A Mitochondrial Phylogenetic Analysis of Sculpin (genus Cottus)
in Montana based on the NADH dehydrogenase subunit 4 gene.
Faculty Mentor: R. Paul Evans, Micro and Molecular Biology
The purpose of this project was to investigate the distribution and phylogenetic status of Cottus
(sculpins) species populations in Montana relative to other species in western North America
using the NADH dehydrogenase subunit 4 (ND4) gene in mitochondrial DNA (mtDNA). This
study of the ND4 gene in sculpin could possibly provide additional insight into the historical
timing of fish invasion events and provide the phylogenetic status of species in the genus Cottus.
Although the distribution of aquatic vertebrates in western North America has been interpreted
relative to late Pleistocene events, genetic studies of cutthroat trout have not supported that
mechanism and have led to hypotheses of much earlier dispersal events. This study was initially
meant to test the possibility of similar studies as trout to decipher hydrogeography, as well as
decipher relationships of sculpin species located in Eastern Montana
The preliminary data I collected for this project indicate that sculpin DNA isolation using a
chaotropic salt treatment is best performed on pectoral fin tissue and not muscle or liver tissue.
The primers for polymerase chain reaction amplification of the ND4 gene using the isolated total
sculpin DNA are functional using the same PCR protocol I used this last year for genetic
analyses of cutthroat trout a year previous. PCR was performed in 50-µL reaction mixtures
consisting of DNA template (100 ng), deoxyribonucleotides (0.125 mM each), primers (10
pmoles each), buffer (10 mM Tris-HCl; 1.5 mM MgCl2, 25 mM KCl), and Taq polymerase (0.5
units) as follows: 4 min at 95°C; 34 cycles of 20 sec at 94°C, 30 sec at 47°C, and 1.5 min at
72°C; and 7 min at 72°C. Purification of the PCR product has been performed using the
QBiogene Gene Clean III protocol (QBiogene, Carlsbad, California). Cycle sequencing has been
performed using the ABI Big Dye terminator protocol (Applied Biosystems, Foster City,
California). Samples were submitted to the Brigham Young University DNA Sequencing Center
and sequenced on an ABI 377 automated sequencer. Sequences have been edited and aligned
with Sequencher 3.0 software (Gene Codes Corporation, Ann Arbor, Michigan). I did not
anticipate the presence of insertions or deletions in the coding region of this mitochondrial gene
so the alignment has been unequivocal.
Eight Montana cottid populations from both sides of the continental divide, as well as 18
populations from other states, have been examined using the DNA sequence of the NADH
dehydrogenase subunit 4 (ND4) gene in mitochondrial DNA (mt DNA). Species from the 18
populations outside of Montana include: Cottus bairdi (WI & UT), C. beldingi (UT & NV), C.
carolinae (AL), C. cognatus (WI), C. confuses (ID), C. rhotheus (OR), and Leptocottus armatus
(WA). Of the eight populations in Montana, five are west, and three are east of the continental
divide. Two of the five western populations from lower Libby Creek and Pleasant Valley Fisher
River, are C. rhotheus. The three eastern sculpin populations, the putative C. bondi, form a
distinct clade basal to C. rhotheus. The remaining three western populations from Pipe Creek,
West Fork Yaak River, and upper Libby Creek appear to be basal to the C. bondi-C. rhotheus
group (see figure 1). Thus far, we have determined sculpin populations in Lower Libby and
Pleasant Valley Fisher located in western Montana, to be C. rhotheus, and have a closer
relationship to their Oregon counterparts than streams as nearby as “Upper” Libby, which exhibit
influence from the bairdi species. Those species located in central Montana exhibit a closer
relationship to rhotheus than bairdi. The challenge from these results had been the inability to
decipher the species of those from Pipe Creek, West Fork Yaak River, and upper Libby Creek.
These have been mistaken for C. rhotheus for a number of years (Hendricks 1997), yet the data I
have gathered shows a distinct difference between Pipe, WF Yaak, upper Libby versus C.
rhotheus as far back as the initial restriction enzyme trial (figure 2). This trial was a precursor to
sequencing to get an idea of possible hypotheses. Although this study wasn’t able to find the
exact species of Pipe, WF Yaak, and Upper Libby, it did show they are distinctly separate and
basal from C. rhotheus and the putative C. bondi, and yet to be identified as a separate species.
Future studies based off the information gained here should be able to determine whether it is a
known, or new species.
Using earlier data from Jared Crowley’s Masters Degree thesis, I was able to expand the
accuracy of the results. My mentor Dr. R. Paul Evans was then able to combine my data, and
further develop the project for presentation in the 2005 American Fisheries Society annual
meeting in Anchorage, Alaska. His results were to the initial clades, and increased the validity
through larger sampling size (see figure 3). Further analyses could include more populations
from Montana and elsewhere. Since the results from the clade analysis aren’t consistent across
the board, (except the shown relationship) one can conclude the ND4 gene is not conserved
enough, and has changed too rapidly throughout time to sufficiently determine all relationships
by itself. Also, sequences other than ND4, including cytochrome B, D-loop, or other more
conserved regions will aid in determining the relationship of the
Montana sculpins to outlying areas.
Figure 1—shows the
preliminary clade with
the unknown Pipe,
WF Yaak, & Upper
Libby systems sculpin
not the same as C.
RFLPs from which the
hypothesis was made.
Figure 3—Final clade
analysis with the same
relationship as in