Genes Genet. Syst. (2010) 85, p. 121–127

Evolution of two Rh blood group-related genes of theamphioxus species Branchiostoma floridae
Takashi Kitano1*, Masahiro Satou1 and Naruya Saitou2
1

Department of Biomolecular Functional Engineering, College of Engineering, Ibaraki University,
4-12-1 Nakanarusawa-cho, Hitachi 316-8511, Japan
2
Division of Population Genetics, National Institute of Genetics,
1111 Yata, Mishima 411-8540, Japan(Received 7 September 2009, accepted 31 March 2010)

We determined cDNAs of two genes that belong to the Rhesus (Rh) blood groupgene family in an amphioxus species (Branchiostoma floridae) and designatedthem Rh-related-1 (RhR-1) and Rh-related-2 (RhR-2). RhR-1 and RhR-2 consisted of 10 and 11 exons, respectively. 3’ UTR sequences of RhR-1 were shorter(220–272 bp) than those of RhR-2 (1,505–1,650 bp). CDS lengths were 1,344 and
1,476 bp for RhR-1 and RhR-2, respectively, and the average nucleotide difference
between their CDS regions was 0.33. The corresponding regions of Rh genes fromexons 2 to 7 were relatively conserved among the chordate species examined inthis study. Length difference numbers were in multiples of three, which implies
that codon frames were conserved among them, and the same exon/intron boundary phases were observed in those regions. This region was used for the phylogenetic analyses. RhR-1 and RhR-2 formed a cluster on the phylogenetic tree of the
Rh gene family. Gene duplication time of RhR-1 and RhR-2 was estimated to be
ca. 500 million years ago. It is likely that the four Rh family genes in vertebrates
emerged by gene duplications in the common ancestor of vertebrates, and functional differentiation has occurred after the first gene duplication.
Key words:

amphioxus, blood, evolution, phylogeny, Rh

INTRODUCTION
The human Rhesus (Rh) blood group plays importantroles in transfusion and clinical medicine, as it has been
known to be involved in hemolytic diseases of newborns,
autoimmune diseases, and mild hemolytic anemia.
Human Rh blood group (also known as Rh30) genes
encode membrane proteins with multiple transmembrane
domains (Avent et al., 1990, 1992) and are expressed only
on erythrocytes (Cherif-Zahar et al., 1990). Three othergenes that are homologous to Rh blood group genes havebeen identified in the human genome. These are, RhAG
(Rh-associated glycoprotein, also known as Rh50), RhBG
(Rh family, B glycoprotein), and RhCG (Rh family, C
glycoprotein). RhAG is an erythrocyte-specific protein
and is known to interact with Rh. RhBG and RhCG proteins are non-erythroid members of the Rh blood groupgene family and are primarily expressed in the kidney.
In humans, the chromosomal locations are 1p36.11 forRh, 6p21.1-p11 for RhAG, 1q21.3 for RhBG, and 15q25 for
RhCG. Kitano and Saitou (2000) suggested that gene
Edited by Chung-I. Wu* Corresponding author. E-mail: tkitano@mx.ibaraki.ac.jp

duplication that produced Rh and RhAG occurred in thecommon ancestor of vertebrates. Moreover, they pointed
out the possibility of two more duplications for Rh bloodgroup genes in jawed vertebrates. Later, all three duplications that produced these four genes were also suggested to have occurred in the common ancestor ofvertebrates (Huang and Peng, 2005; Peng and Huang,
2006).
Vertebrata, Urochordata, and Cephalochordata belong
to Chordata, and Urochordata is the closest neighbor of
Vertebrata (Putnam et al., 2008). The ascidian Ciona
intestinalis is frequently used as a representative of
Urochordata; its genome sequence data are available.
However, it was demonstrated that ascidians have long
branches on a deuterostome phylogeny constructed from
1,090 orthologous genes, indicating higher amino acidsubstitution levels (Putnam et al., 2008), which may posedifficulties for phylogenetic analyses. On the other
hand, Cephalochordata (amphioxus) is the second closest
neighbor of Vertebrata, and it does not have a long
branch.
In order to include Rh-related (RhR) genes of amphioxus
for phylogenetic analysis of the Rh gene family, it is necessary to obtain clear characteristics of the genes. Data

T. KITANO et al.

122of the four genome sequences (ABEP01017616,
ABEP01003689, ABEP01002794, and ABEP01062874) of
RhR genes in amphioxus (Branchiostoma floridae), whichwere obtained from the whole genome shotgun sequence
project (Putnam et al., 2008), were deposited in the
DDBJ/EMBL/GenBank International Nucleotide Sequence
Database, and some EST sequences of amphioxus, whichcontain a part of the RhR gene, were available. To elucidate exon and intron boundaries and coding regions of
RhR genes in amphioxus, we sequenced complete cDNA
sequences of six clones of RhR genes in amphioxus andconducted an evolutionary analysis of the Rh gene family.
MATERIALS AND METHODS
cDNA sequencing Six clones (bfad001a05, bfad037h16,
bfad047k21, bfad049a17, bflv024h10, and bflv041d06) ofRh blood group-related genes in amphioxus (Branchiostoma
floridae) were kindly provided by the Academia DNA
Sequencing Center, National Institute of Genetics in collaboration with the Department of Zoology, Graduate
School of Science, Kyoto University (Yu et al., 2007).
Sequencing were performed using a BigDye Terminatorv3.1 Cycle Sequencing Kit and an ABI PRISM 3130xl

Table 1.

Genetic Analyzer (PE Biosystems). Both strands wereread using sequencing primers. Primers used for RhR-1
were as follows: bfRhR1-1, 5’-CTGGTGGCTGCCTTCGTGCT-3’; bfRhR1-2, 5’-CGCCGCCATCTGGAATTCTG-3’;
bfRhR1-3, 5’-GTTTGTCACCCTTGGAGTC-3’; bfRhR1-4,
5’-AGAAACGTCATCAGGAACC-3’, and for RhR-2 wereas follows: bfRhR2-1, 5’-CAACGAGTGGGTCGGACTCA-3’;
bfRhR2-2, 5’-ATTAGGCAGATTCCTAATAC-3’; bfRhR2-3,
5’-GTGGACATGAACGGCAGTC-3’; bfRhR2-4, 5’-TTGTCTGTATCTAGCCTCC-3’; bfRhR2-5, 5’-CCCAAATAGGGAAACTTGGT-3’; bfRhR2-6, 5’-CGAGTACGGAGACGATAAG-3’; bfRhR2-7, 5’-ATATGATCATACATTTAACGC3’; bfRhR2-8, 5’-TGTGCCCCACCATGTCCTC-3’; bfRhR29, 5’-CACTCTGCGTCCTGAATGAC-3’. The Phred/Phrap
software program (Ewing et al., 1998) was used for basecalling and assembly and for obtaining quality scores for
assembled data. Editing was performed using Consed(Gordon et al., 1998) to identify all low-quality bases andto check that the assembly was correct based on linking
information.
Sequence analyses To compare sequences with thoseof other members of the Rh gene family, human (Homosapiens) and torafugu (Takifugu rubripes) cDNA

List of genes and accession numbers of Rh family genes used in this study
Accession number

Species (Scientific binomen)

Gene

mRNA

GenomeHuman

RhD

NM_016124

NC_000001(Homo sapiens)

RhCE

NM_020485

NC_000001

RhAG

NM_000324

NC_000006

RhBG

NM_020407

NC_000001

RhCG

NM_016321

NC_000015

Torafugu

Rh

NM_001032763

CAAB01002106(Takifugu rubripes)

RhAG

NM_001037867

CAAB01003585

RhBG

AB218980

CAAB01001243

RhCG-1

NM_001032647

CAAB01000968

RhCG-2

NM_001032762

CAAB01003371

RhP2-1

NM_001032644

CAAB01000115

RhP2-2

NM_001032645

CAAB01000285

Ascidian

Rh-A

NM_001032776

AABS01000115(Ciona intestinalis)

Rh-B

NM_001032787

AABS01000034

Rh-C

NM_001032774

AABS01000044

Amphioxus

RhR-1

AB519682a

ABEP01017616a(Branchiostoma floridae)

RhR-2

AB519685a

ABEP01002794a

Purple sea urchin

LOC584796 XM_784645

NW_001467604(Strongylocentrotus purpuratus)a

They are representatives. AB519683 and AB519684 for the mRNA of RhR-1,
AB519686 and AB519687 for the mRNA of RhR-2, ABEP01003689 for the genomeof RhR-1, and ABEP01062874 for the genome of RhR-2 were also used in the study.

Rh-related genes in amphioxus
sequences were used as representatives of vertebrates.
Three genes of ascidian (Ciona intestinalis) were also
used. The genes and GenBank accession numbers of themRNAs and the genomes are listed in Table 1. Exon andintron boundaries of RhR genes in amphioxus were predicted by comparing human, torafugu, and ascidian
sequences. T-Coffee version 5.72 (Notredame et al.,2000) was used for multiple alignments that were madeexon by exon, followed by concatenating thesealignments. Amino acid sequences of exons 2–7 (exonnumbers followed human RhD) were used for thefollowing phylogenetic inferences (see RESULTS ANDDISCUSSION).
Repetitive elements were detected by the RepeatMasker
program (Smit et al., http://www.repeatmasker.org) using
the cross_match search engine with slow speed/sensitivity

123

options, and the DNA source of Branchiostoma floridae
species.
Phylogenetic inferences We estimated phylogenetic
trees using the neighbor-joining (Saitou and Nei, 1987),
the maximum likelihood (Felsenstein, 1981), and theBayesian (Huelsenbeck et al., 2001) tree-building
methods. The sequence data (XM_784645) of purple sea
urchin (Strongylocentrotus purpuratus) was used as an
outgroup. For the neighbor-joining and the maximum
likelihood trees, the JTT model (Jones et al., 1992) with1,000 bootstrap replicates was used, and a gamma shapeparameter was estimated for the maximum likelihood
method. MEGA version 4 (Tamura et al., 2007) and
PHYML version 2.4.4 (Guindon and Gascuel, 2003) wereused to construct the neighbor-joining and the maximum

Table 2.

Exons and introns and their boundaries in amphioxus RhR genes

Exonboundary 5’aExon bp

boundary 3’a

199c

Intron bpb

Phase1) RhR-1…CCWCTgtaag…

450 (468)22…tgcagCMTAG…

164…CCCGAgtacg…

5,201 (1,248)

1

3…cacagTGTTC…

184…AGCAGgtatt…

555 (700)

2

4…yccagCMTGC…

151…TCAAGgtwws… 1,235 (748)05…gacagGCSCA…

145…GATAGgtgag…

761 (873)

1

6…cmcagGTACG…

164…ACATGgtaag…

511 (468)0

1

7…accagGTTCA…

138…TTCAGgtacg…

1,321 (1,942)

0

8…ctcagCCGTW…

128…GAAAGgtaaa…

465 (741)

2

9…ttcagGTCGG…

157…GGGAGgtgcg…

885 (1,569)

0

10…cgcagGTTCC…

362c…TTGAGgtacg…

930 (920)

02) RhR-2
186c

1

a

2

…cmcagGTGGT…

136…CCCGAgtgag…

9,661 (8,584)

1

3…ytcagTGTTC…

184…ACTACgtaag…

1,201 (709)24…tgyagYCTGC…

151…TCAAGgtcag…

411 (424)

05…cacagGCCGT…

145…GATCGgtaag…

1,849 (1,996)

1

6…tacagGTACT…

164…ACATGgtaag…

715 (668)

07…cacagGTTCA…

138…TCACGgtaag…

502 (506)08…tgcagCCCTT…

134…AACAGgtggg…

527 (785)

2

9…cacagYGTGT…

182…CACTGgtaag…

351 (434)

1

10…tstagGCCTC…

74…GGGAGgtgag…

665 (472)011…cycagATACC…

1,775cEach of the five nucleotides of exon and intron are shown; and polymorphic sites
among five sequences (three cDNAs and two genome sequences) are shown byabbreviations of the Nomenclature Committee of the International Union of
Biochemistry.
b
Lengths of introns are from ABEP01017616 for RhR-1 and ABEP01002794 for
RhR-2; and lengths of introns of ABEP01003689 for RhR-1 and ABEP01062874
for RhR-2 are in parentheses.
c
For lengths of the first and last exons, the longest ones among the three cDNAs
are used.

T. KITANO et al.

124

likelihood trees, respectively. For the Bayesian method,
the WAG model (Whelan and Goldman, 2001) with
300,000 generations was used. MrBayes version 3.1.2(Ronquist and Huelsenbeck, 2003) was used to construct
the Bayesian tree.
RESULTS AND DISCUSSION
Structures of amphioxus RhR genes We determined
six mRNAs of RhR genes in amphioxus. We also predicted the codon start position of each sequence, as only
one methionine codon was observed in each putative firstexon of each mRNA. The DDBJ/EMBL/GenBank International Nucleotide Sequence Database accession numbers are AB519682–AB519687. These six mRNAs weredivided into two types of Rh blood group-related
genes. Thus, the amphioxus species probably has two
Rh blood group-related loci, and we designated them:RhR-1 and RhR-2. CDS lengths were 1,344 and 1,476 bp
for RhR-1 and RhR-2, respectively. The average nucleotide difference (p-distance) in the CDS region between
RhR-1 and RhR-2 was 0.33. A large difference wasobserved in the lengths of 3’ UTRs between RhR-1 andRhR-2. 3’ UTR sequences of RhR-1 were shorter (220–272 bp) than those of RhR-2 (1,505–1,650 bp).
Because the whole genome shotgun sequences of theamphioxus genome were available, we predicted exons,
introns, and their boundaries in the two amphioxus RhR
genes (Table 2) by comparing mRNA and genome
sequences. We compared two genome sequences for each

Table 3.

RhR gene (ABEP01017616 and ABEP01003689 for RhR1 and ABEP01002794 and ABEP01062874 for RhR2). RhR-1 and RhR-2 consisted of 10 and 11 exons,
respectively. These are the typical exon numbers for Rh
family genes. All exon/intron boundaries were consistent with the GT/AG rule. The exon lengths were identical among the three mRNAs and in the two genome
sequences for each gene, whereas the intron lengths weredifferent among the two genome sequences. In particular, intron 2 of RhR-1 had larger differences: one was5,201 bp and the other was 1,248 bp (Table 2). This difference was due to the insertion of 4,399 bp-long EnSpmN4_BF type DNA transposon.
Phylogenetic relationships of amphioxus RhR
genes To construct a more reliable phylogenetic tree of
Rh family genes, we considered the following threepoints. First, to eliminate noise from phylogenetic analyses, we constructed a simpler phylogenetic tree and used
only human and torafugu genes as representatives of vertebrates (Table 1). Because of tandem gene duplication,
there are two Rh loci (RhD and RhCE) in humans(Blancher and Socha, 1997). In torafugu, there are two
RhCG loci, probably because of a gene duplication eventin the fish lineage. In addition, torafugu has two additional Rh-related genes (RhP2-1 and RhP2-2). Second,
we used data of amino acid sequences from exons 2 to 7
(Table 3), as these regions were relatively conserved.
The genes used here had the same exon/intron boundary
phases at the 3’ ends as exons 1–7, except exon 7 of purple

Exons and phases of Rh family genes
ExonaSpecies

Gene

8

9

10

Human

RhD

148 (1) 187 (2) 151 (0) 148 (1) 167 (0) 138 (0) 134 (2)80 (2)

74 (0)

27

RhCE

148 (1) 187 (2) 151 (0) 148 (1) 167 (0) 138 (0) 134 (2)80 (2)

74 (0)

27

RhAG

157 (1) 184 (2) 151 (0) 148 (1) 167 (0) 138 (0) 122 (2)71 (1)

74 (0)

18
17

Torafugu

Ascidian

3

4

5

6

7

RhBG

187 (1) 187 (2) 151 (0) 148 (1) 167 (0) 138 (0) 134 (2) 122 (1)75 (1)

184 (1) 187 (2) 151 (0) 148 (1) 167 (0) 138 (0) 137 (2) 125 (1)74 (0) 129

Rh

139 (1) 187 (2) 151 (0) 148 (1) 173 (0) 138 (0) 128 (2)80 (1)

74 (0)

27

RhAG

193 (1) 184 (2) 151 (0) 148 (1) 167 (0) 138 (0) 122 (2)74 (1)

74 (0)

75

RhBG

178 (1) 187 (2) 151 (0) 148 (1) 167 (0) 138 (0) 134 (2) 122 (1)74 (0)

78

RhCG-1223 (1) 193 (2) 151 (0) 148 (1) 167 (0) 138 (0) 137 (2) 125 (1)74 (0) 126

RhCG-2205 (1) 193 (2) 151 (0) 148 (1) 167 (0) 138 (0) 137 (2) 125 (1)74 (0)

Rh-A

148 (1) 193 (2) 151 (0) 148 (1) 167 (0) 138 (0) 137 (2)Rh-C

74 (2)

94 (0) 138

220 (1) 193 (2) 139 (0) 169 (1) 164 (0) 138 (0) 134 (2) 202
5 (2) 149 (1) 193 (2) 139 (0) 175 (1) 164 (0) 138 (0) 134 (2) 187 (0)

67

RhR-1

23 (2) 164 (1) 184 (2) 151 (0) 145 (1) 164 (0) 138 (0) 128 (2) 157 (0)

90

RhR-2

48 (0) 136 (1) 184 (2) 151 (0) 145 (1) 164 (0) 138 (0) 134 (2) 182 (1)74 (0) 120

Purple sea urchin LOC584796
a

2

RhCG

Rh-B

Amphioxus1

154 (1) 193 (2) 151 (0) 145 (1) 164 (0) 138 (0) 127 (1)Exon numbers are followed by the human RhD. Phases are shown in parentheses.

77 (0)72

11

34

Rh-related genes in amphioxus
sea urchin. Moreover, length difference numbers inexons 2–7 were in multiples of three. For example, on
exon 2, human RhD is of 187 bp and human RhAG is of
184 bp; thus, the difference is of 3 bp. Therefore, we canassume that there are gaps that do not affect codon
frames in those regions. Exon 1 was not used because it
had variable base pair lengths, suggesting a variety oftranslation start sites. Phases and base-pair lengths
were not conserved at exon 8 and later exons. It is likelythat many changes occurred in the codon frames of that
region. Thus, the data of amino acid sequences fromexons 2 to 7 were used to make multiple alignments of Rh
family genes. Third, in order to focus attention on the125

relationship among Rh family genes of Chordata (Vertebrata, Urochordata, and Cephalochordata), we used genes
only from these organisms. A gene from purple sea
urchin, which belongs to Echinodermata, was used as an
outgroup, and other invertebrate species were not used
for phylogenetic analyses.
Figure 1A shows the phylogenetic tree of chordate Rh
family genes. Two RhP2 genes of torafugu formed acluster with each other. However, the phylogenetic position of RhP2 genes was unclear with low bootstrap andposterior probabilities. RhP2-1 of torafugu is an intronless gene, and RhP2-2 of torafugu has only one
intron. Their homologous genes have been observed inFig. 1. Phylogenetic trees of chordate Rh family genes, including (A) and excluding (B) two RhP2
genes of torafugu. The root was determined using purple sea urchin as an outgroup. These phylogenetic trees were constructed by the neighbor-joining method. The scale is 0.1 substitutions per site.
The maximum likelihood and the Bayesian methods constructed the same topologies. Numbers on eachbranch show bootstrap values calculated by the neighbor-joining method (normal font), by the maximum likelihood method (bold), and posterior probabilities by the Bayesian method (italic).

126

T. KITANO et al.

other fishes, such as spotted green pufferfish (Tetraodon
nigroviridis; intronless, DQ013062) and zebrafish (Daniorerio; one intron, NM_131547), and in western clawedfrog (Xenopus tropicalis; intronless, NM_001045792), andin platypus (Ornithorhynchus anatinus; two introns,
XM_001507513). RhP2 gene had probably emerged byinsertion of spliced mRNA by reverse transcription.
Because the phylogenetic position of RhP2 genes was
unclear, two RhP2 genes of torafugu were excluded fromfurther analysis.
Figure 1B shows the phylogenetic tree of chordate Rh
family genes, excluding two RhP2 genes of torafugu. It
is important to emphasize that the same topologies wereconstructed by the three tree-building methods. Rh and
RhAG genes, which have erythrocyte specific expression,
formed a cluster, although branch supporting probabilities were low (bootstrap value of 59% by the neighborjoining method, bootstrap value of 51% by the maximum
likelihood method, and posterior probability of 88% by the
Bayesian method), and RhBG and RhCG genes formed acluster with high probabilities (bootstrap value of 99% by
the neighbor-joining method, bootstrap value of 99% by
the maximum likelihood method, and posterior probability of 100% by the Bayesian method). Rh blood group
genes had longer branches than other Rh related genes in
the phylogenetic tree. This pattern is consistent withprevious studies (Kitano et al., 1998; Kitano and Saitou,
1999; Huang and Peng, 2005). In ascidian, three genes(Rh types A, B, and C glycoproteins) formed a cluster.
They probably arose by two gene duplication events(Huang and Peng, 2005), independent from those invertebrates. In amphioxus, two genes (RhR-1 and RhR2) formed a cluster arisen by a gene duplication. This
gene duplication was probably independent of those invertebrate and ascidian lineages.
Emergence time of each Rh family gene To estimate
duplication times between RhR-1 and RhR-2 genes inamphioxus, and among RhAG, RhBG, and RhCG genes invertebrates, we reconstructed a linearized tree (Fig. 2)
assuming equal evolutionary rates in all lineages(Takezaki et al., 2004). Three Rh genes (human D and
CE, and torafugu) were excluded from the tree, as they
have higher evolutionary rates and longer branches, as
indicated above. The divergence time (T) between
human and torafugu was assumed to be 450 million years
ago (MYA) (Kumar and Hedges, 1998). Evolutionary
rates (λ) for RhAG, RhBG, and RhCG were estimated tobe 3.6 × 10–10, 3.4 × 10–10, and 3.8 × 10–10, respectively.
Because these three values were similar, the mean λ(3.6 × 10–10) was used as the rate of amino acid change ofRh family genes. Thus, gene duplication time of the two
amphioxus genes (RhR-1 and RhR-2) was estimated to be
ca. 500 MYA [= d/λ = 0.1852/3.6 × 10–10]. Whether or notthese duplicated genes have functional differentiation

Fig. 2. A linearized tree of chordate Rh family genes. The root
was determined using purple sea urchin as an outgroup. Divergence nodes between human and torafugu are shown by filled
circles. Two gene duplications in the common ancestor of vertebrates and the gene duplication in the amphioxus lineage areshown by filled and gray diamonds, respectively. Other gene
duplications are shown by open diamonds. Because the common
ancestral branch of three ascidian genes had no amino acidchanges in the linearized tree, this branch is shown by a broken
gray line.

requires further investigation, as these two Rh family
genes have existed for quite a long time in amphioxus.
Two gene duplication times for RhBG and RhCG and
for RhAG and RhBG-RhCG in the vertebrate lineage were
estimated to be ca. 650 MYA and ca. 750 MYA,
respectively. This period roughly corresponds to tworounds of genome duplications in the common ancestor ofvertebrates. However, clear syntenies around these four
Rh family genes have not been observed. Thus, further
investigation is required in order to clarify whether or notthese four Rh family genes were duplicated by two roundsof genome duplications in the common ancestor ofvertebrates. In either case, because Rh and RhAG genes
have erythrocyte-specific expression and RhBG and
RhCG proteins are non-erythroid members of the Rh genefamily, it is reasonable to suppose that these functional
differentiations occurred after the first gene duplication.
We thank Dr. Yuji Kohara of the Academia DNA Sequencing
Center, National Institute of Genetics and Dr. Noriyuki Satoh of
the Department of Zoology, Graduate School of Science, Kyoto University for providing us cDNA clones of amphioxus (Branchiostoma
floridae). We also thank two anonymous reviewers for theirhelpful and valuable suggestions. This study was supported by
a Grant-in-Aid for Scientific Research (18770002) from the Ministry of Education, Culture, Sports, Science, and Technology ofJapan to TK.

Rh-related genes in amphioxus
REFERENCES
Avent, N. D., Butcher, S. K., Liu, W., Mawby, W. J., Mallinson,
G., Parsons, S. F., Anstee, D. J., and Tanner, M. J. (1992)
Localization of the C termini of the Rh (rhesus) polypeptides
to the cytoplasmic face of the human erythrocyte membrane.
J. Biol. Chem. 267, 15134–15139.
Avent, N. D., Ridgwell, K., Tanner, M. J., and Anstee, D. J.
(1990) cDNA cloning of a 30 kDa erythrocyte membrane protein associated with Rh (Rhesus)-blood-group-antigen
expression. Biochem. J. 271, 821–825.
Blancher, A., and Socha, W. W. (1997) The Rhesus system. In:
Molecular biology and evolution of blood group and MHC
antigens in primates (eds.: A. Blancher, J. Klein, and W. W.
Socha), pp. 147–218. Springer-Verlag, Berlin, Heidelberg,
New York.
Cherif-Zahar, B., Bloy, C., Le Van Kim, C., Blanchard, D.,
Bailly, P., Hermand, P., Salmon, C., Cartron, J. P., andColin, Y. (1990) Molecular cloning and protein structure of ahuman blood group Rh polypeptide. Proc. Natl. Acad. Sci.
USA 87, 6243–6247.
Ewing, B., Hillier, L., Wendl, M. C., and Green, P. (1998) Basecalling of automated sequencer traces using phred. I. Accuracy assessment. Genome Res. 8, 175–185.
Felsenstein, J. (1981) Evolutionary trees from DNA sequences: amaximum likelihood approach. J. Mol. Evol. 17, 368–376.
Gordon, D., Abajian, C., and Green, P. (1998) Consed: a graphical tool for sequence finishing. Genome Res. 8, 195–202.
Guindon, S., and Gascuel, O. (2003) A simple, fast, and accurate
algorithm to estimate large phylogenies by maximum
likelihood. Syst. Biol. 52, 696–704.
Huang, C. H., and Peng, J. (2005) Evolutionary conservationand diversification of Rh family genes and proteins. Proc.
Natl. Acad. Sci. USA 102, 15512–15517.
Huelsenbeck, J. P., Ronquist, F., Nielsen, R., and Bollback, J. P.
(2001) Bayesian inference of phylogeny and its impact on
evolutionary biology. Science 294, 2310–2314.
Jones, D. T., Taylor, W. R., and Thornton, J. M. (1992) The rapid
generation of mutation data matrices from protein
sequences. Comput. Appl. Biosci. 8, 275–282.
Kitano, T., and Saitou, N. (1999) Evolution of Rh blood group
genes have experienced gene conversions and positive
selection. J. Mol. Evol. 49, 615–626.

127

Kitano, T., and Saitou, N. (2000) Evolutionary history of the Rh
blood group-related genes in vertebrates. Immunogenetics
51, 856–862.
Kitano, T., Sumiyama, K., Shiroishi, T., and Saitou, N. (1998)
Conserved evolution of the Rh50 gene compared to its
homologous Rh blood group gene. Biochem. Biophys. Res.
Commun. 249, 78–85.
Kumar, S., and Hedges, S. B. (1998) A molecular timescale forvertebrate evolution. Nature 392, 917–920.
Notredame, C., Higgins, D. G., and Heringa, J. (2000) T-Coffee:
a novel method for fast and accurate multiple sequencealignment. J. Mol. Biol. 302, 205–217.
Peng, J., and Huang, C. H. (2006) Rh proteins vs Amt proteins:an organismal and phylogenetic perspective on CO2 andNH3 gas channels. Transfus. Clin. Biol. 13, 85–94.
Putnam, N. H., Butts, T., Ferrier, D. E., Furlong, R. F., Hellsten,U., Kawashima, T., Robinson-Rechavi, M., Shoguchi, E.,
Terry, A., Yu, J. K., et al. (2008) The amphioxus genome andthe evolution of the chordate karyotype. Nature 453,
1064–1071.
Ronquist, F., and Huelsenbeck, J. P. (2003) MrBayes 3: Bayesian
phylogenetic inference under mixed models. Bioinformatics
19, 1572–1574.
Saitou, N., and Nei, M. (1987) The neighbor-joining method: a
new method for reconstructing phylogenetic trees. Mol.
Biol. Evol. 4, 406–425.
Smit, A. F. A., Hubley, R., and Green, P. (1996–2004) RepeatMasker Open-3.0. http://www.repeatmasker.org.
Takezaki, N., Rzhetsky, A., and Nei, M. (1995) Phylogenetic testof the molecular clock and linearized trees. Mol. Biol. Evol.
12, 823–833.
Tamura, K., Dudley, J., Nei, M., and Kumar, S. (2007) MEGA4:
Molecular Evolutionary Genetics Analysis (MEGA) software
version 4.0. Mol. Biol. Evol. 24, 1596–1599.
Yu, J. K., Satou, Y., Holland, N. D., Shin-I, T., Kohara, Y.,
Satoh, N., Bronner-Fraser, M., and Holland, L. Z. (2007)
Axial patterning in cephalochordates and the evolution of
the organizer. Nature 445, 613–617.
Whelan, S., and Goldman, N. (2001) A general empirical modelof protein evolution derived from multiple protein families
using a maximum-likelihood approach. Mol. Biol. Evol. 18,
691–699.