The Evolutionary Analysis of Cytochrome P450 in Aquatic Fish Species
Using Phylogenetic Analysis
Devin Radel
Jett Peng
This analysis was co-conducted throughout the entire process.
Introduction:
For our research we decided to examine the evolutionary relationships between different
aquatic fish species from the analyzation of cytochrome p450. Cytochrome p450 is involved with
the metabolism of foreign compounds like pollutants. We are examining different aquatic species
from different habitats (lakes, freshwater streams, the ocean and its coastal environments) to
infer phylogenetic relationships of cytochrome p450 among those species. This research can lead
to further examination and monitoring of these waterways to examine possible mutations in the
species present there. By doing this we may find areas of concern in aquatic environments to
infer possible pollution events, since pollution itself cannot be directly inferred from the obtained
data.
Cytochrome P450 (CYP) is an enzyme that is responsible for the metabolism of foreign
compounds or xenobiotics (environmental contaminants) (Harskamp). An environmental
contaminant can be any foreign substance that is introduced into an environment by pollution,
run-off, or any other anthropogenic means. Environmental pollutants can harm organisms in
many different ways; the most common is through hormonal responses, which are more
commonly referred to as Endocrine Disruptors (Kloas). These toxins (Endocrine Disruptors) are
harmful to aquatic organisms due to their ability to inhibit cytochrome P450 causing the
organism to be susceptible to the effects of the toxin (Kloas). Due to this great threat organisms
in aquatic environments must adapt by means of mutation or selection to ensure their survival.
In a previous study conducted by John Stegeman and Pamela Kloepper-Sams entitled
“Cytochrome P-450 Isozymes and Monooxygenase Activity in Aquatic Animals” researchers
examined the mechanisms and functionality of cytochrome P450 when introduced to different
environmental toxins (Steggman). It was claimed that cytochrome P450 activity was most
observable in fish species. In this study it was found that cytochrome P450 responds similarly to
these toxins in all examined species (Rainbow Trout, Scup, and Cod) (Steggman). As suggested
above, this research examines the biochemistry behind the mechanism of cytochrome P450 to
determine its function in an organism with relation to multiple terrestrial and aquatic species
(Steggman). Our research will be a supplement to this by providing evolutionary and
phylogenetic analysis for aquatic fish species.
Since previous research determined that the functionality of cytochrome P450 is similar
among multiple aquatic species we decided to compare the phylogenetic relationships between
aquatic fish species that live in different ecological habitats and geographical regions. Multiple
research studies have compared cytochrome P450 among aquatic, terrestrial, and microscopic
organisms, but further research has been hinted in all studies for the need of evolutionary
analysis for this specific enzyme. We believe that our results will enable us to determine any
evolutionary process that may have arisen in cytochrome P450.
Materials and Methods:
We collected 13 completed cytochrome P450 1A mRNA sequences from GenBank. The
Yellowhead Catfish was first examined for the sequence then BLAST was used to find other
highly similar and more dissimilar sequences. Lengths of each sequence were between 13002700 base pairs long. The following sequences were obtained:
Table 1:
Species
Accession
Origin
Length (BP)
Author
Yellowhead Catfish
EF584508
Korea
2757
Kim,J.H., Raisuddin,S.,
Ki,J.S., Lee,J.S. and
Han,K.N.
Freshwater Minnow
JN648712
Korea
1536
Kim,J.-H.
Rainbow Trout
AF059711
United States
2678
Carvan,M.J. III,
Ponomareva,L.V.,
Solis,W.A., Matlib,R.S.,
Puga,A. and Nebert,D.W.
Atlantic Salmon
AF059711
Canada
1391
Leong,J., von
Schalburg,K., Cooper,G.,
Moore,R.,
Holt,R.,Davidson,W.S.
and Koop,B.F.
Brook Trout
AF539414
United States
1391
Rees,C.B. and Li,W.
Zebrafish
AF210727
United States
1530
Buhler,D.R.
Lake Trout
AF539415
United States
1391
Rees,C.B. and Li,W.
Three-Spined S.
HQ202281
Sweden
1387
Gao,K., Brandt,I.,
Goldstone,J.V. and
Jonsson,M.E.
Spotted Gar
XM_006628888
United States
1414
Predicted Sequence: Bestplaced RefSeq; Gnomon
Armoured Catfish
HM043798
South America
1703
Parente,T.E.M.,
Rebelo,M.F.,
Woodin,B.R. and
Stegeman,J.J.
Cory Catfish
HM043797
South America
2643
Parente,T.E.M.,
Rebelo,M.F.,
Woodin,B.R. and
Stegeman,J.J.
Turbot
AJ310694
United Kingdom
1319
Craft, J. A.
Yellow Croaker
GQ281041
China
2598
Qian,Y., Qian,L. and
Tong,L.
Table 1 displays the species, accession number, origin, length, and author of the sequences gathered from GenBank.
To examine this data ClustalW was then used to trim and align the sequences for the
longest gaps. The program MEGA6.06 was then used to produce phylogenetic trees. Neighbor
Joining and Maximum Parsimony trees were created with Kimura 2-parameter distance model.
1000 bootstrap replicas were also ran during the creation of the phylogenetic trees.
To create each phylogenetic tree there is a need for the estimation of evolutionary
distances using the pairwise distance method (Figure 1). This method compares the sequences of
the species we decided to examine by calculating the percent difference between their
nucleotides (Li).
The Bootstrap method of phylogeny was used to determine trees that agree on an overall
consensus of how the tree should be arranged. In other words, because the tree was run 1000
times (due to the 1000 replicas) the given tree is arranged due to reliability. Reliability is the
probability that the members of a given clade are indeed member of that clade (Li).
The Neighbor Joining method for phylogeny minimizes the overall branch length for a
tree (Li). This method takes into account the varying rate of nucleotide substitution. One
potential problem with this method is that the tree is subject to the accuracy of the sequences
obtained due to potential statistical errors (Li). The Maximum Parsimony method for phylogeny
calculates and finds a tree that requires the fewest number of changes. This is done by
distinguishing between informative and uninformative sites, which gives us important
information about taxa (Li). A site is only informative if there are only two different kinds of
nucleotides present there.
Kimura’s 2-parameter distance model was used as opposed to Jukes-Cantor distance
model due to its recognition between transitional and transversional substitution rates (Li). Since
transitions are more common than transversions it was decided that Kimura’s model would
compensate for this bias. Also this decision was made due to the varying types of aquatic fish
species present in our analysis.
Results:
From the data obtained from MEGA we observed consistent results of members within a
clade for each tree that was created from the pairwise distance matrix (Figure 1). For each
method used (Neighbor Joining and Maximum Parsimony) bootstrap trees were created as well
(Figures 3 and 5 respectively). The bootstrap tree when compared to the Neighbor Joining tree
did not differ from the parent tree in clade and member groupings, but only in branch lengths and
organization to allow for better understanding. This is not the case for Maximum Parsimony.
For the Kimura Neighbor Joining tree below (Figure 2) we observed a 100 percent
agreement value of the freshwater minnow and the Zebrafish clade. Moving up the tree we
observed a 96 percent agreement value for the clade containing the Armored Catfish, Cory
Catfish, and Yellowhead Catfish. Within this clade there was a distinguishable sub-clade present
that obtained a 98 percent agreement value for the Armored Catfish and Cory Catfish species.
Above this clade a 99 percent agreement value was calculated for the clade containing Spotted
Gar, Yellow Croaker, Turbot, Three-Spined Sickleback, Brook Trout, Atlantic Salmon, Lake
Trout, and Rainbow Trout. Within this clade there were two 100 percent agreement values for
the clades containing Turbot and Yellow Croaker as well as the clade containing Brook Trout,
Lake Trout, Rainbow Trout, and Atlantic Salmon. As mentioned above, the bootstrap tree of
phylogeny for the Neighbor Joining method (Figure 3) contained the exact same results as it’s
parent tree.
For the Maximum Parsimony tree below (Figure 4) we observed the Three-Spined
Sickleback to be the most diverged from all other species because of its distinctively separate
clade. Moving up the tree we observed a 100 percent agreement value for Turbot and Yellow
Croaker clade. Above this clade we obtained a 63 percent agreement value for the remaining
species clades and the Spotted Gar. Within this clade we observed three significant clades. The
first one obtaining a 100 percent agreement value clade for the Atlantic Salmon, Brook Trout,
Rainbow Trout, and Lake Trout. The second clade was found to have a 98 percent agreement
value for the Freshwater Minnow and Zebrafish. The final clade had a 95 percent agreement
value for Armored Catfish, Cory Catfish, and Yellowhead Catfish. Noticeable differences were
observed in the bootstrap tree for Maximum Parsimony for the clade containing the Atlantic
Salmon, Brook Trout, Rainbow Trout, and Lake Trout. This one clade from the Maximum
Parsimony was made into two separate clades when calculated in bootstrap.
Figure 1:
Figure 1 contains the pairwise distance method for Kimura’s 2-parameter model of the species examined obtained from MEGA.
Following each species is a description of their typical habitat (FWS = fresh water stream) and the geographical location the
sample was obtained from.
Figure 2:
Figure 2 contains the Kimura’s 2-parameter model for the Neighbor Joining method of phylogeny. This tree was obtained from
MEGA. Following each species is a description of their typical habitat (FWS = fresh water stream) and the geographical location
the sample was obtained from.
Figure 3:
Figure 3 contains the Kimura 2-parameter model Neighbor Joining Bootstrap method of phylogeny. This free was obtained from
MEGA. Following each species is a description of their typical habitat (FWS = fresh water stream) and the geographical location
the sample was obtained from.
Figure 4:
Figure 4 contains the Maximum Parsimony method of phylogeny. This tree was obtained from MEGA. Following each species is
a description of their typical habitat (FWS = fresh water stream) and the geographical location the sample was obtained from.
Figure 5:
Figure 5 contains the Maximum Parsimony Bootstrap method of phylogeny.This tree was obtained from MEGA. Following each
species is a description of their typical habitat (FWS = fresh water stream) and the geographical location the sample was obtained
from.
Discussion:
Through phylogenetic analysis we observed that the Rainbow Trout, Lake Trout, Brook
Trout, and Atlantic Salmon are closely related. From the Neighbor Joining (Figure 2) method
these species were consistently grouped together in the same clade for both the parent and
bootstrap tree. However, a noticeable change occurred between Maximum Parsimony and its
bootstrap tree. This method grouped these species in separate, distinct clades. This is an
indication that these species are not closely related which disagrees with previous research that
was conducted by Allendorf and Utter stating that these species were all apart of the Salmonidae
family (Allendorf). The reasoning why we observed different results from each tree is due to the
methods used in each process to make a tree (Li). We found this strange due to the fact that the
Neighbor Joining method (and its bootstrap tree) and Maximum Parsimony method (just the
parent tree) agreed on this clade with a 100 percent agreement value.
The next clade we noticed consisted of the Freshwater Minnow and the Zebrafish. These
species were also grouped consistently throughout each tree. Agreement values for Neighbor
Joining and Maximum Parsimony for this clade were 100 and 98 percent, respectively. These
species are known to a part of the Cyprinidae family.
The last clade that contained the same family of fish was the clade with the Yellowhead
Catfish, Armored Catfish, and Cory Catfish. There was a 96 percent agreement value in the
Neighbor Joining tree and a 95 percent agreement value in the Maximum Parsimony tree. These
species are known to be part of the Siluriforms family, which is consistent with our findings.
We observed significant findings in the Turbot and the Yellow Croaker species, which
consistently formed a clade together. The agreement value of their clades (in both methods) was
found to be 100 percent. We believe that this finding is extraordinary due to the two species
sharing no relation between each other except ecological habitat. Although future research is
needed to confirm our assumptions, we believe that cytochrome P450 co-evolved within the two
species.
The remaining two species (Spotted Gar and Three-Spined Sickelback) we believe are
classified as out-groups for this research. For all phylogenetic trees, these two species were
consistently grouped in a branch of their own occasionally placed in relation to the clade
containing the Turbot and Yellow Croaker.
In conclusion, cytochrome P450 can be used as a biomarker. This is due to its important
nature in metabolizing toxins and other foreign compounds. The findings obtained from this
evolutionary and phylogenetic analysis can be used as a stepping-stone for future research
regarding the determination of possible pollution events. This is due to inferences made from the
geographical and ecological habitats of individual fish species and their groupings
(phylogenetically) with other species. Research would involve monitoring of these waterways,
and its species, to classify when cytochrome P450 diverged.
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