The document discusses the conservation genetics of the blueface darter, a rare fish in Northwest Alabama, highlighting its genetic differentiation from the bandfin darter and the effects of isolation on genetic variation across populations. Genetic analyses reveal that blueface darters have a 23% reduction in allelic diversity compared to bandfin darters, but this difference is not statistically significant. The study emphasizes the need for conservation efforts for this species, particularly in isolated populations such as those in Hubbard and Bear Creeks.

1. Conservation Genetics of the Blueface Darter
(Etheostoma sp. cf. zonistium), a rare
undescribed fish in northwest Alabama
Kenny Jones
Brook L. Fluker
Bernard R. Kuhajda

2. Distribution and Characteristics
Bandfin Darter, Etheostoma zonistium
Blueface Darter, Etheostoma cf. zonistium
Fish art: Joseph R. Tomelleri
AL
MS
TN
KY
Gulf Coastal
Plain
Cumberland
Plateau
Bailey and Etnier (1988); Kuhajda and Mayden (upubl. data)
Blueface Darter description is forthcoming (Kuhajda and Mayden).
Once formally recognized, conservation status will need to be determined.

3. Previous Work
 Phylogenetic analysis of
cytochrome b (mtDNA) data
identified Blueface Darters
as a monophyletic group
 Sister to E. zonistium sp.
group
 Approximately 3% sequence
divergence
Kuhajda and Fluker (2009)

4. An Isolated Distribution
 Natural isolation: The
Hubbard Creek population is
potentially isolated from
other populations via natural
isolation.
 Reservoir-induced isolation:
The Upper Bear Creek
systems are possibly
isolated due to the
construction of the Upper
Bear Cr. Reservoir (1978).
Extant
Possibly extirpated
Kuhajda and Mayden (2002)
Tennessee River
Drainage
Black Warrior River
Drainage (Mobile Basin)
Upper Bear
Cr. Reservoir
10.2 km
5.4 km

5. Questions
I. Is there significant genetic structure between Blueface and
Bandfin Darters?
II. Does the Blueface Darter have reduced genetic variation
compared to the Bandfin?
III. Is there significant genetic structure within the Blueface
Darter across the Tennessee/Black Warrior divide?
IV. Does the Black Warrior population of Blueface Darter have
reduced genetic variation compared to the Tennessee River
drainage populations?

6. Methods: Sample collection
Upper Bear
Cr. Reservoir
2
AL
MS
TN
KY
Tennessee River
Drainage
Black Warrior River
Drainage (Mobile Basin)
1
3
5
Upper Bear
Cr. Reservoir 2
6
47
8
9
Kinlock Falls, Hubbard Creek
 20-30 individuals per site (seine)
 Fin-clipped (95% EtOH)
 Photo-vouchered or formalin preserved
specimens
 Extracted DNA using Qiagen DNeasy Kit

7. Methods: Microsatellite DNA analyses
 Genotyped 187 individuals for 8
microsatellite DNA loci:
 Esc132b, Esc68, Esc18, Esc26b (Gabel
et al. 2008)
 Eca46, Eca48, Eca49 (Tonnis 2006)
 EosD107 (Switzer et al. 2008)
 Estimated genetic structure using
AMOVA (ARLEQUIN) and
STRUCTURE
 Estimated genetic variation via
allelic diversity and heterozygosity
expected (ARLEQUIN)
 Evaluated differences in genetic
variation using non-parametric
statistics (Kruskal-Wallis and Mann-
Whitney U tests)
Structure v2.3.4

8. Results
Upper Bear
Cr. Reservoir
2
AL
MS
TN
KY
Tennessee River
Drainage
Black Warrior River
Drainage (Mobile Basin)
1
3
4
Upper Bear
Cr. Reservoir 2
6
57
8
9
Results
1 63 4
Blueface Darter Bandfin Darter
2 5 7 8 9

9.  FCT= 0.02, and an overall P-value of 0.03
1 2 3 4 5 6 7 8 9
Hubbard
Above --
Hubbard
Below 0.003* --
Bear Cr.
(cf. zon) 0.098 0.066 --
Little Bear
Cr. 0.146 0.109 0.060 --
Turkey Cr. 0.079 0.073 0.022* 0.109 --
Big Sandy 0.070 0.041 0.033 0.067 0.064 --
Birdsong 0.073 0.056 0.034 0.076 0.070 0.023 --
Hatchie 0.247 0.228 0.205 0.234 0.247 0.176 0.155 --
Bear Cr. (E.
zon) 0.168 0.140 0.119 0.162 0.158 0.090 0.104 0.246 --
Results cont.

10. Blueface Darters
Bandfin Darters
Results
0
5
10
15
20
25
Blueface darters show a 23% reduction in
genetic variation compared to Bandfin, but not
significant (P = 0.084)
MeanAllelesPerLocus

11. Results
Upper Bear
Cr. Reservoir
2
AL
MS
TN
KY
Tennessee River
Drainage
Black Warrior River
Drainage (Mobile Basin)
1
3
4
Upper Bear
Cr. Reservoir 2
6
57
8
9
Results
1 63 4
Blueface Darter Bandfin Darter
2 5 7 8 9

12. 1 2 3 4 5
Hubbard Above --
Hubbard Below 0.003* --
Bear Cr. (cf. zon) 0.098 0.066 --
Little Bear Cr. 0.146 0.109 0.060 --
Turkey Cr. 0.079 0.073 0.022* 0.109 --
Results cont.

13. MeanAllelesPerLocus
Genetic diversity differed between Blueface populations
(P=0.018). Turkey Creek lower than all others.
0
5
10
15
20
25
Hubbard Above Hubbard Below Bear Cr. Little Bear Cr Turkey Cr.
Results

14. Conclusions
Blueface and Bandfin darters are differentiated with both microsatellite
and mtDNA. Species validity is supported with genetic data, although
microsatellite data indicate retention of ancestral alleles in the Bear
Creek population.
Overall, Blueface Darters do not have lower have lower genetic variation
compared to the Bandfin Darter, even though allelic diversity was 23%
percent lower and was a non-significant value.
Populations of the Blueface darter are isolated across the
Tennessee/Black Warrior divide.
Possible reservoir fragmentation effect seen in Little Bear Creek, i.e.
significant genetic structure and lower genetic variation compared to
Bear Creek.

15. Conservation implications
 After being formally described,
the results generated from this
study can inform state and
federal agencies about the
conservation needs of the
Blueface darter
 Conservation units
 Hubbard Creek
 Bear Creek system
 Conservation priorities
1. Turkey Creek
2. Hubbard Creek
3. Little Bear Creek
4. Bear Creek (source for PTRA)

16. Acknowledgements
 Funding
 Arkansas State University Faculty
Research Award Committee (FRAC).
 Tennessee Aquarium Conservation
Institute.
 Field and Lab Assistance
 Alexandra Hook
 Taylor Lee
 Brittany McCall
 Dave Neely
 Collecting Permits
 Alabama Dept. of Conservation and
Natural Resources
 Tennessee Wildlife Resources Agency

17. Closing Statement