doi: 10.1111/j.1420-9101.2011.02311.x
Rapid loss of MHC class II variation in a bottlenecked population
is explained by drift and loss of copy number variation
J. A. EIMES*, J. L. BOLLMER*, L. A. WHITTINGHAM*, J. A. JOHNSON ,
C. VAN OOSTERHOUTà 1 & P. O. DUNN* 1
*University of Wisconsin-Milwaukee, Milwaukee, WI, USA
University of North Texas, Denton, TX, USA
àUniversity of East Anglia, Norwich, UK
Keywords:
Abstract
bottleneck;
copy number variation;
genetic drift;
major histocompatibility complex;
selection;
Tympanuchus cupido.
Population bottlenecks may reduce genetic variation and potentially increase
the risk of extinction. Here, we present the first study to use historic samples
to analyse loss of variation at the major histocompatibility complex (MHC),
which plays a central role in vertebrate disease resistance. Balancing selection
acts on the MHC and could moderate the loss of variation expected from drift;
however, in a Wisconsin population of greater prairie-chickens (Tympanuchus
cupido), the number of MHC class II B alleles per individual declined by
44% following a population bottleneck, compared to a loss of only 8% at
microsatellites. Simulations indicate that drift likely reduced MHC variation at
the population level, as well as within individuals by reducing the number of
gene copies per individual or by fixing the same alleles across multiple loci.
These multiple effects of genetic drift on MHC variation could have important
implications for immunity and fitness.
Introduction
Changes in genetic variation following a population
bottleneck depend on the interaction of drift, selection,
recombination and gene flow (Crow & Kimura, 1970).
The outcome of these interactions differs fundamentally
between neutral genes and functional genes, which are
under selection and often thought to be less affected
by drift (Hedrick, 2001; Le Corre & Kremer, 2003). For
example, in small populations experiencing genetic drift,
variation at neutral loci may decline, whereas variation
at expressed genes may be maintained by balancing
selection (Hedrick, 2001). Several studies have tested this
idea by comparing levels of genetic variation at putatively
neutral markers (e.g. microsatellites) and those involved
in immune function that are thought to be under
relatively strong selection (Garrigan & Hedrick, 2003).
One of the most common functional gene families used
to examine balancing selection is the major histocomCorrespondence: John A. Eimes, Department of Biological Sciences,
University of Wisconsin-Milwaukee, PO Box 413, Milwaukee,
WI 53201, USA.
Tel.: 414 229 3026; fax: 414 229 3926; e-mail: [email protected]
1
Joint last authors.
patibility complex (MHC), which plays a central role in
vertebrate immunity and may influence population
viability (Piertney & Oliver, 2006; Siddle et al., 2007;
Radwan et al., 2010).
MHC genes are among the most polymorphic coding
loci known in vertebrates (Klein, 1986), which is
important for their role in the recognition and presentation of foreign antigens as part of the adaptive immune
response. MHC molecules differ in their ability to
recognize pathogens due to sequence variation at their
peptide-binding region (PBR) (Potts & Wakeland, 1990;
Apanius et al., 1997), and associations between particular
MHC alleles or haplotypes and parasite resistance have
been demonstrated across vertebrate taxa (Briles et al.,
1983; Langefors et al., 2001; Schad et al., 2005). Variation
in the MHC is generated at multiple levels. First, the
MHC is prone to gene duplication and deletion (Klein
et al., 1993; Nei et al., 1997), which results in copy
number variation (CNV) among individuals (Hosomichi
et al., 2006; Anmarkrud et al., 2010; Doxiadis et al., 2010)
as well as between species (Trowsdale, 1995; Kelley et al.,
2005). Several models of multigene evolution have been
proposed that may explain the evolution of CNV,
including the accordion model (Klein et al., 1993), the
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birth-and-death model (Nei et al., 1997) and Associative
Balancing Complex evolution (van Oosterhout, 2009).
Second, allelic variation at individual loci is generated
by recombination and mutation, and this variation is
maintained by balancing selection acting on the PBR
(Sommer, 2005; Piertney & Oliver, 2006; Spurgin &
Richardson, 2010). Selection may also operate against
deleterious mutations that have accumulated in the MHC
(Shiina et al., 2006), and this can maintain a balanced
polymorphism at the MHC as well (van Oosterhout,
2009).
In multigene families, such as the MHC, CNV could be
lost due to drift in a similar fashion as allelic diversity is
lost at single loci (Cutler et al., 2007; Schrider & Hahn,
2010). Different mechanisms have been demonstrated
to generate CNV, including replication slippage, retrotransposition and recombination between duplicated
sequences (Schrider & Hahn, 2010). Stochastic removal
of particular haplotypes with a relatively large number
of gene copies could eliminate several interacting (and
co-evolving) genes at once, reducing genetic variation
above and beyond that expected from the loss of
heterozygosity at a single locus alone. Furthermore,
duplicated alleles can occur at multiple loci (Chaves et al.,
2010), which means that drift could cause the fixation of
the same allele at different loci within the gene family.
Such drift-across-loci could further exacerbate the loss of
genetic variation during a population bottleneck in
multigene families.
Despite the mechanisms generating variation at the
MHC, many small populations exhibit low levels of
variability, and some are even fixed for single MHC
alleles (Hedrick et al., 2000; Miller & Lambert, 2004;
Babik et al., 2009; Radwan et al., 2010). This suggests that
drift can overwhelm balancing selection, although in
some cases, MHC diversity appears to be maintained
despite the loss of variation at neutral loci (Aguilar et al.,
2004; van Oosterhout et al., 2006a; Mona et al., 2008).
It has also been suggested that if genetic variation is
depleted through a bottleneck, divergent alleles are more
likely to be retained because they will confer resistance to
a wider suite of potential pathogens (Hedrick et al., 2002;
Hedrick, 2003). To date, no studies have directly compared the genetic diversity of the MHC in a wild
population before and after a bottleneck using both
historic and contemporary DNA samples. Furthermore,
theory and computer simulation models have focused
primarily on the effects of bottlenecks on single locus
heterozygosity (Borghans et al., 2004; Ejsmond &
Radwan, 2009) and do not consider CNV, which may
be important in immune function and disease resistance
(Siddle et al., 2010).
Here, we examined genetic variation in a population of
greater prairie-chickens (Tympanuchus cupido pinnatus)
before and after a bottleneck to test the relative effects of
genetic drift and balancing selection on putatively neutral (microsatellite) and functional (MHC class II B)
genes. Over the past century, prairie-chickens have
experienced one of the largest declines in range size of
any bird species in North America (Anderson & Toepfer,
1999; Johnson et al., 2011), primarily because of habitat
loss (Johnsgard, 2002; Johnson et al., 2011). For example, in Wisconsin, the species occurred in nearly every
county in the state in the 1930s, but by the 1950s its
range was reduced to seven (of 72) counties in the centre
of the state. The population in central WI experienced
a further decline of 50% during the 1950s to approximately 1200 individuals, and it has remained at that
population size for the past 50 years (Anderson &
Toepfer, 1999).
Our previous studies of greater prairie-chickens
showed that genetic variation at neutral markers, such
as microsatellites and the mtDNA control region,
declined as a consequence of the bottleneck in Wisconsin
(Bellinger et al., 2003; Johnson et al., 2003, 2004, 2007).
In the present study, we used a subset of the same
contemporary and historic samples used in the microsatellite study to directly measure the effect of the bottleneck on allelic variation in MHC class II B genes. We
found a loss of MHC polymorphism (number of alleles in
the population) similar to microsatellites (Johnson et al.,
2004), a pattern that is consistent with the effect of drift.
However, the loss of MHC variation within individuals
(fewer alleles per individual) significantly exceeded the
rate of loss expected from drift. Here, we show that this
pattern of loss can be explained by drift acting on the
number of loci (CNV) or the fixation of the same
duplicated allele across multiple loci (drift-across-loci).
Materials and methods
Sampling and DNA extraction
To measure the change in MHC polymorphism through
the population bottleneck, we examined sequences from
40 adults (20 contemporary and 20 historic) from Buena
Vista Marsh, Portage County, WI, USA. Our sample
consisted of a subset of the same historic (1951–1952)
and contemporary (1996–1999) DNA samples that were
previously analysed using microsatellites (Bellinger
et al., 2003; Johnson et al., 2004) and mtDNA control
region sequences (Johnson et al., 2004). The historic
DNA was extracted from wings collected by F.N. and
F. Hamerstrom in 1951–1952 during the last hunting
season in Wisconsin (Bellinger et al., 2003). All historic
DNA was extracted in a separate laboratory free of any
other DNA or tissue samples from grouse (Johnson et al.,
2004).
PCR, cloning and sequencing
We generated a 239-bp amplicon (excluding primers) of
the PBR of MHC class II B (BLB) genes using the primers
Blex2F (Eimes et al., 2010) and RNA R1 (Strand et al.,
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Reduced MHC variation following a bottleneck
2007). This fragment begins at position 14 and ends at
position 252 of the 270-bp exon 2.
PCRs contained approximately 100 ng of DNA, 0.2 lM
of each primer (5¢ phosphorylated), 25 lL of Phusion
Flash High Fidelity PCR Master Mix (New England
Biolabs, Ipswich, MA, USA) and water to a total volume
of 50 lL. Cycling conditions included an initial denaturation step at 94 C for two min followed by 30 cycles of
94, 62 and 72 C each for 30 s and a final extension of
72 C for 10 min. Target PCR products were gel excised
before purification in spin columns (Qiagen-QIAquick,
Germantown, MD, USA). PCR amplicons were then
cloned using Clonesmart blunt end high count kanamycin cloning kits with chemically competent cells (Lucigen, Middleton, WI, USA). Colonies were diluted in
20 lL of water, and gene products were amplified by
whole colony PCR using the vector primers SL-1 and
SR-2 in the Clonesmart kit. Whole colony PCR amplifications contained 3 lL of template, 0.625 lM of each
primer, 0.5 mM of each dNTP, 1.5 mM MgCl2, 1· PCR
buffer and 1.0 U of GoTaq Flexi polymerase in a total
volume of 20 lL. The cycling conditions included an
initial denaturation at 94 C for 2 min followed by 30
cycles of 94, 55 and 72 C each for 1 min and a final
extension of 72 C for 10 min. Sequencing was performed on Applied Biosystems (Carlsbad, CA, USA)
3730XL and 3130 automated DNA sequencers (University of Chicago Cancer Research Center). Sequences were
labelled BLB3-BLB26 and were aligned manually using
BI O ED I T v. 7.0.9.0 (Hall, 1999) and GE N E I O U S PR O v.
5.0.4 (Biomatters, Ltd., Aukland, New Zealand).
To avoid the inclusion of erroneous sequences into our
data set that may be caused by heteroduplex formation,
Taq error or deamination (Hansen et al., 2001), all
sequences (alleles) were verified by at least two independent PCR, cloning and sequencing rounds (Cummings et al., 2010). Thus, identical sequences from these
two independent samples are likely to be genuine
autapomorphies (Paabo et al., 2004; Sefc et al., 2007).
Furthermore, none of the eight single nucleotide substitutions that we discovered in historic samples were
distinguished by a cytosine to thymine replacement,
which is often caused by deamination (Paabo et al., 2004;
Sefc et al., 2007).
We sequenced at least 23 colonies per individual for
38 of the 40 individuals; the remaining two individuals
had 17 and 22 clones sequenced because of difficulties
amplifying historic DNA. We chose 23 colonies per
individual, because this number is expected to result in
finding all alleles of an individual with > 90% probability. Assuming there are five loci (with ten equally
frequent alleles), the binomial probability of missing
one allele equals P = 0.089. Overall, there was no
difference (t38 = 0.1; P = 0.90) between the contemporary (25.5 ± 3.4) and historic (25.3 ± 4.3) samples in the
number of colonies screened per individual. All sequences were deposited in Genbank (accession numbers
1849
FJ232512–FJ232514, FJ232516–FJ232520, GQ176848–
GQ176851 and HM011573–HM011586).
RFLP and Southern blot
To estimate the number of MHC class II loci and to verify
CNV, we performed a random fragment length polymorphism (RFLP) analysis using a probe that binds
specifically to MHC class II of eight contemporary
individuals. We digested seven micrograms of gDNA for
3 h with 20 units of PvuII-HF (New England BioLabs
Inc., Ipswich, MA, USA), an enzyme that has been used
successfully in other avian MHC studies (e.g. Miller &
Lambert, 2004; Balakrishnan et al., 2010). The digested
DNA was run on a 0.8% agarose gel for 19 h at 60 V in
0.5· Tris-Borate-EDTA buffer and then transferred by
Southern blotting to a Hybond (GE Healthcare Life
Sciences, Piscataway, NJ, USA) membrane following the
manufacturer’s instructions. The blot was prehybridized
at room temperature overnight in 50% formamide, 5·
standard saline citrate (SSC), 10· Denhardt’s solution,
and 250 lg ⁄ mL yeast RNA. The blot was then hybridized
at 42 C overnight with 32P-labelled 334 bp probe using
random primer labelling. The probe was designed from
an amplicon of a contemporary individual using the
primers Blex2F and E3R (Chaves et al., 2010) that
extended from the 108th bp of exon 2 to the 85th bp
of exon 3 using the first PCR conditions described
previously. The homology of this PCR product to
Galliform MHC Class II genes was verified by cloning
and sequencing as described previously and by performing a B L A S T (NCBI, Bethesda, MD, USA). The hybridization buffer was the same as the prehybridization, with
the addition of dextran sulphate to a concentration of
10%. The blot was washed in 0.5· SSC, 0.1% sodium
dodecyl sulfate (SDS) by rinsing briefly at room temperature, followed by two 30 min washes at 65 C with a
buffer change between each wash. Autoradiography was
carried out for 24 h on Kodak (Rochester, NY, USA)
BioMax film at )80 C using an intensifying screen.
Sequence analysis
Ratios of nonsynonymous (dN) and synonymous (dS)
substitution rates are often used as a test for (historic)
selection at MHC loci, with dN ⁄ dS ratios greater than one
suggesting balancing or positive selection (Garrigan &
Hedrick, 2003). We tested for positive selection at
peptide-binding codons (PBCs) and non-PBCs in MHC
class II B sequences using the modified Nei-Gojobori ⁄
Jukes-Cantor method (Nei & Gojobori, 1986) in M E G A
v. 4 (Tamura et al., 2007). PBCs in our sequences were
identified using the position of PBCs in HLA II (Brown
et al., 1993).
Average pair-wise nucleotide diversity (d) and segregating sites (S) were calculated with DN A SP 4.50.3
(Rozas et al., 2003). In addition, a phylogenetic network
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was constructed using the program SplitsTree4 (Huson
& Bryant, 2006) and employed the Neighbour-Net
method (Bryant & Moulton, 2002) with Jukes-Cantor
distances.
Loss of genetic variation
Genetic variation was expressed as the number of distinct
alleles (or sequences) in the population sample (Ap) and
the number of distinct alleles (sequences) per individual
(Ai). These measures were computed by summing the
alleles across loci, which allowed us to directly compare
the temporal loss at the MHC with that at microsatellite
loci. Mann–Whitney U tests were used to analyse whether
the population bottleneck reduced Ai between temporal
samples. The relative loss of Ai was calculated as the
contemporary Ai divided by mean historic Ai. The relative
loss of Ai was compared between the MHC and microsatellite loci using a randomization test with 10 000 iterations to simulate the loss of MHC and microsatellite alleles.
MHC simulations
We simulated the loss of MHC diversity using an
individual-based model and several demographic scenarios from molecular estimates of Ne for the Buena Vista
population (Johnson et al., 2004). These values ranged
from a maximum value of Ne = 1500 birds before the
bottleneck down to an estimated Ne = 26 in the contemporary population for t = 30 generations (Johnson
et al., 2004). Simulations focused on the loss of number
of unique MHC sequences at the population level (Ap),
as well as the loss of alleles per individual (Ai).
We first examined the hypothesis that drift within loci
can explain our results by testing whether the loss of
MHC variation was due to simulated population bottlenecks with Ne = 26, 50, 100, 500 and 1500 over 30
generations. We assumed there were five loci (based on
the maximum of 10 alleles found in one individual; see
Results later) that were completely linked and present
in all individuals. The simulations took into account the
probability of not observing all alleles per individual due
to sequencing a finite number of clones (N = 23).
Drift within loci did not completely explain our results
(see later), so we tested a second hypothesis that genetic
drift accelerates the loss of genetic variation in multigene
families due to the loss of CNV. For this hypothesis, we
simulated the effects of genetic drift on: (i) the number of
alleles within a single locus, (ii) fixation of the same allele
across multiple loci and (iii) stochastic changes in CNV. If
the same allele or sequence occurs at different loci, drift
may not only reduce gene diversity (i.e. heterozygosity)
within loci, but it can also fix the same allele at different
loci. This can reduce the apparent copy number, and
we refer to this as the ‘drift-across-loci’ hypothesis. For
example, an individual may have five MHC loci, but if drift
fixes all these loci for the same allele, its multilocus
genotype is identical to an individual that is homozygous
for a single locus. Haplotypes were constructed by
randomly allocating 1 £ k £ 5 loci per individual, and
drawing k from a uniform distribution. Alleles were then
randomly allocated to these loci by sampling from the
allele frequency distribution observed in the prebottleneck population. Hence, some alleles could occur at
multiple loci, which introduced the possibility that the
simulated loss of alleles per individual (Ai) could be
affected by fixation or loss of alleles at more than one locus
(drift-across-loci). Next, genotypes were generated by
randomly combining the MHC haplotypes, and simulations were run with the same demographic scenarios and
simulated sequencing effort (23 clones ⁄ individual), as
described previously. The mean (95% CI) simulated Ap
and Ai values were then compared with the values
observed in the historic and contemporary population.
The drift-across-loci hypothesis provided a good fit to
our results, but for completeness we also tested a more
complex third hypothesis that a combination of genetic
drift, balancing selection (s), gene conversion (c) and
migration (m) could explain our results. Ultimately, we
rejected this hypothesis because extremely high levels
of gene conversion were required to explain our results
(Fig. S1). Simulations were run 1000 times using a macro
written for MI N I T A B 12.1 (available from the authors
upon request).
Results
Loss of population variation (Ap)
Genetic drift following the population bottleneck led to a
reduction in MHC class II variation. The contemporary
greater prairie-chicken population had only 14 distinct
MHC class II B alleles, which was significantly fewer than
the 22 alleles in the historic population (randomization
test: P < 0.001). In total, we recovered 24 distinct
sequences (alleles) from the combined populations: 10
were unique to the historic population, two were unique
to the contemporary sample and 12 were present in both
samples (Fig. 1). The loss of less common alleles in the
population was consistent with that expected from
genetic drift (Fig. 2).
The population-level loss of MHC variation, as measured by the loss of alleles across all class II B loci, was
33% (8 lost ⁄ 24 total). We found previously that the
number of haplotypes at the mtDNA control region
declined by 50% from 10 in the historic period (haplotype diversity: 0.889 ± 0.013, N = 19) to five in the
contemporary period [0.511 ± 0.128, N = 20; (Johnson
et al., 2004)]. There was also a 26% reduction in number
of alleles at microsatellite loci from the historic (mean
of 8.2 ± 1.3 alleles ⁄ locus, N = 42) to the contemporary
(mean of 6.1 ± 1.1 alleles ⁄ locus, N = 87) period when
accounting for differences in sample size (Johnson et al.,
2004). Thus, the loss of MHC genetic variation, at least
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Reduced MHC variation following a bottleneck
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Fig. 1 Amino acid alignment of 24 greater
prairie-chicken major histocompatibility
complex (MHC) class II B exon 2 sequences.
Putative peptide-binding codons identified
by Brown et al. (1993) are indicated by plus
signs. Asterisks denote sites under positive
selection with a posterior probability of at
least 95% in the historic and contemporary
samples as identified by omegaMap. The two
sequences present only in the contemporary
population are in bold, whereas the
sequences present only in the historic
population are in italics.
Loss of individual variation (Ai)
Percentage of individuals with MHC allele
100
Contemporary sample
Historic sample
80
60
40
20
0
20
40
Overall, the number of distinct MHC alleles per individual (Ai) declined by 44%, from a mean (±SD) of 6.20
(±2.14) in the historic population to 3.45 (±1.88) in
the contemporary population (Mann–Whitney U test,
Z = 3.48, P < 0.001; Fig. S2). For microsatellites, however, the number of alleles per individual (summed
across six loci) declined by only 8% from 10.19 (±1.17)
in the historic to 9.34 (±1.15) in the contemporary
samples (Fig. S2).
60
80
RFLP analysis
100
2
4
6
8
10
12
14
16
18
20
22
24
26
MHC allele identifier
Fig. 2 The frequency of greater prairie-chicken MHC class II B
sequences (alleles) expressed as the percentage present in individuals in the contemporary population sample (black bars) and
the historic population sample (grey bars).
Individuals had between two and four RFLP fragments,
indicating at least four loci in some individuals. The
banding pattern suggested MHC class II CNV as there
were at least three distinct haplotypes among the eight
individuals screened (Fig. S3).
Computer simulations
at the population level, was comparable to that found
previously using putatively neutral markers in many of
the same individuals (Johnson et al., 2004).
The historic and contemporary populations did not
differ in average nucleotide divergence at either the PBCs
(mean p-distance for both populations = 0.02 ± 0.03) or
over the entire 239-bp fragment (mean p-distance for
both populations = 0.09 ± 0.01). The contemporary and
historic sequences intermixed in the network showing
similar divergence among alleles in both samples (Fig. 3).
The ratio of nonsynonymous and synonymous substitution rates (dN ⁄ dS) at the PBCs (2.02) was significantly
larger than one (codon-based Z test, Z = 2.01; P = 0.047)
for the contemporary population and approached significance for the historic sample (codon-based Z test,
Z = 1.74; P = 0.062; Table S1).
We first tested the hypothesis that genetic drift at five loci
in combination with failed amplification of some alleles
could explain the empirical results mentioned earlier
(within-locus drift hypothesis). The simulation showed
that even in the most extreme bottleneck scenario
(Ne = 26), the simulated number of alleles per individual
(Ai = 5.61) remained considerably higher than the
observed value (Ai = 3.45; Fig. 4). Therefore, we rejected
the hypothesis that drift within loci in combination with
the stochastic failure to amplify alleles could explain our
results.
Next, we examined the drift-across loci hypothesis
by simulating the effects of genetic drift on CNV and
allowing for variation in the number of loci (between 1
and 5 per individual drawn from a uniform distribution).
In this simulation, identical alleles can occur at multiple
loci and drift can act across these loci. These simulations
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0.01
Gaga-BLB1
Coja-DBB1
Coja-DBB1
Mega-IIB1
Gaga-BLB1 Gaga-BLB2
Gaga-BLB1
Gaga-BLB2
Gaga-BLB1
Gaga-BLB2
Coja-DBB1 Coja-DBB1
Coja-DBB1
Gaga-BLB2
Coja-DAB1
Gaga-BLB1
Coja-DAB1
Tycu08
Tycu16
Mega-IIB3
Mega-IIB2
Tycu20
Tycu21
Tycu14
Coja-DCB1
Tycu13
Tycu03
Tycu04
Tycu22
Tycu12
Tycu11
Tycu26
Tycu19
Tycu25
Tycu06
Tycu05
Tycu10 Tycu23
Tycu18
Tycu17
Tycu07
Tycu24
Tycu09
Tycu15
showed that the observed value of Ai (3.45) in the
contemporary post-bottleneck population fell within
the distribution of simulated values (mean = 4.69, 95%
CI = 1.7–7.9; Fig. 5). The observed variance in Ai
between individuals (3.53), which is a measure of CNV,
also fell within the 95% CI (1.63–4.62) of variances in
Ai of individuals from the simulations. Furthermore, the
distribution of simulated Ap values (mean = 11.5, 95%
CI = 5–16) included the observed Ap (14) of the contemporary population. Thus, our computer simulations
support the hypothesis that genetic drift reduced variation both within and between loci to produce the large
decline in number of MHC alleles per individual (Ai) and
the relatively moderate decline in the number of alleles
in the population (Ap).
Discussion
Greater prairie-chickens in Wisconsin lost a significant
number of MHC class II alleles following a population
bottleneck in the late 1950s. This finding is consistent
with our previous study of the same population using
microsatellites and mtDNA (Johnson et al., 2004), and
it is consistent with random genetic drift. However, we
also observed a dramatic loss of MHC variation at the
individual level (the number of unique sequences per
individual, Ai), and we showed that this loss was
significantly greater for the MHC (44%) than for six
Fig. 3 Phylogenetic network of major histocompatibility complex class II B sequences
(based on 239 bp) from four Galliform
species: the greater prairie-chicken (Tycu;
Tympanuchus cupido), red jungle fowl (Gaga;
Gallus gallus), Japanese quail (Coja; Coturnix
japonica) and domestic turkey (Mega; Meleagris gallopavo). Greater prairie-chicken
sequences found only in the contemporary
population are in boxes, sequences occurring
only in the historic population are in bold
and the remaining sequences are present
in both time periods. All of the Gallus
(AM489767–AM489775), Meleagris
(DQ993255) and Coturnix (AB078884,
AB265804 and AB282649–AB282651)
sequences were downloaded from Genbank
and originate from mapped loci.
(neutral) microsatellite loci (8%). We then used an
individual-based model to test whether this loss of Ai
could be explained by drift acting simply within loci,
but rejected this hypothesis. We tested a more complex
multilocus drift hypothesis, which proposes that drift
reduced gene diversity (heterozygosity) within loci, as
well as diversity between loci, by either fixing the same
allele at different loci or by eliminating haplotypes with
high gene copy number. Our simulations show this can
result in a loss of CNV, and our empirical data appear to
be best explained by this hypothesis.
The observed loss of MHC diversity at the population
level is consistent with the results from other fragmented
or bottlenecked populations (Radwan et al., 2010), as
well as with simulations of bottlenecked populations
(Ejsmond & Radwan, 2009). These studies show that
selection at the MHC does not necessarily maintain
variation over relatively short time scales. In contrast,
other studies of bottlenecked populations have found
evidence that selection may have maintained MHC
variation (Aguilar et al., 2004; van Oosterhout et al.,
2006a). In greater prairie-chickens, the dN ⁄ dS ratios
suggested selection at the MHC class II B region, but it
is probably the result of selection over an evolutionary
time scale (Eimes et al., 2010), rather than just the
50 years between our historic and contemporary
samples. It has been suggested that if allelic variation is
lost through a bottleneck, then alleles that are more
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Reduced MHC variation following a bottleneck
Historic sample
Contemporary sample
Simulated populations with 26 < N < 1500
Number of alleles per individual (Ai)
7
6
5
4
3
2
0
0
10
15
20
Number of alleles per population (Ap)
25
Fig. 4 The mean (±95% CI) observed number of major histocompatibility complex (MHC) alleles per population (Ap) vs. the mean
(±95% CI) observed number of MHC alleles per individual (Ai) for
the historic sample (solid circle) and contemporary sample (open
circle). Also shown is the mean (±SE) numbers of Ap and Ai in
simulated populations with effective size Ne = 26, 50, 100, 500 and
1500. Genetic drift alone without loss in copy number variation
cannot explain the large decline in Ai.
12
Contemporary observed
Historic observed
Contemporary simulated
Historic simulated
11
Number of individuals
10
9
8
7
6
5
4
3
2
1
0
1
2
3
4
5
6
7
8
9
10
Number of MHC alleles per individual (Ai)
Fig. 5 Number of observed greater prairie-chicken major histocompatibility complex (MHC) alleles per individual (Ai) detected
in the contemporary sample (black bars) and historic sample (grey
bars). The number of alleles per individuals was significantly lower
in the contemporary sample than in the historic sample (Mann–
Whitney test, W = 282, P = 0.0005). Also shown are the distributions of the mean (±95% CI) simulated Ai values for the historic
(grey circles) and contemporary (black circles) populations. In
simulations, the individuals have a variable number of MHC loci
(range: 1–5), the historic (prebottleneck) population has a size of
Ne = 1500 and the contemporary (post-bottleneck) population has
been through a bottleneck with Ne = 26 for t = 30 generations
(see text for details).
1853
divergent are more likely to persist, assuming they
increase the chance of recognizing new pathogens
(Hedrick et al., 2002; Hedrick, 2003). However, the
contemporary alleles in our population were no more
divergent from each other than the historic alleles
(Fig. 3), similar to results from a previous simulation
study (Ejsmond & Radwan, 2009). This finding suggests
that the loss of MHC variation is governed by stochastic
processes rather than by selection.
The MHC appears to follow a birth-and-death model
of evolution, in which new genes arise via gene
duplication and genes may be lost by deletion (Nei et al.,
1997; Nei, 2005). This results in variation in MHC gene
number between haplotypes both within (Hosomichi
et al., 2006; Doxiadis et al., 2010) and across species
(Trowsdale, 1995; Kelley et al., 2005). In the Japanese
quail (Coturnix japonica), Hosomichi et al. (2006) identified between one and six transcribed class II B loci per
haplotype. Our RFLP analysis indicates that MHC class II
locus number is variable in greater prairie-chickens.
Therefore, drift could have caused an increase in the
relative frequency of haplotypes with fewer loci and a
decrease in individual level MHC variability (Fig. 5).
Additionally, identical alleles may have been present at
multiple loci, as has been found in turkeys (Chaves et al.,
2010) and guppies (McMullan, 2010), and drift could
have caused multiple loci to become homozygous for the
same alleles.
The roles of selection and drift in generating CNV
are just beginning to be studied, particularly in natural
populations (Nozawa et al., 2007; Schrider & Hahn,
2010). MHC copy number has been reduced and become
fixed among inbred strains of laboratory mice (Cutler
et al., 2007) and isolated populations of ornamental
guppies (Poecilia reticulata; van Oosterhout et al., 2006b).
In the Tasmanian devil (Sarcophilus harrisii), which is
endangered as a consequence of a contagious cancer,
MHC class I diversity is low and most of the variation is
attributable to CNV, rather than sequence polymorphism
(Siddle et al., 2010). Interestingly, Siddle et al. (2010)
speculated that devils with a lower number of gene
copies might be most resistant to the cancer, possibly as
a consequence of a selective sweep. Our study suggests
that a reduction in the number of gene copies may also
occur simply through drift in the absence of any
selection.
In conclusion, there was a significant loss of MHC class
II B diversity following a population bottleneck in this
Wisconsin population of greater prairie-chickens. The
bottleneck decreased both the number of alleles in the
population (Ap) and the average number of alleles
present in individuals (Ai). Computer simulations suggest
that these results can be explained by drift acting on the
within- and between-locus gene diversity, as well as CNV
between haplotypes. The loss of alleles could have been
caused by fixation of the same allele across multiple loci
or a reduction in the number of haplotypes with high
ª 2011 THE AUTHORS. J. EVOL. BIOL. 24 (2011) 1847–1856
JOURNAL OF EVOLUTIONARY BIOLOGY ª 2011 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY
1854
J . A . E I M E S E T A L.
gene copy numbers. Changes in CNV at the MHC are
related to disease in humans and laboratory animals
(Aldhous et al., 2010; Cahan & Graubert, 2010; Ye et al.,
2010), but they are relatively unstudied in wild populations and could be an important contributor to population viability.
Acknowledgments
Funding was provided by grants from the Research
Growth Initiative, University of Wisconsin-Milwaukee
Graduate School, National Science Foundation (DEB0948695), American Ornithologists’ Union, The American Museum of Natural History, and a Ruth Walker
Research Award from the University of WisconsinMilwaukee. We thank J. Toepfer and R. Bellinger for
tissue collection and processing, and C. Wimpee for
advice and assistance in the laboratory.
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Supporting information
Additional Supporting Information may be found in the
online version of this article:
Figure S1 The mean (±95% CI) observed number of
MHC alleles per population vs. the mean (±95% CI)
observed number of MHC alleles per individual for the
ª 2011 THE AUTHORS. J. EVOL. BIOL. 24 (2011) 1847–1856
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J . A . E I M E S E T A L.
historic sample (solid circle) and contemporary sample
(open circle).
Figure S2 The relative loss in number of alleles per
individual (Ai) for MHC (solid circles) and microsatellites
(open circles) expressed as Ai divided by mean historic Ai
of the corresponding gene.
Figure S3 RFLP ⁄ Southern blot of eight contemporary
greater prairie chickens.
Table S1 Variation in MHC class II B sequences of
greater prairie-chickens before (Historic) and after (Contemporary) the population bottleneck.
As a service to our authors and readers, this journal
provides supporting information supplied by the authors.
Such materials are peer-reviewed and may be reorganized for online delivery, but are not copy-edited
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addressed to the authors.
Received 23 February 2011; revised 10 April 2011; accepted 28 April
2011
ª 2011 THE AUTHORS. J. EVOL. BIOL. 24 (2011) 1847–1856
JOURNAL OF EVOLUTIONARY BIOLOGY ª 2011 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY