Introduction
The
banded darter (Etheostoma zonale) is
a small nongame fish named for the brilliant green bands that encircle the
bodies of breeding males. It is a member of the family Percidae, a group that
contains the popular sport fishes yellow perch and walleye as well as nearly
200 species of the smaller darters. While the family has a Holarctic
distribution, all darters are limited in range to North America with the
majority of species endemic to very small ranges in the Central Highland
regions east and west of the Mississippi River. The banded darter is one of the
relatively few darters with a widespread distribution; it ranges from the
highlands of Arkansas and Tennessee
northeast through the Ohio River valley, and reaches the northwest portion of
its range in the southern half of Minnesota.
This large distribution makes the banded darter a good species for studying
population genetics questions, such as amount and patterns of migration, both
past and present.
The
Pleistocene was an epoch of glacial ebb and flow, with glaciers reaching their
maximum southern extent in North America during the Illinoian advance between
65,000 and 122,000 years before present (YBP), and with the most recent
(Wisconsinan) glaciers retreating from the northern United States about
10,000–11,000 YBP (Dawson, 1992). The banded darter, along with other aquatic
life, was confined to glacial refugia during these onsets of ice, and had to
colonize the northern parts of its range postglaciation. Accordingly,
populations of banded darters currently inhabiting Minnesota are relatively recent in origin,
and should be most closely related to populations of banded darters from one or
more refugium areas.
The
current distribution of the banded darter provides insight about the timing of
its postglacial colonization. Because the banded darter is present only in the
Minnesota River and lower Mississippi River drainages in Minnesota,
it is thought to be a relatively recent migrant into the state, becoming
established only after the southern outlet to glacial Lake Agassiz
had closed (Underhill, 1957). The banded darter’s Minnesota distribution also
suggests its colonization from the lower Mississippi River refugium (Underhill,
1989), but whether colonizing individuals came from the Central Highlands
regions east or west of the Mississippi River (or both) is not known.
The
field of phylogeography interprets population genetic data in a historical and
geographic context (Avise, 1994); it allows the analysis of intraspecific
variation in DNA sequences to provide insight into processes like gene flow or
colonization of new areas. For example, Strange and Burr (1997) compared
intraspecific phylogenies of five Central Highlands fish species to distinguish
between vicariance and dispersal as processes explaining current population
distributions. Similarly, Near et al. (2001) examined DNA sequences of a
widespread darter (Percina evides) to
hypothesize how these processes have affected not only Central Highlands
distribution but also the species’ presence in glaciated areas of the Midwest.
Mitochondrial
DNA has provided the data sets for the majority of recent phylogeographic
studies. In general, the mitochondrial genome accumulates nucleotide
substitutions more rapidly than its nuclear counterpart (Brown, 1987), and its
uniparental inheritance and lack of recombination result in a lower effective
population size, allowing for the possibility of faster fixation or extinction
of haplotypes via drift (Moore,
1995). The cytochrome b protein spans
the inner membrane of the mitochondrion (Degli Esposti et al., 1993), and its
gene has been used extensively in phylogenetic studies, including those of
fishes (Lydeard and Roe, 1997). Although the phylogenetic utility of cytochrome
b has been the subject of some debate
(e.g., Graybeal, 1993; Meyer, 1994), it is still widely used in a variety of
studies ranging from intergeneric (e.g., Dunn et al., 2003) to intraspecific
(e.g., Van Houdt et al., 2003).
Mitochondrial
NADH dehydrogenase genes, including NADH dehydrogenase subunit 2 (ND2), have
also been widely used in phylogenetic analyses. Results from studies that have
obtained sequences for both cytochrome b
and ND2 are mixed; the two genes exhibit comparable variability in dabbling
ducks (Johnson and Sorenson, 1998), but ND2 is more variable than cytochrome b in Myotis
bats (Cooper et al., 2001) and fishes such as suckers (McPhail and Taylor,
1999) and logperches (George, 2003).
The
purpose of this study was to investigate the phylogeography of Minnesota banded darters in order to elucidate the
pattern of this species’ radiation in Minnesota
over the past 10,000 years. Banded darters were sampled from seven sites in the
state, and mitochondrial DNA sequences from the cytochrome b and ND2 genes were analyzed. These genes were chosen because of
their past success in inferring intraspecific phylogeny, and because darter
sequences for these genes are readily available for comparison.
MateriAls
and Methods
Collection of
specimens
Banded
darters were collected throughout southern Minnesota during 2001-2002, and the seven
sites from which sequence data were obtained are mapped in Figure 1. Locality
information and number of individuals sequenced (with GenBank accession
numbers) are provided in Table 1. Fish were collected using a ten-foot seine
under State of Minnesota Department of Natural Resources Division of Fisheries
Special Permit No. 10962, and frozen in liquid nitrogen or preserved in 95%
ethanol onsite. Specimens are stored at St.
Olaf College.
DNA Sequence
Data
Total
DNA extractions were performed on muscle tissue using a protocol modified from
the Puregene DNA Purification Kit
(Gentra Systems) by T. Near (personal communication). The polymerase chain
reaction (PCR) was used to amplify a 550-nucleotide region of the cytochrome b gene of banded darters using primers
THR (Song et al., 1998) and ZON635F (5’-ACT CCG ACG CCG ATA AAG TGT C-3’). PCR
was also used to amplify the entire ND2 gene using primers ND2Met and ND2Trp
(Kocher et al., 1995). Primers were annealed at 50 C in all reactions. The
primers THR (for the cytochrome b
gene) and ND2Met (for the ND2 gene) were used in sequencing reactions of clean
PCR product (QIAquick PCR Purification Kit, Qiagen, Inc.), which were done at
the DNA Synthesis and Sequencing Laboratory of the University of Medicine and
Dentistry of New Jersey’s Robert Wood Johnson Medical School (cytochrome b sequences) or the Auburn University
Genomics and Sequencing Laboratory (ND2 sequences).
Sequence
Analysis
Chromatograms
were viewed and sequences were edited using EditView (version 1.0.1, Applied
Biosystems), and ClustalW (Thompson et al., 1994) was used to align sequences.
A cytochrome b DNA sequence from an Illinois banded darter (Porterfield, 1998) was aligned
along with the sequences from Minnesota
individuals. Uncorrected pairwise distances among individuals were calculated
with PAUP* version 4.0b10 (Swofford, 2000), and MacClade version 3.03 (Maddison
and Maddison 1992) was used to assess variable nucleotide and amino acid
positions.
Results
and Discussion
Cytochrome
b sequence data were obtained for 29 Minnesota banded darters, which resulted in a data set of
30 individuals (including the individual from Illinois). While the amount of sequence
obtained per individual varied, the data set was reduced to the length of the
shortest high-quality sequence (426 nucleotides). These nucleotides correspond
to nucleotide positions 637 to 1062 of this 1140-nucleotide gene (amino acid
positions 213 to 354 out of 380). ND2 sequences of 712 nucleotides in length
were obtained for eight of the 29 banded darters (representing seven Minnesota localities;
see Table 1); these nucleotides represent the first 712 positions (first 237 amino
acids) of this 1047-nucleotide gene. Only single-stranded sequence was obtained
in this study; although double-stranded sequence is more desirable, the lack of
variation found when examining the single-strand data set suggested that
obtaining double-stranded sequence would not be cost-effective (since
sequencing the other strand is more likely to contradict putative substitutions
than to identify more substitutions).
Of
the 426 nucleotides of cytochrome b
for all 30 individuals, only two were variable (not including the 14 positions
where an undetermined nucleotide [N] was present in one individual). This
resulted in an average uncorrected genetic distance of only 0.0007 in all
pairwise comparisons among the 30 sequences. One of the variable nucleotides
(position 768) was a third-position silent substitution unique to the
individual from the Zumbro
River. The other
(position 1061) was a second-position substitution found in four individuals
(two from Cobb River
and two from Yellow
Medicine River);
it resulted in an amino acid change from alanine to glycine. These amino acids
are very similar in structure and biochemical properties, with alanine
exhibiting slightly more hydrophobicity than glycine. Of the 712 nucleotides of
ND2 for nine banded darters, the only variable position was nucleotide 48 where
an undetermined nucleotide (N) was present in one individual. Contamination is
not likely based on other PCR and sequencing reactions that were performed at
the same time.
Based
on the cytochrome b sequence data,
three haplotypes were found among Minnesota
banded darters, and the most common haplotype is shared with the banded darter
from Illinois.
This high degree of similarity not only within Minnesota
fish but also among Minnesota and Illinois fish is consistent with the glacial history of
the Midwest. All 30 individuals were collected
from regions that experienced the last (Wisconsinan) glacial retreat just
10,000–11,000 YBP, so they represent relatively recently established
populations. Near et al. (2001) also found high genetic similarity among
central Minnesota and northern Wisconsin gilt darters (Percina evides), with the three
individuals from this region exhibiting pairwise sequence distances of only
0.1–0.2% (based on all 1141 nucleotides of the cytochrome b gene). Both the banded darter and the gilt darter are
hypothesized to be relatively recent migrants (Underhill, 1957). In contrast,
Kassler et al. (1997) found some among-population mtDNA variation in their
study of 36 Minnesota and Wisconsin populations of johnny darters (Etheostoma nigrum). The johnny darter is
hypothesized to be one of the earliest postglacial arrivals to Minnesota (Underhill,
1957).
While
the data from this study are too homogeneous for phylogeographic analysis, the
lack of variation among Minnesota
banded darter mitochondrial DNA sequences is informative about other aspects of
this species’ colonization history. Patterns of low nucleotide and haplotype
diversity are consistent with historical bottlenecks during colonization (e.g.,
Gamache et al., 2003; Wang et al., 2000). Considerably more variation is found
within and among Central Highlands populations of the banded darter
(Porterfield, unpublished data), but if there were relatively small numbers of
colonizing banded darters, it is probable that genetic diversity among
colonizers was low. Estimates of the rate of cytochrome b evolution range from 0.70%/Myr for ectothermic vertebrates (Johns
and Avise, 1998) to 0.76%/Myr for European cyprinid fishes (Zardoya and
Doadrio, 1999). Based on these estimates, banded darters would be expected to
exhibit little cytochrome b variation
after 10,000 years in Minnesota,
presuming genetic uniformity among colonizing fishes.
A
different molecular marker is necessary to address phylogeographic patterns
among Minnesota
banded darters. Microsatellite loci have proven valuable for uncovering genetic
variation within and among populations (Koskinen et al., 2002; Pope et al.,
2000), and may be an appropriate direction for the study of Minnesota freshwater fishes including the
banded darter. For now, the high degree of genetic similarity among Minnesota banded
darters, even in the fast-evolving mitochondrial genome, provides some insight
into the postglaciation colonization of these fish into the state. This
information is also important in the context of the future evolution and
conservation of Minnesota
populations, as these populations represent the northern edge of the banded
darter’s range.
Acknowledgments
Funding
for this research was provided by a St. Olaf College Faculty Development Grant
to Jean Porterfield and by a Howard Hughes Medical Institute Undergraduate
Science Education grant to St. Olaf College.
Matt Berkseth and Kathryn Huber helped with sequencing of the ND2 gene. Figure
1 was compiled by Charles Umbanhowar Jr., and Diane Angell, Patrick Ceas, Jay
Hatch, Gary Phillips, and Charles Umbanhowar Jr. provided helpful comments on
the manuscript.
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