Journal of Heredity, 2014, 395–402
doi:10.1093/jhered/esv022
Brief Communication
Advance Access publication April 23, 2015
Brief Communication
Diversity of MHC DQB and DRB Genes in the
Endangered Australian Sea Lion (Neophoca
cinerea)
Quintin Lau, Natalie Chow, Rachael Gray, Jaime Gongora, and
Damien P. Higgins
From the Faculty of Veterinary Science, The University of Sydney, B14 McMaster Building, Camperdown, New South
Wales 2006, Australia.
Address correspondence to Damien P. Higgins at the address above, or e-mail: [email protected].
Data deposited at Dryad: http://dx.doi.org/doi:10.5061/dryad.n4g95
Received December 22, 2014; First decision February 19, 2015; Accepted March 25, 2015.
Corresponding Editor: Scott Baker
Abstract
Major histocompatibility complex (MHC) class II molecules have an important role in vertebrate
adaptive immunity, being responsible for recognizing, binding, and presenting specific antigenic
peptides to T lymphocytes. Here, we study the MHC class II DQB and DRB exon 2 genes of the
Australian sea lion (Neophoca cinerea), an endangered pinniped species that experiences high
pup mortality. Following characterization of N. cinerea DQB and DRB by molecular cloning, and
evaluation of diversity in pups across 2 colonies using variant screening (n = 47), 3 DQB alleles and
10 DRB variants (including 1 pseudogene allele) were identified.The higher diversity at DRB relative
to DQB is consistent with other studies in marine mammals. Despite overall lower MHC class II
allelic diversity relative to some other pinniped species, we observed similar levels of nucleotide
diversity and selection in N. cinerea. In addition, we provide support for recent divergence of MHC
class II alleles. The characterization of MHC class II diversity in the Australian sea lion establishes
a baseline for further investigation of associations with disease, including endemic hookworm
infection, and contributes to the conservation management of this species.
Subject area: Conservation genetics and biodiversity
Key words: Australian sea lion, hookworm, MHC, pinnipeds
Intensive commercial sealing has had a significant impact on population numbers of many pinniped species worldwide and Australia’s
only endemic pinniped species, the Australian sea lion (Neophoca
cinerea), is no exception. Neophoca cinerea belongs to the Otariidae
family (eared seals) and is listed as vulnerable (EPBC Act 1999, listed
2005) and Endangered (IUCN Red List, 2008), due to the species’
limited recovery post-sealing and the risk of extinction from fishery
by-catch. The estimated population size of the species is less than
15 000 individuals spread across 80 colonies (Shaughnessy et al.
2011) and 2 of the largest colonies, Seal Bay (approximately 250
pups born/breeding season) and Dangerous Reef (approximately
500 pups born/breeding season), in South Australia (Figure 1a), regularly experience pup mortality rates which exceed 30% and 40%,
respectively (Goldsworthy et al. 2009). One of the major causes of
pup mortality is haemorrhagic enteritis (unpublished data) caused
by hookworm (Uncinaria sanguinis) infection (Marcus et al. 2014a).
© The American Genetic Association. 2015. All rights reserved. For permissions, please e-mail: [email protected]
395
Journal of Heredity, 2015, Vol. 106, No. 4
396
ψ
ψ
Figure 1. (a) Location of Seal Bay and Dangerous Reef, South Australia. Following survey of N. cinerea pups, (b) DQB allele and genotype (smaller inset)
frequencies are presented due to single-locus amplification, while (c) DRB variant frequencies are presented due to multi-loci amplification.
Uncinaria sanguinis occurs endemically in 100% of N. cinerea pups
at Seal Bay and Dangerous Reef, with pups acquiring infection via
the transmammary route shortly after birth (Marcus et al. 2014b).
Similar to findings in other pinniped species whereby greater hookworm infection intensity is associated with sandy versus rocky substrates and higher density colonies (Sepúlveda 1998; Lyons et al.
2005), fluctuations in hookworm infection intensity in N. cinerea
may be mediated by colony-specific seasonal differences in host
aggregation, influencing the level of exposure to infective free-living
hookworm larvae and subsequent transmission to pups (Marcus
et al. 2014b). However, the effects of host genetics and immune
responses on hookworm infection intensity and the clinical manifestation of hookworm disease in pinnipeds are not well known.
Reduced population size from historical sealing, along with colony
isolation and strong female natal site fidelity, have led to decreased
genetic diversity of N. cinerea, including fixation of mtDNA haplotypes within many colonies and a high degree of mtDNA allele differentiation between sites (Campbell et al. 2008; Lowther et al. 2012).
While diversity in neutral genes are useful for understanding species
and population history, it is important to study adaptive loci such as
the major histocompatibility complex (MHC) to assess how balancing
selection maintains diversity relevant for immunity against pathogens.
MHC genes are highly polymorphic with an important role in disease
resistance and susceptibility (reviewed by Sommer 2005). MHC proteins recognize, bind and present antigens to the adaptive immune system; pathogen-driven selection is one of the major mechanisms that
maintain genetic diversity (Doherty and Zinkernagel 1975; Takahata
and Nei 1990, reviewed by Spurgin and Richardson 2010). MHC
class II molecules consist of noncovalently associated alpha (α) and
beta (β) chains, each of which has 2 extracellular domains designated
α1 and α2, and β1 and β2, respectively. The majority of eutherian mammals investigated to date have 2 or 3 pairs of functional class α and β
chain genes, designated DR, DP, and DQ (Beck and Trowsdale 1999).
Generally, the peptide binding region of the β1 chain shows the highest
degree of polymorphism; ultimately, diversity of this region determines
the repertoire of antigenic determinants to which an individual can
respond (Brown et al. 1993). Most of the research on genetic diversity
in relation to disease has therefore focused on the DR and DQ β1
genes encoded by the second exon (DRB and DQB, respectively).
There were previous thoughts that marine mammals have lower
levels of MHC class II diversity relative to terrestrial mammals, attributed to reduced pathogen-driven selection in a marine environment
(Slade 1992; Murray et al. 1995); more recently, balancing selection
in DQB was suggested to be more variable and weaker in cetaceans
Journal of Heredity, 2015, Vol. 106, No. 4
397
Table 1. DQB and DRB allelic and nucleotide diversity and selection in 2 Neophoca cinerea colonies compared with other pinniped
populations
Species/colony
n
DQB
na
π
Average ω (CI)
0.010 ± 0.006
0.008 ± 0.006
0.011 ± 0.007
0.004 ± 0.004
3.27 (0.89–46.62)
3
2.04 (0.28–65.19)
2
9 (1)
9 (1)
7 (1)
28
0.004 ± 0.003a
2.38 (0.40–61.55)
4
11 (1) 0.029 ± 0.015a 6.05 (0.95–47.72) 14
3
0.007 ± 0.005a
2.45 (0.76–61.27)
1
27
8
0.016 ± 0.010a
2.90 (0.29–44.07)
4
5
0.054 ± 0.028a 7.10 (1.56–40.72) 17
2
0.007 ± 0.006a
2.35 (0.28–63.31)
2
3
0.037 ± 0.020a 4.86 (0.94–36.66)
5
0.040 ± 0.021a
3.86 (0.82–32.10) 12
Australian sea lion, Neophoca cinerea
Overall
47 3
Seal Bay
27 3
Dangerous Reef
20 3
New Zealand sea lion,
87 2
Phocarctos hookeri
California sea lion,
2 8 (3)
Zalophus californianus
Galápagos sea lion,
506
Zalophus wollebaeki
Southern elephant seal, 109b
Mirounga leonina
Northern elephant seal, 110b
Mirounga angustirostris
Walrus, Odobenus
2
rosmarus
Reference
DRB
# PSS na
π
Average ω (CI)
0.025 ± 0.014
0.025 ± 0.014
0.026 ± 0.014
0.020 ± 0.010
5.03 (1.17–46.70)
9
This study
5.01 (1.24–43.74)
7
Osborne
et al. (2013)
Bowen
et al. (2002,
2004)
Lenz et al.
(2013)
Hoelzel
et al. (1999);
Weber et al.
(2004)
Sonsthagen
et al. (2014)
0.042 ± 0.022
# PSS
6.51 (2.90–37.95) 11
9
n, number of individuals; na, number of alleles (number of pseudogene alleles); average ω (CI), average dN/dS value per codon (confidence interval), # PSS = number of positively selected codon sites. Calculation of π (nucleotide diversity) excludes pseudogenes.
a
Allele frequencies not available for calculating π, thus equal frequencies were used.
b
Highest number of elephant seal individuals are presented as they differ between the 2 studies.
(Villanueva-Noriega et al. 2013). However, low diversity at the DQB
locus is not universal among marine mammals, with relatively high
diversity observed in the southern elephant seal (Mirounga leonina)
and California sea lion (Zalophus californianus) (Hoelzel et al. 1999;
Bowen et al. 2002). Nonetheless, greater allelic diversity has been identified at the DRB locus in pinnipeds (Table 1), with extensive diversity
in the New Zealand sea lion (Phocarctos hookeri) and Galápagos sea
lion (Zalophus wollebaeki) (Lenz et al. 2013; Osborne et al. 2013)
despite low DQB diversity in the former (Lento et al. 2003). A recent
study of pinniped MHC demonstrated limited phylogenetic clarity
between species, suggesting that recent allele diversification and transspecies polymorphism cannot be ruled out (Osborne et al. 2013). In
contrast to cetaceans, pinnipeds breed on land, often at high densities,
and are therefore likely to be exposed to a range of potential pathogens
from conspecifics, other wildlife and both the marine and terrestrial
environments. The fragmentation of the N. cinerea population into
small colonies suggests that diversity may be limited, but as colonies
differ in terms of size, density, and substrate, there is potential for diversity to vary among colonies. In the present study, we used previously
developed exon 2 primers (Lau et al. 2014a) to characterize N. cinerea
DQB and DRB alleles through molecular cloning, and conducted a
preliminary analysis of diversity across 2 of the largest and biogeographically diverse, N. cinerea colonies, Seal Bay and Dangerous Reef
in South Australia, using a high throughput screening method.
Materials and Methods
Sample Collection and Preparation
Characterization of exon 2 of N. cinerea DQB and DRB from
genomic DNA (gDNA) was performed using species-specific exon
2 primers (Lau et al. 2014a) in 2 stages: 1) molecular cloning and
sequencing to characterise alleles from a subset of individuals
(DQB n = 15, DRB n = 21); and 2) establishing high-throughput
methods to survey DQB and DRB genetic distribution and diversity across two N. cinerea colonies (n = 47). Blood samples were
collected from N. cinerea pups from the brachial vein into lithium
heparin or EDTA, at 2 N. cinerea colonies in South Australia: Seal
Bay, Kangaroo Island (n = 27), and Dangerous Reef, Spencer Gulf
(n = 20) (Figure 1). These individuals were selected randomly from
a larger health study (>500 pups between 2006–2013), and represented animals exhibiting a range of health status. Blood samples
were stored at −20 °C until gDNA extraction using the DNeasy
blood and tissue kit (Qiagen, Doncaster, VIC, Australia).
Identification of DQB and DRB Exon 2 Alleles
For DQB, the 214 bp exon 2 fragment was amplified from gDNA
of pups from Seal Bay (n = 10) and Dangerous Reef (n = 5), using
primers NeciDQBex2F (5′-CCCATAGTTGTGTCTGCACAC-3′)
and
NeciDQBex2R
(5′-CGAGTGCTACTTCACCAACGG-3′)
following PCR conditions described by Lau et al. (2014a). PCR
products were visualized by electrophoresis on a 1.5% agarose
gel, and bands of appropriate length excised and purified using the
UltraClean GelSpin DNA Extraction kit (Mo Bio, Carlsbad, CA)
and sequenced by Macrogen, Inc. (Seoul, South Korea). Sequences
were edited using Sequencher® (Gene Codes, Ann Arbor, MI), and
a secondary heterozygous peak in sequence chromatograms that
either exceeded 40% of the primary peak height or occurred in more
than 1 animal was considered evidence of multiple nucleotides at
that position. Based on direct sequence data, PCR products from
3 DQB heterozygotes from Seal Bay and 1 from Dangerous Reef,
incorporating all apparent polymorphisms, were subjected to cloning and sequencing to resolve the multiple DQB alleles and generate
standards for use in subsequent higher throughput diversity studies. Purified PCR products were ligated into a pCR®2.1-TOPO®
Journal of Heredity, 2015, Vol. 106, No. 4
398
cloning vector (Invitrogen, Mulgrave, VIC, Australia). From each
reaction, 5 positive clones were cultured overnight in LB broth, then
plasmid was purified using the UltraClean Mini Plasmid Prep Kit
(Mo Bio). The resulting nucleotide sequences were compared with
the direct sequence obtained from the original PCR to confirm that
all polymorphisms were accounted for.
For DRB, the 268 bp exon 2 fragment was amplified from gDNA
of pups from Seal Bay (n = 11) and Dangerous Reef (n = 10) using
primers NeciDRBEx2F (5′-CATTTCTTGCACCTGTKTAAGG-3′)
and NeciDRBEx2R (5′-CTCGCCGCTGCRCCRKGAAG-3′). As
direct sequence data indicated more nucleotide polymorphisms relative to DQB, molecular cloning was performed on all individuals in
this first stage using the same methods as for DQB, except that 20
positive clones were screened by single strand conformation polymorphism (SSCP, methods below) assay and 2 representative clones
of each SSCP “clone genotype pattern” were cultured, plasmid purified and sequenced. This approach accounted for all single-nucleotide polymorphisms identified in direct sequence results.
DQB and DRB Diversity
Diversity and distribution of DQB and DRB was surveyed in a
larger sample size (Seal Bay: n = 27; Dangerous Reef: n = 20) using
SSCP for DQB or one-strand conformation polymorphism (OSCP)
for DRB following methods detailed in Lau et al. (2013). Briefly,
SSCP analysis of DQB involved PCR-generated exon 2 amplicons
denatured at 95 °C for 5 min, rapid chilling to 4 °C, followed by
electrophoresis on a 5–10% polyacrylamide gel at 4 °C for 5 h,
then staining with SYBR® Gold (Invitrogen) and visualization with
UV illumination. For DRB, OSCP analysis was used due to a large
number of bands observed in initial SSCP screening of DRB amplicons. Conditions were the same as for DQB SSCP analysis, except
that the NeciDRBEx2F primer was 5′ phosphorylated, and lambda
exonuclease (New England Biolabs, Ipswich, MA) was used to
digest the forward strand from PCR amplicons prior to electrophoresis. For both DQB SSCP and DRB OSCP analyses, amplicons
from individuals characterized by molecular cloning were included
in each electrophoresis run as standards to infer genotypes. In addition, the inferred genotypes in many individuals not screened by
molecular cloning (n = 20) was validated using direct sequencing.
Sequence Analyses
Phylogenetic analysis among DQB and DRB sequences of N. cinerea
and other pinniped and mammalian species was performed by alignment of nucleotide sequences using ClustalW and analysis using
Neighbor-Joining method with 1000 bootstrap replicates in MEGA
6.0 (Tamura et al. 2013). The Tamura three-parameter model (with
gamma distribution) was determined as the best-fit substitution
model in MEGA 6.0 for phylogenetic reconstruction.
As it appeared that a single locus of DQB was amplified, allele
and genotype frequencies were calculated, and the Hardy–Weinberg
equilibrium tested using the Markov chain method. Expected heterozygosities (HE) were calculated using GENEPOP 4.0 (Raymond
and Rousset 1995). Allelic richness (R) was calculated using GenAlEx
vers. 6.4 (Peakall and Smouse 2012). For DRB, heterozygosity could
not be inferred due to amplification of multiple loci, and thus variant frequencies were estimated using Arlequin (Excoffier and Lischer
2010). Arlequin was also used to estimate pairwise FST as ΦST to compare DQB or DRB genetic differentiation between the 2 N. cinerea
colonies, as well as nucleotide diversity (π) in both colonies (allele
sequences and frequencies used as input data). In addition, DQB and
DRB nucleotide diversity was calculated in other pinniped species,
including the New Zealand sea lion, California sea lion, Galápagos
sea lion, walrus (Odobenus rosmarus), southern elephant seal, and
northern elephant seal (Mirounga angustirostris), with allele frequencies taken into consideration when available (Hoelzel et al.
1999; Bowen et al. 2002, 2004; Weber et al. 2004; Lenz et al. 2013;
Osborne et al. 2013; Sonsthagen et al. 2014).
To identify molecular evidence of balancing selection, we followed
the approach of Jaratlerdsiri et al. (2012) and Lau et al. (2014b) to
test for an excess of nonsynonymous (dN) to synonymous (dS) substitutions among MHC nucleotide sequences using omegaMap version
5.0 (Wilson and McVean 2006). Briefly, we performed Bayesian inference (BI) on independent alignments of NeciDQB and DRB variants
(excluding 1 identified pseudogene), measured the ratio of nonsynonymous to synonymous substitution rates (ω = dN/dS), and inferred
positively selected codon sites (ω > 1 with a posterior probability
greater than 0.9), even in the presence of recombination. Both the
selection parameter (ω) and recombination rate (ρ) were co-estimated
and allowed to vary along the sequence. For each gene, two independent omegaMap runs were performed with 5 × 105 MCMC iterations, 50 000 burn-in iterations, 100 thinning iterations, independent
model of ω, codon frequency 1/61, and 10 orderings. Results from the
independent runs were combined and graphed using R version 3.0.2
(http://www.r-project.org). The ratio of nonsynonymous to synonymous substitution rates (ω = dN/dS) and positively selected codon sites
were also determined in omegaMap from published data in other
pinniped species for comparison alongside nucleotide diversity (π).
We estimated the divergence time (T) between pairs of DQB and
DRB alleles within Australian sea lions and pinnipeds, as well as
90% confidence limits, using the number of nonsynonymous differences among alleles (kN) and the substitution rate (LNµ) per LN
nonsynonymous sites per year. We assumed a Poisson distribution of
kN, µ = 10–8 per nonsynonymous site per year (Takahata et al. 1992),
and the formula T = kN/2LNµ for a pairwise comparison. Exploration
of allele divergence provide insights into the evolutionary history
shaping MHC diversity, and here all estimations are based on nonsynonymous differences (dN) observed in the Australian sea lion and
other pinnipeds, due to the absence of synonymous differences.
Data Archiving
In fulfillment of data archiving guidelines (Baker 2013), we have
deposited the primary data underlying these analyses as follows:
• MHC class II DQB and DRB genotypes: Dryad
• DNA sequences: GenBank accessions KP127612 to KP127614
Results and Discussion
We have applied species-specific primers developed by Lau et al. (2014a)
to characterize the allelic diversity of the DQB and DRB from gDNA
originating from N. cinerea pups at 2 key colonies, sampling sites which
differ in density and substrate type. SSCP and OSCP analysis resolved
all retrieved NeciDQB and NeciDRB alleles, with agreement with
results of direct sequencing and molecular cloning. For DQB, despite
amplification coverage of 79.0% of the exon and prior scrutiny of
species-specific primer sites for potential locus-specific polymorphisms
(Lau et al. 2014a), we identified only 3 alleles in multiple independent
PCR reactions; designated NeciDQB*01 to NeciDQB*03 (GenBank
accession numbers KP127612 to KP127614). These alleles had an average 2.33% nucleotide and 3.51% amino acid differences, and a total of
6 nonsynonymous substitutions, which corresponded to polymorphic
sites in other species (Figure 2, Supplementary Figure S1 online).
Journal of Heredity, 2015, Vol. 106, No. 4
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.
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|
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NeciDQB*01
NeciDQB*02
NeciDQB*03
PhhoDQB*0101
PhhoDQB*0102
ZawoDQB*01
ZawoDQB*02
ZacaDQB*10
ZacaDQB*11
HagrDQB*03
HagrDQB*04
TutrDQB*02
MomoDQB*0201
DeleDQB*0202
HLA-DQB1
DLA-DQB1
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NeciDRB*01
NeciDRB*02
NeciDRB*03
NeciDRB*04
NeciDRB*05
NeciDRB*06
NeciDRB*07
NeciDRB*08
NeciDRB*09
PhhoDRB*0402
Phho-DRB1
ZawoDRB*01
ZawoDRB*02
ZacaDRB*01
ZacaDRB*02
DLA-DRB1
HLA-DRB1
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Figure 2. Multiple amino acid sequence alignment of the 3 DQB and 9 DRB exon 2 alleles isolated from Neophoca cinerea compared with other pinniped
and mammalian species. The conserved CD4-binding motif (RFDS) is boxed. Dots represent agreement with NeciDQB*01. Positively selected sites with
posterior probabilities >0.9 identified by BI are indicated by *. Accession numbers are New Zealand sea lion (Phocarctos hookeri) PhhoDQB 2 (doi:10.5061/
dryad.2kt7s), PhhoDRB clone 1 (KC544970); Galápagos sea lion (Zalophus wollebaeki) ZawoDQB*01 (HE663126.1) and ZawoDRB*01 and *18 (HE663098.1,
HE663115.1); California sea lion (Zalophus californianus) ZacaDQB*01 (AF503397.1) and ZacaDRB*01 and *08 (AY491456.1, AY491463.1); walrus (Odobenus
rosmarus) OdroDQB*01 (KJ004394); grey seal (Halichoerus grypus) HagrDQB*01 (HQ456122.1); southern elephant seal (Mirounga leonina) MileDQB*05
(clone 5, AF111035.1) and MileDRB1*01 (Weber et al. 2004); narwhal (Monodon monoceros) MomoDQB*0201 (U16991); beluga whale (Delphinapterus leucas)
DeleDQB*0202 (U16990); bovine (Bos taurus) BLA-DQB (NM_001034668) and BLA-DRB3 (NP_001012698.2); and human (Homo sapiens) HLA-DQB1 (NM_002123)
and HLA-DRB1*01 (NM_002124.3).
For DRB, we found greater diversity, identifying 9 variants designated NeciDRB*01 to NeciDRB*09 (GenBank accession numbers KP127615 to KP127623), and 1 pseudogene allele designated
NeciDRB*10 (GenBank accession number KP127624). The pseudogene had a nucleotide sequence similar to NeciDRB*02 but with a
substitution at position #23 (G to C) and a single nucleotide deletion (G) at position #27 (Supplementary Figure S1 online); it was
excluded from all subsequent analyses. The 9 NeciDRB variants had
a mean 2.60% nucleotide and 6.19% amino acid differences and
a total of 13 nonsynonymous substitutions previously identified in
other species (Figure 2, Supplementary Figure S1 online). All DQB
and DRB alleles amplified in this study displayed the RFDS CD4binding motif typical of MHC class II alleles which is essential for T
cell activation; interestingly, there were no synonymous substitutions
in this region, suggestive of recent divergence.
Phylogenetic analyses demonstrated that DQB and DRB allele
sequences from N. cinerea clustered with respective genes from other
pinniped and mammalian species (Figure 3). However, the relationships among alleles in pinnipeds were not strongly resolved at either
locus, with limited clusters with over 60% bootstrap values. No synonymous substitutions were observed in N. cinerea DQB or DRB
sequences, and this is suggestive of a relatively young age of alleles.
From pairwise comparisons of alleles, the mean divergence time
(Tmean) and the 90% confidence limits in parenthesis was estimated
as 0.8 (0.1, 2.0) MYA at the DQB locus and 1.4 (0.4, 5.5) MYA
at the DRB locus. We also estimated TMRCA (the time back to the
most recent common ancestor) to be 1.2 (0.2, 2.5) MYA at the DQB
locus and 3.0 (1.4, 5.5) MYA at the DRB locus. As the divergence
time of the Australian sea lion with the most closely related New
Zealand sea lion is 6.7 MYA as calculated using TimeTree (Hedges
et al. 2006), it is unlikely that any MHC class II alleles have diverged
before speciation. The ratio of TMRCA/Tmean is 1.5 and 2.1 for DQB
and DRB, respectively, and reflects the structure of the allelic genealogy. Since similar values of the ratio are expected in the case of the
neutral coalescence process under a stable population size and little
effects of intragenic recombination (Takahata and Satta 1998), it
seems possible that N. cinerea has kept a fairly constant population
size over millions of years and that intragenic recombination has
played little role in diversifying DQB and DRB alleles.
Furthermore, we estimated Tmean of MHC class II alleles across
multiple pinniped species (Figure 3 for sequence accession numbers)
to be 2.4 (0.7, 6.3) MYA for DQB and 3.2 (1.4, 6.3) MYA for DRB,
and a TMRCA of 8.1 (3.9, 14.6) and 6.4 (3.5, 10.6) MYA for DQB and
DRB, respectively. As Phocidae (true seals) and Otarioidea (sea lions,
fur seals, and walruses) split around 23 MYA (Higdon et al. 2007),
the absence of synonymous substitutions in N. cinerea MHC class II
sequences and limited species-specific phylogenetic groups, supports
recent diversification of alleles in otariids. While disease could drive
a recent diversification of alleles, it is more likely to influence allele
frequencies and the strength of hookworm as a selective agent and
the time scale of action need further investigation.
As no more than 2 NeciDQB alleles per individual were identified, DQB genotypes (homozygotes and heterozygotes) could be
characterized in pups from the 2 colonies (Figure 1b). Both colonies
Journal of Heredity, 2015, Vol. 106, No. 4
400
ZacaDRB*01-03,05-07
ZawoDRB*17
ZacaDRB*04
ZawoDRB*05
ZawoDRB*01,02,04,16
ZawoDRB*08,20
ZawoDRB*06
MileDRB1*02,05
ZacaDRB*10-12
ZawoDRB*11,12,15,19,21,24,25
MileDRB1*04
MianDRB1*01
DRB
MileDRB1*01
MianDRB2*01,02
NeciDRB*04
NeciDRB*03
NeciDRB*05
NeciDRB*07
ZacaDRB*08,09
ZawoDRB*18,26
NeciDRB*02
NeciDRB*01
NeciDRB*09
NeciDRB*08
NeciDRB*06
PhhoDRB clone 01-28, ZawoDRB*14
ZawoDRB*03,07,09,10,13,22,23,27
ZawoDQB*02
ZawoDQB*01
NeciDQB*01
NeciDQB*02
PhhoDQB2
OdroDQB*01-03,05
MileDQB*07
MileDQB*05
NeciDQB*03
MileDQB*08
HagrDQB*03,04
MileDQB*06
DQB
MileDQB*01
MileDQB*03,04
MianDQB*01
MianDQB*02
OdroDQB*04
PhhoDQB1
ZawoDQB*03
ZacaDQB*01,02,04-06,10,11
HagrDQB*01,02
DeleDQB*0202
MomoDQB*0201
BLA-DQB
HLA-DQB1
0.02
Figure 3. Neighbor-Joining tree of MHC class II DQB and DRB alleles of the Australian sea lion compared with other pinniped, cetacean and mammalian species.
Shaded circles indicate bootstrap percentages above 80% (1000 replicates). NeciDQB and DRB alleles identified in this study are highlighted by shaded boxes.
Sequences from other species are those from Figure 2 with addition of PhhoDRB clone 2–28 (KC544971–KC544997), ZawoDQB*02–03 (HE663127.1–HE663128.1),
ZawoDRB*02–28 (HE663099.1–HE663124.1), ZacaDQB*02–11 (AF503398.1–AF503407.1, excluding pseudogenes), ZacaDRB*02–12 (AY491457.1–AY491467.1),
HagrDQB*02–04 (HQ456124.1–HQ456125.1), OdroDQB*02–05 (KJ004395–KJ004398), MileDQB*01–08 (AF111032.1–AF111038.1). MileDRB and northern elephant
seal (Mirounga angustirostris) MianDRB sequences are from Weber et al. (2004). HLA-DQB1 (NM_002123) from humans was used as an outgroup.
Journal of Heredity, 2015, Vol. 106, No. 4
shared all 3 NeciDQB alleles, although the number of heterozygotes
at Dangerous Reef (HE= 0.528, HO = 0.700) was significantly higher
than at Seal Bay (HE= 0.297, HO = 0.259, nonparametric Mann–
Whitney U test P < 0.01; Figure 1b). While there is a difference in
heterozygosity between the 2 colonies, there is no difference in hookworm prevalence and intensity or any other pathogens contributing to
significant pup mortality (Marcus et al. 2014b). Neither colony deviated significantly from Hardy–Weinberg equilibrium (P > 0.10), and
allelic richness (R) of DQB was not different between the 2 colonies
(R = 2.99 at Seal Bay, R = 3.00 at Dangerous Reef) due to the low
number of alleles. In contrast to DQB, at least 2 DRB loci were amplified from N. cinerea, with 1–4 variants identified per individual. Six
NeciDRB variants and the pseudogene were shared between the 2
colonies, although the 2 rare variants found in Seal Bay (NeciDRB*08
and NeciDRB*09) were retrieved only from 2 separate animals and
may be identified in further surveys of the Dangerous Reef colony
(Figure 1c, Supplementary Table S1 online). The NeciDRB*10 pseudogene was more common in Dangerous Reef (60% of animals) compared with Seal Bay (25.9% of animals), although this allele is likely
nonfunctional due to the single base deletion at the 5′ end, and was
not amplified in preliminary screening of cDNA (Lau et al. 2014a).
All 6 and 13 segregating sites identified in the remaining NeciDQB
and NeciDRB alleles, respectively, are observed in cDNA (Lau et al.
2014a) and thus likely expressed. As a result of predominantly shared
MHCII alleles between the 2 N. cinerea colonies, there was no genetic
differentiation between the Seal Bay and Dangerous reef colonies at
DQB (ΦST = −0.001, P = 0.38) or DRB (ΦST = −0.012, P = 0.77).
Although N. cinerea at Seal Bay and Dangerous Reef share only
one mitochondrial DNA haplotype at a low frequency (<5%) with
significant neutral genetic differentiation (Lowther et al. 2012),
common DQB and DRB alleles in both colonies and the absence of
significant MHC class II genetic differentiation might indicate substantial gene flow between populations. Since females exhibit strong
natal site fidelity (Campbell et al. 2008), this gene flow may be malemediated. Alternatively, the allelic conservation of DQB and DRB
between colonies could be due to balancing selection maintained by
a universal, functionally important role in adaptive immunity, and
a single species of hookworm across colonies (Haynes et al. 2014;
Marcus et al. 2014b) could play a role. The characterization of DQB
and DRB diversity and the development of high throughput genotyping methods undertaken in the current study will facilitate future
studies of MHC-disease association and inter-colony gene flow.
Evidence of positive selection was identified among the functional DQB and DRB alleles. The NeciDQB alleles had an average
ratio of nonsynonymous to synonymous substitution rate (ω = dN/dS)
of 3.27 per codon (CI: 0.89–46.62) although only 3 amino-acid residues were positively selected sites (ω > 1; Figure 2, Supplementary
Figure S2 online). The 9 NeciDRB variants had an average ω of 5.03
per codon (CI: 1.17–46.70) and a total of 9 positively selected sites
(Figure 2, Supplementary Figure S2 online). Overall, the pattern of
nucleotide diversity and positive selection in N. cinerea is similar to
other pinnipeds (Table 1). Expansion of MHCII in the DRB locus
relative to DQB in N. cinerea is similar to what has been reported
for the New Zealand sea lion (Osborne et al. 2013) and Galápagos
sea lion (Lenz et al. 2013). While the DQB allelic diversity is higher
in the California sea lion and southern elephant seal, with 8 functional alleles in each species, the level of nucleotide diversity (π) and
average ω (dN/dS) is not markedly different to N. cinerea. Of interest,
DQB nucleotide diversity and average ω is highest in the walrus
amongst all pinnipeds studied to date, and while the MHC diversity
is labelled “low,” variation at DRB in the walrus was not studied
401
(Sonsthagen et al. 2014). The lower DRB allelic diversity (9 variants) in N. cinerea, compared with the diversity in New Zealand and
Galápagos sea lion species (28 and 27 alleles, respectively), may be
attributed to variation in effective population size, demographic history or sealing pressures between the species. Despite this, NeciDRB
nucleotide diversity (π = 0.025 ± 0.014) and average ω (5.03 per
codon) is similar to many other pinnipeds (Table 1) with the exception of higher values in Z. wollebaeki, either due to large-scale sampling or minimal inbreeding (Lenz et al. 2013).
While the occurrence of U.sanguinis hookworm is 100% for
pups born at both Seal Bay and Dangerous Reef, there are fluctuations in hookworm infection intensity of pups between breeding
seasons (Marcus et al. 2014b). This is hypothesized to be due to
colony-specific seasonal differences in host behavior that influences
exposure to free-living infective hookworm larvae during the prior
season. Factors influencing inter-individual differences in hookworm infection intensity, pup health and survival have not been
elucidated in N. cinerea, thus maternal and pup MHC genetics and
immunity should be further investigated. Additionally, the role of
MHC in influencing the effectiveness of the adult immune system
and its implications for individual health, reproductive success, and
survival in this species is unknown. Even though MHC diversity
appears adequate in N. cinerea, conservation and population management should consider the adaptive potential of this species, given
their small and fragmented populations, the proximity of many
remaining colonies to anthropogenic activities, and their terrestrial
breeding and coastal foraging habits that increases their risk from
extant and emerging pathogens. The characterization of NeciDQB
and NeciDRB alleles and diversity using high throughput methods
undertaken in the current study provides the springboard for future
investigation of MHC-disease associations and determining the role
of key factors in influencing host–pathogen–environment relationships and the manifestation of health and disease.
Supplementary Material
Supplementary material can be found at http://www.jhered.oxfordjournals.org/.
Funding
Australian Marine Mammal Centre, Department of the
Environment, Australian Government (09/17); the Winifred Violet
Scott Foundation (2007).
Acknowledgments
All sample collection procedures were approved by Government of South
Australia Department of Environment, Water and Natural Resources
Wildlife Ethics Committee (3-2008 and 3-2011 and modifications), Scientific
Research Permits (A25088/1–8, A25088/4–8) and the University of Sydney
Animal Ethics Committee (NOO/3-2006/3/4333). We thank the Department
of Environment, Water and Natural Resources staff at Seal Bay, Kangaroo
Island for field assistance, in particular Clarence Kennedy and Janet Simpson;
Alan Marcus, Liisa Ahlstrom, Loreena Butcher, Michael Edwards, Simon
Goldsworthy, Claire Higgins, Janet Lackey, Zoe Larum, Theresa Li, Andrew
Lowther, Rebecca McIntosh, Paul Rogers, Laura Schmertmann, Adrian
Simon, Ryan Tate, Michael Terkildsen, Mark Whelan, Peter White, Sy Woon,
and Mariko Yata for field assistance. We thank Weerachai Jaratlerdsiri and
Amanda Lane from The University of Sydney for assistance with genetic characterization and population analysis, Alan Marcus for checking the manuscript, and Naoyuki Takahata from the Graduate University for Advanced
Studies (Japan) for assistance with evolutionary analysis.
402
References
Baker CS. 2013. Journal of heredity adopts joint data archiving policy. J
Hered. 104:1.
Beck S, Trowsdale J. 1999. Sequence organisation of the class II region of the
human MHC. Immunol Rev. 167:201–210.
Bowen L, Aldridge BM, Gulland F, Woo J, Van Bonn W, DeLong R, Stott JL,
Johnson ML. 2002. Molecular characterization of expressed DQA and
DQB genes in the California sea lion (Zalophus californianus). Immunogenetics. 54:332–347.
Bowen L, Aldridge BM, Gulland F, Van Bonn W, DeLong R, Melin S, Lowenstine LJ, Stott JL, Johnson ML. 2004. Class II multiformity generated by
variable MHC-DRB region configurations in the California sea lion (Zalophus californianus). Immunogenetics. 56:12–27.
Brown JH, Jardetzky TS, Gorga JC, Stern LJ, Urban RG, Strominger JL, Wiley
DC. 1993. Three-dimensional structure of the human class II histocompatibility antigen HLA-DR1. Nature. 364:33–39.
Campbell RA, Gales NJ, Lento GM, Baker CS. 2008. Islands in the sea:
extreme female natal site fidelity in the Australian sea lion, Neophoca
cinerea. Biol Lett. 4:139–142.
Doherty PC, Zinkernagel RM. 1975. Enhanced immunological surveillance in
mice heterozygous at the H-2 gene complex. Nature. 256:50–52.
Excoffier L, Lischer HE. 2010. Arlequin suite ver 3.5: a new series of programs
to perform population genetics analyses under Linux and Windows. Mol
Ecol Resour. 10:564–567.
Goldsworthy SD, Mckenzie J, Shaughnessy PD, Mcintosh RR, Page B, Campbell R. 2009. An update of the report: understanding the impediments to
the growth of Australian sea lion populations. Report to the Department
of the Environment, Water, Heritage and the Arts. Contract No.: SARDI
Aquatic Sciences Publication Number F2008/00847-1.
Haynes BT, Marcus AD, Higgins DP, Gongora J, Gray R, Šlapeta J. 2014.
Unexpected absence of genetic separation of a highly diverse population
of hookworms from geographically isolated hosts. Infect Genet Evol.
28:192–200.
Hedges SB, Dudley J, Kumar S. 2006. TimeTree: a public knowledge-base
of divergence times among organisms. Bioinformatics. 22:2971–
2972.
Higdon JW, Bininda-Emonds OR, Beck RM, Ferguson SH. 2007. Phylogeny
and divergence of the pinnipeds (Carnivora: Mammalia) assessed using a
multigene dataset. BMC Evol Biol. 7:216.
Hoelzel AR, Stephens JC, O’Brien SJ. 1999. Molecular genetic diversity and
evolution at the MHC DQB locus in four species of pinnipeds. Mol Biol
Evol. 16:611–618.
Jaratlerdsiri W, Isberg SR, Higgins DP, Gongora J. 2012. MHC class I of saltwater crocodiles (Crocodylus porosus): polymorphism and balancing
selection. Immunogenetics. 64:825–838.
Lau Q, Jobbins SE, Belov K, Higgins DP. 2013. Characterisation of four major
histocompatibility complex class II genes of the koala (Phascolarctos
cinereus). Immunogenetics. 65:37–46.
Lau Q, Wilkin T, Payne E, Gray R, Gongora J, Higgins DP. 2014a. Primers for
amplifying major histocompatibility complex class II DQB and DRB exon
2 in the Australian sea lion (Neophoca cinerea). Conserv Genet Resour.
6:813–816.
Lau Q, Jaratlerdsiri W, Griffith JE, Gongora J, Higgins DP. 2014b. MHC
class II diversity of koala (Phascolarctos cinereus) populations across their
range. Heredity (Edinb). 113:287–296.
Lento GM, Baker CS, David V, Yuhki N, Gales NJ, O’brien SJ. 2003 Automated single-strand conformation polymorphism reveals low diversity of
a major histocompatibility complex class II gene in the threatened New
Zealand sea lion. Mol Ecol Notes. 3:346–349.
Lenz TL, Mueller B, Trillmich F, Wolf JB. 2013. Divergent allele advantage
at MHC-DRB through direct and maternal genotypic effects and its
Journal of Heredity, 2015, Vol. 106, No. 4
consequences for allele pool composition and mating. Proc Biol Sci.
280:20130714.
Lowther AD, Harcourt RG, Goldsworthy SD, Stow A. 2012. Population structure of adult female Australian sea lions is driven by fine-scale foraging site
fidelity. Anim Behav. 83:691–701.
Lyons ET, Delong RL, Spraker TR, Melin SR, Laake JL, Tolliver SC. 2005.
Seasonal prevalence and intensity of hookworms (Uncinaria spp.) in California sea lion (Zalophus californianus) pups born in 2002 on San Miguel
Island, California. Parasitol Res. 96:127–132.
Marcus AD, Higgins DP, Šlapeta J, Gray R. 2014a. Uncinaria sanguinis sp.
n. (Nematoda: Ancylostomatidae) from the endangered Australian sea
lion (Neophoca cinerea) (Carnivora: Otariidae). Folia Parasitol. (Praha).
61:255–265.
Marcus AD, Higgins DP, Gray R. 2014b. Epidemiology of hookworm (Uncinaria sanguinis) infection in free-ranging Australian sea lion (Neophoca
cinerea) pups. Parasitol. Res. 113:3341–3353.
Murray BW, Malik S, White BN. 1995. Sequence variation at the major histocompatibility complex locus DQ beta in beluga whales (Delphinapterus
leucas). Mol Biol Evol. 12:582–593.
Osborne AJ, Zavodna M, Chilvers BL, Robertson BC, Negro SS, Kennedy MA,
Gemmell NJ. 2013. Extensive variation at MHC DRB in the New Zealand
sea lion (Phocarctos hookeri) provides evidence for balancing selection.
Heredity (Edinb). 111:44–56.
Peakall R, Smouse PE. 2012. GenAlEx 6.5: genetic analysis in Excel. Population genetic software for teaching and research—an update. Bioinformatics. 28:2537–2539.
Raymond M, Rousset F. 1995 GENEPOP (Version 1.2): Population genetics
software for exact tests and ecumenicism. J. Hered. 86:248–249.
Sepúlveda MS. 1998 Hookworms (Uncinaria sp.) in Juan Fernandez fur
seal pups (Arctocephalus philippii) from Alejandro Selkirk Island, Chile.
Endanger. Species Res. 84:1305–1307.
Shaughnessy PD, Goldsworthy SD, Hamer DJ, Page B, Mcintosh RR. 2011
Australian sea lions Neophoca cinerea at colonies in South Australia: distribution and abundance, 2004 to 2008. Endanger. Species Res. 13:87–98.
Slade RW. 1992 Limited MHC polymorphism in the southern elephant seal:
implications for MHC evolution and marine mammal population biology.
Proc. R. Soc. Lond. B Biol. Sci. 249:163–171.
Sommer S. 2005. The importance of immune gene variability (MHC) in evolutionary ecology and conservation. Front Zool. 2:16.
Sonsthagen S, Fales K, Jay C, Sage G, Talbot S. 2014 Spatial variation and low
diversity in the major histocompatibility complex in walrus (Odobenus
rosmarus). Polar Biol. 37:497–506.
Spurgin LG, Richardson DS. 2010. How pathogens drive genetic diversity:
MHC, mechanisms and misunderstandings. Proc Biol Sci. 277:979–988.
Takahata N, Nei M. 1990. Allelic genealogy under overdominant and frequency-dependent selection and polymorphism of major histocompatibility complex loci. Genetics. 124:967–978.
Takahata N, Satta Y, Klein J. 1992. Polymorphism and balancing selection at
major histocompatibility complex loci. Genetics. 130:925–938.
Takahata N, Satta Y. 1998. Selection, convergence, and intragenic recombination in HLA diversity. Genetica. 102–103:157–169.
Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. 2013. MEGA6:
Molecular Evolutionary Genetics Analysis version 6.0. Mol Biol Evol. doi:
10.1093/molbev/mst197.
Villanueva-Noriega MJ, Baker CS, Medrano-González L. 2013. Evolution of
the MHC-DQB exon 2 in marine and terrestrial mammals. Immunogenetics. 65:47–61.
Weber DS, Stewart BS, Schienman J, Lehman N. 2004. Major histocompatibility complex variation at three class II loci in the northern elephant seal.
Mol Ecol. 13:711–718.
Wilson DJ, McVean G. 2006. Estimating diversifying selection and functional
constraint in the presence of recombination. Genetics. 172:1411–1425.