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Paucity of Genetic Variation at an MHC Class I Gene in Massachusetts
Populations of the Diamond-backed Terrapin (Malaclemys terrapin): A Cause for
Concern?
Author(s): S. Shawn McCafferty , Amanda Shorette , Julia Simundza , and Barbara Brennessel
Source: Journal of Herpetology, 47(2):222-226. 2013.
Published By: The Society for the Study of Amphibians and Reptiles
DOI: http://dx.doi.org/10.1670/11-069
URL: http://www.bioone.org/doi/full/10.1670/11-069
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Journal of Herpetology, Vol. 47, No. 2, 222–226, 2013
Copyright 2013 Society for the Study of Amphibians and Reptiles
Paucity of Genetic Variation at an MHC Class I Gene in Massachusetts Populations of the
Diamond-backed Terrapin (Malaclemys terrapin): A Cause for Concern?
S. SHAWN MCCAFFERTY,1 AMANDA SHORETTE, JULIA SIMUNDZA, AND BARBARA BRENNESSEL
Department of Biology, Wheaton College, Norton, Massachusetts 02766 USA
ABSTRACT.—The Diamond-backed Terrapin (Malaclemys terrapin), endemic to the brackish marshes of the eastern and Gulf of Mexico coasts
of the United States, is a threatened species in Massachusetts with populations suffering drastic declines in the late 19th and early 20th
centuries. To assess the potential effects of population bottlenecks on contemporary levels of genetic variation, we analyzed 219 bp of a major
histocompatibility complex class I gene region (MHCI) by direct sequencing and single-strand conformational polymorphism analysis and six
microsatellite loci from three locations around Cape Cod, Massachusetts. No variation was found at the MHCI, despite finding appreciable
levels of variation within and among populations at the microsatellite loci. We discuss alternative explanations for these results, and we propose
that the lack of variation at the MHCI may be due to the effects of selection rather than demographic changes in terrapin populations.
A prevailing view in conservation genetics is the importance
of preserving levels of genetic variation within populations of
threatened and endangered species. Decreases in population
size and changing demographics can result in a loss of genomic
variation, potentially decreasing mean population fitness and
viability (Hedrick, 2001; Allendorf and Luikart, 2007). This
effect is particularly true for genes that are thought to be of
adaptive importance where low levels of variation may have
profound implications on the long-term adaptability of a species
to changing environmental conditions (Moritz, 2002; Kohn et
al., 2006; Gebremedhin et al., 2009).
Genes at the major histocompatibility complex (MHC) have
become increasingly popular targets for studying levels of
adaptive molecular variation in nonmodel organisms (Bernatch-
ez and Landry, 2003; Hedrick, 2004; Sommer, 2005; Acevedo-
Whitehouse and Cunningham, 2006; Piertney and Oliver, 2006).
With an increasing awareness of the threat of emergent
pathogens on wildlife populations (Daszak et al., 2000; Morens
et al., 2004), the MHC loci have become important candidate
gene regions for studying the affects of changing population
sizes on levels of adaptive molecular variation in threatened and
endangered species.
The Diamond-backed Terrapin (Malaclemys terrapin) is a
brackish water–adapted terrapin with a wide distribution
ranging from Cape Cod, Massachusetts, to Corpus Christi,
Texas (Ernst et al., 1994). Because of a directed fishery dating
from before the 1800s, excessive habitat loss, by-catch from crab
fisheries, predation, and road mortalities, terrapin populations
in Massachusetts are considered threatened and are highly
regulated (e.g., Brennessel, 2006). Here, we report the results of
a study estimating the level of variation at the MHC class I gene
region (MHCI) within and among local Cape Cod populations
of the Diamond-backed Terrapin by using direct sequencing and
single-strand conformational polymorphism (SSCP) analysis.
We compare the level of variation found at MHCI to estimated
levels of variation at six microsatellite loci to infer the effects of
population bottlenecks on levels of adaptive genetic variation.
MATERIALS AND METHODS
Fifty-nine samples were collected from terrapins at three
breeding sites in Massachusetts, the northern limits of the
species (Fig. 1). Blood samples were collected from Wellfleet
Harbor (n = 22); Sandy Neck, Barnstable (n = 7); and Sippican
Harbor, Marion (n = 22) by syringe and preserved on Whatman
FTA cards (Whatman Inc., Piscataway, NJ). An additional eight
individuals from Sandy Neck were sampled using tail clips
stored at -808C. Whole genomic DNA was extracted using the
QIAmp DNA Blood Mini-Extraction kit or the QIAmp
DNAeasy kit (QIAGEN, Valencia, CA).
A 219-bp fragment of the MHCI was amplified from 34
samples (13 Wellfleet, 4 Sandy Neck, and 17 Buzzard’s Bay) by
using the primers PSMHCIa2-f (5 0-CAGCTGTATGGGTGT-
GATCT-30) and PSMHCIa2-r (50-TTTAAGCCACTCGATGC-30)
designed from Pelodicus sinensis (GenBank accession AB022885).
Polymerase chain reaction (PCR) was performed in 25-ll
reactions by using 2.0 ll of DNA, 0.5 lM of each primer, and
GoTaq Green Master Mix (Promega) under the following
conditions: 2-min denaturing at 948C; 35 cycles of 948C
denaturing, 568C annealing, and 728C extension for 30 sec each;
728C elongation for 4 min. The resulting PCR products were
cleaned (AMPure PCR Purification kit, Agencourt Bioscience,
Beverly, MA) and directly sequenced in both directions using
the DTCS Quickstart kit (Beckman Coulter, Fullerton, CA). The
resulting sequencing products were cleaned by ethanol precip-
itation and analyzed on a CEQ8000 Genetic Analyzer (Beckman
Coulter) following the manufacturer’s recommendations. The
resulting sequences were edited and aligned using Sequencher
4.2 (Gene Codes Corporation, Ann Arbor, MI). An additional 25
individuals (9 from Wellfleet, 11 from Sandy Neck, and 5 from
Sippican Harbor) were amplified as described above, cleaned
using EXOSAP-IT (Invitrogen, Carlsbad, CA), and analyzed by
SSCP. Cleaned PCR reactions were denatured in a formamide-
NaOH solution at 958C for 5 min, snap-cooled for 3 min, and
separated on a GMA gel by using an Origins system (Elchrom
Scientific AG, Cham, Switzerland) following the manufacturer’s
recommendations. The resulting fragment patterns were visu-
alized using SYBER Green II. Two control samples of known
sequence were run on each SSCP gel, and any samples that were
not clearly resolved were rerun with appropriate controls. Ten
samples that were sequenced previously for MHCI also were
analyzed using SSCP to verify the relationship between SSCP
fragment profile and DNA sequence.
The edited MHC sequences were checked for homology to
MHC by using tblastn against all vertebrate nucleotide
sequences in GenBank. A multiple sequence alignment of
terrapin MHC sequences to other known MHCI sequences
(Glaberman et al., 2008) was performed by first translating the
1Corresponding Author. E-mail: smccaffe@wheatonma.edu
DOI: 10.1670/11-069
nucleotide data into amino acid data, aligning the amino acid
data by using CLUSTALX, and then reverting the amino acid
alignment back into nucleotide data by using the online version
of TranslatorX (Abascal et al., 2010). We tested for evidence of
selection on the terrapin MHCI based on the ratio of the number
of nonsynonymous substitutions per nonsynonymous site to the
number of synonymous substitutions per synonymous site
(dN/dS) by using the program MEGA 4.1 (Kumar et al., 2008).
Site-specific tests for selection were performed based on
maximum likelihood estimates by using the online service
Datamonkey (Kosakovsky and Frost, 2005). We used the fixed
effects likelihood method incorporating the general reversible
substitution model with the phylogenetic tree inferred using
neighbor joining.
In addition, the same 59 individuals were analyzed at six
microsatellite loci (GmuB08, GmuD28, GmuD51, GmuD55,
GmuD87, and GmuD121) by using the primers described in
King and Julian (2004). Each locus was amplified individually
with only the forward primer fluorescently labeled using the
WellRead dyes D2, D3, or D4 (Beckman Coulter). PCR was
performed in 15-ll reactions by using 1.5 ll of a 1:10 dilution of
the genomic DNA, 0.5 lM of each primer, and GoTaq Master
Mix (Promega) under the following conditions: 2-min dena-
turing at 948C; 42 cycles of 948C denaturing for 45 sec, 568C
annealing for 45 sec; 728C extension for 90 sec. The resulting
fragments were separated on a CEQ8000 Genetic Analyzer
(Beckman Coulter) following the manufacturer’s recommen-
dations, and fragment sizes were determined using a Fragment
Analyzer. Genotyping errors and the presence of null alleles
were assessed using MicroChecker 2.2.3 (Oosterhout et al.,
2004). Estimates of allele frequencies, levels of heterozygosity,
tests of Hardy–Weinberg equilibrium, and estimates of Fst
were performed using GenePop version 3.4 (Raymond and
Rousset, 1995). A Baysian approach was taken to estimate the
number of populations in the data based on multilocus
genotypes by using the program STRUCTURE (Pritchard et
al., 2000). The default values for most parameters were used
with sample location as a prior based on both the admixture
and correlated allele frequency models. Three independent
runs of 1,000,000 generations, with a burn-in at 50,000
generations were conducted for each value of K (the number
of populations) from 1 to 3.
RESULTS
Thirty-four individuals from Wellfleet, Sandy Neck, and
Sippican Harbor were sequenced for 219 bp of the MHCI. A
tblastn search of a representative sequence (GenBank accession
GQ495891) had a highest match to the P. sinensis (AB185243),
with all top 100 hits corresponding to MHCI from other
vertebrates. Alignment of the M. terrapin MHCI sequence to
Gala´pagos Marine Iguana (Amblyrhynchus cristatus; EU604309)
shows that the region amplified is homologous to the MHCI a-
2 region. An amino acid alignment of the putative terrapin
MHCI to other reptiles can be found in Figure 2. We found no
evidence for heterozygosity or polymorphisms in the 34
individuals sequenced. The 25 additional samples analyzed
using SSCP also showed no evidence for variation. All
fragment patterns were invariant for all SSCP run samples
and corresponded to the fragment pattern seen in the 10
sequenced samples.
The dN/dS ratio was significantly different from 1 when
comparing terrapin MHCI to the other reptile MHCIs (Fig. 2).
We found strong evidence for the effects of purifying selection
(HA: dN < dS ; P< 0.05 for all pairwise comparisons with Green
Iguanas (Iguana iguana), Galapagos Land Iguanas (Conolophus
subcristatus), Galapagos Marine Iguanas, and Pelodiscus turtles;
P = 0.072 for comparison with Ameiva lizards) but no evidence
for positive election (Ho: dN > dS; P  0.05 for all pairwise
comparisons with other reptiles). The results from the site
specific tests for selection are also summarized in Figure 2. Two
sites showed limited evidence for positive selection, whereas 15
sites showed evidence for negative or purifying selection. Six of
these sites showed evidence for selection specifically along the
terrapin branch.
All six loci showed appreciable levels of variation within and
among populations comparable to Hauswaldt and Glenn (2005)
and Hart (2005) (Table 1). Mostly, the populations were found to
be at Hardy–Weinberg equilibrium except Wellfleet and Sandy
Neck at GmuD87 and Sippican Harbor at GmuD28. There was
no evidence for null alleles or other genotyping artifacts based
on MicroChecker, and the levels of divergence among popula-
tions were similar to that described by Hauswaldt and Glenn
(2005) and Hart (2005) (Table 1). Estimates of Fst (Table 2) show
a substantial level of divergence between the Cape Cod Bay
(Wellfleet and Sandy Neck) and Buzzard’s Bay samples
(Sippican Harbor), with a lower level of divergence between
Wellfleet and Sandy Neck. The results from the Baysian analysis
for population structure (Fig. 3) are consistent with the Fst
results, suggesting that these data are optimally structured into
two clusters (posterior probabilities: K = 1, Pr(XjK)  0.01; K =
2, Pr(XjK) > 0.999; K = 3, Pr(XjK)  0.01), a Cape Cod Bay
population consisting of Wellfleet and Sandy Neck and a
Buzzards Bay population consisting of Sippican Harbor. The
Sandy Neck locale is somewhat intermediate as evidenced by a
proportion of individuals from Sandy Neck having a high
probability of falling into the Sippican cluster (Table 3; 3 of 15
individuals have an assignment probability of <0.6 to the Cape
Cod Bay population). This result may be due to relatively recent
migration between Buzzard’s Bay and Sandy Neck.
FIG. 1. Sampling location of M. terrapin. (1) Wellfleet Harbor, (2)
Sandy Neck, Barnstable, and (3) Sippican Harbor, Marion (Buzzards
Bay).
MHC IN DIAMOND-BACKED TERRAPINS 223
DISCUSSION
We were unable to detect any variation at the MHCI based on
direct sequencing and SSCP analysis of 59 individuals derived
from three Massachusetts populations, suggesting that M.
terrapin populations in this region are genetically depauperate
at this potentially important immune locus. However, an
analysis of six microsatellite regions showed substantial levels
of variation within and among these three terrapin populations,
with sufficient variation to suggest that these three locales may
represent two distinct populations, one population in Cape Cod
Bay and the other population south of Cape Cod in Buzzard’s
Bay.
A possible explanation for the observed lack of variation at
the MHCI is that the region amplified was from a nonclassical
MHCI, a region usually characterized by low levels of
nucleotide variation (e.g., Glaberman et al., 2008), although
several studies have shown the MHCI a-2 region to be variable
in other reptiles (Madsen et al., 2000; Glaberman and Caccone,
2008; Miller et al., 2010). An alignment of M. terrapin MHCI with
other MHCI a-2 domains clearly shows that the M. terrapin
MHCI shares several key conserved residues with reptiles and
other species (Fig. 2), suggesting the region sequenced may be a
classical MHCI (Kaufman et al., 1994). However, Glaberman et
al. (2008) suggest that sharing of conserved sites may not be
sufficient evidence for determining whether an MHC region is
classical or nonclassical. Repeated attempts to amplify MHCII
regions or other MHCI regions proved unsuccessful (McCaff-
erty et al., unpubl. data), and we have yet to assess tissue
expression patterns, evidence that would go far in resolving
whether we are looking at a nonclassical MHCI. Therefore, we
cannot say for certain whether the MHCI sequences presented
here are classical or nonclassical. However, this distinction may
not be a particularly important distinction because nonclassical
MHCI loci also may act as part of the innate immune system;
they only function in ways that differ from classical loci
(Glaberman and Caccone, 2008). Evidence for conserved
binding sites and purifying selection argue that the region we
are studying is an adaptive gene region and that it may be
involved in antigen binding, although perhaps in a manner that
differs from classical MHCI.
Another explanation for the lack of variation at the MHCI is
that purifying selection acted recently on this gene region, with
a lack of variation at synonymous sites due to linkage effects
(selective sweep). To test this possibility, we compared the dN/
dS ratio to other reptile MHCIs and found significant evidence
for purifying selection. Site-specific tests also suggest purifying
selection at several sites. Unfortunately, we were not able to
compare our results with other MHC gene regions in M.
terrapin, and little is known concerning levels of variation at
MHC in Testudines in general. As far as we are aware, this is the
first population study of any turtle MHC gene region.
TABLE 1. Variation at six microsatellite loci from Massachusetts
populations of the Diamond-backed Terrapin. Na, number of alleles; Ho,
observed heterozygosity; and He, expected heterozygosity.
Locus Na Ho He
Wellfleet (22)
GmuD28 7 0.792 0.766
GmuB08 3 0.417 0.398
GmuD87* 8 0.818 0.685
GmuD51 8 0.826 0.734
GmuD55 3 0.542 0.624
GmuD121 4 0.333 0.327
Sandy Neck (15)
GmuD28 6 0.500 0.592
GmuB08 4 0.500 0.679
GmuD87** 7 0.933 0.691
GmuD51 12 0.900 0.880
GmuD55 3 0.400 0.451
GmuD121 4 0.600 0.516
Sippican (22)
GmuD28*** 7 0.636 0.752
GmuB08 4 0.636 0.611
GmuD87 9 0.905 0.842
GmuD51 12 0.762 0.858
GmuD55 7 0.591 0.638
GmuD121 5 0.727 0.617
*, 0.05 > P > 0.01; **, 0.01 > P > 0.001; ***, P < 0.001; Hardy–Weinberg test.
FIG. 2. Amino acid alignment of MHCI a-2 domain. Conserved sites are marked by a D (disulfide bridge forming cysteine), S (salt bridge forming
residue), or P (conserved peptide-binding residue of antigen N- and C-terminal binding site). After Glaberman et al. (2008). Results for site specific
tests for selection also are shown with a dash (-) marking sites that show evidence for negative selection and a plus (+) for positive selection. The
probabilities resulting from the maximum likelihood test for each site for reptiles only are as follows: positive selection (42, P = 0.081; 62, P = 0.040);
negative selection (3, P = 0.015; 10, P = 0.049; 15, P = 0.018; 18, P = 0.0004; 21, P = 0.058; 22, P = 0.098; 30, P = 0.055; 35, P = 0.009; 43, P = 0.001; 47, P
= 0.016; 49, P = 0.028; 54, P = 0.053; 66, P = 0.067; 68, P = 0.040; and 69 P = 0.010). Sites 15, 21, 30, 42, 47, and 66 were along the branch leading to
terrapins. Mate, Malaclemys terrapin; Pesi, Pelodiscus sinensis; Amcr, Amblyrhynchus cristatus; Cosu, Conolophus subcristatus; Igig, Iguana iguana; Amam,
Ameiva ameiva; Sppu, Sphenodon punctatus; Gaga, Gallus gallus; Hosa, Homo sapiens; Mumu, Mus musculus; Maru, Macropus rufogriseus; Trvu, Trichosurus
vulpecula; Oran, Ornithorhynchus anatinus; Xela, Xenopus laevis.
224 S. S. MCCAFFERTY ET AL.
Our results implicate the role of selection in the lack of
variation observed at MHCI. However, recent population
bottlenecks or small effective population size also may have
acted to reduce the overall level of variation in the terrapin
genome. If this were the case, then we would expect reductions
in levels of variation genome-wide, including at microsatellite
loci. However, our microsatellite results show appreciable levels
of genetic variation consistent with Hauswaldt and Glenn (2005)
and Hart (2005). In fact, levels of microsatellite variation were
sufficiently large within and among locales such that we were
able to distinguish two populations of terrapins in Massachu-
setts with limited gene flow.
Based on these observations, we propose that the observed
lack of variation found at the MHCI in M. terrapin is not due to
recent population bottlenecks or demographic changes but is
the result of natural selection acting some time in the recent
past. The presence of substantial levels of variation at the
microsatellite loci suggests that the lack of variation at MHCI
may not be reflective of the genome in general. However, this
conclusion may not necessarily be the case. It is well known that
microsatellite variation is driven by a very different mutational
process than nucleotide variation (Ellegren, 2004) and therefore
may not reflect overall levels of variation at genomic regions
other than simple sequence repeats. If this were the case, then
the lack of variation at MHCI may reflect overall low levels of
genomic variation in terrapins (e.g., Avise et al., 1992; Lamb and
Avise, 1992; Parham et al., 2008) and may not be due to the
effects of selection alone. To assess this possibility requires
studying other genomic regions unlinked to the MHC; we are
currently addressing this possibility by using random portions
of the genome anchored by retrotransposons.
Irrespective of the cause, it is reasonable to ask whether this
lack of variation at a potentially important immune locus is a
concern for the long-term viability of these populations.
Although examples can be found where low levels of MHC
variation correlate with reduced population fitness (e.g.,
Hedrick, 2001; Siddle et al., 2007), there is also evidence for
species with low variation at MHC that apparently remain
viable over the long term (Ellegren et al., 1993; Mikko et al.,
1999; Weber et al., 2004; Babik et al., 2009). Determining why
there is limited variation at this potentially adaptive gene region
is important in understanding the factors driving levels of
genetic variation in Diamond-backed Terrapins and may give
direction to the development of a sound conservation strategy
for these threatened populations.
Acknowledgments.——We thank D. Lewis, S. Wieber Nourse,
and R. Nourse for collecting the Sippican Harbor samples; P.
Auger for providing access to Sandy Neck; L. Fleck for efforts in
developing the primers used; and all the students from
Wheaton College who worked on the Terrapin Project. All
samples were collected under permit 045.07SCRA from the
Division of Fisheries and Wildlife, the Commonwealth of
Massachusetts in compliance with Wheaton College animal
care guidelines. The MHC analyses and microsatellite data
analysis were performed by A. Shorette and J. Simundza,
respectively, as part of their BIO500 honor’s research at
Wheaton College. Funding for this project was provided for
by the Wheaton College Faculty Research Grant, Mars Student
Faculty Research Collaboration funds, the Wheaton Foundation,
and the Richard White and Sons Science Fund.
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Accepted: 13 November 2011.
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