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BMC Evolutionary Biology
BioMed Central
Open Access
Research article
A comparison of variation between a MHC pseudogene and
microsatellite loci of the little greenbul (Andropadus virens)
Andres Aguilar*1,2,3, Thomas B Smith1,2 and Robert K Wayne1,2
Address: 1Department of Ecology and Evolutionary Biology, University of California, Los Angeles, CA 90095 USA, 2Center for Tropical Research,
Institute of the Environment, 1609 Hershey Hall, University of California, Los Angeles, CA 90095 USA and 3Southwest Fisheries Science Center &
Department of Ocean Sciences, 110 Shaffer Road, University of California, Santa Cruz, California 95060 USA
Email: Andres Aguilar* - [email protected]; Thomas B Smith - [email protected]; Robert K Wayne - [email protected]
* Corresponding author
Published: 13 September 2005
BMC Evolutionary Biology 2005, 5:47
doi:10.1186/1471-2148-5-47
Received: 15 March 2005
Accepted: 13 September 2005
This article is available from: http://www.biomedcentral.com/1471-2148/5/47
© 2005 Aguilar et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Background: We investigated genetic variation of a major histcompatibility complex (MHC)
pseudogene (Anvi-DAB1) in the little greenbul (Andropadus virens) from four localities in Cameroon
and one in Ivory Coast, West Africa. Previous microsatellite and mitochondrial DNA analyses had
revealed little or no genetic differentiation among Cameroon localities but significant differentiation
between localities in Cameroon and Ivory Coast.
Results: Levels of genetic variation, heterozygosity, and allelic diversity were high for the MHC
pseudogene in Cameroon. Nucleotide diversity of the MHC pseudogene in Cameroon and Ivory
Coast was comparable to levels observed in other avian species that have been studied for variation
in nuclear genes. An excess of rare variants for the MHC pseudogene was found in the Cameroon
population, but this excess was not statistically significant. Pairwise measures of population
differentiation revealed high divergence between Cameroon and Ivory Coast for microsatellites
and the MHC locus, although for the latter distance measures were much higher than the
comparable microsatellite distances.
Conclusion: We provide the first ever comparison of variation in a putative MHC pseudogene to
variation in neutral loci in a passerine bird. Our results are consistence with the action of neutral
processes on the pseudogene and suggest they can provide an independent perspective on
demographic history and population substructure.
Background
Portrayed as the paradigm of neutral evolution [1], pseudogenes are thought be free of selective forces that constrain functional genes and this single feature should
make pseudogenes highly attractive for population
genetic studies. Pseudogenes may be more appealing than
introns for population genetic studies, as introns may be
closely linked to functional gene regions [2] and therefore
may often be under the influence of selection [3]. Though
pseudogenes have been the focus of molecular evolutionary studies at the species level, there is a paucity of
research that utilizes them for analysis of populations [see
[4,5]]. The main reason for the lack of pseudogenes in
population level studies may be that few have been isolated for non-model taxa.
Levels of population differentiation and variability
depend on the type of molecular marker used. Modes of
Page 1 of 7
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BMC Evolutionary Biology 2005, 5:47
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Table 1: Observed (Ho) and expected (He) heterozygosities, allelic richness (A) for the Anvi-DAB1 and 6 microsatellite loci. Mean
allelic richness (A) is give for the 6 microsatellite loci. DNA sequence polymorphism statistics are reported for the Anvi-DAB1 gene
(nucleotide diversity – Π; Tajima;s D – D; Fu and Li's F* – F).
Microsatellites
He
Ho
Cameroon
Luna
Nkwouak
Ndibi
Wakwa
Ivory Coast
0.51
0.70
0.62
0.60
0.42
0.62
0.68
0.62
0.66
0.55
A
Ho
He
A
6.8
6.8
6.4
7.1
5.5
0.38
0.65
0.71
0.30
0.41
0.57
0.77
0.79
0.69
0.71
5.0
5.7
7.1
5.7
4.7
inheritance [6], mutation rate [7], mutation models [8,9],
recombination [10], and natural selection [11] are important factors that can affect estimates of genetic variability,
and consequently measures of population differentiation
[12]. Microsatellites are 2–5 base pair (bp) repetitive elements found throughout eukaryotic genomes and are
hypervariable genetic markers that are commonly used in
molecular genetic studies of natural populations [7]. The
use of nuclear sequences in population genetic studies is
becoming more common in evolutionary studies [13-15].
However, nuclear sequences are often not as attractive for
population genetic studies as they generally have much
lower mutation rates than microsatellite loci and consequently are less variable. Most recent population genetic
studies have utilized non-coding nuclear markers such as
microsatellites or nuclear length variants such as amplified fragment length polymorphisms [15-17,19].
The little greenbul (Andropadus virens) is a small passerine
that inhabits rainforests in Sub-Saharan Africa [20]. Previous research on the little greenbul with di- and tetranucleotide microsatellite loci has revealed extensive gene
flow among Cameroon localities [21,22] and showed
Cameroon and Ivory Coast populations to be genetically
distinct [22]. Analysis of mitochondrial DNA control
region variation found Cameroon and Ivory Coast populations define two distinct sequence clades [23]. These
phylogeographic units correspond to putative rainforest
refugia in lower (Cameroon) and upper (Ivory Coast)
Guinea [23,24].
We assessed genetic variation in Anvi-DAB1, a putative
MHC pseudogene in the little greenbul. This designation
was based on the presence of frame-shift mutations
within the reading frame of exon 2 (Aguilar et al., in
review). Genetic variation in Anvi-DAB1 should be correlated with that of neutral loci. To test this prediction, we
compared genetic variation in Anvi-DAB1 to variation in
six microsatellite loci in little greenbuls from Cameroon
and Ivory Coast.
Anvi-DAB1
k
S
11
5
8
11
6
6
13
5
9
9
6
9
Π
D
F*
0.007
0.006
0.007
0.007
0.006
0.007
-0.84
-0.03
-0.36
-0.82
-0.36
-0.76
-1.42
0.37
-1.05
-1.86
-0.83
-0.05
Results
We sequenced 16 individuals from Ivory Coast and 55
individuals from Cameroon for variation in the AnviDAB1 MHC gene. A total of 17 alleles were found (Table
1) and three alleles, Anvi-DAB1*07, Anvi-DAB1*08, and
Anvi-DAB1*14, were shared between Cameroon and
Ivory Coast. Eleven of 17 alleles were unique to Cameroon and three were unique to Ivory Coast. Cameroon
exhibited much more allelic diversity than Ivory Coast for
Anvi-DAB1 (Table 1). An allele previously found containing a frame shift mutation (Anvi-DAB1*05) was found at
a frequency of 0.23 in Nkwouak and 0.03 in Ndibi.
Observed heterozygosity (Ho) for the Cameroon sites varied from 0.30 (Wakwa) to 1.0 (Tibati) for Anvi-DAB1
(Table 1). Two of the Cameroon populations, Ndibi and
Wakwa, exhibited significant deviations from H-W equilibrium (p < 0.05) for the Anvi-DAB1 locus. Per site nucleotide diversity (π) for Cameroon and Ivory Coast was
0.007 and 0.004 (Table 1). The number of segregating
sites (S) for the 14 and 4 alleles found in Cameroon and
Ivory Coast was 13 and 3, respectively (Table 1). Within
Cameroon, the Ndibi site possessed the greatest number
of alleles (k = 11) and nucleotide diversity was 0.006 or
0.007 at each site (Table 1).
Tajima's D and Fu and Li's F* were both negative for the
pooled Cameroon (D = -0.885 and F = -1.330; Table 1)
and Ivory Coast sample (D = -0.431 and F* = -0.798; Table
1). However, these values were not significantly different
from zero (p > 0.1). All of the sites sampled possessed negative values of Tajima's D (Table 1) whereas all but one of
the sites had a negative value for Fu and Li's F* (Luna;
Table 1). None of the values for Tajima's D or Fu and Li's
F* were significantly different from zero.
Pairwise measures of population divergence based on
Anvi-DAB1 and microsatellite data were in general agreement (Table 2). For microsatellite loci and Anvi-DAB1,
Ivory Coast had the highest degree of differentiation from
all other populations (Table 2). All pairwise FST measures
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Table 2: Measures of pairwise population differentiation for the 6 microsatellite loci (above diagonal) and for Anvi-DAB1 (below
diagonal). For the Anvi-DAB1 FST measures, the numbers on top are based on allelic data and the numbers on bottom are based on
sequence data (see Methods). Numbers in bold indicate measures that are significantly different from zero (see Methods).
Luna
Luna
Nkwouak
Ndibi
Wakwa
Ivory Coast
Nkwouak
Ndibi
Wakwa
Ivory Coast
0.036
0.039
0.036
0.037
-0.041
0.027
0.301
0.285
0.016
-
0.021
0.027
0.0
0.008
0.080
0.101
0.019
0.034
-0.003
0.001
0.229
0.214
-
0.003
0.084
-0.024
0.016
0.168
0.196
-
0.077
0.191
0.248
-
Table 3: Results of the linkage disequilibrium test for ongoing gene flow versus recent divergence among Cameroon sites for the AnviDAB1 locus. Reference group for comparison is listed in rows. NA indicated that less than four pairs of sites were compared and
means were not taken.
Reference Population
Luna
Ndibi
Nkwouak
Wakwa
Luna
Ndibi
Nkwouak
Wakwa
1.167
0.867
1.167
1.167
0.381
NA
1.167
0.762
NA
1.167
0.733
0.400
-
for Anvi-DAB1allelic data between Ivory Coast and Cameroon site were statistically greater than zero (Table 2).
Likewise, all four pairwise FST measures between Ivory
Coast and Cameroon sites for the six microsatellite loci
were significantly greater than zero (Table 2). However,
FST values between Cameroon and the Ivory Coast were
larger for Anvi-DAB1 (allelic: 0.222 +/- 0.06 [s.d.];
sequence: 0.236 +/- 0.04 [s.d.]) than for the six microsatellite loci (0.086 +/- 0.01 [s.d.]). In contrast, the mean FST
for all pairwise comparisons within Cameroon is lower
for Anvi-DAB1 allelic (0.004 +/- 0.03 [s.d.]) than for microsatellite data (allelic: 0.012 +/- 0.01 [s.d.]) whereas
sequence data has the highest levels of FST (0.026 +/- 0.01
[s.d.]). The FST measures from microsatellites were not significantly different from Anvi-DAB1 allelic (p = 0.23; t = 1.24) and Anvi-DAB1 sequence FST (p = 0.08; t = -1.82).
However, all pairwise values within Cameroon are low
suggesting high rates of gene flow. The results of the linkage disequilibrium (LD) test for recent divergence versus
ongoing gene flow are all positive, indicating that gene
flow is most likely occurring among the sampled sites
within Cameroon (Table 3). Allelic richness was not
highly correlated between the two marker types (r2 =
0.11).
Pairwise values of FST for allelic and sequence Anvi-DAB1
information were highly correlated (r2 = 0.944) and both
statistics were correlated with values of FST for microsatellite loci (r2 = 0.889, r2 = 0.876, respectively). However,
none of these relationships were significantly based on
the Mantel's test likely reflecting the small number of
matrix entries (n = 4). The Mantel's test was also preformed omitting the pairwise measures from Lamto, and
a non-significant positive correlation was still found (r2 =
0.864; p = 0.167).
Population level relationships based on genetic distance
measures varied with distance measure used and with
locus type (Figure 1). All neighbor-joining trees showed
that Ivory Coast is divergent from the Cameroon sites
(Figure 1). However, high bootstrap support distinguishing Ivory Coast from Cameroon is only evident in the tree
constructed using DS with Anvi-DAB1 sequence data (Figure 1B). There was not any support within trees or consistency among trees with regard to the relationships among
the Cameroon sites (Figure 1).
Discussion
We have shown that measures of population differentiation for a MHC pseudogene, Anvi-DAB1, are not significantly differently different from those of six unlinked
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A
Lamto
100
Ndibi
Wakwa
Nkwouak Luna
0.1
B
Lamto
Wakwa
Luna
0.1
Ndibi
Nkwouak
Figure 1
from using Nei's standard trees for the five A. allelic data
tions the neighbor-joining(A) and (Ds) for the virens populaUnrootedAnvi-DAB1 locus distance 6 microsatellite loci (B)
Unrooted neighbor-joining trees for the five A. virens populations using Nei's standard distance (Ds) for the allelic data
from the Anvi-DAB1 locus (A) and 6 microsatellite loci (B).
Bootstrap support above 50% is shown (see methods).
neutral microsatellite loci. A high degree of differentiation
for the Anvi-DAB1 pseudogene as measured by FST was
found between sites in Cameroon and Ivory Coast, a
result that has been previously found in studies that utilized microsatellite loci [22], and mitochondrial DNA
[23]. Within sampled Cameroon sites, high levels of gene
flow, as evidenced by low pairwise FST measures, was
found for the Anvi-DAB1 locus. This again is concordant
with results from microsatellite and mitochondrial DNA
[21-23].
Evidence for Anvi-DAB1 being a pseudogene is based on
the observation that an allele containing a frame-shift
mutation (Anvi-DAB1*05) is homozygous in three individuals, nearly equal rates of synomonous and nonsynomonous substitutions, absence from a survey of
transcribed genes in the little greenbul, high divergence in
sequence type when compared to classical transcribed
MHC sequences, and a lack of conserved MHC class II vertebrate amino acid residues (Aguilar et al., in review).
Pseudogenes are rarely used in studies of natural populations, yet they may be valuable tool for quantifying
genetic variation and differentiation. For example, polymorphism at the psGBA pseudogene in humans was
found to be concordant with previous studies of neutral
genes [5]. Nucleotide diversity in Anvi-DAB1 was found
to be low, and was similar to that found for another avian
MHC pseudogene (Came-DAB1: π = 0.03 [38]). This level
of polymorphisms is also low compared to functional
MHC genes isolated from other birds and vertebrate taxa
[25]. However, study of a human MHC class I pseudogene
(HLA-H) found elevated levels of genetic variation, and
this was attributed to the linkage of HLA-H to functional
HLA loci [26]. Therefore, although pseudogenes maybe
useful loci in population genetic studies, comparison of
their genetic variability to neutral markers is needed to
determine if levels of genetic variability may be influenced
by selection.
Negative D and F* values suggest an excess of rare mutants
in the pooled Cameroon population though these values
were not statistically significant. Similarly, all individual
sites possessed negative values for Tajima's D and Fu and
Li's F* but again these were not statistically significant. An
overabundance of rare mutants in a sample can be caused
by recent population expansions [27,28], selective sweeps
[29,30], or from pooling samples [31,32]. Further sampling of sites within Cameroon and Ivory Coast, the inclusion of other loci, and establishing fine-scale patterns of
population structure will elucidate the significance of the
excess of rare mutants for the Anvi-DAB1 gene.
Levels of differentiation were high and significant
between Ivory Coast and Cameron for the MHC pseudogene (allelic and sequence data) and microsatellite loci sug-
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BMC Evolutionary Biology 2005, 5:47
gesting long-term isolation. In contrast, low mean FST for
within Cameroon comparisons, for both Anvi-DAB1 and
microsatellites, is indicative of a high level of gene flow
among the sampled Cameroon sites. The results of the LD
test, coupled with the low FST values, indicate that gene
flow is still occurring within the samples Cameroon sites.
The positive correlation of pairwise measures of population differentiation for Anvi-DAB1 and the six microsatellite loci and the lack of significant differences in levels of
within and between population variation supports drift
and migration are the primary influences on the observed
genetic variation at Anvi-DAB1.
The lack of any significant correlation between allelic richness measures from microsatellites and the MHC gene
could be due to the small-observed differences in allelic
richness across populations and/or the low number of
populations sampled. High gene flow, as well as large
effective population sizes, could account for low discrepancy in allelic richness. To determine if drift is an important factor affecting allelic richness at the two marker sets
we would need to sample populations with low effective
population size, where we would expect a concordant
decrease in microsatellites and MHC variation.
Null alleles could account for the deficiency of heterozygotes observed in many samples. Other factors that could
contribute to the deviations from Hardy-Weinberg expectations include sampling artifacts, family structure, and
non-random mating. Further work that could elucidate
the role of null alleles in generating the observed pattern
in heterozygosity would include the re-designing of PCR
primers and the use of less stringent PCR conditions.
However, such modifications could lead to the amplification of non-orthologous closely related loci.
The unrooted neighbor-joining dendrograms showed that
Ivory Coast was topologically distinct from Cameroon
localities for both Anvi-DAB1 and microsatellite data. The
main difference in the neighbor-joining trees was the
degree of genetic distance observed for both marker types,
as the Anvi-DAB1 dendrogram constructed with Ds
showed much lower differentiation between Ivory Coast
and Cameron than the dendrogram based on microsatellite loci (Figure 1). This difference is most likely due to
both the limited sample from Ivory Coast and the effect of
using a single locus. Unrepresentative allele frequencies as
well as the biases associated with a single locus might suggest the average distance measures based on the six microsatellite loci more accurately reflect population history.
The observed genetic differences between Cameroon and
Ivory Coast little greenbul populations are a result of geographic isolation two million years ago [23]. Reciprocally
monophyletic clades representing the Upper and Lower
http://www.biomedcentral.com/1471-2148/5/47
Guinea refugia were found using mitochondrial NADH
dehydrogenase subunit 2 sequence data [23]. The corrected sequence divergence between the two clades was
4.7%, and the estimated time of gene divergence was 2
mya. A more rigorous analysis of 10 microsatellite loci
revealed elevated FST between Cameroon and Ivory Coast
and these two population groups were also recovered
using a Bayesian clustering approach [22]. Examination of
population differentiation within Cameroon sites has
revealed low levels of gene flow among lowland forest
sites [21-23]. Similar results, based on Anvi-DAB1, indicate that this locus is reflecting historical population separations and the contemporary effects of gene flow.
Conclusion
Comparable measures of population differentiation and
similarity in population level phylogenetic trees indicate
that the processes that are operating on Anvi-DAB1 are
analogous to those acting on the typed microsatellite loci.
These results suggest that pseudogenes may be useful as
molecular tools in population level studies. However, several pseudogenes should be used to decrease locus specific
effects and comparisons should be made to other nuclear
loci that are unlikely to be under selection (such as
microsatellites) so that the influences of selection on
pseudogenes can be evaluated. Though pseudogenes may
not be as readily available for use, they may become more
common as researchers continue large scale sequencing
projects on non-model organisms (see [38] and others).
Methods
Little greebul blood samples were collected by T. B. Smith
in Cameroon and Ivory Coast. A total of 71 individuals
were genotyped at Anvi-DAB1, 55 were from Cameroon,
and 16 from Ivory Coast. From Cameroon, localities Luna
(n = 8), Nkwouak (n = 20), Ndibi (n = 17), and Wakwa (n
= 10) were sampled. The lone site from Ivory Coast was
Lamto (n = 16) (see [22] for locality detail). DNA was
extracted from blood samples by digestion with proteinase-K followed by phenol-choloroform extraction [33] or
by use of a commercially available DNA extraction kit
(Qiagen Inc.). The microsatellite dataset used here was
from Smith et al. [22] and contained scores on six tetranucleotide microsatellite loci.
The nuclear pseudogene used was the Anvi-DAB1 MHC
gene isolated from the little greenbul (Aguilar et al. in
review). SSCP [34] was used to identify unique alleles.
Briefly, both primers were end-labeled with α-32P [33]
and these radio-labeled primers were used in a PCR reaction with the following conditions: 10 ng genomic DNA,
1 mM of each primer (Anviex2F.1 [TGC CAT GGA CGC
TTA CAC T] and Int2R.1 [CCG AGG GGA CAC GCT CT]
[35], 1 mM dNTPs, 1 x PCR buffer (Sigma), 0.5 units of
Taq polymerase (Sigma) and 1.0 mM MgCl2 in a 25 µL
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BMC Evolutionary Biology 2005, 5:47
reaction volume. These primer pairs target a 267 bp portion of exon 2 from the Anvi-DAB1 pseudogene. Reactions
were run with the following temperature cycles: an initial
3 min denaturing step at 94°C, 30 sec at 94°C, 30 sec at
58°C, 30 sec at 72°C, and a final 5 min extension at 72°C.
Five µL of the PCR reaction were mixed with two µL of
stop solution (95% formamide and 0.05% bromophenol
blue), heated for 5 min at 95°C then cooled immediately
on ice. Two µL of this cocktail were loaded into a 5% nondenaturing polyacrylamide gel containing 5% glycerol (v/
v) and run at 20 W for 8–10 hours at room temperature.
Gels were transferred to 3 M Whatman paper, dried, and
exposed to autoradiographic film for 12–48 hours
(depending upon activity of 32P). Unique alleles, as identified from SSCP, were isolated from dried gels and reamplified [34]. PCR products were separated on 1% agarose gels, isolated, and sequenced using forward and
reverse primers. Alleles having the same confirmation
were sequenced from multiple individuals to assure identity in sequence. Sequencing was done either on an ABI
377 or a Beckman CEQ2000 following manufacture's protocols.
Sequences
were
then
imported
into
SEQUENCHER (GeneCodes, Inc.) and aligned. Sequences
have been deposited in [Genbank: AY437894-AY437899;
DQ113429-DQ113439].
Observed and expected heterozygosity for Anvi-DAB1 and
the microsatellite loci were calculated using GENETIX
[36]. Deviations from Hardy-Weinberg equilibrium were
assessed with the exact test implemented in GENEPOP
[37]. We also calculated Tajima's D [38] and Fu and Li's F*
[39] for each site and pooled samples from Cameroon to
assess any deviations from neutral evolution using DNAsp
[40]. Both Tajima's D and Fu and Li's F statistics test for
deviations from neutrality by examining the frequency
spectra of mutations in the sample. Statistical significance
from neutrality was assessed for Tajima's D and Fu and
Li's F* using 10,000 coalescent simulations in DNAsp
[40].
http://www.biomedcentral.com/1471-2148/5/47
We used the approach of Machado et al. [44] to distinguish between ongoing gene flow and recent divergence
among the Cameroon populations. This method compares the difference in LD between all shared polymorphisms (DSS) between two populations and the LD from
pairs of nucleotide sites that are shared between populations and exclusive to one reference population (DSX).
This difference has previously been reported as x. LD, was
estimated as D', and x were estimated with the program
SITES [45]. Ongoing gene flow is expected to produce positive x values, while the lack of gene flow will produce x
values close to zero [44].
Unrooted neighbor-joining dendrograms also were constructed from genotype data using Nei's standard genetic
distance (Ds) [46] calculated between each population
pair with the program POPULATIONS [47]. Five hundred
bootstrap replicates were preformed to assess the support
for branching nodes.
Authors' contributions
This work started out of a collaborative effort between the
laboratories of TBS and RKW. AA designed the study,
carried out the laboratory work and statistical analyses,
and drafted the manuscript. TBS collected samples and
TBS and RKW participated in the design and drafting of
the manuscript. All authors read and approved the final
manuscript.
Acknowledgements
We thank the Government of the Republic of Cameroon for permission to
conduct the field research. This research was supported by grants from the
National Geographic Society, National Environmental Research Council,
Royal Society, and the National Science Foundation grants DEB-9726425
and IRCEB9977072 to T.B.S, an Academic Senate grant (UCLA) to A.A. and
R.K.W. and an NSF-DDIG to A.A. We thank J. Pollinger for laboratory
assistance and S. V. Edwards and an anonymous reviewer for helpful comments on the manuscript.
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