Journal of Great Lakes Research 35 (2009) 163–167
Genetic variation at the mtDNA ND-1 locus among North American wild and hatchery
brown trout (Salmo trutta)
Shelby S. Johnson1*, Mark R. Luttenton2,3 and Alexey G. Nikitin1,2
1
Cell and Molecular Biology Program, Grand Valley State University, Allendale, Michigan
49401, USA
2
Biology Department, Grand Valley State University, Allendale, Michigan 49401, USA
3
Annis Water Resources Institute, Grand Valley State University, Muskegon, Michigan 49441,
USA
Running title: Mitochondrial polymorphism in brown trout
*Correspondence:
Shelby Johnson
Biology Department
118 Padnos Hall of Science
Grand Valley State University
1 Campus Drive
Allendale, MI 49401
E-mail: johnshel@gvsu.edu
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Journal of Great Lakes Research 35 (2009) 163–167
Abstract
Despite extensive knowledge of mitochondrial DNA (mtDNA) variation in European brown
trout (Salmo trutta) populations, little is known about their nucleotide sequence variation in
North America. The objective of this study was to quantify single nucleotide polymorphisms
(SNPs) at the ND-1 mtDNA locus of 62 brown trout from hatcheries in Michigan and Wisconsin
as well as Michigan streams and Lake Michigan. We identified 25 SNPs that characterized nine
distinct mtDNA haplotypes in the Wild Rose, Gilchrist and Seeforellen brown trout strains.
Although most SNPs were represented by synonymous nucleotide substitutions, three individuals
of the Seeforellen strain had non-synonymous nucleotide changes. MtDNA haplotypes identified
in North American brown trout in this study showed nucleotide similarity at the ND-1 locus to
brown trout from northern Europe.
Index words: brown trout, Wild Rose, Gilchrist, Seeforellen, mtDNA, single nucleotide
polymorphism, ND-1 locus, population genetics
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Journal of Great Lakes Research 35 (2009) 163–167
Introduction
The brown trout (Salmo trutta) is native to Europe, parts of Asia, and North Africa; and
introductions to North America in the late 1800s and early 1900s expanded its range. It first was
imported to the USA in 1883 from Germany and stocked in the Pere Marquette River, Michigan,
by the U.S. Fish Commission (Westerman 1977), then subsequently introduced into nearly every
state in the USA from several European sources (MacCrimmon 1968). Across its native and
introduced ranges, the brown trout exhibits considerable life history variation (Bernatchez et al.
1992). A population may have both anadromous (migratory) and resident forms, with migration
influenced by genetic and environmental factors (Cucherousset et al. 2005). Adult migrant and
resident forms may appear similar morphologically and may interbreed. However, brown trout
often display significant variation in morphology.
Early studies characterized brown trout populations on the basis of morphology and life
history tactics, leading to the proposal of numerous species names for the various forms (Laikre
1999). However, many differences between their forms can be influenced by environmental and
phenotypic plasticity; therefore, delineation of populations for taxonomic as well as for
conservation and management purposes should be based on genetic differences (Laikre 1999).
In recent years, mtDNA has been used to study the genetic relatedness of brown trout forms,
including within and among natural and hatchery-reared populations (Apostolidis et al. 1996,
1997, Hanson and Loesche 1996, Ruzzante et al. 2004).
For management purposes, different strains of brown trout are maintained by fish
hatcheries (Kincaid 1981). For example, the State of Michigan currently maintains and stocks
three hatchery strains; Gilchrist, Seeforellen, and Wild Rose (MDNR 2007). Brown trout
hatcheries in the US have considerable interest in maintaining and improving the performance of
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Journal of Great Lakes Research 35 (2009) 163–167
hatchery strains (see Avery et al. 2001), thus knowledge of the genetic composition of hatchery
and wild brown trout strains is of key importance for improving fisheries.
The Wild Rose and Seeforellen strains used to stock Michigan waters, have been
maintained in hatcheries for nearly two decades, are considered domesticated, and have been
used in other hatcheries across the U.S. (Wills 2006). The Seeforellen strain was originally
brought to Michigan in 1989 as eggs from the Caledonia State Fish Hatchery in New York (Dan
Sampson, MDNR, personal communication). Offspring of this original shipment were used to
establish a Seeforellen broodstock from which all subsequent Seeforellen broodstock lots were
developed (D. Sampson, personal communication). The Sefforellen broodstock at Caledonia
were progeny of eggs transferred from the Catskill State Fish Hatchery (New York), which
originally received eggs directly from Germany in 1985. In Germany, the Seeforellen strain was
reported to survive well and grow up to 27 kg in large lake environments, although they grow
more slowly in hatchery conditions in comparison to the other two Michigan strains (D.
Sampson, personal communication). Seeforellen is generally considered to be a lake strain and
is primarily used to stock the Great Lakes (MDNR 2007).
The Wild Rose strain was originally brought to Michigan in 1987 as eggs from the
Wisconsin Wild Rose State Fish Hatchery (WRSFH), where they are believed to have been
established by the mid-20th century from an unknown source (Randal Larson, Wisconsin
Department of Natural Resources, personal communication). This strain has been found to
perform very well in the Oden State Fish Hatchery (OSFH), where they grow quickly and readily
consume artificial foods (D. Sampson, personal communication).
A third hatchery strain, Gilchrist, was derived recently from a naturalized stream
population captured from Gilchrist Creek, Montmorency County, Michigan, whose broodstock
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Journal of Great Lakes Research 35 (2009) 163–167
were first established at the Oden State Fish Hatchery (OSFH) in 1995 (Wills 2005). The origin
of brown trout in Gilchrist Creek is unknown, although it likely resulted from unrecorded
plantings of European brown trout earlier in the 20th century (Wills 2005). In the OSFH, the
Gilchrist strain retains “wild” behavioral characters. It also grows more slowly in the hatchery
than does the Wild Rose strain (D. Sampson, personal communication).
These three hatchery strains of brown trout (Gilchrist, Wild Rose and Seeforellen) are
nearly identical in appearance and are difficult to differentiate phenotypically (Tiano 2005).
Recent analysis of the mtDNA-encoded ND-1 and ND-5/6 subunits of mtDNA NADH
dehydrogenase of Michigan brown trout from the OSFH, Muskegon River (Newaygo Country),
Rogue River (Kent County), and Lake Michigan near Ludington using polymerase chain
reaction-restriction fragment length polymorphism (PCR-RFLP) techniques revealed two
haplotypes for the Gilchrist strain, two for the Wild Rose strain, and two previously unknown
haplotypes in the Rogue River (Tiano et al. 2007). An additional haplotype was found in three
Lake Michigan individuals, which may belong to the Seeforellen strain according to Michigan
Department of Natural Resources (MDNR) stocking records (Tiano 2005). Because the three
strains differ substantially in survival and growth (Wills 2006), further study of their mtDNA
composition was undertaken here to evaluate their genetic basis.
Materials and Methods
Sample Origin and Collection
Samples of Wild Rose, Gilchrist and Seeforellen strain hatchery fry and broodstock used
in this study were obtained from the OSFH, and wild-caught samples were collected from the
Muskegon River, Rogue River, and Lake Michigan (Tiano et al. 2007). Samples of young-of-
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Journal of Great Lakes Research 35 (2009) 163–167
the-year (YOY) of the Wild Rose and Seeforellen strain were obtained from the WRSFH
(Wisconsin) in 2007. We analyzed a total of 62 brown trout (Table 1).
Based on alternative RFLP patterns produced by Alu I and Hae III restriction
endonucleases, three mtDNA haplotypes previously were identified in Michigan brown trout
(Tiano et al. 2007). Further screening of samples with other restriction enzymes, notably Hpa II,
revealed additional polymorphism (Tiano 2005). Thus, representatives of each previously
identified RFLP haplotype were selected for sequencing in this study, along with fish samples
obtained from the OSFH and the WRSFH, whose RFLP haplotypes had not been determined
(Table 1).
DNA Amplification and Sequencing
Genomic DNA was extracted from fin clips using the Qiagen DNeasy Tissue Kit
(Valencia, CA) with final DNA elution in 60µl of distilled water. The ND-1 mtDNA segment,
along with a portion of the 16S rRNA region and the leucine tRNA directly preceding it, was
amplified using an Eppendorf Mastercycler (Westbury, NY) with NADH-dehydrogenase 1
forward and reverse primers described by Nielsen et al. (1998). PCR reactions totaled 50 µl of
1X Thermopol buffer (New England Biolabs (NEB, Ipswich, MA)), 100 pmoles of each primer,
2.5 units of Taq DNA polymerase (NEB), 20 mM each of dATP, dCTP, dGTP, and dTTP
deoxynucleotides, and 2 µl DNA. PCR conditions were: 5 min at 94oC, followed by 30 cycles of
94oC denaturation for 30 sec, 62.4oC annealing for 45 sec, and 2.5 min of 72oC extension, ending
with a termination step of 7 min at 72oC (Tiano et al. 2007). PCR products were separated on a
1% agarose gel with ethidium bromide staining to verify amplification, purified with the Qiagen
MinElute PCR Purification Kit, and outsourced to the University of Michigan Sequencing Core
laboratory (Ann Arbor, MI) for direct DNA sequencing.
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Journal of Great Lakes Research 35 (2009) 163–167
Sequence Analysis
The DNA sequence analysis initially was performed using the forward PCR primer, ND1F, yielding ~1,000 nucleotides. Based on the initial sequence data, a new internal sequencing
primer (5’- AGAAGGGGCCCATGCTTAAGG -3’) was generated for subsequent analyses
using the Primo 3.4 Sequencing Primer Design algorithm from Chang Bioscience
(http://www.changbioscience.com/primo/primoseq.html). All samples also were sequenced using
the ND-1R primer, yielding ~1,100 base pairs analyzed for each sample.
DNAStar Lasergene Sequence Analysis (version 6.0, DNAStar Inc., Madison, WI)
software was used for sequence alignment and identification of SNPs. Sequence alignments for
each designated mtDNA haplotype were submitted to the National Center for Biotechnology
Information (NCBI) sequence database (www.ncbi.nlm.nih.gov, Genbank EU003592-602). A
search of the NCBI database to assess sequence similarity between our samples and other brown
trout ND-1 sequences followed. The search returned several sequences of Danish brown trout
with various degrees of sequence similarity to our samples (Genbank AF117716-19).
Results
Haplotypes and Occurrence in Populations
A total of eight haplotypes and 25 SNPs within the ND-1 region were detected among the
62 brown trout samples sequenced in this study. The Seeforellen strain had four haplotypes
(Genbank EU 003598-003601). All but one SNP (2925; in the first codon position,
characterizing haplotype G) of the synonymous polymorphic sites occurred in the third codon
position (Table 2). Haplotype E (GenBank 003598) was found in hatchery fish from Michigan
and Wisconsin, as well as Lake Michigan and a single individual from the Rogue River. This
haplotype shared identical SNPs with a Danish brown trout (Denmark Gudenaa 2, GenBank
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Journal of Great Lakes Research 35 (2009) 163–167
AF117716). Haplotype F (GenBank EU003599) appeared unique to Michigan broodstock and
appeared to be more closely related to the Gilchrist haplotypes than to the other three haplotypes
found in the Seeforellen strain. Haplotype G (GenBank 003600) was found in individuals from
both Michigan and Wisconsin hatcheries and contained 15 SNPs, of which two were nonsynonymous substitutions (SNP 3315 in the first codon position and 3736 in the second; Table
2). Both substitutions occurred in the extramembrane loop portions of the ND-1 gene (data not
shown). SNP 3315 replaced serine with glycine and SNP 3736 replaced serine with asparagine.
Haplotype G was similar in sequence with two Danish brown trout strains, differing by two
SNPs from Denmark Romania isolate D1(GenBank AF117720) and by seven from the Danish
D2 Romania isolate (GenBank AF117721); which all shared the characteristic non-synonymous
SNPs. Haplotype H (GenBank EU003601) was found only in Wisconsin hatchery fish of the
Seeforellen strain. A single individual from the Rogue River (haplotype I) had a unique
haplotype (GenBank EU003597) that could not be assigned to any of the hatchery strains, but
was nearly identical to a Danish brown trout sample from the GenBank database (Denmark
Gudenaa 7, GenBank AF117718).
Wild Rose fish from both Michigan and Wisconsin hatcheries shared a single Wild Rosespecific haplotype (A; GenBank EU003596), confirming the previous RFLP-based observations
of the strain’s haplotype sequence uniformity assessed on 161 samples of Wild Rose (Tiano,
2005; Tiano et al. 2007). The Gilchrist hatchery strain had a common haplotype (B; GenBank
EU003592) which also was found in Wild Rose strain samples from both hatcheries and was
common in wild samples from the Rogue River, Muskegon River, and Lake Michigan (Table 1,
2). This haplotype appeared identical to the ND-1 sequence of a brown trout sample from
Denmark (Denmark Gudenaa 4, GenBank AF117719). Two additional haplotypes occurred in
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Journal of Great Lakes Research 35 (2009) 163–167
the Gilchrist strain, including haplotype C (GenBank EU003593) in samples from the Michigan
hatchery and the Rogue and Muskegon Rivers, and haplotype D (GenBank 003594) in a single
individual from the OSFH (Table 1).
Discussion
Although brown trout populations have been studied throughout Europe using PCRRFLP and sequencing techniques, this is the first study to quantify their mtDNA nucleotide
sequence variation in the United States. Our results corroborate the findings of previous studies
reporting appreciable genetic variation in European and North American brown trout populations
(Ferguson 1989; Ferguson et al. 1995; Apostolidis et al. 1996, Bernatchez 2001).
Previous analysis of brown trout from the OSFH, the Muskegon and Rogue Rivers, and
Lake Michigan using PCR-RFLP techniques indicated that there were at least four polymorphic
sites within the ND-1 mtDNA region and at least two polymorphic sites in the ND-5/6 mtDNA
region (Tiano 2005; Tiano et al. 2007). Furthermore, it was found that a single RFLP was
sufficient to differentiate between the haplotype found in the Wild Rose strain (A; Genbank
EU003596) and the three haplotypes found in the Gilchrist strain (B-D; Genbank EU003592-94)
(Tiano et al. 2007). In the current study, DNA sequence analysis of the entire ND-1 segment
further evaluated its component polymorphism. Our study indicated that RFLP analysis detected
50% of the total genetic variation present. RFLP techniques are cost-effective for screening of
genetic markers and typically allow a greater range of genetic variation to be surveyed, which is
useful for fisheries management. However, DNA sequencing allows detection of a greater
number of nucleotide variations and may allow more in-depth comparison among putative
populations.
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Journal of Great Lakes Research 35 (2009) 163–167
Our study clarified the results of RFLP analysis reported by Tiano et al. (2007). For
example, one of two Rogue River individuals from the Tiano et al. (2007) study that did not
match any of the hatchery RFLP types was here assigned to haplotype E (Genbank EU003598)
characteristic of the Seeforellen strain based on mtDNA sequencing. According to the Fish
Stocking Database maintained by the MDNR, the Seeforellen strain was not stocked into this
river until 2006, three years following sample collection (Tiano et al. 2007). In addition, the
sampling site where this fish was caught is located upstream from a dam making it virtually
impossible that this individual migrated to the upper Rogue River from Lake Michigan. Its
presence in the upper Rogue River may indicate that this haplotype also occurred in either the
Wild Rose or Gilchrist strains that were used for the Rogue River stocking before 2003, or that it
characterized strains used in earlier stocking events (prior to 1995) and has persisted due to
natural reproduction.
Haplotypes characterizing the Seeforellen strain (E-H; Genbank EU3592-94) had the
greatest number of SNPs, including two non-synonymous substitutions. Our results indicated
that the Wild Rose strain has low variation in the ND-1 region which may reflect drift in this
long-term hatchery strain. Wild Rose samples from Wisconsin and Michigan hatcheries also
exhibited haplotype B, which is characteristic of fish belonging to the Gilchrist strain. This
haplotype likely also was present in the source population used to establish the Wisconsin Wild
Rose strain.
The genetic identity of brown trout used for stocking may provide valuable information
on how a particular trout strain may adapt to a particular environment. Knowledge of the
original habitat and its associated environmental conditions may be used to “match” the stocked
habitat of a genetic type with its origin. For example, our DNA sequence analysis of strains
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Journal of Great Lakes Research 35 (2009) 163–167
suggested that Gilchrist and Seeforellen strains from Michigan and Wisconsin may trace to
northern European waters, for which further study is warranted. Stocking these strains into
systems with similar environmental characteristics may improve survival. This approach would
reduce the degree of trial and error in fisheries management.
Post stocking growth rates and survival of Michigan strains of brown trout differ
substantially in Michigan streams. Wills (2005, 2006) found that the Gilchrist strain, a relatively
new hatchery strain, survives >100X better than the Seeforellen strain and 6X better than Wild
Rose (Wills 2006). Enhanced survival of wild brown trout versus domesticated hatchery strains
also was reported in a longitudinal study in Wisconsin (Avery et al. 2001) as well as in several
European studies (Hansen 2002, Hansen et al. 2000). Thus, differential survival of these strains
may have a genetic basis, and some may better adapt to local environmental conditions. The
results of the current study uncovered a significant amount of mtDNA nucleotide polymorphism
in the Gilchrist strain, which could be used as markers for tracing fish with differential survival
after stocking.
Possible adaptive significance of non-synonymous substitutions detected in haplotype G
found in the Seeforellen strain merits testing, since the ND-1 region codes for the mitochondrial
respiratory chain and altering energy production may have adaptive value. In conclusion,
additional genetic studies should include larger sample sizes of these brown trout strains and
examine both mitochondrial and nuclear DNA variation, especially high-resolution microsatellite
loci.
Acknowledgements
We thank Randal Larson from the Wild Rose State Fish Hatchery and Dan Sampson from the
Oden State Fish Hatchery for fish samples and for information about brown trout stocks in their
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Journal of Great Lakes Research 35 (2009) 163–167
care. This study was funded by the Graduate Student Presidential Research Grant to SSJ and by
graduate research support funds from the Cell and Molecular Biology Program, GVSU.
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Avery, E.L., Niebur, A., and Vetrano, D. 2001. Field performance of wild and domesticated
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historical and contemporary samples. Mol. Ecol. 11:1003-1015.
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mitochondrial DNA segments. J. Fish Biol. 48:422-436.
Kincaid, H.L. 1981. Trout strain registry. FWS/NFC-L/81-1. National Fisheries CenterLeetown, Kearneysville, WV 25430. 118 pages.
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MacCrimmon, H.R., and Marshall, T.L. 1968. World distribution of brown trout, Salmo trutta. J.
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Michigan Department of Natural Resources (MDNR). 2007. State Fish Hatcheries.
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mitochondrial DNA polymorphism. M.Sc. thesis, Grand Valley State University,
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Journal of Great Lakes Research 35 (2009) 163–167
Table 1. Numbers of brown trout sequenced with the source of samples and corresponding mtDNA
haplotypes. RFLP haplotype designations are per Tiano (2005). Abbreviations: GC, Gilchrist Creek
strain; WR, Wild Rose strain; SF, Seeforellen strain; YOY, young of the year fish. Order of enzymes
in RFLP haplotype designation: Alu I, Hae III, Hpa II.
RFLP/SNP haplotype Genbank # MI
hatchery
YOY
(strain of
origin)
AAA/Haplotype B
EU003592
ABA/Haplotype C
ABA/Haplotype D
BAA/Haplotype A
BAB/Haplotype E
Haplotype F
Haplotype G
Haplotype H
BBA/Haplotype I
Total
EU003593
EU003594
EU003596
EU003598
EU003599
EU003600
EU003601
EU003597
8 (6 GC,
2 WR)
1 (GC)
1 (GC)
2 (WR)
12
MI
WI
Rogue Muskegon Lake
hatchery hatchery River River
Michigan
broodstock YOY
(strain of (strain of
origin)
origin)
3 (2 GC,
1 WR)
2 (GC)
2 (WR)
1 (SF)
3 (SF)
2 (SF)
13
Total
4 (WR)
3
3
2
23
4 (WR)
2 (SF)
1 (SF)
2 (SF)
13
3
2
1
1
10
1
3
7
2
3
7
7
1
15
7
3
3
2
1
62
15
Journal of Great Lakes Research 35 (2009) 163–167
Table 2. Identification of polymorphic sites and site discrimination in mitochondrial ND-1 sequences among representative Michigan
and Wisconsin brown trout. See Table 1 for haplotype designations. Nucleotide positions designations are based on the reference
mitochondrial genome sequence of brown trout (Salmo trutta) NC_010007. Variable nucleotides within the codon are underlined.
Non-synonymous substitutions are in bold.
Haplotype
Haplotype A
SNP Position: 2882
2925
2984
3041
3080
3104
3119
3149
3164
Codon Position:
3
1
3
3
3
3
3
3
3
Genbank #
n
EU003596
15 CTC - L TTA - L GGT - G CCG - P GCT - A CTC - L TGA - W ACG - T GGG - G
Haplotype B
Haplotype C
Haplotype D
EU003592
EU003593
EU003594
22
7
1
CTC - L TTA - L GGT - G CCG - P GCT - A CTC - L TGA - W ACA - T GGA - G
CTC - L TTA - L GGT - G CCG - P GCT - A CTC - L TGG - W ACA - T GGA - G
CTC - L TTA - L GGT - G CCG - P GCT - A CTC - L TGA - W ACA - T GGA - G
Haplotype E
Haplotype F
Haplotype G
Haplotype H
EU003598
EU003599
EU003600
EU003601
7
3
3
2
CTC - L
CTC - L
CTT - L
CTC - L
Haplotype I
EU003597
1
CTC - L TTA - L GGT - G CCG - P GCT - A CTC - L TGG - W ACC - T GGG - G
TTA - L
TTA - L
CTA - L
TTA - L
GGT - G
GGT - G
GGT - G
GGC - G
CCA - P
CCG - P
CCA - P
CCG - P
GCT - A
GCT - A
GCT - A
GCC - A
CTC - L
CTC - L
CTT - L
CTC - L
TGA - W ACG - T
TGA - W ACA - T
TGA - W ACG - T
TGA - W ACA - T
GGG - G
GGA - G
GGA - G
GGG - G
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Journal of Great Lakes Research 35 (2009) 163–167
3170
3
3314
3
3315
1
3356
3
3413
3
3422
3
3428
3
3434
3
3470
3
3587
3
3596
3
3713
3
3716
3
CTG - L CTG - L AGC - S TTC - F ATA - M ATC - I ACC - T GCT - A GGA - G TTT - F GCG - A CAG - Q CTT - L
CTG - L CTC - L AGC - S TTC - F ATA - M ATC - I ACC - T GCC - A GGA - G TTT - F GCG - A CAG - Q CTC - L
CTG - L CTC - L AGC - S TTC - F ATA - M ATC - I ACC - T GCC - A GGA - G TTT - F GCG - A CAG - Q CTC - L
CTA - L CTC - L AGC - S TTC - F ATA - M ATC - I ACC - T GCC - A GGA - G TTT - F GCG - A CAG - Q CTC - L
CTG - L
CTG - L
CTA - L
CTG - L
CTC - L
CTC - L
CTC - L
CTC - L
AGC - S
AGC - S
GGC - G
AGC - S
TTC - F
TTC - F
TTT - F
TTC - F
ATA - M
ATA - M
ATG - M
ATA - M
ATC - I
ATC - I
ATT - I
ATC - I
ACC - T
ACC - T
ACT - T
ACC - T
GCT - A GGA - G
GCC - A GGA - G
GCT - A GGG - G
GCT - A GGA - G
TTT - F
TTT - F
TTC - F
TTT - F
GCG - A CAG - Q
GCG - A CAA - Q
GCA - A CAG - Q
GCA - A CAG - Q
CTC - L
CTC - L
CTC - L
CTC - L
CTG - L CTC - L AGC - S TTC - F ATA - M ATC - I ACC - T GCT - A GGA - G TTT - F GCG - A CAG - Q CTC - L
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Journal of Great Lakes Research 35 (2009) 163–167
3736
2
3767
3
3770
3
AGC - S CTA - L TGA - W
AGC - S CTA - L TGA - W
AGC - S CTA - L TGA - W
AGC - S CTA - L TGA - W
AGC - S
AGC - S
AAC - N
AGC - S
CTA - L
CTA - L
CTG - L
CTA - L
TGA - W
TGA - W
TGG - W
TGA - W
AGC - S CTA - L TGA - W
18
Journal of Great Lakes Research 35 (2009) 163–167
Figure 1. Map of Michigan, with sample locations on the Muskegon River (A), Rogue River (B), and Lake Michigan adjacent to
Ludington, MI (C).
19