Zoological Studies 49(1): 132-151 (2010)
Phylogenetic Analyses of MHC Class II Genes in Bottlenose Dolphins
and Their Terrestrial Relatives Reveal Pathogen-Driven Directional
Selection
Wei-Cheng Yang, Jer-Ming Hu, and Lien-Siang Chou*
Institute of Ecology and Evolutionary Biology, National Taiwan University, 1 Roosevelt Rd., Sec. 4, Taipei 106, Taiwan
(Accepted June 3, 2009)
Wei-Cheng Yang, Jer-Ming Hu, and Lien-Siang Chou (2010) Phylogenetic analyses of MHC class II genes
in bottlenose dolphins and their terrestrial relatives reveal pathogen-driven directional selection. Zoological
Studies 49(1): 132-151. The mammalian order Cetacea is believed to have made the challenging evolutionary
transition from land to sea early in the Eocene, > 50 million years ago (Mya). With the shift in habitat, cetaceans
had to evolve a range of specializations, including immune response. The major histocompatibility complex
(MHC) multigene families have proven to be ideal candidates for research on the timing of selection and the
spatial heterogeneity of selection pressures experienced by the ancestors of cetaceans because MHC multigene
families consist of a mixture of divergent genes and play key roles in immune responses. Herein, we report
the evolutionary relationship and estimates of divergence times of cetartiodactyls using MHC class II β-chain
genes. In both the DQB and DRB phylogenies, cetaceans (Tursiops truncatus and T. aduncus) and artiodactyls
(pig, hippo, and ruminants) formed 2 distinct clades, and the estimated divergence time was about 60 Mya or
slightly earlier. Furthermore, our results showed that T. truncatus and T. aduncus diverged about 24 Mya, which
greatly predates the emergence of the oldest dolphin (approximately 11 Mya). These findings are explained
by postulating gene duplications in MHC genes and pathogen-driven directional selection during cetacean
evolution. This research provides new information for studying the dynamics of MHC-pathogen co-evolution.
http://zoolstud.sinica.edu.tw/Journals/49.1/132.pdf
Key words: Major histocompatibility complex, Bottlenose dolphins, Pathogen-driven directional selection.
R
ecent molecular and morphological
studies suggested that the order Cetacea may
be more closely related to the order Artiodactyla
than to other orders of ungulates (Kumar and
Hedges 1998, Arnason et al. 2000, Murphy et
al. 2001, Boisserie et al. 2005), supporting a
hypothesized order Cetartiodactyla (Montgelard
et al. 1997). In addition, cetaceans and the
hippopotamus (Hippopotamus amphibious) form
a monophyletic group deeply nested within the
Cetartiodactyla, while camels and pigs are basal
to this order (Gatesy et al. 1996, Gatesy 1997,
Nikaido et al. 1999, Boisserie et al. 2005). The
molecular clock estimate for the divergence of
the artiodactyls and cetaceans is about 60 million
years ago (Mya) (Arnason and Gullberg 1996), a
time slightly predating the oldest known cetacean,
Himalayacetus, dated in the early Eocene, at 53.5
Mya (Bajpai and Gingerich 1998). It is believed
that early cetaceans initially lived in freshwater
habitats as terrestrial quadrupeds and were
partially dependent on fresh water at some stages
of their life before they gradually adapted to the
marine environment and ultimately became fully
aquatic marine mammals (Thewissen and Williams
2002). Adaptations from the land to sea life in
cetaceans have been studied by comparative
analyses of morphological traits (Thewissen et al.
*To whom correspondence and reprint requests should be addressed. Jer-Ming Hu and Lien-Siang Chou contributed equally to this
work. Tel: 886-2-33662468. Fax: 886-2-23639902. E-mail:[email protected]
132
Yang et al. – MHC Genes in Dolphins and Relatives
2001, Bejder and Hall 2002, Spoor et al. 2002,
Nummela et al. 2004) as well as protein function
(Naylor and Gerstein 2000, McClellan et al. 2005)
among cetaceans and closely related species.
However, these adaptations have never been
investigated in terms of evolution of immune genes
in cetaceans. This issue nonetheless had to be
critical for cetaceans during their move from land
to water, which is an enormous shift in habitat
environments. Since major qualitative differences
in microorganisms and infectious diseases are
believed to exist between marine and terrestrial
environments (McCallum et al. 2004), the immune
genes of primitive cetaceans are supposed to have
adapted for defending against distinct pathogens
in aquatic environments.
Among immune genes, several major histocompatibility complex (MHC) genes are
suggested to be under strong pathogen-driven
selective forces (Hughes and Yeager 1998). The
mammalian MHC related to peptide expression
is divided into two main regions, class I and II
gene regions (Hughes and Yeager 1998). Both
class I and class II MHC gene families include a
large number of loci that encode heterodimeric
peptide-binding proteins consisting of extracellular
domains (including the peptide-binding region;
PBR), a connecting portion, a transmembrane
portion, and a cytoplasmic tail. These multigene
families consist of mixtures of divergent genes
including large numbers of functionally closely
related genes and pseudogenes resulting from
birth-and-death evolution (Nei et al. 1997). In
this evolutionary process, new genes are created
by repeated gene duplications, while some
genes may later become pseudogenes or even
be deleted from the genome. The evolutionary
longevity of new genes can vary from gene to
gene in the MHC. MHC class II loci undergo a
slower rate of birth-and-death evolution than do
class I loci, and the longevity of class II genes
is much greater than that of class I genes (Klein
and Figueroa 1986). Consequently, many class
II homologous loci are shared by different orders
of mammals, while it is difficult to determine the
homologous relationships of class I genes among
different orders of mammals (Hughes and Nei
1990). These characteristics make MHC class II
loci more-appropriate subjects for comparative
evolutionary studies and for estimating divergence
times of various loci (Klein and Sato 1998,
Takahashi et al. 2000).
Divergence times between different
gene clusters of mammalian class II genes
133
were estimated (Klein and Figueroa 1986,
Hughes and Nei 1990, Takahashi et al. 2000).
Estimates by Takahashi et al. (2000) are more
reliable because many more gene sequences
were available at that time. However, only 3
species from the Cetartiodactyla (pig, cattle,
and sheep) were studied by Takahashi et al.
while evolutionary relationships of MHC class II
genes among the Cetartiodactyla remain
unresolved. Because the problem is important
for understanding how immune system genes
evolved in the Cetartiodactyla, herein we
constructed phylogenetic trees and estimated the
divergence times of clades using complementary
(c)DNA sequences of MHC class II β-chain genes
from bottlenose dolphins (Tursiops truncatus
and T. aduncus), the hippo (Hippopotamus
amphibious), other mammals, chicken, frog, and
fish. By interpreting phylogenetic relationships and
divergence time estimates of MHC class II genes
in bottlenose dolphins and their closely related
terrestrial species, we attempted to reveal how and
when the habitat shift, accompanied by changes
in foreign antigens, affected MHC evolution in
cetaceans and other closely related mammals
when they moved from land to water.
MATERIALS AND METHODS
Sample preparation and rapid amplification of
cDNA ends (RACE) polymerase chain reaction
(PCR) amplification
Tissue sample from 1 captive hippopotamus
was collected and stored at -70°C. RNA was
isolated by silica-based gel membranes supplied
in the RNeasy Fibrous Tissue Mini Kit (Qiagen,
Valencia, CA, USA). The isolated RNA was also
stored at -70°C prior to the RACE cDNA synthesis.
A cDNA population was constructed using
SMART RACE cDNA amplification kits (Clontech,
Mountain View, CA, USA) in order to facilitate the
amplification of full-length gene transcripts. In
brief, RNA samples from the hippopotamus were
used as templates for cDNA synthesis. Adaptorlike sequences were added to either the 5’ or 3’
end of the cDNA fragments in 2 separate reactions.
These modified cDNAs were generated from
cellular RNA by Moloney murine leukemia virus
(MMLV) reverse transcriptase-driven 1st-strand
synthesis using lock-docking oligo(dT) primers
and the SMART II oligonucleotide. The resulting
5’- and 3’-modified cDNA fragments were used
134
Zoological Studies 49(1): 132-151 (2010)
as templates for the subsequent PCR and RACE
PCR analyses.
We used published degenerate oligonucleotide primers (Bowen et al. 2002 2004)
which recognize conserved regions of each of 2
MHC class II genes, DQB and DRB. DQB and
DRB genes were shown to be polymorphic in
some terrestrial carnivores and domestic animals
(Schook and Lamont 1996, Yuhki and O’Brien
1997, Wagner et al. 1999). PCR amplifications
using these degenerate class II primers were
performed on 20 ng of each RACE cDNA library
in 50 ml volumes containing 20 pmol of each
primer (either DQB-U172 and DQB-L769, or
DRB-U182 and DRB-L729), 40 mm Tris-KOH
(pH 8.3), 15 mm KOAc, 3.5 mm Mg(OAc)2, 200
mM each dNTP, and 5 U of Advantage 2 Taq
polymerase (Clontech). For the 5’ gene transcript
amplifications, the reactions contained 20 pmol
of either DRB-L729 (for DRB gene products) or
DQB-L769 (for DQB gene products), along with 1-5
pmol of the universal primer mix (UPM, SMART
RACE cDNA amplification kit; Clontech). For
3’ gene transcript amplifications, the reactions
contained 20 pmol of either DRB-U182 (for DRB
gene products) or DQB-U172 (for DQB gene
products), along with 1-5 pmol of the UPM (SMART
RACE cDNA amplification kit; Clontech). The PCR
was performed on a thermal cycler and consisted
of 30 cycles at 94°C for 30 s, 68°C for 30 s and
72°C for 2 min, followed by a single extension step
of 72°C for 10 min. The products of these reactions
were electrophoresed on 1.5% agarose gels and
visualized by ethidium bromide staining. Bands
representing PCR products of the predicted size
were excised from the gel, extracted, and purified
using a commercially available nucleic acidbinding resin (Qiaex II gel extraction kit, Qiagen).
These isolated RACE fragments were then ligated
into a T/A-type cloning vector (pGEM-T Easy
Vector System, Promega, San Luis Obispo, CA,
USA). Following transformation, growth, and bluewhite selection in DH5α-competent cells, the DNA
from positive clones was isolated. The nucleotide
sequences of both strands were determined
by dideoxynucleotide methodology using an
automated sequencer. The nucleotide sequences
of these amplicons were compared using MEGA
vers. 3.0 (Kumar et al. 2004). However, we could
not obtain the predicted product of the 5’ gene
transcript amplifications using 5’-modified cDNA
fragments from the hippopotamus, possibly due
to RNA degradation during tissue preservation.
Therefore, we only used sequences from the
3’ gene transcript amplifications, the nucleotide
number of the coding region of which was around
660, for the subsequent phylogenetic analysis.
The full-length nucleotide sequences of
DQB and DRB genes of bottlenose dolphins (T.
truncatus and T. aduncus) (Yang et al. 2007) used
in this study were obtained from blood samples by
similar methods described above.
Phylogenetic analysis of MHC class II β-chain
genes in vertebrates
We used nucleotide sequences of the class II
β-chain genes consisting of the extracellular
domain, connecting peptide, transmembrane,
and part of the cytoplasmic tail obtained from
RACE product sequences and GenBank (Table
1). Several different species including placental
mammals, chicken, frog (Xenopus), and bony fish
(zebrafish Danio rerio) were studied. Tursiops
truncatus and T. aduncus were representatives
of the Cetacea, and H. amphibious was representative of the Hippopotamidae. The choice of
other species and loci followed Takahashi et al.
(2000) with modifications. Pseudogenes were not
used. When allelic variants were found at a locus,
only a single typical allele was used to represent
that locus. The nucleotide sequences obtained
were aligned using the CLUSTAL W method
(Thompson et al. 1994), taking into account
the deduced amino acid sequences. Minor
adjustments were made to the alignments after
visual inspection. The number of nucleotides was
616 (205 codons). We identified positions 1-218
as the segment including the PBR and positions
219-616 as the non-PBR segment following
Bowen et al. (2002 2004). A test for recombination events between sequences was
conducted with the program GENECONV 1.81
(Sawyer 1999). The program runs 10,000
permutations of the original data in global and
pairwise tests for the occurrence of possible inner
and outer fragments involved in recombination
or gene-conversion events. We used all codon
positions and only the 1st + 2nd codon positions
in the following analysis. The appropriate model
was selected with Modeltest (Posada and Crandall
1998), using the Akaike information criterion (AIC)
(Posada and Buckley 2004) with a parsimonious
tree chosen as the basis for Modeltest. The best
model was GTR + Г + I (Rodríguez et al. 1990,
Yang 1994). A phylogenetic analysis was carried
out using the Bayesian inference (BI) implemented
in MRBAYES 3.1 (Ronquist and Huelsenbeck
Yang et al. – MHC Genes in Dolphins and Relatives
2003) with the following settings. The maximum
likelihood model employed 6 substitution types
(nst = 6), with base frequencies estimated from the
data. Rate variations across sites were modeled
using a γ-distribution (rates = invgamma). The
prior probability distribution on branch lengths was
specified as the birth-death clock model. A Markov
chain Monte Carlo (MCMC) search was run with 4
chains for 106 generations, sampling the Markov
chain every 100 generations, and the sample
points of the 1st 2500 generations were discarded
as “burn-in” after which the chain reached
stationarity. We used the Shimodaira-Hasegawa
(SH) test (Shimodaira and Hasegawa 1999), which
assumes a resampling estimated log-likelihood
(RELL) approximation, to compare the likelihood
scores of potential topologies derived from the
BI tree. To test for topological consistency, we
used 3 more methods to construct phylogenetic
trees: Neighbor-joining (NJ), maximum-parsimony
(MP), and maximum-likelihood (ML) using PAUP*
4.0b10 (Swofford 2003). Parsimony analyses
were performed using 100 replicates with random
addition of taxa, tree-bisection-reconnection branch
swapping, and a transversion: transition weighting
of 2: 1. The ML analysis was performed using
the GTR+ Г + I model with estimated parameters.
The NJ analysis was performed using the Kimura-
135
2-parameter model of Takahashi et al. (2000).
We performed the SH test to compare scores of
trees resulting from the above searches. We also
conducted Tajima’s relative rate test (Tajima 1993)
and likelihood-ratio test as described by Swofford
et al. (1996) to test the molecular clock hypothesis
of this dataset.
Divergence time estimation
To estimate the divergence time, zebrafish
sequences were used as outgroups in the trees
obtained from 4 inferences. We estimated the
divergence times of MHC class II gene clades
of the BI ultrametric tree by performing age
calibration in r8s vers. 1.7 (Sanderson 2003) using
the bird-mammal divergence time estimate (310
Mya) (Hedges et al. 1996, Kumar and Hedges
1998, Wang et al. 1999a) as a calibration point.
For comparison, we used a linearized tree method
under the assumption of a molecular clock to
estimate the times of divergence of different
clades in the NJ tree as described by Takahashi
et al. (2000). However, we used 310 Mya as the
calibration point in the linearized tree method,
while Takahashi et al. (2000) used 300 Mya.
Table 1. Major histocompatibility complex (MHC) class II β-chain genes used in this study with respective
GenBank accession numbers
Species
Gene [accession no.] Species
Gene [accession no.] Species
Gene [accession no.]
Zebrafish
DAB1 [L04805]
DAB4 [U08870]
DCB [U08873]
DEB [U08874]
B(1) [D13685]
B(2) [D13684]
BL(1) [M26306]
BL(2) [M29763]
BL(3) [M26307]
DPB [M21468]
DRB [U51575]
DRB [AY491464]
DQB [AF503406]
DRB [M29611]
DQB [AF043908]
DQB [L33910]
DRB [EF017817]
DQB [EF017815]
DRB [EF017818]
DQB [EF017816]
DRB [EF017820]
DQB [EF017819]
DRB [M93432]
DQB [L08792]
DOB [AB117946]
DRB [U77067]
DQB [M30003]
DRB [AB008346]
DRB [M55165]
DQB [AY459301]
DPB [M16685]
DQB(BB) [X56596]
DRB1(EB1) [M12382]
DOB [NM010389]
DQB(AB1) [M13538]
DRB(EB1) [K01145]
DRB4 [M60062]
DRB1 [M60059]
DRB3 [M77153]
DRB1 [M77154]
DRB5 [M77152]
DRB1 [M11161]
DRB3 [NM022555]
DRB4 [NM021983]
DRB5 [NM002125]
DPB1 [M57466]
DOB [X03066]
DOB [M24358]
DRB [M76488]
Frog
Chicken
Rabbit
Cat
Sea lion*
Dog
Horse
Dolphin (Tt)*
Dolphin (Ta)*
Hippo
Sheep
Cattle
Goat
Pig
Mole rat
Rat
Mouse
Macaque
Gorilla
Human
Chimpanzee
Tamarin
Tt, Tursiops truncatus; Ta, Tursiops aduncus; Sea lion, California sea lion Zalophus californianus.
Zoological Studies 49(1): 132-151 (2010)
136
RESULTS
Identification of MHC genes in the hippo and
bottlenose dolphins
3’ RACE cDNA clones were obtained from
muscle tissue of a captive hippo. These partiallength transcripts were characterized as hippoDQB (647 bp) or hippo-DRB (653 bp) based on
comparisons with bovine lymphocyte antigen
(BoLA) and human lymphocyte antigen (HLA)
DQB and DRB sequences. The numbers of the
deduced amino acids of hippo-DQB and DRB
gene products were 213 and 216, respectively.
No sequence compatible with a class II MHC
pseudogene was identified.
Clones containing full-length bottlenose
dolphin MHC class II sequences were obtained
from the RACE cDNA products of 2 T. truncatus
and 2 T. aduncus (Yang et al. 2007). The
nucleotide sequence of the 780 (DQB-primer
derived) and 801 bp (DRB-primer derived) products
were typical of transcripts from mammalian class
II genes. These transcripts were characterized
as Tt-DQB, Ta-DQB, Tt-DRB, or Ta-DRB based
on comparisons with HLA and BoLA DQB and
DRB sequences. No sequence compatible with a
class II MHC pseudogene was identified.
Phylogenetic analysis of MHC class II β-chain
genes in vertebrates
No evidence for genetic-recombination or
gene-conversion events between the aligned
sequences was found by using the program
GENECONV 1.81 with gscale values of 0, 1 and
2. The phylogenetic trees were obtained by BI
using 3 data sets: 1-218 (PBR), 219-616 (nonPBR), and 1-616 nt positions of MHC class II
β-chain genes from various vertebrate species
(Fig. 1). The BI trees were constructed under
the GTR + Г + I model incorporating the birthdeath clock model, and all 3 codon positions were
used. When 616 nt was used, the tree revealed
that all genes from mammalian species formed
a monophyletic group separated from the clades
of chicken, frog, and zebrafish (Fig. 2a). In the
mammal group, there were 4 clades of genes
corresponding to DRB, DPB, DQB, and DOB,
respectively. The DOB clade was the sistergroup
of the much-larger DRB/DPB/DQB clades. The
DPB and DQB clades were more closely related to
each other than to the other clades. The DRB and
DQB clades both showed well-defined clustering of
sequences from major mammalian lineages, such
as rodent, primate, carnivore, and ungulate clades.
Comparable clustering patterns were observed in
the ML, MP, and NJ trees using all codon positions
(Fig. 3a). The SH test showed that there were
no significant differences among trees obtained
from the BI, ML, MP, and NJ algorithms (p > 0.5).
When only the 1st and 2nd codon positions were
used, the topologies of the trees obtained by each
method slightly differed from those using all codon
positions, but the evolutionary relationships among
the major clades remained the same (results not
shown). Sequences from ungulates and dolphins
formed a monophyletic group that was separated
from other mammals with a posterior probability
of 1.00 in the DQB and DRB clades (Fig. 2a). In
addition, dolphins diverged from ungulates with
moderately high (0.82 for DQB) or high (0.98 for
DRB) posterior probabilities. Ruminants (cattle,
sheep, and goat) were grouped with high posterior
probabilities in both the DQB (0.92) and DRB (1.00)
clades. However, relationships among the pig,
horse, hippo, and ruminants were not conclusively
resolved due to low posterior probabilities
(0.50-0.69). The alternative hypothesis that
dolphins represent a sistergroup of the hippo in
both the DQB and DRB genes was rejected by the
SH test (p < 0.001). Nevertheless, a basal position
of the horse among the dolphin/ungulate group
in the DQB clade was not rejected in the tree
constructed using either the original data matrix (p
> 0.3) or only DQB sequences (p > 0.3).
The BI tree based on PBR sequences only
(positions 1-218) (Fig. 2b) showed that β-chain
genes of mammals diverged from those of nonmammalian species with a low posterior probability
(0.53). However, the DPB, DQB, and DRB clades
formed a monophyletic group which was separated
from the DOB clade with a posterior probability of
1.00. The DQB and DRB sequences of dolphins
were sistergroups within the clade containing
all other DRB sequences with high posterior
probability (0.97). The DQB and DRB sequences
of the California sea lion Zalophus californianus
were sistergroups and clustered with the dog
DQB sequence with a high posterior probability
(0.96). DQB sequences of ungulates were
grouped with rodent DQB sequences with a high
posterior probability (0.98), but their relationships
were not conclusively resolved. In the DRB
clade, the hippo was grouped with ruminants with
a low posterior probability (0.68), while the pig
was grouped with rodents and tamarin although
the posterior probability was low as well (0.68).
Yang et al. – MHC Genes in Dolphins and Relatives
(A)
137
[
[
[
1]
1
2
3
4
5
6
7
8
9
0]
1234567890 1234567890 1234567890 1234567890 1234567890 1234567890 1234567890 1234567890 1234567890 1234567890]
TtDRB
TaDRB
HippoDRB
PigDRB
SheepDRB
CattleDRB
GoatDRB
CatDRB
DogDRB
ZacaDRB
RatDRB1
MouseDRB
TamarinDRB
MacaqueDRB4
MacaqueDRB1
GorillaDRB5
GorillaDRB3
GorillaDRB1
HumanDRB1
HumanDRB3
HumanDRB4
HumanDRB5
TaDQB
TtDQB
PigDQB
HippoDQB
CattleDQB
SheepDQB
HorseDQB
RatDQB
MouseDQB
DogDQB
ZacaDQB
RabbitDPB
HumanDPB
MoleratDPB
HumanDOB
MouseDOB
ChimpanzeeDOB
CattleDOB
ChickenBL1
ChickenBL2
ChickenBL3
FrogB2
FrogB1
ZebrafishDAB1
ZebrafishDAB4
ZebrafishDCB
ZebrafishDEB
GGTTCGTGGA
.........T
.....C....
....AT..C.
....GC....
.....C....
..C.TC....
..C.TC....
..........
..C..C...T
..C.TC...C
..C.TC...T
.CC..C....
A..A.C..AT
.....C....
...ATC..C.
...A.C....
.....C....
....GC....
...A.C....
..AA.C..AT
.....C..C.
..C.......
..GG.A..AG
..C.....AC
...A....AC
...A....AC
..C.....AC
..C.....AC
..GAT...AT
..C.T...AT
..C.TC..AC
...C.C..AC
.C..GC....
.C...C.A..
.C.A......
A...T....T
AC..GC...T
A...T....T
....T....T
....TC....
...ATC..C.
...ATC....
...ATC....
..C.TC...T
T...GC.T..
T..ATC.T.C
A.C..A.CTT
.TG.TCAT..
[
[
[
1
2]
0
1
2
3
4
5
6
7
8
9
0]
1234567890 1234567890 1234567890 1234567890 1234567890 1234567890 1234567890 1234567890 1234567890 1234567890]
TtDRB
TaDRB
HippoDRB
PigDRB
SheepDRB
CattleDRB
GoatDRB
CatDRB
DogDRB
ZacaDRB
RatDRB1
MouseDRB
TamarinDRB
MacaqueDRB4
MacaqueDRB1
GorillaDRB5
GorillaDRB3
GorillaDRB1
HumanDRB1
HumanDRB3
HumanDRB4
HumanDRB5
TaDQB
TtDQB
PigDQB
HippoDQB
CattleDQB
SheepDQB
HorseDQB
RatDQB
MouseDQB
DogDQB
ZacaDQB
RabbitDPB
HumanDPB
MoleratDPB
HumanDOB
MouseDOB
ChimpanzeeDOB
CattleDOB
ChickenBL1
ChickenBL2
ChickenBL3
FrogB2
FrogB1
ZebrafishDAB1
ZebrafishDAB4
ZebrafishDCB
ZebrafishDEB
CGAGTACTGG
..........
.A........
.A.....C..
.A........
....C.....
..........
.A........
T....C....
T.........
.......A..
....A.....
G.........
..........
..........
..........
..........
..........
..........
.....C....
T.........
T.........
.AC.......
........TC
...C......
.A........
..........
..........
..........
........TT
..........
T.........
T.........
.....C....
G..C......
...CG...TC
T...C.G...
T..CC.G...
T...C.G...
....CGG...
T..AAT....
T..A......
T..A......
GA....T...
G..TA.T...
ACGAA...TC
A...A...TC
T..AA...AT
AC.A.TA...
GAAACACACG
..G.TG....
C.G.T..TTC
..GGA.TTGC
A.G.T..TTC
C.G....TTC
C.G.T..TTC
C.GGT..TTC
A.G.T...TC
C.G.....TC
C.G.TTA.TC
A.G.T..TTC
A.G.T..TTC
C.G.T..TTC
C.G.T..TTC
C.G.G...TC
C.G.T..TTC
C.G.T..TTC
A.G.TG..TC
C.G.T..TTC
C.G.T...TC
C.G.G...TC
A.G.T...TC
C.G.T...TC
C.G.T...TC
C.G.TTG.TC
C.G.T...TC
C.G.T...TC
C.G.T...TC
C.G.T...TC
C.G.T...TC
T...T...TC
C.G.T...TC
C.G.TG.G.C
..GTT...TC
..G.T...TC
C.G.TT..TC
C.GGTT..TC
C.G.TT..TC
C.G.TT..TC
..GG....TC
A.GGT...TC
ACGGG.A.TC
AGGCT..G.T
.CGCT...TT
ATC.GGTT.T
TTC.T.TT.C
CTC.TT..TC
T..TT.T.TC
AACAGCCAGA
..........
..........
..........
..........
..........
..........
...G.G....
...G.G....
...C......
....AA....
.........C
.......G..
...G......
...G......
..........
..........
..........
..........
..........
..........
..........
...G.....G
..........
..........
.......G..
..........
..........
...G.G....
....AG.....T.AG......C.A....
..........
...------C
..........
.......G..
.......G.C
....AA.GCC
.......G.C
....A..G.C
......A.CG
......A.CG
......A.CG
..........
..........
....AAA.CC
....AAG.CC
..A.ATA.C..GCAA.GAT
TATAACCGGG
..........
......G.A.
......G.A.
.....TG.A.
.....TG.A.
C....TG.A.
......G...
C.........
..........
..C...A...
..C....T..
..C....A..
.......AA.
.......A..
.......AA.
C......AA.
..C....AA.
.......AA.
C......A..
.......AA.
.......AA.
..........
..........
..C....A..
.......A..
..C....A..
..C.......
..C.......
..C....A..
..C.......
..........
..........
..C...A...
..C.......
..C....AA.
.T....TT..
.TC....T..
.T....TT..
.TC....T..
..C......C
..C......C
..C......C
A....T.A..
AG...T.A..
.TC..TAAA.
.TC..TAAA.
.TC...AA.A
.TC..TAAA.
AGGACCTCCT
..........
.....T..A.
..........
....TT....
.....T....
....GA....
.....T..A.
....GA..T.
.....G..A.
....GT..A.
C...GT....
.....A....
..........
.....A....
.....G....
......A...
.....A....
..........
..........
..........
.....T....
..........
.....A....
.....A....
.....A....
.....A....
.....A..AC
.....G....
-----TA...
-----TA...
.....GAGA.
.....A....
T...ATA...
.....A....
....GGC...
T...T...T.
T...T.....
T...T...T.
C...TA.A..
CC..GA.T..
CC..G..T..
CC..G..T..
....T.....
....GAC.G.
...CATA...
...C.TA.T.
--ACAT.TG.
.CC.G..T..
AGGAGTACGT
..........
..........
..........
.A........
.A....T...
.A...AT...
.......T..
......T...
..........
.........C
.....A..C.
......TG..
..........
..........
.....G..T.
......T...
..........
......C...
......T.C.
.........C
.....G..T.
......T...
..........
.........C
......T..C
.........C
.........C
..........
........C.
..........
......T...
..........
......T...
......T...
..........
.......T..
.......TT.
.......T..
....T..T.C
..C...T.A.
..C...T.AC
..C......C
.........C
......T...
TT.T.G.T..
TT.T.G.TAC
TT..A...A.
.T.T....A.
GGAGCAGAAA
.....G.C.C
.........G
........GG
....AGC.GG
.........G
....AGC.GG
.........G
.......G.G
.....G..GG
.....G..GG
......A..G
........C.
........GG
...AG.CGCG
...AG.CG.G
.........G
...AG.C.GG
.........G
.........G
.....G..GG
...AG.C.GG
........G.
.......G..
........CG
.....G..CG
........CG
.....G.GTG
........CG
........CG
.....GA.CG
...C.G.GT.
.....G..CG
.C.....GCG
....G..G.G
.........G
....AG..GC
....AC..GC
....AG..GC
..CCAG..GT
....G.CG..
....A.CCG.
....A.CCT.
A..T......
A....G.T.C
.C....ACGG
.C.T..ATTG
.CTAGTCCTG
C.....AG.G
GCGCTTCGAC
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
.....A....
..........
..........
..........
..........
..........
..........
.....A.A..
..........
..........
..........
..........
.G...A....
..........
..........
..........
.....A....
..........
..........
..........
..........
..........
..........
A..T......
A.A.......
A..T......
A..T......
..A.......
..A.......
..A.......
CTA...T...
CTA...T...
T.AG.A.A..
T.AG...A..
TA.A.A.A..
A..A...A..
CAGGCCAAGG
.G....G...
.G....G..C
.G...GGC..
AG.A..GC..
.G....G...
AG.A..GC..
.GC...G...
.G...A.C..
.G....G...
.G....GC..
.G....G...
.G....GC..
.G....G...
.G....GC..
.G....TC..
.G..G.CG..
.G....TC..
.G..G.C...
.G..G.CG..
.G....G...
.GC...GC..
.G....GC.C
.G....GC..
.G...AG..C
.G....G..A
TG....G...
.G....G...
.G....G..T
.G....G..C
.G....G..C
.G....G..C
.G....G...
.G....G...
.G...AGT.C
.GC...GC.C
AGACAGGCC.
AGA..TGCT.
AGACAGGCC.
AGA...TCT.
AT.AATGCA.
AT.AATG.A.
AT.AAT.TA.
.G...TGT..
.G...TG...
A.A..TG...
A.A..TC.A.
GCT.AGTTT.
AGA..TC.C.
AGCGACGTGG
..........
..........
..........
......TG..
......TG..
......TG..
.....A....
..........
..........
..........
..........
..........
........C.
..........
..........
..........
..........
..........
..........
..T...C...
..........
..........
..........
..........
..........
......TG..
......TG..
..........
..........
..........
..........
..........
......A.C.
..........
..........
..T..T....
..T...C...
..T..T....
......T...
..........
..........
..........
..T..T....
..T..A....
...ACT....
...TCT....
..TACT.ATC
...ACTT...
TGGACACGTA
..........
........GT
..........
..........
......G.GT
..........
.......CCT
.......C..
........GT
..........
........GT
.......C.T
.......CGT
.......C..
.......C..
......AT..
.......C..
......AT..
......AT..
.......C..
.......C..
........GT
........GT
........GT
........GT
......G.GT
........GT
......GAGT
........GT
........GT
........GT
C.......GT
......G.GT
C.....G.GT
CC......GG
....TGG.GT
..A...T.GT
....TGG.GT
......T.CT
....T....T
......G..T
C....GG.CC
....T..AGT
....T..AGT
....A.GT.T
.......A.T
G.ATTTACA.
A...TCGA.T
GCGAGTACCG
......T...
..........
..........
......T...
......T...
......T...
.G....T...
.G....T...
.G........
..........
......T...
.G........
.G........
.G........
.G........
.G........
.G........
.G........
.G........
.G.......A
.G........
......T...
......T...
.G........
..........
..........
..........
.G........
..........
..........
.G....T...
.G........
.G....T...
.G....T...
...T..T...
.GAT..TTGT
.GAT..TTGT
.GAT..TTGT
.GAT..TTGT
.GA.A...GT
.GA.A.TTGT
.GA.A.TTGT
.G..A..TAA
.TTTC.TTAT
.GA....TGT
.GA...TTGT
AGA.AATTGT
.GT.C.TTGT
CTGCAGGCAC
......AT..
G....AA...
......A...
......A...
G.....A...
......A...
......A...
......A...
G.....A...
......A...
G.....A...
......A...
G.....A...
......A...
T.....A...
......A...
T.....A...
......A...
......A...
......AT..
......A...
G.....A...
G.....A...
G....AA...
G.....A...
G.....AA..
G.....A...
G.....AT..
......A...
G.....A...
G.....A...
G.....A...
G.....A...
A.....A.G.
G.....A...
...T..A...
.........G
...T..A...
......A.G.
....C.....
....C.....
....C.....
......AT..
......A...
T.....A..T
......A..T
.....AAA.A
.....AAT..
GGCGGTGACC
..........
..........
..........
.......G..
..........
...A......
.........G
......C..G
.C.......G
C.........
C.........
.........G
.........G
.......T.G
.........G
.........G
.........G
......TGAG
.........G
.........G
.........G
..........
..........
..........
........AT
....C.....
.........G
....C.....
C...C.....
C.........
......C..G
.C.......G
C..C..C.G.
.........G
C.........
...AT.....
...TC....G
...AT.....
...CT....G
...C.AT..A
...C.ATT.A
...C.AT..A
A..TAAA.A.
C..TAAA.A.
A.G.TAC..T
..G.TAC..T
T.GCTAC..T
T.G.TAC..T
------AACT
------....
------....
------....
------....
------....
------....
------....
------....
------....
------....
------....
------....
------....
------....
------....
------....
------....
------....
------....
------....
------....
------....
------....
------....
------....
------....
------....
------....
------....
------....
------....
------....
------....
------....
------...C
------....
------..G.
------....
------....
------....
------....
------....
------....
------....
------..TG
------..TG
ATTGCA..GG
------..TG
AAGCTGGGCC
G.........
G.......G.
G.......G.
G.......G.
G.......G.
G.......G.
G.......G.
G....C..G.
G.......G.
G.......G.
G.......G.
G.......G.
G.......G.
G.......G.
G.......G.
G.......G.
G.......G.
G.......G.
G.......G.
G.......G.
G.......G.
G.........
G.........
CC......G.
G.......G.
CGC---T.G.
---.....G.
G.......G.
G.......G.
G.......G.
G....C..G.
G.......G.
G....C....
G.......G.
G.......G.
........G.
G.......GG
........G.
G.......GA
CC......TG
CC......TG
CC......TG
G.TT.T..AG
G..TG...GA
G...A...AG
G.A.A...A.
G.AT.T..AG
G.A.AT..AG
ACGGG---GT
..A..---..
.TC..---A.
..A..---A.
.....---..
.....---.G
.....---..
....T---..
.....---..
.....---..
...A.---A.
.T.A.---A.
...A.---A.
.....---..
..C..---A.
.....---..
.....---..
.....---..
.....---..
.....---..
.....---..
.....---..
..A..---A.
..A..---A.
..CA.---A.
..CA.---C.
..CA.---..
..CA.---.C
..CA.---T.
...A.GGGTC
...A.AAGAC
.....---AG
.T...---A.
...A.---T.
...A.---C.
...A.---C.
..A..---C.
..AA.---C.
..A..---C.
..TAT---C.
.....---..
.....---.G
.....---A.
.TCA.---C.
.TCA.---TC
CTCA.---A.
CTCA.---A.
CACTA---A.
CT.A.---A.
GGCCGGACGC
..........
.....TCA..
....A.....
..........
......C...
.......T..
....A.....
....C.T...
..........
....CTCA..
....A.....
....T....A
....T.....
...GTAG...
....T.....
....T.T...
....T..T..
....T..T..
....T.T...
....T.....
....T.....
..........
..........
......C...
..TG.CCA..
......C...
...G.C.A..
.....AC...
....CTCA..
....A.....
....C.....
..........
...A..T...
....T..T.A
..T..TGG..
A...A..T..
AA..T..T..
A...A..T..
A.........
A..GTC.G..
A....C.A..
A....C.A..
AAGT.C.G..
AA.....T..
T.ATTTTT..
T.ATATTT..
A.AAATTT.T
TATATA.T..
TGTGGAGAGC
..G.......
.C.T......
CT....T.CA
C.GT.....T
.........T
C..T.....T
...T..C...
GA.T......
G.GT......
.T.T..T..A
CT.C..T.A.
.T.A.....G
...T......
..GT......
..........
..........
..G.......
..GT......
..GT......
..........
..GT......
.CATA...C.
..AT....C.
A.A...AG..
G.A..TC.T.
G.AA.CCCC.
A.A.CTC.T.
G.A..TCCC.
G.A..TCC..
G.A.ACCCC.
G.AA...CT.
..A......A
GT.CC..G.T
G.AC...GC.
GAACC..C..
G.GC.CACC.
G.G..CCCC.
G.GC.CACC.
G.GT.CACC.
..G....TC.
.......TC.
.C.....TC.
ATATA.ACC.
C..TA.ACC.
CTC...CTCA
CTG...CTCA
CTCT..TG.A
..ACT.C.A.
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[97]
[97]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[100]
[191]
[191]
[191]
[191]
[191]
[191]
[191]
[191]
[191]
[191]
[191]
[191]
[191]
[191]
[191]
[191]
[191]
[191]
[191]
[191]
[191]
[191]
[191]
[191]
[191]
[191]
[188]
[188]
[191]
[188]
[188]
[191]
[191]
[185]
[191]
[191]
[191]
[191]
[191]
[191]
[191]
[191]
[191]
[191]
[191]
[191]
[191]
[194]
[191]
Fig. 1. Nucleotide sequence identity between Tursiops truncatus DRB sequence (Tt-DRB) and other major histocompatibility complex
(MHC) class II β-chain sequences from T. aduncus, artiodactyls, other mammals, chicken, frog, and fishes. Tt, Tursiops truncatus; Ta, T.
aduncus; Zaca, California sea lion Zalophus californianus.
Zoological Studies 49(1): 132-151 (2010)
138
(B)
[
[
[
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0
1
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3
4
5
6
7
8
9
0]
1234567890 1234567890 1234567890 1234567890 1234567890 1234567890 1234567890 1234567890 1234567890 1234567890]
TtDRB
TaDRB
HippoDRB
PigDRB
SheepDRB
CattleDRB
GoatDRB
CatDRB
DogDRB
ZacaDRB
RatDRB1
MouseDRB
TamarinDRB
MacaqueDRB4
MacaqueDRB1
GorillaDRB5
GorillaDRB3
GorillaDRB1
HumanDRB1
HumanDRB3
HumanDRB4
HumanDRB5
TaDQB
TtDQB
PigDQB
HippoDQB
CattleDQB
SheepDQB
HorseDQB
RatDQB
MouseDQB
DogDQB
ZacaDQB
RabbitDPB
HumanDPB
MoleratDPB
HumanDOB
MouseDOB
ChimpanzeeDOB
CattleDOB
ChickenBL1
ChickenBL2
ChickenBL3
FrogB2
FrogB1
ZebrafishDAB1
ZebrafishDAB4
ZebrafishDCB
ZebrafishDEB
TTCACGGTGC
..........
..........
...CT.....
.....T....
.....T....
.....T....
...CT.....
..........
..........
...CTT....
...CTT....
...CTA....
.....A....
.....A....
.....A....
.....A....
.....A....
.....A....
.....A....
.....A....
.....A....
..........
..AG......
ACG..CC...
ACGT.CT...
.....CTG..
AC.T.CT...
AGGT.CT...
AC.T.CC...
AC.T.CC...
AC....T...
ACG.TCT...
C..C.C....
G.G..CC...
C..T.CCA.T
.....T...G
.....T...G
.....T...G
.....C...G
..........
..........
..........
.A...T..AG
.A...T..AG
GCTGTCCGTG
GCTGTACGTG
A.GCT.AAT.
AA..TTC.AG
[
[
[
3
4]
0
1
2
3
4
5
6
7
8
9
0]
1234567890 1234567890 1234567890 1234567890 1234567890 1234567890 1234567890 1234567890 1234567890 1234567890]
TtDRB
TaDRB
HippoDRB
PigDRB
SheepDRB
CattleDRB
GoatDRB
CatDRB
DogDRB
ZacaDRB
RatDRB1
MouseDRB
TamarinDRB
MacaqueDRB4
MacaqueDRB1
GorillaDRB5
GorillaDRB3
GorillaDRB1
HumanDRB1
HumanDRB3
HumanDRB4
HumanDRB5
TaDQB
TtDQB
PigDQB
HippoDQB
CattleDQB
SheepDQB
HorseDQB
RatDQB
MouseDQB
DogDQB
ZacaDQB
RabbitDPB
HumanDPB
MoleratDPB
HumanDOB
MouseDOB
ChimpanzeeDOB
CattleDOB
ChickenBL1
ChickenBL2
ChickenBL3
FrogB2
FrogB1
ZebrafishDAB1
ZebrafishDAB4
ZebrafishDCB
ZebrafishDEB
TCTATCCAGG
..........
..........
....C.....
....C.....
....C.....
....C.....
..........
..........
..........
....C..T..
....C..T..
....C.....
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
........A.
........A.
.......G..
..........
........A.
....C....C
....C....C
..........
....C.....
..........
....C...A.
....C.....
..........
....C..C..
..........
....C.....
....C..GCC
....C..GCC
....C..GCC
...T...TCC
...T...TCC
....C..TAA
....C..TAA
.T..C..CAA
.......CAA
AGCGGCGAGT
..........
..........
C........C
..........
..........
..........
..........
..........
..........
C....A....
C....A....
C....A....
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
....C.A...
......A...
..........
...AC.A...
G.....TT.A
G.....TT.A
..........
..........
T..TC.A.AC
....C.....
C..TCAT..C
G.A.AAA...
..A.AAAT..
G.A.AAA...
G.A..A....
..A..A.C..
..A..A.C..
..A..A.C..
.CA..AA.TC
.CA..AA.TC
.TAAAGC...
.TAAAGC...
.TGT.AC...
GAAAAAC...
CCACATTGAA
..........
.A........
....G.G..G
..........
..........
..........
...T.....G
..........
..........
.AG.......
.A........
.AG.......
.AG.......
.AG.......
.AG.......
.AG.......
.AG.......
.AG.......
.AG.......
.AG.......
.AG.......
...G..CA..
...G..CA..
...GG.CA..
...G..CA..
...G..CA.G
..GG..CA.G
...G..CA..
...G..CA..
.A.G..CA..
...G..CA..
...G..CA..
....G..C..
.AG....C..
.A....CC..
GG.T..CA.G
GG....AAGT
GG.T..CA.G
AG....CA.G
GG.G..C..G
GG.G..C..G
GG.G..C..G
AATG..AA.G
TATG..AA.G
AA.A...A..
AA.A..CA..
AGC....A..
AAGA..CA..
GGCACCTACA
...G......
CAAG......
T.AG..C...
..AG....T.
..AG......
..AG......
T.AG..G...
C.AG......
T.AG......
T.A...C.AG
T.AG.....G
CCA.....AG
TCA.....AG
CCA.....AG
T.AG....AG
CCAT...CAG
CCA.....AG
T.AG....AG
CCAT...CAG
CCA.....AG
T.AG....AG
..A.......
..A.......
.CA.......
..A.......
..A.......
..A.......
..A.......
.CAG..C.AT
ACAG..C.GT
..A...G...
..A.......
CCAG..CCG.
CCAG....AG
TCAG....AG
.CA...AGAG
.C.T..AGAG
.CA...AGAG
ACA...AGAG
..AG..C.AG
..AG..C.AG
..AG..C.AG
TCA...CGAT
TCA...C.AT
AAA...G.AG
ACT...AGAG
.AA...AGA.
AAA...ACAG
GTCAGGTGGT
..........
....A.....
..........
..........
..........
..........
....A.....
.........C
..........
.....A....
.....A....
....A.....
..........
..........
..........
..........
..........
..........
..........
..........
..........
..TC......
..TC......
..TCA.....
..TC......
..TC......
..TC......
..TC......
..GC.C....
..GC.C....
...C......
..TC......
...C.T....
...C.A....
...C.C....
A...A.....
....A.....
A.........
....CC....
..G.A.....
..G.A.....
..G.A.....
..G.CT...C
..G.CT...C
A.GTCT...C
A.GTCC...C
C.G.C....A
....CC....
GTGACTGTGT
..........
..........
.....G....
..........
..........
..........
.....C....
..........
.....C....
.....C....
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
.....CA.C.
.....CA.C.
.....CA.C.
.....CA.C.
.....CA.C.
.....CA.C.
.....CA.C.
...G.CA.C.
..CGTCA.C.
.....CA.C.
.....CA.C.
....GC..C.
....AC..T.
..ACAC....
.....A....
.....A....
.....A....
.....G....
....GG..C.
....GG..C.
....GG..C.
....AGA.TG
....AGA.TG
..A..AA.TC
..A..AA.CA
..C.T.A.TC
..T.AGC.TA
TCCGGAACGG
..........
..........
.......T..
..........
..........
........A.
..........
.......T..
.T.....T..
.......T..
.......T..
.........A
..........
.......T..
..........
...A......
.......T..
..........
..........
..........
........A.
.......T.A
.......T.A
.......T..
.......T.A
.......T.A
.......T.A
.......T.A
.......T..
.......T..
.T.....T.A
.T.....T.A
...T...T..
...T...T..
........AA
...T...T..
.......T..
...T...T..
.......T..
...T......
...T......
...T......
.GAA...T..
.GAA...T..
.GA.AG.T.A
.GA.AG.T..
.GA....T.A
.AA.A..T..
ATCCTGCAAA
..........
..........
.C........
..........
..........
..........
TC..CT.G..
.....A.G..
.....A.G..
.C...T....
.C..CA....
..........
..........
.....T....
.........G
..........
.....T...G
.....T....
..........
.....T....
.........G
CC..AT.C.G
CC..A..C.G
CC..AT.C..
CC..AT.C.G
CC..GT.C.G
CC..AA.C.G
CC..AT.C..
CC.TGT.C.G
CC.TGT.C.G
TC..GT.C..
CC..GT.C.G
CC..CT.C..
CC..CT.C..
CG..CT.C..
.C..A.AG.G
....A.AG.G
.C..A.AG.G
....G.AG..
CGG.GCTGC.
CGG.GCTGC.
CGG.GCTGC.
TAAACA.G..
TAAACA....
.GT.A.TG.T
.GT.A.TG.G
GGT.A.TT.C
..T.A.TG..
CCAGGAAGAG
..........
..........
..........
...C......
...T......
...T......
......G...
..........
..........
.A....G..A
.A....G...
T.....G...
..........
..........
..........
..........
..........
..........
..........
..........
..........
......G...
......G...
......G...
......G...
..G...G...
......G...
......G...
......G...
......G...
......GC..
......G...
A.G...G..A
A.....G..A
......G..A
G.....G...
A.....G...
G.....G...
G......C..
G.G...G...
G.G...G...
G.G...G...
GATT......
GATT......
TA.A.TG.T.
TA.A..G.T.
TA.AAGG.TT
TA.ACC..T.
GACACAGCCC
..........
...C......
...C......
...C......
...T......
...C......
...G......
...T...A..
...C......
...G......
...G......
...C......
...C......
..TC......
...C...A..
...T......
...C..A.T.
...C......
...C......
...C......
...C...A..
....G..G.T
....G..G.T
AG..G..G.T
...TG..G.T
....G..G.T
...TG..G.T
....G..GTT
....G..G..
....G..G..
....G..GTT
....G..GTT
..AGGG....
..AGGG....
AGG.GG.A..
...C.CA.T.
...C.C.TTG
...C.CA.T.
...C.CAG..
.T.GGGCT..
.T.GGGCT..
.T.GGGCT..
.....T.GAT
...G.T.GAT
.CAGGCTGAA
.CAGGCTGAA
TGA.GCTAAA
.CAGGCTGGT
GAGGCAGGGG
..........
.....T....
.C...G....
.....T....
.....T....
.....T..A.
...A.T....
..A..T....
.....T....
A...AT..AC
A.AA....AA
A....T....
A....T....
A....T....
A....T....
A....T....
A....T....
A....T....
A....T....
A....T....
A....T....
ACA..T..C.
ACA..T..C.
ACA..T..C.
ACA..C..CA
ACA..C..C.
ACA..C..A.
ACA..T..C.
AC...G....
AC..TG....
ACA..T..T.
AGA..T..T.
ACA..C....
ACA..T....
ACAA.T...A
AGA..T....
AG.T.T....
AGA..TC...
AGA.AG...A
AC..AGC.C.
AC..AGC.C.
AC..AGC.C.
AGA.A.CAA.
.GA.A.CA..
ACCT...AT.
ACCT...AT.
ACA..T.AT.
ACCA.T.AT.
CTGCAGCACC
..........
..........
..........
..........
..........
..........
..........
T.......T.
..........
...G.A....
...G.A....
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..AA.C....
..AA.C....
..AA.C..T.
..AA.C....
..AA.C....
..AA.C....
..AA.C....
..CA.C....
..CA.C....
...A.C....
...A.C....
..........
..........
...A.C..T.
.....C..G.
........G.
.....C..G.
..........
....CCG.AA
....CCG.AA
....CCG.AA
T.AG.A..TG
T..G.A..TG
GGTA.A..T.
GGCAGA..T.
GGCA.T..GA
GGCAGA..G.
TGGTCTCCAC
..........
.......T..
..........
..A.......
..A.......
..A.......
.C..G.....
.T..G.....
.C..G.....
.C..G.....
.T..G.....
....G.....
....G.....
....G.....
....G.....
....G.....
....G.....
....G.....
....G.....
....G.....
....G.....
.T..G.....
.T..G.....
.T..G.....
.T..G.....
.T..G.....
.T..G.....
.T..G.....
.C..G.....
.CTCG.....
.C..G.....
.C..G.....
.C..G.....
.C..G.....
.CTCAA....
.CA.G.....
.CA.G.....
.CA.G.....
.CA.G.....
....G.....
....G.....
....G.....
.CACA...T.
.CACA...T.
..AC......
..AC......
..ACG....T
.AAC......
ACAACCTCCT
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
...C......
..........
..........
..........
..........
..........
..........
..........
..........
.......G..
.......G..
.......G..
.......G..
.......G..
.......G..
.......G..
.....T.G..
.....ACG..
....T..G..
.......G..
...G.T.G..
.......G..
.......G..
.T..T..G..
.......G..
.T..T..G..
G...T..G..
C.G...GT..
C.G...GT..
C.G...GT..
.A..T..GA.
.A.....GA.
CAGCTA.G..
CAGCTG.GT.
.AGCTG....
CAGCTG.GT.
AGGCCTGATC
..........
..........
..........
..........
..........
..........
..........
......A...
....T.....
C.........
G......G..
..........
..........
..........
.........T
G.........
..........
G.........
G.........
..........
.........T
CCCT..T..T
CCCT..T..T
TCCT..T..T
CCCT..C..T
CCCT..T..A
.CCT..T..T
CCC...T..T
.CAG..T..T
.CAG..T..T
TCCA..T..T
TCCA..T..T
CCA..C....
CAA.......
CAA..C....
T....CT...
T.....TG.T
C....CT...
T..T..C...
G.A.G....G
G.A.G....G
G.A.G....G
..CAT.AC.A
..AGT.AC.A
TATAGA...G
TATGGA...G
T.AGGA...G
T.AGGA.C.G
GGTCTGCTCT
....C.....
..........
..........
..........
..........
..........
.........C
..........
..........
..........
..........
..........
..........
..........
..........
..........
......T...
..........
..........
..........
..........
.........G
.......A.G
.......G..
...T.....G
.........G
.........G
.........A
.........A
.........A
.........A
.........A
C......CAC
T......CAC
C......CAG
.CA.......
.C........
.CA.......
.C.T....TG
..CG....AC
..CG....AC
..CG....AC
AAC....ATA
AAC.....T.
.C.G...GA.
.C.G...AG.
...G...AG.
...G...AG.
CCTAATGGAG
G.........
..........
..........
.AG.......
.AG.......
.AG.......
.G........
.G........
.GG.......
.GA.......
.GA.......
.AG.......
.AG.......
.AC.......
.AG.......
.AG.......
.AG.......
.AG.......
.AG.......
.AG.......
.AG.......
AGA.....G.
AGA.....G.
AGG..C....
AGG..C..G.
GGG.....G.
AGG..C..G.
AGA..C..G.
AGG..C..G.
AGG.....G.
AGG.....G.
AGG.....G.
.A........
.G........
.GC.......
AGG.......
AGG.......
AGG.......
AGG.......
.AG..C..G.
.AG..C..G.
.AG..C..G.
AAG.......
..G.......
G.......TA
G..G......
G..G......
G..G....G.
GTGAATGGTT
..........
..........
....C...G.
........A.
.....C....
........A.
..........
..........
.....C....
....G..AC.
....G..AC.
..A.G.....
.....C....
.....C....
..........
....G.....
....G.....
....G.....
....G.....
..........
..........
....CA.A..
....CA.A..
....CA.A..
...GCA.A..
....CG.A..
....CA.A..
....CA.A..
....CA.A..
....CA.A..
....CA.A..
....CA.A..
....CA.A..
....CA.A..
....CA.A..
....CA..C.
....CA..C.
....CA..C.
....CA....
....CG..C.
....CG..C.
....CG..C.
..AG....G.
..AG....G.
.CAT...AG.
.CAT...AA.
.AAT...AC.
.CAT...AC.
ACTGGACCTT
..........
..........
..........
..........
..........
..........
..........
..........
..........
.T........
..........
..........
..........
..........
..........
..........
..........
.T........
..........
..........
..........
..........
..........
.........A
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
.......G.A
.......G.A
.......G.A
.......A..
.......A..
.....TA..A
.....TA..A
.....TAT.A
.....TA..A
[291]
[291]
[291]
[291]
[291]
[291]
[291]
[291]
[291]
[291]
[291]
[291]
[291]
[291]
[291]
[291]
[291]
[291]
[291]
[291]
[291]
[291]
[291]
[291]
[291]
[291]
[288]
[288]
[291]
[288]
[288]
[291]
[291]
[285]
[291]
[291]
[291]
[291]
[291]
[291]
[291]
[291]
[291]
[291]
[291]
[291]
[291]
[294]
[291]
[391]
[391]
[391]
[391]
[391]
[391]
[391]
[391]
[391]
[391]
[391]
[391]
[391]
[391]
[391]
[391]
[391]
[391]
[391]
[391]
[391]
[391]
[391]
[391]
[391]
[391]
[388]
[388]
[391]
[388]
[388]
[391]
[391]
[385]
[391]
[391]
[391]
[391]
[391]
[391]
[391]
[391]
[391]
[391]
[391]
[391]
[391]
[394]
[391]
Fig. 1. Nucleotide sequence identity between Tursiops truncatus DRB sequence (Tt-DRB) and other major histocompatibility complex
(MHC) class II β-chain sequences from T. aduncus, artiodactyls, other mammals, chicken, frog, and fishes. Tt, Tursiops truncatus; Ta, T.
aduncus; Zaca, California sea lion Zalophus californianus.
Yang et al. – MHC Genes in Dolphins and Relatives
(C)
139
[
[
[
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4
5
6
7
8
9
0]
1234567890 1234567890 1234567890 1234567890 1234567890 1234567890 1234567890 1234567890 1234567890 1234567890]
TtDRB
TaDRB
HippoDRB
PigDRB
SheepDRB
CattleDRB
GoatDRB
CatDRB
DogDRB
ZacaDRB
RatDRB1
MouseDRB
TamarinDRB
MacaqueDRB4
MacaqueDRB1
GorillaDRB5
GorillaDRB3
GorillaDRB1
HumanDRB1
HumanDRB3
HumanDRB4
HumanDRB5
TaDQB
TtDQB
PigDQB
HippoDQB
CattleDQB
SheepDQB
HorseDQB
RatDQB
MouseDQB
DogDQB
ZacaDQB
RabbitDPB
HumanDPB
MoleratDPB
HumanDOB
MouseDOB
ChimpanzeeDOB
CattleDOB
ChickenBL1
ChickenBL2
ChickenBL3
FrogB2
FrogB1
ZebrafishDAB1
ZebrafishDAB4
ZebrafishDCB
ZebrafishDEB
CCAGACCATG
..........
..........
..........
..........
..........
..........
.......C..
.....T.C..
......TC.A
....TTGC..
......AC..
......AC..
.......C..
.......C..
.....TTC..
.......C..
.......C..
.......C..
.......C..
.......C..
.......C..
.....TGC.T
.....TGC.T
....GTGC.T
.....T.C.C
.....T.C.C
.....T.C.C
.....T.C.T
.....T.C..
....GT.C..
.....T.C.T
.....T.C..
.....T.C..
.....T.C..
.....T.C..
T.....TG..
......A.CA
T.....TG..
T....TG.C.
....GTGC..
....GTGC..
....GTGC..
TG.A.T.CAT
TG.A.T.CAT
T....TTCAC
T....TTCAT
T....TTCAC
...A.TTCAT
[
[
[
5
6]
0
1
2
3
4
5
6
7
8
9
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1234567890 1234567890 1234567890 1234567890 1234567890 1234567890 1234567890 1234567890 1234567890 1234567890]
TtDRB
TaDRB
HippoDRB
PigDRB
SheepDRB
CattleDRB
GoatDRB
CatDRB
DogDRB
ZacaDRB
RatDRB1
MouseDRB
TamarinDRB
MacaqueDRB4
MacaqueDRB1
GorillaDRB5
GorillaDRB3
GorillaDRB1
HumanDRB1
HumanDRB3
HumanDRB4
HumanDRB5
TaDQB
TtDQB
PigDQB
HippoDQB
CattleDQB
SheepDQB
HorseDQB
RatDQB
MouseDQB
DogDQB
ZacaDQB
RabbitDPB
HumanDPB
MoleratDPB
HumanDOB
MouseDOB
ChimpanzeeDOB
CattleDOB
ChickenBL1
ChickenBL2
ChickenBL3
FrogB2
FrogB1
ZebrafishDAB1
ZebrafishDAB4
ZebrafishDCB
ZebrafishDEB
AGGGCACAG................G........G........G........G........G......T......................AA.......AA........A....GA..A........A....G...A........A...AG..AA....G...A....G...A....G...T....G...A......T........T........C........C........C........C........C.A......C........C........C..................A........A............C.....A..T....T...T.....A..T.....A..T...GA.C.T.C.GA.C.G.C.GA.A.T.C.GAAC..G.TG
GA.C..G.TG
.ATC.T..TA
.ATC.C..TA
GATC.CTCTC
GATT..TCTC
GTGATGCTTG
..........
..........
..........
..........
..........
..........
........G.
........G.
........G.
........G.
........G.
........G.
........G.
........G.
........G.
........A.
........G.
........G.
........A.
........G.
........G.
........G.
........G.
........A.
........G.
........G.
........G.
........G.
..C.....G.
..C.....G.
........G.
........G.
........G.
........G.
....CA..G.
........A.
..A.....G.
........A.
........G.
...G....G.
...G....G.
...G....G.
........G.
........G.
TCTCAC....
TCTCAC..G.
TCCCAC..G.
TCCCAC..G.
--TCTGAATC
--........
--........
--........
--.....C..
--.....C..
--.....C..
--........
--.....T..
--.....G..
--..CACG..
--..CAC...
--..C.....
--........
--........
--........
--........
--........
--........
--........
--........
--........
--........
--....G...
--........
--........
--........
--........
--........
--..C..G..
--..C..G..
--........
--........
--.....T..
--.....T..
--.....C..
--.......A
--.......A
--.......A
--.......A
--G.G..CG.
--G.G..CG.
--G.G..CG.
TA........
TA.....T..
TC.....G..
TC.....G..
TCC.C..GA.
TC.....GC.
AAACAGTCCC
..........
..........
....G..T..
.......T..
.......T..
.......T..
.......T..
.G.T...T..
.G.....T..
.G..G..T..
.G..G..T..
....T..T..
.......T..
.......T..
.......T..
.......T..
.......T..
...T...T..
.......T..
.......T..
.......T..
...TGACT..
...TGACT..
.G.TGAAT.T
...TGACT..
...TGAC...
.G.TGAC...
...TGACT..
.G.TGACG..
.G.TGAC...
.G.TGACT..
...TTACT..
...TGAC...
...TGAC...
...TGAC...
...TGACT..
...TGA....
...TGACT..
CC.TGACT..
.G..C.....
.G..C.....
.G..C.....
.G...AC.AT
.G...AC.AT
..TACACT..
..TACAC...
..TATT.T..
..TACAC...
TGCTCAGAGC
..........
..........
.......G..
A.........
..........
A.........
...A......
...A......
...G......
...A..A.AT
...A....A.
...A.....T
...A......
...A......
...A......
...A......
..GA......
...A......
...A......
...A......
...A......
...C......
...C......
...C......
...C......
...C......
...C......
.T.C......
...C......
...C.G....
...C......
...C......
C..C.G....
...C.....T
...C.G..A.
.T..TG...A
.T.CTG..AA
.T..TG..AA
.T..TG...A
G.GCAG....
G.GCAG....
G.GCAG....
.....GC.AT
.....GC.AT
C.ACAG..AT
..ACAG..AT
..AAAGATCT
..AGAG..AT
TCAGAGTGGA
..........
..........
..........
..........
..........
..........
..........
......C...
..........
...AG.....
..........
..........
..........
..........
..........
..G.......
..G.......
..G.......
..G.......
..G.......
..GA......
C...CAA...
C...CAA...
C...C.A...
C...C.A...
C...C.A...
C...C.A...
C...C.A...
....C.G...
..G.C.G...
C...C.A...
C...C.A...
C..CCAG...
C...CAG...
....C.G...
.G.ACT....
AG..CT...T
.G.ACT....
.G..CT....
G.G.C.C..G
G.G.C.C..G
G.G.C.C..G
.A.ACA...T
.A.ACA...T
CA.ATC....
.A.ATC....
...ACC....
CA.ATC....
AAGATGCTGA
..........
......A...
......A...
......A...
......A...
......A...
..........
..........
..........
....A.A...
......T...
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
......T...
..........
..........
........CG
....CAT...
....CA....
..........
.....A....
..........
.....C....
...C......
...C......
...C......
......T...
..........
..AT.TGCC.
..AT.TGCC.
.....CA.TC
..A..TGCT.
GAGGTCTACA
..........
..........
..........
..........
..........
..........
..........
..........
..........
.....T....
.....T....
.....T....
.....T....
.....T....
.....T....
.....T..T.
.....T....
.....T....
.....T....
.....T....
.....T....
..T.......
..T.......
..T.......
..T.......
..T.......
..T..G....
..T.......
.....G....
..........
..T.......
..T.......
..C.......
..C.......
..C.......
C.T.......
..CA......
C.T.......
..........
..CAG....G
..CAG....G
..CAG....G
..CACT.T..
..CACT.T..
...AAGAT.C
...AAGAT.C
...AAGAT.T
..AAAGATTT
GTGGAATCGG
..........
.....G.T..
....GG....
.....G.T..
.....G....
.....G....
..........
..........
.......T..
.....G....
.....G.T..
.....G....
.....G....
.....G....
.....G....
.....G....
.....G....
.....G....
.....G....
.....G....
.....G....
....TG.T..
....TG.T..
.C..TG....
....TG.T..
....TG.T..
....TG.T..
....CG....
.C..C.....
.C..C.....
....C..T..
....C.....
C...CG....
CG...GCT..
C....G.T..
....C..T.C
.....GCT.C
....C..T.C
.....GCT.C
CG..CG.G..
CG..CG.G..
CG..CG.G..
C...T..T.T
C...T..T.T
TA...GC.TC
TC...GCATC
T...GGCT.T
T....GCATC
CCTGCCACGT
..........
.......A..
G.....GA..
.......A..
.......A..
.......A..
..........
.......A..
.......A..
.......G..
.......G..
T......A..
.......A..
.......A..
.......A..
.......A..
.......A..
.......A..
.T.....A..
.......A..
.......A..
.......T..
.......A..
......G...
..........
......G...
......G...
..........
T......T..
.......T..
.T.....T..
.......T..
..........
T......A..
..........
......TT..
G.....T...
......TT..
......T...
TG....GG..
TG....GG..
TG....GG..
.......A..
.......G..
AA..TGTG..
AA..TTTG..
....TGTG..
....TATG..
GGCCTTTGTT
..........
..G.......
..G...C..C
..G.......
..G...C...
..G...C..G
..G.......
..G......C
A.G.......
..G.A.C..G
..G...C..G
..G......G
..G......G
..G.A....G
..G......G
..G......G
..G......G
..G...C..G
..G......G
..G......G
..G......G
..G......G
..G......G
..G...C..G
..G...C..G
..G...C..G
..G...C..G
..G...C..G
..G......G
T.G..GC..G
T.G...C..G
A.G......G
..GG..A..G
..G...C..G
..G.C.G..G
A.....CC.A
A.TG..CC.G
A.....CC.A
A.....CC.G
..G...C..G
..G...C..G
..G...C..G
..G......G
T.GG.....G
T.GTC.G..G
T.GTC.G..G
..GGC.GC.G
T.GT..G..G
GGAGCACCCC
..........
..........
..........
...T......
...T......
..........
......T..A
.........A
.........A
......T..A
......T...
.......G.A
.........A
.........A
...C.....A
......G..A
.........A
.........A
.........A
......T..A
.........A
..........
..........
.......T..
..........
..........
..........
..........
T..C......
......T...
.......G..
..........
..........
.......A..
..........
C..T...T..
..........
C..T...T..
T..C..T...
.......G..
.......G..
.......G..
.......AG.
.......AG.
.......G..
.......G..
...T..TG..
...T..TG..
CTGGGTCTGC
..........
.....G....
..........
..........
..........
..........
..........
..........
..........
..........
.....C....
..........
.....C....
.....C....
.....C....
.....C....
.....C....
.....C....
.....C....
.....C....
.....C....
.....G...A
.....G...A
.....G...A
.....G...A
.....G...A
.....G...A
.....G..CA
.....GG..A
..T..GG..A
.....G...A
.....G...A
.....G..CG
.....G..CA
.....G..CA
..T..G..AA
..T..G...A
..T..G..AA
G.C..G..AG
.....G..CG
.....G..CG
.....G..CG
.....ATCCA
..T..ATCCA
.....AA.TA
.....AA.CA
A....GA.CT
.....AA.CA
AGCCAGACCA
..........
........G.
....T...G.
....G...G.
.AG.G...G.
....G...G.
..T.GC..G.
..TTT...A.
...TT..TG.
....T.C...
....T....G
...AT...G.
...GT...G.
...GT.....
...GT...G.
...GTA..G.
...GT...A.
..TGT...G.
...GTA..G.
...AT..TG.
...GT...G.
....TCCAG.
....TCCAG.
....TCCAG.
....TCCAG.
....TCCAG.
....TCCAG.
....TCCAG.
....TTGAG.
....T..AG.
....TCCAG.
....TCCAG.
....T.GA..
....T.GA..
....T.GA..
....T.CTG.
G...TCCTG.
....T.CTG.
...TT.CTG.
....T.CGGC
....T.CGGC
....T.CGGC
....TTCAAC
....TTCAAC
...TCA..TC
...TTA..TC
...TTCCAT.
...TCA..TG
TCTTCCTTGG
..........
.........C
....TG...C
.........C
.........C
.........C
.......G.T
.........C
.......C.T
.......G..
.......G..
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
.......C..
.......C..
.......C..
.......C..
.......C..
.......C..
.......C..
.......C..
.......C..
..........
.......C..
...CG..G.C
..A..TG...
.......G.C
........CT
.TG..T.CCT
........CT
.....T..CT
.......G.C
.......G.C
.......G.C
.....A.CAT
.T...A.CAT
.TA.AGCGAT
..A.AGCGAT
.TACAGCA.C
..A.TGCCAC
GCCCTGTCAC
..........
.....T....
....C.....
.....A....
.....A....
.....A....
.....A....
..........
.T........
.........G
A.........
.....C....
.....C....
.....C....
T....C....
.....C....
.....C....
.....C....
..G..C....
.....C....
.....C....
.T..CA...T
.T..CA...T
....CA..TT
....CA...T
....CA...T
....CA...T
....CA....
....C.....
.T..CA....
....CA....
....CA....
.T..CA....
.T........
.A..CA....
.......TT.
.A.....TT.
.......TT.
.......TT.
AG..CA...G
AG..CA...G
AG..CA...G
AA.....AT.
AA.....AT.
AA...A.T..
AA...C.T..
AA..CA.G.T
AA..CA...T
GGTGGGGCTG
..........
A.........
T.........
.........C
C........C
.........C
..........
A.........
..........
A.CC......
A.C.......
A.CA......
..CC......
..CC......
..CC......
..CC......
..CT......
..CC......
..CC......
.ACA......
..CC.....A
.C....T..C
.C....T..C
.C....C..T
.C....C..C
.C....C..C
.C....C..C
AC....C..T
.C.T..C..T
.C.T..C..T
.C.T..C..T
.C....C..T
A.....CG.C
A.....CA.C
A.....C..C
......AA.C
.......G.T
......AA.C
.....AAA.T
.C.......C
.C.......C
.C.......C
A..T..C..T
T..C..C..T
T.CT..A..C
T.CT..A..C
T.CA..AG..
T.CT..A..C
AGTGGAATGG
..........
..........
..........
..........
...A......
...A......
C.....G...
C.........
C.........
......G...
G..C..G...
....C.....
..........
.A........
..........
..........
..........
..........
..........
G...C.....
..........
G.....G...
G.....G...
G.....G...
G.....G...
G.....G...
G.....G...
....C.G...
......G...
C.....G...
....C.G...
......G...
T.....G...
C.....G...
C.....G...
T.....G...
T....C....
T.....G...
T.....G...
CCA..CG...
CCA..CG...
CCA..CG...
T...A.C...
T...A.C...
TAAA......
TAAA..T...
CTATT.C...
CA.A.CC...
TTCATCTACT
..........
..........
..........
..........
..........
..........
..........
..........
G.........
...G......
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
..........
A.....CGTC
A.....CGTC
......CGTC
A.....C.TC
A.T...CGTC
A.T...CGTC
A.....CGTC
......CGTC
......CGTC
A.....CGTC
A.....CGTC
.....GC..A
.....GC..A
A....GC..G
G.....C.GC
G.T...C.TC
G.....C.GC
G..G..C..A
...G.G.T.C
...G.G.T.C
...G.G.T.C
G.AG.T...C
G.AG.T...C
A.TTA....A
G.TTA....A
A..TAT..TA
A.TTAT...A
[491]
[491]
[491]
[491]
[491]
[491]
[491]
[491]
[491]
[491]
[491]
[491]
[491]
[491]
[491]
[491]
[491]
[491]
[491]
[491]
[491]
[491]
[491]
[491]
[491]
[491]
[488]
[488]
[491]
[488]
[488]
[491]
[491]
[485]
[491]
[491]
[491]
[491]
[491]
[491]
[491]
[491]
[491]
[491]
[491]
[491]
[491]
[494]
[491]
[588]
[588]
[588]
[588]
[588]
[588]
[588]
[588]
[588]
[588]
[588]
[588]
[588]
[588]
[588]
[588]
[588]
[588]
[588]
[588]
[588]
[588]
[588]
[588]
[588]
[588]
[585]
[585]
[588]
[585]
[585]
[588]
[588]
[582]
[588]
[588]
[588]
[588]
[588]
[588]
[588]
[588]
[588]
[591]
[591]
[591]
[591]
[594]
[591]
Fig. 1. Nucleotide sequence identity between Tursiops truncatus DRB sequence (Tt-DRB) and other major histocompatibility complex
(MHC) class II β-chain sequences from T. aduncus, artiodactyls, other mammals, chicken, frog, and fishes. Tt, Tursiops truncatus; Ta, T.
aduncus; Zaca, California sea lion Zalophus californianus.
Zoological Studies 49(1): 132-151 (2010)
140
DRB sequences of primates were grouped with a
posterior probability of 1.00. Clades with relatively
high posterior probabilities (> 0.90) in the BI tree
were also significantly supported by bootstrap
values on the MP and NJ trees (Fig. 3b).
The phylogenetic relationships of the BI
tree based on non-PBR sequences (position
219-616) (Fig. 2c) were nearly identical to those
of the tree using 616 nt (Fig. 2a) except that the
horse was placed outside cetartiodactyls in the
DQB clade with a moderate posterior probability
(0.82). Furthermore, the artiodactyl clade had
moderate posterior probabilities in both the DQB
and DRB clades (0.85 and 0.88, respectively).
The ML, MP, and NJ trees showed essentially the
same clustering patterns compared to the BI tree,
although the bootstrap values of the cetartiodactyl
group in the DQB clade and the artiodactyl group in
(D)
[
[
[
6
]
0
1
]
1234567890 123456]
TtDRB
TaDRB
HippoDRB
PigDRB
SheepDRB
CattleDRB
GoatDRB
CatDRB
DogDRB
ZacaDRB
RatDRB1
MouseDRB
TamarinDRB
MacaqueDRB4
MacaqueDRB1
GorillaDRB5
GorillaDRB3
GorillaDRB1
HumanDRB1
HumanDRB3
HumanDRB4
HumanDRB5
TaDQB
TtDQB
PigDQB
HippoDQB
CattleDQB
SheepDQB
HorseDQB
RatDQB
MouseDQB
DogDQB
ZacaDQB
RabbitDPB
HumanDPB
MoleratDPB
HumanDOB
MouseDOB
ChimpanzeeDOB
CattleDOB
ChickenBL1
ChickenBL2
ChickenBL3
FrogB2
FrogB1
ZebrafishDAB1
ZebrafishDAB4
ZebrafishDCB
ZebrafishDEB
TCAGGAATCA
..........
..........
...A......
..........
..........
..........
..C.......
....A.....
..........
......GC..
..........
..........
..........
..........
...A......
......G...
..........
..........
..........
..........
...A......
A.....GC..
A.....GC..
A.....G...
A.....GC..
A.....GC..
A.....GC..
A.....GCA.
A..A.CG...
A.....G...
A.....GC..
A.....GC..
GG....GCA.
GG....GCA.
.G....GCA.
.A...GC...
...A.GC...
.A...GC...
.....GCCTG
.GC.CGG...
.GC.CGG...
.GC.CGG...
.AC...G.A.
..C...G.A.
AG.A...ATC
AG.A...ATC
AA..A.....
AG.A...ATC
GAAAGG
......
......
......
......
......
......
......
...G..
...G..
......
......
......
......
......
......
......
......
......
......
......
......
...G..
...G..
...G..
...G..
...G..
...G..
...G..
......
......
......
......
.....C
.....T
.....C
......
.....C
......
......
......
......
......
...GAC
A..GAC
A.C...
A.C...
A.C...
ATC...
[604]
[604]
[604]
[604]
[604]
[604]
[604]
[604]
[604]
[604]
[604]
[604]
[604]
[604]
[604]
[604]
[604]
[604]
[604]
[604]
[604]
[604]
[604]
[604]
[604]
[604]
[601]
[601]
[604]
[601]
[601]
[604]
[604]
[598]
[604]
[604]
[604]
[604]
[604]
[604]
[604]
[604]
[604]
[607]
[607]
[607]
[607]
[610]
[607]
Fig. 1. Nucleotide sequence identity between Tursiops
truncatus DRB sequence (Tt-DRB) and other major
histocompatibility complex (MHC) class II β-chain sequences
from T. aduncus, artiodactyls, other mammals, chicken, frog,
and fishes. Tt, Tursiops truncatus; Ta, T. aduncus; Zaca,
California sea lion Zalophus californianus.
the DQB and DRB clades ware low (< 70%) under
the MP and NJ criteria (Fig. 3c). The phylogenetic
relationships of non-PBR sequences should be
more reflective of the actual evolutionary history
of the mammalian MHC genes since they are
governed by purifying selection. For this reason,
in figure 2a, it is advisable to regard the horse as
a sistergroup of the cetartiodactyls in the DQB
phylogeny. This postulation was further supported
by the result of our SH tests on the position of the
horse as mentioned above. Therefore, we used
this postulated position of the horse DQB gene in
the following estimation of divergence times.
Divergence time estimations
The likelihood scores of the BI trees using
616 nt with and without a molecular clock did
not significantly differ (p = 0.374) in the SH test;
therefore the null hypothesis that the rate of
evolution was homogeneous among all branches
in the phylogeny was not rejected. The results of
Tajima’s relative rate test showed that there was
no significant evolutionary rate difference between
the 2 species of bottlenose dolphins, between the
dolphin/ungulate pair, or between the DQB/DRB
pair. By using the birth-death clock model in the
BI tree (Fig. 2a), we estimated the divergence
times between clades under the assumption
that mammals and chicken diverged 310 Mya.
We obtained an estimate of 249.36 Mya for the
divergence between the DOB and other clades
(Table 2). The estimated time of divergence
between the DRB and DQB/DPB clades was close
to that between the DQB and DPB clades, as
expected from the tree in figure 2a. Divergence
times between several major mammalian
lineages (rodents, primates, carnivores, and
cetartiodactyls) were also estimated. Rodents
and other mammals diverged 109-122 Mya in
the DOB, DPB, DQB, and DRB clades; primates
and fereuungulates (carnivores + cetartiodactyls)
diverged around 98 and 88 Mya in the DOB
and DRB clades, respectively; carnivores and
cetartiodactyls diverged around 78 and 76 Mya
in the DQB and DRB clades, respectively. When
cetartiodactyl DQB sequences were constrained
as a monophyletic group, the estimated divergence
time between dolphins and artiodactyls was
60.03 Mya, which was similar to that in the DRB
clade. The 2 dolphin species (T. truncatus and T.
aduncus) were separated by 26 and 22 Mya in the
DQB and DRB clades, respectively. The BI tree
using position 219-616 was also validated as being
Yang et al. – MHC Genes in Dolphins and Relatives
(a)
141
SheepDRB
GoatDRB
0.98
CattleDRB
PigDRB
0.50
1.00
HippoDRB
TtDRB
1.00
1.00
TaDRB
DogDRB
0.96
SealionDRB
1.00
CatDRB
MacaqueDRB1
GorillaDRB1
1.00
1.00
1.00
DRB
GorillaDRB5
HumanDRB5
GorillaDRB3
HumanDRB3
HumanDRB1
MacaqueDRB4
HumanDRB4
1.00
1.00
1.00
0.92
0.69
0.51
0.82
1.00
1.00
1.00
1.00
1.00
1.00
1.00
0.97
1.00
1.00
1.00
0.80
1.00
1.00
1.00
1.00
1.00
1.00
1.00
SheepDQB
HippoDQB
HorseDQB
PigDQB
TaDQB
TtDQB
DogDQB
DQB
SealionDQB
RatDQB
MouseDQB
RabbitDPB
1.00
1.00
TamarinDRB
RatDRB1
MouseDRB
CattleDQB
HumanDPB
MoleratDPB
ChimpanzeeDOB
DPB
HumanDOB
CattleDOB
MouseDOB
ChickenBL2
DOB
ChickenBL3
ChickenBL1
FrogB2
FrogB1
ZebrafishDAB1
ZebrafishDAB4
ZebrafishDEB
ZebrafishDCB
0.1
Fig. 2. Bayesian trees for major histocompatibility complex (MHC) class II β-chain genes using all codon positions with the GTR + Г + I
model and birth-death clock model. The nucleotide positions for tree construction were (a) positions 1-616, (b) 1-218, and (c) 219-616.
The posterior probabilities are shown in parentheses for the nodes of interest. Tt, Tursiops truncatus; Ta, T. aduncus; Sealion,
California sea lion Zalophus californianus.
Zoological Studies 49(1): 132-151 (2010)
142
(b)
MacaqueDRB1
GorillaDRB1
GorillaDRB5
HumanDRB5
1.00
GorillaDRB3
HumanDRB3
HumanDRB1
1.00
0.72
MacaqueDRB4
HumanDRB4
CatDRB
DogDRB
SheepDRB
GoatDRB
1.00
1.00
0.95
CattleDRB
HippoDRB
RatDRB1
0.68
0.71
0.87
0.68
0.99
0.97
1.00
1.00
MouseDRB
TamarinDRB
PigDRB
TaDQB
TtDQB
TtDRB
TaDRB
1.00
0.98
0.98
0.50
DogDQB
HumanDPB
MoleratDPB
RabbitDPB
0.63
0.91
1.00
0.59
1.00
1.00
ChimpanzeeDOB
HumanDOB
MouseDOB
DOB
ChickenBL1
FrogB2
FrogB1
1.00
1.00
0.61
DPB
CattleDOB
ChickenBL2
ChickenBL3
1.00
0.97
DQB
CattleDQB
SealionDQB
SealionDRB
0.71
0.96
0.64
RatDQB
MouseDQB
SheepDQB
HippoDQB
HorseDQB
PigDQB
0.96
0.53
DRB
ZebrafishDAB1
ZebrafishDAB4
ZebrafishDEB
ZebrafishDCB
0.1
Fig. 2. Bayesian trees for major histocompatibility complex (MHC) class II β-chain genes using all codon positions with the GTR + Г + I
model and birth-death clock model. The nucleotide positions for tree construction were (a) positions 1-616, (b) 1-218, and (c) 219-616.
The posterior probabilities are shown in parentheses for the nodes of interest. Tt, Tursiops truncatus; Ta, T. aduncus; Sealion,
California sea lion Zalophus californianus.
Yang et al. – MHC Genes in Dolphins and Relatives
(c)
0.94
1.00
0.88
1.00
1.00
1.00
143
SheepDRB
GoatDRB
CattleDRB
HippoDRB
PigDRB
TtDRB
TaDRB
DogDRB
0.97
SealionDRB
0.98
CatDRB
GorillaDRB3
HumanDRB3
HumanDRB1
0.99
1.00
GorillaDRB1
HumanDRB4
MacaqueDRB1
1.00
1.00
1.00
0.98
0.85
0.66
0.82
0.87 1.0
1.00
1.0
1.00
1.00
1.00
1.00
DRB
RatDRB1
MouseDRB
CattleDQB
SheepDQB
HippoDQB
PigDQB
TaDQB
TtDQB
HorseDQB
1.00
0.76
1.00
DQB
DogDQB
SealionDQB
RatDQB
MouseDQB
RabbitDPB
HumanDPB
MoleratDPB
0.99
1.00
1.00
TamarinDRB
GorillaDRB5
HumanDRB5
MacaqueDRB4
ChimpanzeeDOB
HumanDOB
CattleDOB
DPB
DOB
MouseDOB
ChickenBL1
1.00
ChickenBL3
1.00
1.00
1.00
1.00
1.00
ChickenBL2
FrogB2
FrogB1
ZebrafishDAB1
ZebrafishDAB4
ZebrafishDEB
ZebrafishDCB
0.1
Fig. 2. Bayesian trees for major histocompatibility complex (MHC) class II β-chain genes using all codon positions with the GTR + Г + I
model and birth-death clock model. The nucleotide positions for tree construction were (a) positions 1-616, (b) 1-218, and (c) 219-616.
The posterior probabilities are shown in parentheses for the nodes of interest. Tt, Tursiops truncatus; Ta, T. aduncus; Sealion,
California sea lion Zalophus californianus.
Zoological Studies 49(1): 132-151 (2010)
144
(a)
69
96 SheepDRB
99
GoatDRB
51
CattleDRB
84
HippoDRB
74
PigDRB
100 TtDRB
41
TaDRB
CatDRB
54
66 SealionDRB
DogDRB
MacaqueDRB1
82
GorillaDRB1
GorillaDRB5
HumanDRB5
GorillaDRB3
HumanDRB3
73
HumanDRB1
90
MacaqueDRB4
HumanDRB4
TamarinDRB
100
RatDRB1
MouseDRB
73
SheepDQB
82
HippoDQB
82
54
CattleDQB
84
HorseDQB
68
PigDQB
78 100 TaDQB
TtDQB
96
DogDQB
SealionDQB
92
71
100
RatDQB
MouseDQB
68 71
RabbitDPB
98
HumanDPB
MoleratDPB
75
82 ChimpanzeeDOB
58
HumanDOB
100
MouseDOB
100
99 CattleDOB
100 ChickenBL2
ChickenBL3
ChickenBL1
100
FrogB2
FrogB1
ZebrafishDAB1
ZebrafishDAB4
ZebrafishDEB
ZebrafishDCB
100
0.1
94
100
10
NJ
(b)
0.1
MP
ML
34
19
44
36
RatDRB1
MouseDRB
PigDRB
TamarinDRB
CatDRB
47
99
SheepDRB
77
GoatDRB
56
CattleDRB
31
HippoDRB
DogDRB
43
83
TaDQB
TtDQB
74 90
TtDRB
TaDRB
GorillaDRB5
61
HumanDRB5
GorillaDRB1
MacaqueDRB1
GorillaDRB3
78
HumanDRB3
HumanDRB1
MacaqueDRB4
72
HumanDRB4
44
CattleDQB
36
HorseDQB
66
SheepDQB
28
HippoDQB
69
PigDQB
93
RatDQB
MouseDQB
66
DogDQB
33
75 SealionDQB
SealionDRB
31
RabbitDPB
44
HumanDPB
MoleratDPB
67
99 ChimpanzeeDOB
74
HumanDOB
89
MouseDOB
CattleDOB
95
ChickenBL2
96
ChickenBL3
ChickenBL1
98
FrogB2
FrogB1
55
50
98
82
69
ZebrafishDAB1
ZebrafishDAB4
26
ZebrafishDEB
35
51
13
RatDRB1
MouseDRB
TamarinDRB
PigDRB
CatDRB
97
SheepDRB
GoatDRB
11 53
CattleDRB
HippoDRB
72
TaDQB
TtDQB
55
TtDRB
84
TaDRB
32
MacaqueDRB1
GorillaDRB1
GorillaDRB5
HumanDRB5
21
MacaqueDRB4
42
HumanDRB4
GorillaDRB3
HumanDRB3
HumanDRB1
DogDRB
35
CattleDQB
13
HorseDQB
54
76
SheepDQB
33
HippoDQB
53
91
RatDQB
23
MouseDQB
29
PigDQB
35
SealionDQB
DogDQB
SealionDRB
38
47
47
HumanDPB
49
MoleratDPB
RabbitDPB
100 ChimpanzeeDOB
39
HumanDOB
96
MouseDOB
CattleDOB
68 ChickenBL2
100
ChickenBL3
ChickenBL1
FrogB2
FrogB1
ZebrafishDAB1
ZebrafishDAB4
ZebrafishDCB
23
62
43
100
100
68
100
ZebrafishDCB
0.1
MacaqueDRB1
GorillaDRB1
GorillaDRB5
HumanDRB5
GorillaDRB3
HumanDRB3
HumanDRB1
MacaqueDRB4
HumanDRB4
RatDRB1
MouseDRB
TamarinDRB
SheepDRB
GoatDRB
CattleDRB
PigDRB
HippoDRB
TtDRB
TaDRB
CatDRB
DogDRB
SealionDRB
CattleDQB
SheepDQB
HippoDQB
HorseDQB
PigDQB
TaDQB
TtDQB
DogDQB
SealionDQB
RatDQB
MouseDQB
RabbitDPB
HumanDPB
MoleratDPB
ChimpanzeeDOB
HumanDOB
CattleDOB
MouseDOB
ChickenBL2
ChickenBL3
ChickenBL1
FrogB2
FrogB1
MacaqueDRB1
GorillaDRB1
TamarinDRB
GorillaDRB3
HumanDRB3
HumanDRB1
69
MacaqueDRB4
HumanDRB4
GorillaDRB5
62
96 HumanDRB5
SheepDRB
98
GoatDRB
52
CattleDRB
59
62 PigDRB
HippoDRB
65
TtDRB
54
100 TaDRB
DogDRB
91
56
SealionDRB
CatDRB
100
RatDRB1
MouseDRB
62
SheepDQB
72
HippoDQB
28
CattleDQB
86
54
HorseDQB
PigDQB
74
TaDQB
66
TtDQB
76
DogDQB
96
SealionDQB
71
100
RatDQB
56
MouseDQB
63
RabbitDPB
98
HumanDPB
MoleratDPB
94
100 ChimpanzeeDOB
46
HumanDOB
99
CattleDOB
MouseDOB
100
61 ChickenBL1
100 ChickenBL2
ChickenBL3
100
FrogB2
FrogB1
ZebrafishDAB1
ZebrafishDEB
ZebrafishDAB4
ZebrafishDCB
ZebrafishDEB
ZebrafishDAB4
ZebrafishDCB
ZebrafishDAB1
ZebrafishDEB
10
NJ
MP
Fig. 3. Neighbor-joining (NJ), maximum-parsimony (MP), and maximum-likelihood (ML) trees for major histocompatibility complex
(MHC) class II β-chain genes using all codon positions. The nucleotide positions for tree construction were (a) positions 1-616, (b) 1-218,
and (c) 219-616. The ML tree was constructed only using nucleotide positions 1-616. The bootstrap values are shown in parentheses
for the nodes of interest. Tt, Tursiops truncatus; Ta, T. aduncus; Sealion, California sea lion Zalophus californianus.
Yang et al. – MHC Genes in Dolphins and Relatives
clock-like by performing the SH test and Tajima’s
relative rate test as described above, and we
obtained estimates of divergence times of several
nodes that were the same as those in the BI tree
using all 616 nt (Table 2). The estimates were
notably younger than those in the BI tree using 616
nt, except those for DRB/(DQB, DPB) and primate/
fereuungulates in the DOB clade.
The Kimura distance was then employed to
construct linearized trees using positions 1-616
and 219-616 to estimate divergence times of the
same nodes in the BI trees. The bird/mammal
divergence time (310 Mya) was also used as a
calibration point. Similarly to the scenario in the
BI trees, most of the divergence times in the NJ
tree using position 219-616 were younger than
those in the NJ tree using position 1-616 (Table
2). Moreover, in trees using the same nucleotide
positions (1-616 or 219-616), estimates of the
(c)
100
93
99
divergence time between the DOB and other
mammal clades and between the DRB and DQB/
DPB clades in the NJ tree ware close to those
in the BI tree, but those of DQB/DPB, rodents/
other mammals, primates/fereuungulates, and
carnivores/cetartiodactyls in the NJ tree were older
than those in the BI tree. In addition, estimates of
the dolphin/artiodactyl divergence time for the DQB
and DRB genes in the NJ tree were slightly older
than those in the BI tree. For the T. truncatus/T.
aduncus divergence time, estimates for the DQB
and DRB genes in the NJ and BI trees were nearly
the same.
DISCUSSION
Klein and O’hUigin (1994) demonstrated that
substitutions of PBR sequences of MHC genes
47
CattleDRB
100 GoatDRB
61
SheepDRB
82
61 PigDRB
HippoDRB
TtDRB
40
100 TaDRB
82 DogDRB
51 SealionDRB
CatDRB
GorillaDRB5
HumanDRB5
78
MacaqueDRB4
GorillaDRB3
HumanDRB3
HumanDRB1
97
MacaqueDRB1
GorillaDRB1
96
HumanDRB4
TamarinDRB
100
RatDRB1
MouseDRB
71
CattleDQB
80
SheepDQB
56
58
HippoDQB
66
PigDQB
TaDQB
70
100 TtDQB
49
90
HorseDQB
DogDQB
97
87
SealionDQB
100
RatDQB
49
MouseDQB
84
RabbitDPB
99
98
HumanDPB
MoleratDPB
100 ChimpanzeeDOB
71
HumanDOB
99
CattleDOB
MouseDOB
ChickenBL1
100 ChickenBL2
ChickenBL3
100
FrogB2
FrogB1
ZebrafishDAB1
ZebrafishDAB4
ZebrafishDEB
ZebrafishDCB
99
97
0.1
145
GorillaDRB3
HumanDRB3
HumanDRB1
TamarinDRB
HumanDRB4
GorillaDRB1
MacaqueDRB1
73 MacaqueDRB4
GorillaDRB5
HumanDRB5
50 SheepDRB
41
97
GoatDRB
45
CattleDRB
55
PigDRB
49
HippoDRB
69
TtDRB
100 TaDRB
59
DogDRB
67
SealionDRB
93
CatDRB
98
RatDRB1
MouseDRB
78 CattleDQB
67
SheepDQB
61
HippoDQB
PigDQB
65
TaDQB
74
100 TtDQB
44
86
HorseDQB
DogDQB
95
57 SealionDQB
100
RatDQB
37
MouseDQB
69
77
RabbitDPB
95
HumanDPB
MoleratDPB
100 ChimpanzeeDOB
98
51
HumanDOB
97
CattleDOB
MouseDOB
100
ChickenBL1
100
ChickenBL3
ChickenBL2
100
FrogB2
FrogB1
ZebrafishDAB1
ZebrafishDAB4
ZebrafishDEB
ZebrafishDCB
10
NJ
MP
Fig. 3. Neighbor-joining (NJ), maximum-parsimony (MP), and maximum-likelihood (ML) trees for major histocompatibility complex
(MHC) class II β-chain genes using all codon positions. The nucleotide positions for tree construction were (a) positions 1-616, (b) 1-218,
and (c) 219-616. The ML tree was constructed only using nucleotide positions 1-616. The bootstrap values are shown in parentheses
for the nodes of interest. Tt, Tursiops truncatus; Ta, T. aduncus; Sealion, California sea lion Zalophus californianus.
Zoological Studies 49(1): 132-151 (2010)
146
occur in a trans-specific manner. They suggested
that closely related species often share some
of the same pathogens which only differ in the
strain, and some pathogens have co-evolved
trans-specifically in parallel with their hosts. In
our study, a similar scenario was generally
observed for phylogenetic relationships based on
PBR sequences (Fig. 2b). However, the distinct
grouping pattern of dolphin DQB and DRB genes
Cetartiodactyls
(a)
Horses
from those of other artiodactyls was noted despite
dolphins and artiodactyls being closely related.
Possible explanations for this pattern include
analytical artifacts and an evolutionary hypothesis
of convergent evolution. Analytical artifacts
were considered unlikely because the expected
homologous relationship of the DQB and DRB
sequences of other mammals was shown in the
same phylogenetic tree. Convergent evolution may
Pigs
Ruminants
Hippos
x
x
Dolphins
x
Mya
x
60
(b)
Ta (coastal)
Tt (offshore)
Mya
x
x
1.5
24
Fig. 4. Hypothesized birth-and-death process of major histocompatibility complex (MHC) class II DQB and DRB genes in (a)
cetartiodactyls and (b) bottlenose dolphins. The broad trees represent the species tree, and the thin trees represent the MHC gene
tree. The times were taken from summary times in table 2 except the time of speciation of these 2 bottlenose dolphins which was
estimated by Wang et al. (1999b). ●, gene duplication; X, gene was lost or became a pseudogene. Tt, Tursiops truncatus; Ta, T.
aduncus; Mya, million years ago.
Yang et al. – MHC Genes in Dolphins and Relatives
be relatively common in the MHC for which several
mechanisms are potentially responsible: point
mutations, large-scale intergenic recombinations,
small-scale intergenic conversions, and strong
selection (Yeager and Hughes 1999). The last
mechanism is the most likely because it was
shown that MHC alleles observed in populations
have been selectively favored (Hughes et al.
1993), and no evidence for genetic recombination
or gene conversion events between sequences
was found in our dataset using the program
GENECONV. This unusual grouping pattern of
dolphin DQB and DRB sequences is probably
due to the requirement that both genes defend
against marine pathogens, which is evidence for
permanent adaptations to marine environments
by cetaceans. In sum, we showed the homoplasy
of the PBR between DQB and DRB genes, which
can best be explained by convergent evolution. A
similar conclusion was reached in the humpback
whale (Baker et al. 2006) and canids (Seddon and
Ellegren 2002). Therefore, the PBR alone might
not be a good indicator of the phylogenetic history
of MHC genes because it is prone to homoplasy in
different loci and species. The evolutionary history
of the PBR in cetartiodactyl MHC genes could be
a good example of pathogen-driven directional
selection, which infers the varying selective
advantages of MHC alleles in accordance to the
diversity and abundance of pathogens in different
environments (Bernatchez and Landry 2003).
Potential homoplasy was noted in sequences
from the California sea lion (Fig. 2b) although the
support was moderate (0.71). Since the unique
grouping pattern of PBR sequences was only
found in marine mammals in this study, more
sequences from other species are needed for
further comparative investigations of PBR evolution
147
in marine mammals.
We compared estimates of divergence
times obtained using either non-PBR sites or all
sites in the BI and NJ trees with previous MHC
and multigene studies (Kumar and Hedges
1998, Takahashi et al. 2000). It was suggested
that reliable estimates of divergence times for
MHC genes can be obtained by using all sites
with a birth-death clock model in the BI tree.
Employing the birth-death clock model is likely
more appropriate for estimating divergence times
of MHC genes in view of the fact that MHC genes
have evolved through a birth-and-death process
(Nei et al. 1997). Furthermore, many studies on
phylogenies and divergence times of mammalian
MHC class I or II genes used longer nucleotide
sequences, including the PBR and other regions,
for phylogenetic reconstruction (Klein and Figueroa
1986, Hughes and Nei 1990, Takahashi et al.
2000, Piontkivska and Nei 2003). Inclusion of the
PBR in these analyses and our study still provided
plausible estimates of divergence times, in support
of the previous suggestion for the use of as many
nucleotide sites as possible (Nei and Kumar 2000).
The BI trees using all sites or non-PBR sites
both showed that phylogenetic relationships in
the respective cetartiodactyl group in the DQB
and DRB clades did not correspond to those in
previously accepted species trees. It was striking
to observe that cetaceans (bottlenose dolphins)
and artiodactyls (pig, hippo, and ruminants)
formed 2 distinct clades in both the DQB and
DRB phylogenies, rather than being of the same
clade with the hippo and dolphin as the closest
relatives (Gatesy and O’Leary 2001). The general
lack of a match between the gene and species
trees was also reported for MHC class I genes of
cetartiodactyls (Holmes et al. 2003), although no
Table 2. Estimates of the times of divergence between different β-chain gene clades
DOB/
(DRB,
DQB,
DPB)
Approach (nucleotide positions)
BI (1-616)
249.36
BI (219-616)
220.04
NJ (1-616)
263 ± 12
NJ (219-616)
207 ± 8
Takahashi et al.●
254 ± 16
Kumar and Hedges○
N/A
DRB/
(DQB,
DPB)
DQB/DPB Rodents/other
mammals
(four gene
clades)
191.79
168.26
193.52
157.31
195 ± 11 190 ± 17
194 ± 12 185 ± 13
189 ± 12 179 ± 12
N/A
N/A
109.30 - 122.03
83.43 - 111.22
126.00 - 147
118.00 - 145
N/A
114
Primates/
ferungulates
(DOB; DRB)
Carnivores/
Dolphins/
Tt/Ta*
cetartiodactyls artiodactyls (DQB; DRB)
(DQB; DRB) (DQB; DRB)
0097.53; 88.43
0097.41; 77.97
138 ± 11; 92 ± 9
120 ± 10; 75 ± 9
N/A
92
78.14; 76.45
62.74; 51.09
84 ± 9; 91 ± 8
61 ± 3; 76 ± 5
N/A
79
The numbers after the ± signs are standard errors. Tt, Tursiops truncatus; Ta*, Tursiops aduncus.
2000 ○From figure 2 and 3 in Kumar and Hedges 1998.
●
60.03; 62.54 26.47; 21.58
38.42; 44.99
7.38; 9.31
67 ± 6; 68 ± 4 25 ± 3; 22 ± 3
47 ± 7; 49 ± 3 07 ± 2; 06 ± 2
N/A
N/A
58
N/A
From Table 3 in Takahashi et al.
148
Zoological Studies 49(1): 132-151 (2010)
sequence was included from cetaceans or hippo
in that study. Possible sources contributing to
the incongruence between the MHC gene tree
and species tree include (1) an incorrect model of
sequence evolution, (2) different evolutionary rates
across the tree, and (3) utilization of paralogous
sequences (Page and Holmes 1998). The 1st
source is unlikely in our case because we obtained
very similar phylogenetic relationships using
different models with different algorithms. The
possibility of the 2nd source can be excluded
since our dataset was clock-like based on the
likelihood-ratio and relative-rate tests. The 3rd
source is very likely to be important because the
gene tree can be discordant with the species tree
if the species relationships are based on mixed
orthologous and paralogous sequences (Page and
Holmes 1998), and it has not yet been confirmed
whether the respective DQB and DRB sequences
of cetartiodactyls are paralogous or orthologous.
While orthologous genes are separated by
speciation events, paralogous genes are separated
by gene duplication events, which were proposed
as being a major force in MHC evolution (Klein
et al. 1998). Gene duplications were observed
in the genetic organization of MHC genes in
many mammals. In bovine MHC class II genes,
for example, 2 DQB genes and 9 DRB genes
were detected, with eight of the DRB genes likely
being pseudogenes (Ellis and Ballingall 1999). In
conclusion, we presumed that the sequences of
cetaceans and artiodactyls were paralogous in
DQB and DRB genes, respectively. We explained
the paralogy between sequences of cetaceans
and artiodactyls by postulating gene duplications
in MHC genes and pathogen-driven directional
selection during cetacean evolution.
Gene-duplication events are hypothesized
to have occurred at least once in both the DQB
and DRB genes, prior to the cetacean/artiodactyl
divergence (Fig. 4a). Gene duplication gave rise
to 2 sets of paralogous genes. Given the 2 sets
named A and B sets, the A sets of all artiodactyls
have survived to the present day, while the B sets
were lost or became pseudogenes; in contrast, the
B sets in cetaceans were preserved, while their A
sets were lost or became pseudogenes. Through
this process, we obtained the phylogenetic
relationship that cetaceans and artiodactyls are
sistergroups in the DQB and DRB phylogenies.
According to the estimates in table 2, the
duplications took place 60 Mya or slightly earlier,
which is comparable to the 1st appearances of
fossil cetaceans at around 53.5 Mya, artiodactyls
at 55 Mya, and another molecular estimate of the
divergence time of cetaceans/artiodactyls at 60
Mya (Arnason and Gullberg 1996, Arnason et al.
2000, Arnason et al. 2004, Theodor 2004). As
already pointed out, gene duplication is a common
feature of MHC genes, and duplicated MHC loci
are usually governed by diversifying selection,
which may favor their specialization (Hughes
1999). Since natural selective pressures of
infectious diseases between terrestrial and aquatic
(especially marine) environments differ (McCallum
et al. 2004), pathogen-driven directional selection
is a plausible driving force for preserving or losing
MHC genes by cetaceans and their terrestrial
relatives in the period during which cetaceans
progressively moved from land to water. Several
other mammal groups also made the evolutionary
transition from land to sea, such as pinnipeds,
sea otters, polar bears, and sirenians. Studying
MHC genes of these marine mammals and their
terrestrial relatives should provide further insights
into the evolution of MHC genes.
We selected T. truncatus and T. aduncus,
which are closely related but inhabit different
environments, to investigate MHC gene evolution
in toothed whales. Tursiops truncatus (Montagu
1821) is a larger form of bottlenose dolphin, which
is generally found in deep, offshore waters. The
smaller form, T. aduncus, inhabits shallow, tropical,
coastal waters (Ehrenberg 1832). Shallow waters
along the coast may be influenced by terrestrial
runoff, and the pathogen diversity and abundance
in coastal waters may differ from those in oceanic
areas. An investigation of stomach contents of
stranded and by-caught specimens in Taiwan
revealed that the diet and microflora presumably
differ between T. truncatus and T. aduncus (Wang
2003), indicating different selective pressures from
pathogens between these 2 species. Yang et al.
(2007) reported the molecular characterization of
DQB and DRB genes in bottlenose dolphins and
found a clear phylogenetic division between T.
truncatus and T. aduncus. This was an intriguing
result compared to the general trans-specific
pattern of evolution observed for MHC loci (Hughes
1999), i.e., different species and different genera
are clustered within the same allelic lineages. It
was assumed that the species-specific MHC
allelic lineages in Tursiops resulted from different
selective pressures from pathogens which exist
in oceanic (T. truncatus) and coastal (T. aduncus)
environments. Nonetheless, it was still not clear
when the species-specific allelic lineages emerged
and the subsequently evolutionary processes
Yang et al. – MHC Genes in Dolphins and Relatives
occurred. The general evolutionary understanding
is that species pairs of closely related delphinids
(true dolphins), such as T. truncatus and T.
aduncus, likely evolved during Pleistocene
glaciation events (< 2 Mya; Wang et al. 1999b).
However, our estimated divergence time of T.
truncatus and T. aduncus according to the DQB
and DRB genes (>20 Mya) was much earlier than
the separation date of these 2 species, and the
earliest fossils identifiable as Tursiops date to only
4-7 Mya (Barnes 1990), as well as the emergence
of the oldest delphinid which is possible 11 Mya in
the late Miocene (Barnes 1977). After conducting
Tajima’s relative rate test, we ruled out a possible
error for the time estimate caused by accelerating
substitution rates of Tursiops. The significant
time gap between speciation and MHC gene
estimates can be explained by postulating a birthand-death process (Fig. 4b), similar to that in the
cetartiodactyl clade we described above. These
allelic lineages of Tursiops MHC genes may have
emerged by gene duplication during the period of
early radiation of small toothed whales (from the
late Oligocene to early Miocene, ~22 Mya (Arnason
et al. 2004). It was shown that MHC alleles
can persist over extremely long time periods by
balancing selection (Bernatchez and Landry 2003).
Long persistence times allow the accumulation of
multiple substitutions between allelic lineages of
MHC genes, which may enable them to possess
different assignments for pathogen defense. Baker
et al. (2006) suggested the scenario that baleen
whales and early divergent toothed whales having
duplicate DQB genes is an ancestral condition
shared with ruminants, while the more-derived
cetaceans such as true dolphins (of the family
Delphinidae) lost this condition and only have 1
DQB locus. The ancestor of Tursiops was possibly
equipped with these 2 ancestral lineages at
different loci, and these lineages were maintained
for a long evolutionary period through speciation
events. Tursiops truncatus and T. aduncus
consequently kept only one of the 2 lineages
expressed. Pathogen-driven directional selection
may have been responsible for T. truncatus
and T. aduncus maintaining different expressed
lineages due to a heterogeneity of natural
selective pressures by infectious diseases found
in oceanic and coastal waters. Since the extant
cetacean fauna consists of more than 80 species
which live in various habitats, such as oceans,
estuaries, polar regions, and rivers, the evolution
of cetacean MHC genes may be more complicated
than previously realized. It would be interesting to
149
elucidate this by obtaining more sequences and
loci from a variety of cetacean species.
Our phylogenetic analysis and divergence
time estimates in cetartiodactyl MHC class II genes
provide a new aspect for discussing the history of
co-evolution between MHC genes and pathogens
when cetaceans moved from land to water. To
the best of our knowledge, this is the first study
on the evolutionary history of cetartiodactyls using
MHC class II genes. More sequences and loci
from other cetacean species and closely related
mammals should enable us to further study the
birth-and-death process of cetartiodactyl MHC
genes. Comparisons with MHC evolution of other
marine mammals would also help elucidate the
factors affecting the immune defense system
in relation to evolutionary change of foreign
antigens. Furthermore, our results show that
cetacean MHC genes are adapted to marine
environments. Their ability to defend against
terrestrial pathogens needs investigation and close
monitoring, especially in these times when there
are potential risks of epidemics in cetaceans, when
they have more occasions to encounter terrestrial
pathogens through human exploitation of marine
environments or, directly, by keeping dolphins in
captivity.
Acknowledgments: This study could not be
conducted without blood samples of bottlenose
dolphins provided by Hong Kong Ocean Park
and Hualien Ocean Park and tissue samples
from a hippopotamus provided by Leofoo Village
Theme Park and Taipei City Zoo. We appreciate
colleagues in the laboratories of JMH and LSC for
useful comments and technical support. We are
grateful to many scholars at the Fifth Conference
on Secondary Adaptation of Tetrapods to Life
in Water held in Tokyo in 2008 for providing
constructive comments on this paper. This study
was funded by grants to LSC from the Council of
Agriculture of Taiwan (94AS-9.1.7-FB-e1(8) and
95AS-11.1.3-FB-e1(10)) and Mission Biotech Corp.
of Taiwan.
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