nearly 

average. Even the least size-dependent variable set assigned 

the holotype to other populations.

These findings were consistent with molecular results 

indicating little coherent structure among the sampled 

populations. The conchological variation was consistent with 

an interpretation that none of the sampled populations showed 

the type of ontogenetic divergence typically associated with 

different species. The findings were further consistent with 

an interpretation that none of the sampled populations were 

reproductively isolated from one another. Therefore, according 

to the biological species concept (Mayr, 1940), the sampled 

populations belonged to the same species.

Soft Tissue Anatomy

Eleven Oxyloma and one Catinella, populations sampled 

for this study, were readily distinguished by the presence in 

the Catinella of a broad penial appendix midway along the 

length of the penis. Oxyloma had a penial sheath; Catinella

did not.

Within Oxyloma, all anatomical characters showed 

substantial variation. Franzen (1963) found a similar level of 

variation in Oxyloma retusum (Lea, 1837) from 11 localities 

in the Midwest from Minnesota to Kansas. The anatomical 

variations Franzen detected were not restricted geographically 

but occurred in all samples collected. Franzen (1964) reported 

a similar amount of variation in the penis of the other 

purported conspecific subspecies of Oxyloma haydeni (W.G. 

Binney, 1858), O. h. haydeni, from 13 localities in Nebraska.7

Örstan (2010) reported variably straight and coiled epiphallus 

and penis in O. retusum from Maryland.

No soft parts are preserved from the type lot of Oxyloma 

haydeni kanabense (Paul Callomon, Collections Manager 

Malacology Department, Academy of Natural Sciences, 

Philadelphia, Pennsylvania, written commun., 2007). The 

only information on the anatomy of the type lot was that 

contained in the original description (Pilsbry, 1948, p. 798, 

figs. 419A and 419a)8. Pilsbry’s (1948) anatomical description 

was difficult to interpret taxonomically. The size of the 

hermaphrodite duct in succineid and other stylommatophoran 

snails is subject to great variation with time and reproductive 

readiness. The large size of the duct in his illustrated specimen 

may depict peak reproductive state. The prostate gland is said 

to be long, but its length, or relative length, is not quantified. 

The length of the prostate gland, in common with that of other 

soft tissues, is difficult to measure objectively. Specimens 

dissected for this study showed substantial variation. The 

fact that the penis was bent near the summit is a character 

of “section” (we would now say “subgenus”) Neoxyloma 

7

The only other anatomical information on O. h. haydeni appears to be the 

drawings by Stevens and others (2000), consisting of a sketch of the talon, 

hermaphrodite duct, and (apparently) part of the ovotestis (upper right in 

figure), and one depicting part of the epiphallus, penial retractor muscle, and 

penis (apparently cut free from the penial sheath, which appears as a collar 

around its base). The figures are unlabeled. No comparison is made with 

16 other specimens identified as “Oxyloma haydeni” (Stevens and others, 

2000). The upper end of the penis bears a small, tubular appendix, like O. 

h. kanabense and many other Oxyloma, but no diagnostic details are presented.

8

“Mantle over the lung not pigmented. Genitalia, Fig. 419A. The 

hermaphrodite duct is very large and swollen in specimens examined, and 

with the talon, is covered with a thin light gray membrane. The prostate gland 

is long. Penis short, somewhat swollen above, where it is abruptly bent, gives 

off a small, tapering appendix and the epiphallus, which makes about one 

spiral turn. (Fig. 419a). The vagina is nearly as long as the penis.” (Pilsbry, 

1948: 798).
Discussion 53

(Pilsbry, 1948, p. 775), which contains nearly all North 

American species of Oxyloma. The observation that the 

epiphallus “makes about one spiral turn” (presumably, within 

the penial sheath) was probably specific to the individual 

Pilsbry examined. We found the extent and deployment of the 

epiphallus within the penial sheath to be variable, from merely 

a small jog to between two and three turns before exiting the 

sheath. The relative lengths of penis and vagina may be a good 

taxonomic character in Oxyloma, if a way of measuring it 

without distorting the elastic tissues could be found.

Available information did not allow dispositive 

comparison between O. h. kanabense and O. h. haydeni.

Franzen’s (1964, fig. 1) illustrations of variation in the content 

of the penial sheath of seven specimens of O. h. haydeni, 

showed some forms of the penial summit and appendix and 

the disposition of the epiphallus that were like those we 

observed in O. h. kanabense; however, others were different. 

A study that included individuals of both nominal subspecies 

would be more informative than trying to draw conclusions 

from figures in the literature.

In summary, the soft anatomy proved relatively 

uninformative for the purposes of this study. It may have 

been more useful in a broader study in which more taxa (with 

more differentiated reproductive systems) were included. 

On the other hand, the absence of compelling differences 

among the Oxyloma samples studied was consistent with the 

lack of differentiation by shell characters and, likewise, with 

an interpretation that none of the sampled populations were 

reproductively isolated from one another.

Morphology and Anatomy—

Taxonomic-Nomenclatural Considerations

Solely on the basis of this study of shells and anatomy, 

the holotype of O. h. kanabense plausibly could be regarded as 

a member of the same species as the populations of Oxyloma

analyzed herein. Oxyloma haydeni kanabense was, on that 

basis, the correct name for this group of populations.

If future studies showed no difference between 

O. h. kanabense and O. h. haydeni, the latter name would be 

the valid name for the species, having priority (by 90 years) 

over the former. Franzen’s (1964) comparison of O. h. haydeni

and O. retusum, although reporting differences in the penial 

appendix and body coloration, leaves open the possibility 

that the still earlier name O. retusum may be yet another 

senior synonym.

On the question of whether O. h. kanabense is a species 

or subspecies: much modern taxonomy would consider this a 

non-issue. In a highly regarded proposal by de Queiroz (2005), 

a species is a separately evolving metapopulation lineage, 

where a metapopulation is an inclusive population made up of 

a set of connected subpopulations and a lineage is a population 

extended through time or an ancestral-descendant series of 

time-limited (instantaneous) populations. Such a species may 

or may not be fully isolated in a reproductive sense from other 

such entities—the essential condition of the biological species 

concept of Mayr (1940). Although theoretically possible, 

observations of presence or absence of interbreeding among 

land snail populations are limited. More often, the judgment 

of what is a given land snail species has been based on degree 

of difference criteria and the distribution of character states 

through an array of specimens. Moreover, reproductive 

isolation is but one possible contingent property of a species 

defined as an evolving metapopulation lineage. Other authors 

have called attention to the apparent maintenance of separation 

between metapopulation lineages (as evidenced by distinctive 

ecologies and morphologies) that appear to exchange genes 

more or less freely (de Queiroz, 2005). A corollary of the 

metapopulation lineage proposal is that all separately evolving 

metapopulation lineages are species. The taxonomy of North 

American land mollusks inherited from Pilsbry and his 

successors includes numerous subspecies, also recognized on 

degree of phenetic difference, subjectively judged.9 There is 

no generally agreed-on rule regarding their formal taxonomic 

recognition. Because under the species concept argued 

here, all separately evolving metapopulation lineages are 

species, the least inclusive taxon in formal taxonomy would 

be the species. This also is the conclusion of proponents of 

phylogenetic species concepts (Mishler and Theriot, 2000; 

Wheeler and Platnick, 2000). Where they exist, well-defined, 

diagnosable “subspecies” of the Pilsbryan system are simply 

called species. What is “well defined” remains a taxonomist’s 

judgment call, but arbitrary decisions on rank (and guesses 

about reproductive isolation) are less a factor than before.

Genetics

In a molecular genetic study, it is essential to analyze 

the markers separately prior to combining them. This allows 

an examination of marked changes (an undesirable result) 

upon combining into large concatenated datasets. Combining 

markers should not change the results but should strengthen 

the support.

9

“Most subspecies are recognizably differentiated populations which are not 

considered sufficiently distinct to be called species” (Pilsbry, 1939, p. xiv). “In 

ambiguous situations it is ... advantageous to treat allopatric populations of 

doubtful rank as subspecies.” (Mayr, 2000, p. 26).
54 Anatomical and Genetic Variation of Western Oxyloma Concerning the Endangered Kanab Ambersnail in Arizona and Utah

Each Marker Separate

The failure of many samples to amplify DNA fragments 

for subsequent sequencing was noteworthy. DNA template 

degradation could be ruled out for most of our samples, as 

samples that dropped for one locus readily amplified for other 

loci. Amplification failures could be caused by SNPs in the 

primer regions. If a SNP occurs at or close to the 3-prime 

end of a primer sequence, the DNA polymerase is unable 

to “sit down” on the template and begin extension. As we 

developed and optimized our primers from sequences derived 

from Vaseys Paradise specimens, no such “drop-outs” were 

observed in this population (or the Elves Chasm population). 

Consequently, “drop-outs” likely bear some SNP information. 

Unfortunately, this information was difficult to quantify other 

than that one or more SNPs between the Vaseys Paradise 

population and the “drop-out” specimens or populations were 

likely in one or both primer sequences. Conclusions of the 

position of such specimens or populations in a phylogenetic or 

population genetics context could not be drawn.

Likely for the same reason, outgroup specimens from 

the genus Catinella failed to amplify for all nuclear loci. This 

was not surprising, as the probability of a SNP at a certain 

position increases with increasing phylogenetic distance. It 

was especially challenging to find conserved areas for primers 

and adjacent, sufficiently variable areas with resolution, 

at the genera-species-population level in the nuclear 

genome. However, the outgroups of Catinella, Succinea and 

Neosuccinea provided the ability to root the mtDNA tree 

and indicated that Indian Garden was the first population to 

branch after the outgroup, followed by Sevier River, Glendale 

Highway, Center Creek, Three Lakes, Escalante River, 

Panguitch Creek, Upper Panguitch (Pole Creek), Lees Ferry, 

The Greens, and Vaseys Paradise. Vaseys Paradise was the 

most recent branch or lineage among these Utah and Arizona 

populations (along with the three outlier specimens from The 

Greens), when an outgroup root was used.

Based on genetic distance (Fst), about three-fourths of the 

observed variability was among populations, which indicated 

some, but limited, gene flow (table 8). When comparing 

pairwise Fst values for all populations and visualizing the 

results in MDS plots (not shown), no consistent population 

groupings over several loci were observed, with the exception 

of the Vaseys Paradise population that appeared more 

distant from other populations in the mitochondrial loci. 

The significance of this observation was unclear in light of 

evidence provided by other loci; it was possible that this 

pattern simply reflected the stochastic evolutionary process. 

Given the range of pairwise Fst values for all populations fell 

somewhere on a continuum from the lowest of 0.29 (Sevier 

and Three Lakes) to the highest of 0.99 (Vaseys Paradise and 

Lees Ferry), we estimated the relative migrations rates to be 

zero per generation between Vaseys Paradise and Lees Ferry, 

indicating almost complete subdivision and greater than or 

equal to 1 per generation between Sevier and Three Lakes. 

If interpreted literally, the results indicated a gene flow rate 

between Sevier River and Three Lakes that was sufficient to 

prevent complete differentiation, which could be as much as 

one per generation (Mills and Allendorf, 1996). However, if 

long-distance dispersal occurred from one source population 

into two different recipient populations, this process would 

mimic gene flow between the recipient populations when 

they instead only shared a recent connection to the source 

population. Overall, we observed a significant positive 

correlation between genetic and geographic distance between 

populations at one mtDNA locus and two of the nDNA loci. 

This indicated that the isolation-by-distance model applies to 

some extent, although only weak patterns existed.

Significant results for population expansion (Tajima’s D, 

Fu’s Fs, R2 and mismatch distribution) were seen in all 

populations except Indian Garden, The Greens, and to some 

degree, Sevier River. These results were most consistent 

across tests in the Lees Ferry, Vaseys Paradise, and Glendale 

Highway populations. As population expansions usually 

followed bottlenecks (such as recent population founder 

events), this scenario is likely for the latter three populations, 

and to a lesser degree for all populations with the possible 

exception of Indian Garden, The Greens, and Sevier River.

After much technical difficulty, we were able to 

successfully amplify one microsatellite DNA locus in the 

Oxyloma populations for this study (fig. 23). Although one 

microsatellite marker was not sufficient to draw scientifically 

rigorous conclusions, the analysis produced some useful basic 

information. The microsatellite results, with The Greens and 

Three Lakes containing most of the known allelic diversity, 

were consistent with our suggestion that The Greens may 

represent most of the genetic diversity for this area and may be 

a potential “hub” for long-distance dispersal events. The other 

populations showed a relatively reduced allele diversity, with 

four populations that are monomorphic (homozygous) and 

Vaseys Paradise showing only two alleles (the second allele 

only in heterozygous specimens).

Our phylogenetic analyses show that the nominal Kanab 

ambersnail, defined in the literature during the 1900s, was 

not monophyletic because The Greens (type locality) was 

not monophyletic with the Vaseys Paradise and Three Lakes 

populations (the only known Kanab ambersnail populations at 

the time of this study). This result agreed with a cyt b study on 

this group of snails with a much broader geographic sampling 

scheme (Stevens and others, 2000). 

We observed predominately shallow structure among 

the 11 Oxyloma populations sampled in Arizona and Utah 

using 2 mitochondrial DNA markers and 4 nuclear loci 

(which included Vaseys Paradise, Arizona, Three Lakes, 

Utah, and the type locality of The Greens, Utah). Likewise, 

STRUCTURE analyses of the AFLP data highlighted the 

general differentiation of populations, although some mixing 

of populations was apparent (fig. 23). The single nuclear 

microsatellite locus suggested similar patterns, as there 

tended to be limited sharing of microsatellite alleles among 

most populations (fig. 21). Overall, these patterns suggested 

that southwestern Oxyloma populations generally tended to 
Discussion 55

be genetically differentiated. Furthermore, these findings 

suggested that the “Kanab ambersnail” taxonomic designation 

was not valid. If it were valid, we would have expected to 

detect genetic clusters of specimens from Vaseys Paradise, 

Three Lakes, and The Greens. Instead, our findings tended to 

indicate the relatively similar differentiation of all populations.

Using DNA sequences from four nuclear genes, we did 

not observe structure consistent with that of the mitochondrial 

DNA, microsatellites, or AFLPs. Because mitochondrial 

DNA has a more rapid evolutionary rate (about ten times as 

rapid in humans) and a smaller effective population size than 

nuclear DNA, it is not unusual to see more structure using 

mitochondrial markers relative to nuclear markers. Likewise, 

microsatellite loci can show stronger genetic structure patterns 

than most nuclear genes because of their high mutation rates 

compared to non-microsatellite nuclear DNA (Ellegren, 

2004). Our analyses of AFLP markers adopted the standard 

practice of focusing solely on variable loci and excluded all 

monomorphic bands (Bensch and Åkesson, 2005; Foll and 

others 2008). Consequently, AFLP marker datasets generally 

tended to reflect more diverse, faster-evolving loci than 

background nuclear genomic regions. 

It is not considered scientifically rigorous to make 

conclusions on species or other major taxonomic statuses 

(such as ESUs) based on only one molecular marker. Our 

criterion for defining significant structure or taxonomic 

category was a corroboration of mitochondrial and nuclear 

markers; this criterion is well accepted as a taxonomic 

standard (Moritz, 1994). Because the nuclear markers 

indicated gene flow among populations, our combined results 

are consistent with this group of populations belonging to the 

same species. However, within a species, certain populations 

that show monophyletic origins and are geographically 

separated from other populations would potentially fit the 

definition of a separate ESU. In this study, although our 

nuclear genes and random regions did not support distinct 

populations, our more rapidly evolving markers (AFLP, 

microsatellite, mtDNA genes) indicated the presence of 

distinct populations at levels below the species level. In 

particular, two populations, Vaseys Paradise and Escalante 

River, showed monophyletic origins for mitochondrial 

markers, whereas AFLPs and microsatellite alleles suggested 

the presence of distinct groups.

From the structure observed using both mitochondrial 

DNA markers, the Vaseys Paradise population was the most 

distant population from the others. However, 3 samples from 

The Greens (in Utah) were even more distant (by a factor 

of about 10) than the Vaseys Paradise population; the other 

22 samples from The Greens cluster with the other Oxyloma

populations in this study area. Additionally, one sample from 

the Three Lakes population (9-12) did not cluster with the 

population from which it was sampled in any of our mtDNA 

analyses. All four of these samples were included in the 

nuclear dataset and showed no signs of distinctiveness, but 

shared haplotypes with a number of other samples. Stevens 

and others (2000) found one specimen (21.185ALB) from an 

Alberta, Canada, population showing almost exactly the same 

pattern and distance as samples from Arizona and Utah in 

their cyt b analysis. In a follow-up to this study, we sampled 

museum specimens of Oxyloma haydeni from Canada and 

several of those individuals clustered closely with the 3 The 

Greens outliers and also with the Three Lakes outlier. This 

indicated that the presence of divergent lineages in these 

populations (The Greens and Three Lakes) resulted from 

long-distance dispersal events from as far away as Canada. 

Although snails appear comparatively immobile, longdistance dispersal over several thousand kilometers has been 

demonstrated for pulmonate gastropods (Gittenberger and 

others, 2006). Migrating birds are possible vectors by carrying 

the snails in their feathers, feet, or digestive tract (Green and 

Figuerola, 2005; Wada and others, 2011). As for the three The 

Greens specimens and the single Three Lakes specimen, the 

most likely explanation was an immigration event involving 

one or very few specimens from a distant population.

A different type of dispersal event observed in this dataset 

was short-distance dispersal. An example of short-distance 

dispersal of an mtDNA haplotype could be seen in our cyt b

dataset: one specimen from Vaseys Paradise (4-20) was placed 

within the Lees Ferry population clade and shared the same 

haplotype with 20 individuals from that location. Lees Ferry is 

located upstream of Vaseys Paradise along the Colorado River 

within the Grand Canyon. The Lees Ferry cyt b haplotype may 

have reached Vaseys Paradise on drifting plant material, by 

bird transportation, or by other means. 

The Vaseys Paradise population had longer branch 

lengths than the other populations sampled in many of our 

mtDNA analyses. However, our combined analyses indicated 

that the Vaseys Paradise group was the most recent lineage 

of all the Arizona and Utah populations after outgroups 

were included and a rooted tree topology was obtained. We 

suspected the Vaseys Paradise population had experienced 

unusual population demographic events such as a founder 

event followed by one or more bottlenecks. Multiple lines of 

evidence supported this viewpoint: (1) population expansion 

was indicated by a number of tests for the Vaseys Paradise 

population (as it was for Lees Ferry and Glendale Highway); 

(2) repeated population expansion and contraction events 

could have led to lineage sorting (as observed in the Elves 

Chasm population, which was a known human-mediated 

bottleneck) and could explain the observed greater diversity 

of other populations when compared to the Vaseys Paradise, 

Elves Chasm, Lees Ferry, and Glendale Highway populations; 

and (3) the comparatively isolated small habitat situated 

on a steep slope at the waterline of the Colorado River (at 

Vaseys Paradise) likely allowed for repeated flooding of the 

habitat. Considering this scenario, the natural Colorado River 

dynamics in the Grand Canyon may have contributed to the 

genetic distinctiveness of the Vaseys Paradise population. The 

lack of nuclear and mitochondrial marker variability in the 

Lees Ferry and Glendale Highway populations also suggested 

relatively recent founder events or severe bottlenecks.
56 Anatomical and Genetic Variation of Western Oxyloma Concerning the Endangered Kanab Ambersnail in Arizona and Utah

Markers Combined

In this study, primarily AFLPs, mtDNA, and even the 

microsatellite locus showed structuring among populations. 

However, the distribution of all other genetic markers 

and morphological characters among the samples could 

be interpreted as no isolation. Our taxonomic conclusion, 

therefore, is that all studied samples of Oxyloma were drawn 

from populations of the same species.

The conclusions of this study are far more definitive and 

comprehensive than those provided by Stevens and others 

(2000). The current investigation relied on a greater numerical 

sampling of locations and individuals, a more focused and 

taxonomically relevant set of southwestern ambersnail 

populations, and a substantially larger panel of genetic 

markers. Although similar patterns have been identified in all 

studies, the more substantial data associated with the current 

project allowed us to understand better the relevance of the 

allele frequency differences. Our findings suggested that the 

genetic structure patterns may partially reflect periodic longdistance migration and establishment of populations by one or 

a few individuals rather than long-term population divergence. 

In studies of another snail species, Succinea caduca, 

an endemic ambersnail from Hawaii, Holland and Cowie 

(2007) used the mtDNA COI and 16S ribosomal ribonucleic 

acid genes to examine genetic lineages and population 

diversification among islands. They expected to find a pattern 

of lineage evolution representing a progressive linear dispersal 

from island to island. Instead, they found that the S. caduca

lineage predated the age of the islands and likely arose on 

an older island, and later dispersed to the Hawaiian Islands. 

This result was confirmed by Holland and Cowie (2007), 

using the same mtDNA markers, and one nuclear DNA 

marker (18S ribosomal ribonucleic acid). The 2008 paper also 

revealed multiple dispersals onto several of the islands. The 

dispersal they observed, island to island, sometimes occurring 

multiple times on the same island, is similar to our inferred 

“long-distance” dispersal with locations such as The Greens 

and Three Lakes receiving higher rates of dispersal than other 

populations (based on our single microsatellite locus; fig. 21).

The observed genetic patterns were in agreement with 

the species’ reproductive and dispersal strategies. Passive 

short-distance and long-distance dispersal in snails also was 

documented as occurring in amber snails from the Hawaiian 

Islands (Holland and Cowie, 2007), where suitable habitat 

and populations over large geographic areas contributed to 

panmixia. In this study, less suitable habitat areas with small, 

island-like, and comparatively isolated, habitat patches, such 

as the habitats for Oxyloma in Arizona and Utah, contribute to 

much of genetic structure observed in AFLP and mitochondrial 

DNA. However, Oxyloma are capable of selfing (Patterson, 

1970), which enables a single specimen to found a population. 

Subsequent gene flow by new immigrants contributed to 

increased genetic diversity, which countered reproductive 

isolation. How frequently gene flow between populations 

occurred is a function of factors such as geographic isolation, 

concordance with bird migratory routes, river flow directions, 

and so on. The pattern observed in this study agrees with a 

model of different populations showing varying degrees of 

isolation with limited gene flow. The populations of Lees 

Ferry and Glendale Highway showed a low genetic diversity, 

possibly a result of a recent demographic event. That no such 

signals were seen in the nuclear loci is not surprising, as all the 

tests are either completely or partly based on the occurrence 

of singletons in the dataset. In a recent expansion event, not 

enough time may have passed for a sufficient number of 

singletons to occur. Similarly, demographic events likely have 

led to the genetic structure observed in the Vaseys Paradise 

population from this study, as described in Mitochondrial 

DNA and Nuclear DNA in the Results section. On the other 

side of the spectrum, The Greens showed a comparatively 

large amount of genetic diversity and signs of recent 

population expansion. This could have been caused by a large 

population size, frequent immigrants from source populations 

possibly situated outside the geographic context of this study 

(long-distance dispersal events), or both. Furthermore, The 

Greens population could be seen as a source population 

(“hub”) for migrants to other populations within this study 

area. Similar to The Greens, the Sevier River population 

showed much diversity of haplotypes with relationships to 

many other populations in all genetic markers. This suggested 

that Sevier River might be another source population like The 

Greens. The observed genetic diversity at the Sevier River 

location could be accounted for by short-distance dispersal 

events between populations in this study. These hypotheses, 

incorporating a mixture of long-distance and short-distance 

dispersal events, were consistent with the observed haplotype 

diversity and distribution in haplotype analyses results. 

Additionally, the phylogenetic pattern of shallow structure 

and recent lineages was more consistent with an explanation 

of long-distance dispersal versus ancient vicariance and 

long-term evolution in situ. This is a story of dispersal, 

sometimes over very long distances, and colonization of 

populations, occasionally by single individuals.

Conservation Implications

The validity of the Kanab ambersnail taxonomic unit 

(Oxyloma haydeni kanabense) was not supported because 

Vaseys Paradise did not cluster with the type localities of 

Three Lakes and The Greens. We suggest that the ambersnail 

populations in this study area generally were undergoing a 

dynamic process of long-distance and short-distance dispersals 
Acknowledgments 57

with most lineages being fairly recent as reflected in the 

shallow structure of our trees. Additionally, the ambersnail 

life history supports frequent colonization events with snail 

dispersal over short and long distances. The ambersnail’s 

ability for selfing, and, therefore, for colonization, by one 

single individual may explain genetically distinct populations, 

which arise in a short period of time. If this were the case, the 

question arises of whether these genetically distinguishable 

populations should be characterized as ESUs or subspecies. 

Going back to the O’Brien and Mayr (1991) definition of a 

subspecies, a subspecies would share (a) a unique geographic 

range, (b) a set of “phylogenetically concordant phenotypic 

characters,” (c) a suite of molecular genetic similarities, and 

(d) derived adaptations relative to other subspecies. Some 

populations from this study arguably fit criteria a, and c; 

however, we did not find evidence for divisions below the 

species level to support criteria b or d. The O’Brien and 

Mayr definition applies mainly to population histories over 

extended time periods and with vicariance events that give 

rise to independently evolving lineages and metapopulations. 

Therefore, the definition does not fit the proposed model of 

colonization and possible extinction (“boom and bust”) of 

ambersnail populations in the Southwest, with its history 

of random, “non-phylogenetic” signatures. A consequential 

question arises here: Is it worthwhile to protect, as 

endangered, each population of ambersnails that may be 

founded by recent dispersal events that may be an integral 

part of these organisms’ life history, rather than populations 

that exist as long-time unique evolutionary lineages? We 

suggest that, by protecting a habitat and its dynamics to allow 

the continuation of future dispersals and colonizations to 

occur, this dynamic process may continue uninterrupted just 

as well as by protecting each genetically unique population. 

Some genetically unique populations in this system may be 

naturally short-lived, as dispersal and colonization strategies in 

marginal habitats may suggest. Some of the observed genetic 

uniqueness (for example, in the Vaseys Paradise population), 

may actually be caused and managed by re-occurring dramatic 

events such as floods, which have severe demographic effects.

Future Research 

Future research would usefully include similar analyses 

of additional populations from the species’ range (farther north 

[Canada] and farther south [Mexico]) to better understand 

dispersal patterns, and additionally, should examine larger 

microsatellite or SNP marker sets to address fine-scale 

population genetics and parentage questions (such as the role 

of selfing). These areas of inquiry are less relevant for the 

Grand Canyon conservation effort than for snail conservation 

and biology in general.

Summary

Because of controversy regarding taxonomy of Oxyloma

and the endangered Kanab ambersnail at the time this study 

began, new detailed and scientifically rigorous analyses 

were completed of morphological and genetic variation from 

ambersnail specimens collected at 12 locations in Arizona 

and Utah and some comparison individuals from farther 

north including Canada. These new data have allowed for the 

evaluation of Kanab ambersnail taxonomy from the standpoint 

of shell morphology and reproductive anatomy as well as 

from molecular genetics. Morphology and anatomy indicate 

that the holotype of Oxyloma haydeni kanabense plausibly 

can be regarded as a member of the same species as the 

populations of Oxyloma analyzed in this study. Genetic data 

indicate the presence of gene flow among all populations, 

evidence that they are members of the same species. Almost 

all the observed genetic diversity can be accounted for by 

short- or long-distance dispersal events between populations 

in this study. Our major taxonomic conclusion is that all 

samples collected for this study were drawn from populations 

of the same species. The combined analyses in this study and 

resulting conclusions produced some useful basic information 

for taxonomic classification and management of Oxyloma in 

Arizona and Utah.

Acknowledgments 

We would like to thank the USGS Grand Canyon 

Monitoring and Research Center for funding this study and 

encouraging us to write our grant proposal. The Bureau of 

Reclamation and U.S. Fish and Wildlife Service also partially 

funded this study. We thank Larry Stevens of the Grand 

Canyon Wildlands Council for his help on previous Kanab 

ambersnail work, the entire Kanab Ambersnail Working 

Group for helpful discussions at working group meetings, 

and Darren Bolen, Dan Cox, and Clay Nelson of the Arizona 

Game and Fish Department for collection of many of the 

samples. Finally, we thank Anna Carlson for help with genetic 

laboratory work and Mickey Reed for cartographic products.
58 Anatomical and Genetic Variation of Western Oxyloma Concerning the Endangered Kanab Ambersnail in Arizona and Utah

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Alleles Different Forms Of A Gene, Or Region 

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Amplicon Double-stranded DNA product 

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Amplified Fragment Length Polymorphism 

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method to compare genetic variation within 

versus among populations.

Autosomal locus A position on a chromosome, 

other than a sex chromosome. 

Bayesian A statistical phylogenetic treebuilding method that assigns probabilities or 

distributions after obtaining experimental data 

(posterior probability).

Bidirectional sequencing DNA sequencing 

from the forward and reverse directions; 

combining these sequences confirms the 

sequence because the forward and reverse 

sequences are not identical but are actually 

complementary sequences.

Bp Base pairs of deoxyribonucleic acid. 

Codominant Both alleles of a gene, or region of 

DNA, are expressed.

Denature DNA Double-stranded DNA melts 

into single-stranded strands.

Diploid A double set of homologous 

chromosomes. 

DNA Deoxyribonucleic acid, the molecular 

make-up of chromosomes.

DNA sequencing Sequence of nucleotide pairs 

along the DNA molecule.

Dominant Only one allele of a gene, or region 

of DNA, is expressed.

Electrophoresis Separating molecules based on 

different rates of travel in an electric field. 

EtOH Ethanol, an alcohol used to precipitate 

DNA in the process of extracting and purifying 

DNA from cellular material.

Evolutionarily Significant Unit (ESU) A 

population of organisms that are distinct for the 

purpose of conservation.

Fluorometer An instrument for detecting and 

measuring fluorescence; for the purpose of 

quantifying DNA. Fluorescent dye is first bound 

to the DNA, and then measured to quantify the 

amount of DNA.

Fu’s F statistic A test for population expansion. 

A significant Fu’s F means population expansion 

has occurred.

Genome The complete makeup of genetic 

material for an organism.

Haplotype Unique combination of linked 

mutations on a section of DNA.

Heterozygous Containing two different alleles 

at the same locus.

Homozygous Containing two of the same allele 

at the same locus.

Indel Refers to an insertion or deletion 

mutation.

ITS Internal transcribed spacer, refers to a piece 

of nonfunctional RNA (ribonucleic acid) located 

between structural rRNAs (ribosomal RNAs).

Kbp Kilo base pairs .

Locus The position of a specific gene, or region 

of DNA, on a chromosome.

Mantel test A statistical test of the correlation 

between two matrices, such as genetic distance 

and geographic distance.

Maximum Likelihood A statistical phylogenetic 

tree-building method used to fit a statistical 

model to data and to provide estimates for the 

model’s parameters.

Maximum Parsimony (MP) A minimum 

evolution tree-building method used to estimate 

phylogenies with the fewest number of 

mutational steps.

Glossary of Molecular Genetic Terms
64 Anatomical and Genetic Variation of Western Oxyloma Concerning the Endangered Kanab Ambersnail in Arizona and Utah

Median-joining network A phylogenetic graph 

used to visualize evolutionary relationships 

from sequences.

Microsatellite DNA locus A single location 

in the genome where a microsatellite occurs 

(tandem array of 2–6 base pairs).

Microsatellite genotype All fragments 

generated from PCR of a microsatellite locus, 

and subsequent electrophoresis.

Mitochondrial DNA (mtDNA) DNA located 

in mitochondria organelle or eukaryotic cells, 

which converts energy from food into a form that 

cells can use. Mitochondrial DNA is maternally 

inherited and more rapidly evolving than 

nuclear DNA.

Mendelian inheritance How hereditary 

characteristics are passed from parent organisms 

to their offspring.

Metapopulation A group of spatially separated 

populations of the same species that interact at 

some level.

Molecular genetics Study of gene, or region of 

DNA, at the molecular level.

Molecular (genetic) marker An informative 

gene, or region of DNA, used for genetic study.

Monomorphic Having all alleles the same, with 

no variation.

Multilocus marker Genetic marker that 

simultaneously detects multiple loci throughout 

the genome.

Neighbor Joining (NJ) A phenetic- or genetic–

distance-based method used for construction of 

phylogenetic trees that allows branches of the 

tree to have varying evolutionary rates.

Orthologous Genes evolved directly from a 

common ancestor.

Outgroup Group of organisms that serves as a 

reference group for determination of relationships 

of a monophyletic group of organisms (ingroup), 

and also forms the root of a phylogenetic tree.

Paralogous Genes evolved by duplication 

within a genome.

Phylogenetics A method to infer 

relationships among taxa, usually by building 

phylogenetic trees.

Polymerase Chain Reaction (PCR) Technique to 

make many copies of a specific region of DNA 

using oligonucleotide primers on each end of 

sequence to amplify.

Polymorphic Having many different alleles; 

variation among alleles.

Restriction Fragment Length 

Polymorphism Variation in length of DNA 

fragments generated by endonuclease digestion.

Roza’s value A test of neutrality for a set of 

DNA sequences, which tests for population 

growth versus population stability.

Single locus marker Genetic marker that 

detects only one locus in the genome at one time.

Single Nucleotide Polymorphism (SNP) 

“snips” DNA sequence variant where one 

nucleotide differs from one individual to the next.

Species Taxonomic unit, group of organisms 

that are reproductively isolated from other such 

groups of organisms, and basic unit of biological 

classification.

Subspecies Taxonomic subdivision below the 

species level.

Tajima’s D A test for population expansion 

or selection. A significant negative Tajima’s D 

can mean population expansion or positive 

purifying selection.

Taxon A group of organisms.

Unweighted Pair Group Method with Arithmetic 

mean (UPGMA) A phenetic- or geneticdistance-based method used to build phylogenetic 

trees that assumes equal rates of evolution on all 

branches of the tree.

Variable Number of Tandem Repeat 

(VNTR) Region of DNA whose alleles contain 

different numbers of tandemly repeated 

minisatellite oligonucleotide sequences.

Vicariance The separation of a group of 

organisms by a geographic barrier.
Appendixes 65

Appendix A. Genbank Accession Numbers

Data are available for download at http://pubs.usgs/gov/sir/2013/5164/AppendixA.pdf

Appendix B. DNA Sequences for Random Ambersnail Nuclear Clone 356

Data are available for download at http://pubs.usgs/gov/sir/2013/5164/AppendixB.pdf

Appendix C. DNA Sequences for Ribosomal First and Second Internal 

Transcribed Spacer

Data are available for download at http://pubs.usgs/gov/sir/2013/5164/AppendixC.pdf
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Publishing support provided by the U.S. Geological Survey

Publishing Network, Tacoma Publishing Service Center

For more information concerning the research in this report, contact the 

Leader, Arizona Cooperative Fish and Wildlife Research Unit

U.S. Geological Survey

325 Biosciences East 

Tucson, Arizona 85721

http://www.coopunits.org/Arizona/
Culver and others— Anatomical and Genetic Variation of Western Oxyloma Concerning the Kanab Ambersnail, Arizona and Utah—Scientific Investigations Report 2013–5164