Biological Boundaries and 

Conservation of the Kanab Ambersnail

 

Melanie Culver, Hans-Werner Herrmann, Anna Carlson, Mark 

Miller, Barry Roth, Jeff Sorenson

•Mitochondrial DNA sequence

•Nuclear DNA sequence (gene 

and SNP)

•Anatomical analyses
Molecular Taxonomy: Kanab ambersnail

• Genetic and anatomical variation in Kanab 

ambersnail - USGS (GCMRC)

 Hans-Werner Herrmann, Postdoc (UA)

 Anna Carlson, Postdoc (UA)

 Collaborators: Mark Miller (USU), Barry Roth

 Sampling: Jeff Sorenson (AGFD)

1. Resolve Kanab ambersnail taxonomy in Oxyloma AZ/

UT populations (AZ, UT) and Canada individuals

2. Explain prior discordance of morphological and genetic 

results
Background

• Oxyloma genus, terrestrial snail

– >12 species in North America

– Also occurs in Europe and Africa

• Oxyloma haydeni occurs in western US and Canada

– Taxa not well defined

– Morphology and anatomy have limitations

• Kanab Ambersnail, Oxyloma haydeni kanabense

– Type specimen in Utah, occurs into Canada

– ESA listed as endangered subspecies 

• Family SUCCINEIDAE, Beck, 1837

– Shells offer little indication if genus or species identity

– Genera are identified by anatomical traits (reproductive)

– Species-level resolution poorly understood
Oxyloma haydeni haydeni Oxyloma haydeni kanabense

(Niobrara ambersnail) (Kanab ambersnail) 

- Distribution pattern are disjunct for both subspecies

- Sympatric distribution in Arizona and Alberta
Minus 9 Mile

Vasey’s Paradise

Kanab, Utah

Niobrara 

Kanab 

Utah/Arizona,

Distribution

NOT sympatric

btwn subspecies

Upper Elves Chasm 

Indian Garden 
Previous Molecular Studies

(Miller et al.)

• AFLP

– 3 Lakes, Indian Garden

• High diversity

– Vasey’s, -9 mile

• Low diversity

• mtDNA CoxI and Cytb

– All equally different taxa?

– What taxonomic level?

More research is needed!

KAS

KAS

NAS

NAS

KAS

KAS KAS

NAS

NAS

NAS

KAS

NAS
Current Study

• Molecular Methods

– mtDNA sequencing - CoxI, Cytb (Mark Miller, USU)

– Nuclear DNA - SNPs, genes, STR (Culver, Herrmann)

• Morphological and Anatomical Methods

(Barry Roth)

– Shell characteristics

– Reproductive characteristics

• Management Implications

– River hydro dynamics?

– What taxa occur in the Grand Canyon?
Sampling

(Jeff Sorenson and 

Dan Cox, AGFD)

• AZ and Utah sampled

• 11 sampling locations

• 15-25 individs./pop.

• KAS type locality; 

Greens

• “Named” KAS at 4 

locations
Molecular Markers Overview

• Mitochondrial DNA (extra- nuclear)

– Medium to high resolution marker (one single marker)

– Used to resolve species, subspecies, populations, gene flow

– Genes and non-coding d-loop (control region)

• Autosomal single copy DNA (nuclear)

– Low resolution markers (many independent)

– Used to resolve species or higher order

– Genes, non-coding (DNA sequence or SNP)

• Autosomal microsatellite STR DNA (nuclear)

– High resolution markers (many independent)

– Highly polymorphic (repetitive) motifs [ATATA]....[GCGCG]

– Used for individual ID, relatedness, gene flow, populations 

subdivision, subspecies-level resolution

– Non coding (Fragment length polymorphism)
Nuclear DNA Marker Development

• Nuclear genes

– Tested 6 genes from mollusc literature

– 2 produced PCR product

• SNP development in species with no genome information

– Genomic library

– Sequenced 150 random clones (65kb), design primers

• Most primers did not amplify across populations (species/subspecies)

• Those that did amplify (conserved region), most showed no variability

– 2 regions found with polymorphic sites

• 356 - 25 variable (15 parsimony informative) sites

• 458 - 36 variable (32 parsimony informative) sites, and 5 indels

• STR development

– Genomic library enriched for simple tandem repeat elements

– Sequenced 100 random clones, design primers

• Most primers did not amplify across populations 

• 1 region, KS6511, amplified with 23 polymorphic alleles
DNA Methods Summary

• Mitochondrial DNA

– DNA sequence for Cox1 (366 bp) and CytB (510 bp)

• Nuclear Genes

– 2 polymorphic genes

• S7 (486 bp)

• ITS (625 bp)

• Nuclear SNPs

– 2 polymorphic regions out of 150 clones

• 356 - 25 variable sites

• 458 - 36 variable sites, and 5 indels

• Nuclear STR

– 1 polymorphic regions out of 100 clones

• KS6511 - 23 polymorphic alleles
Results - Oxyloma ingroup

Outgroups of Catinella, Succinea

And Neosuccinea
Mitochondrial DNA phylogeny

CoxI (366bp) & Cytb (510bp)

– KAS not distinct taxa

– One Vasey’s indiv. has LF 

haplotype in Cytb gene

• Short distance dispersal?

– Shallow clades

• Bottlenecks?

• Genetic Drift?

• Gene flow?

– Greens individuals with 

very long branch length
Nuclear DNA S7 gene 

phylogeny S7(486bp)

•KAS not distinct taxa

•“Mixed lineages”

many lineages 

in most geographic

locations
Nuclear DNA gene 

phylogeny ITS (625bp)

•KAS not distinct taxa

•“Mixed lineages”

many lineages 

in most geographic

locations
Nuclear DNA clone 

phylogeny 356(199bp)

• KAS not distinct

• Mixed lineages
Nuclear DNA clone

phylogeny 458(528bp)

• KAS not distinct

• Mixed lineages
STR Alleles in 

Oxyloma

Sample/Allele 10 11 15 18 19 20 22 24 25 26 27 28 30 32 35 36 39 43 45 46 47 48 53

2_1

2_2

2_5

2_11

2_12

2_16

2_19

2_23

3_12

3_21

3_25

A3

A7

4_1

4_2

4_8

4_9

4_12

4_13

4_14

4_16

4_17

4_18

4_19

4_21

C2

C4

C5

C6

C7

C8

C9

D4

D9

5_5

5_6

5_7

5_8

5_9

5_10

5_11

5_12

5_13

5_14

5_15

5_16

5_17

5_18

5_19

5_20

5_21

5_22

5_24

5_25

6_5

6_13

6_17

6_19

6_20

6_21

6_23

6_24

6_25

7_3

7_4

7_5

7_6

7_7

7_8

7_9

7_10

7_11

7_12

7_13

7_14

7_15

7_16

7_17

7_18

7_19

7_20

8_9

8_10

8_12

8_18

9_1

9_2

9_3

9_4

9_5

9_6

9_7

9_8

9_9

9_10

9_11

9_12

9_13

9_14

9_15

9_16

9_17

9_18

9_19

9_20

10_18 Pass Creek

11_2

11_4

12_4

12_8

12_10

12_11

12_12

12_14

12_15

12_16

9

65

102

103

162

178

224

229

285

315

326

351

405

Escalante River Northern Samples Lee's Ferry

Indian Gardens Vasey's Paradise

Elve's Chasm The Greens Glendale Highway Centry Creek

Seveir River Three Lakes

Panguitch Creek

-Canada has full range of alleles

-Arizona and Utah populations 

mirror alleles found in Canada

In phylogeny, the Greens outliers 

clustered with Oxyloma samples 

from Canada

Alleles.......................
AFLP Bayesian STRUCTURE analysis

supplement for STR data

K = 9 genetic clusters (Pass Crk and Indian gardens are 

not distinguishable)
Morphology and Anatomy

• Morphology

– Used mantle digital images (more reliable method than 

measurements from the small and fragile shells themselves)

– Estimated extent and pattern of black pigment – an apparently 

significant character and possibly a useful field mark

– Devised set of standard measurements to be taken from shell 

images

– Large number of shell characters examined

• Anatomy

– Used reproductive characters less susceptible to the effects of 

age, preservation, and individual variation

– Large number of anatomical characters examined

• No significant differences among populations
PCA for Oxyloma samples for 

second and third components
Conclusions

• Extensive gene flow observed among Oxyloma

populations in AZ and UT

• One taxonomic group (species-level) indicated

• Evidence for short and long distance dispersal

• How to explain dispersal?

– Short distance may be river flow mediated (Lee’s - Vasey’s)

– Long distance may be bird transport (highly divergent lineages 

found in same geographic location)

• Unique dispersal strategy

– Oxyloma are hermaphrodites

– Oxyloma are capable of self fertilization

– One immigrant can found a population

– Genetic bottlenecks (and drift) are probably a common 

occurence
Additional support - 458 Network

• 3 Greens “outliers”

– clustered with 

Canada

• 2 - 4 “hubs”

– Where more 

lineage mixing 

occurred
Conservation Implications

• Populations should continue to be monitored 

for stability/decline in numbers

• Important to protect habitat to allow dynamic 

process of colonizations to continue

• Managed as one species group

– Metapop. with historical extinctions/

recolonizations?

– Ongoing colonizations?
Implications for ESA

• Genetics Policy states:

– Genetic differences must be addressed during the listing 

process to determine the taxa being listed

– No further revision of taxa is allowable, due to genetic data

• Under this policy of no revisions - only delisting or relisting as different taxa is allowable.
Questions?
Museum samples from Canada

• Oxyloma haydeni (long distance dispersal)

• Oxyloma retusa (outgroup)

• Oxyloma nuttalina (outgroup)
Mitochondrial DNA, all Canada 

samples added to current samples

• Supports theory of 

bird transport

– Canada haydeni

• Greens

• Outgroup species are 

ingroups

– Oxyloma retusa

• Greens, Escalante

– Oxyloma nuttalina

• Greens, 3 Lakes