Comparative genomic analysis of teleost fish bmal genes

Han Wang

Received: 17 May 2008 / Accepted: 29 September 2008 / Published online: 14 October 2008

� Springer Science+Business Media B.V. 2008

Abstract Bmal1 (Brain and muscle ARNT like 1) gene is

a key circadian clock gene. Tetrapods also have the second

Bmal gene, Bmal2. Fruit fly has only one bmal1/cycle gene.

Interrogation of the five teleost fish genome sequences

coupled with phylogenetic and splice site analyses found

that zebrafish have two bmal1 genes, bmal1a and bmal1b,

and bmal2a; Japanese pufferfish (fugu), green spotted

pufferfish (tetraodon) and Japanese medaka fish each have

two bmal2 genes, bmal2a and bmal2b, and bmal1a; and

three-spine stickleback have bmal1a and bmal2b. Syntenic

analysis further indicated that zebrafish bmal1a/bmal1b,

and fugu, tetraodon and medaka bmal2a/bmal2b are

ancient duplicates. Although the dN/dS ratios of these four

fish bmal duplicates are all\1, implicating they have been

under purifying selection, the Tajima relative rate test

showed that fugu, tetraodon and medaka bmal2a/bmal2b

have asymmetric evolutionary rates, suggesting that one of

these duplicates have been subject to positive selection or

relaxed functional constraint. These results support the

notion that teleost fish bmal genes were derived from the

fish-specific genome duplication (FSGD), divergent reso-

lution following the duplication led to retaining different

ancient bmal duplicates in different fishes, which could

have shaped the evolution of the complex teleost fish

timekeeping mechanisms.

Keywords Circadian clocks � Conserved synteny �Genome duplication � Differential gene loss �Ratio of nonsynonymous to synonymous substitutions and

relative evolutionary rates

Abbreviations

Bmal1 Brain and muscle ARNT like protein 1

bHLH Basic helix-loop-helix

PAS Period-Aryl hydrocarbon receptor nuclear

translocator-Single mind

dN The numbers of nonsynonymous nucleotide

substitutions per nonsynonymous site

dS The numbers of synonymous nucleotide

substitutions per synonymous site

FSGD Fish-specific genome duplication

Introduction

Circadian rhythms, biological rhythms with an approxi-

mately 24-hour periodicity, are ubiquitous displays of the

intrinsic timekeeping mechanisms of life on the Earth

(Bell-Pedersen et al. 2005; Dunlap 1999; Panda et al.

2002; Pittendrigh 1993). The molecular genetic machinery

underlying the timekeeping mechanisms has been

remarkably conserved among major phyla of living

organisms (Bell-Pedersen et al. 2005; Dunlap 1999; Panda

et al. 2002; Yu and Hardin 2006). Central to the operation

of the circadian clocks is a transcription/translation nega-

tive feedback loop composed of a group of circadian clock

genes (Bell-Pedersen et al. 2005; Dunlap 1999; Hardin

2005; Ko and Takahashi 2006; Panda et al. 2002; Yu and

Hardin 2006). Bmal1 (Brain and muscle ARNT like1) is one

of these canonical circadian clock genes and encodes a

Electronic supplementary material The online version of thisarticle (doi:10.1007/s10709-008-9328-9) contains supplementarymaterial, which is available to authorized users.

H. Wang (&)

Department of Zoology and Stephenson Research & Technology

Center, University of Oklahoma, Norman, OK 73019, USA

e-mail: hwang@ou.edu

123

Genetica (2009) 136:149–161

DOI 10.1007/s10709-008-9328-9

highly conserved bHLH (basic-Helix-Loop-Helix)-PAS

(Period-Arylhydrocarbon receptor nuclear translocator-

Single mind) protein. Mouse (Mus musculus) Bmal1 was

identified in a yeast two-hybrid screen using mouse Clock

(Circadian locomotor output cycle kaput), another bHLH-

PAS containing circadian clock gene, as bait (Gekakis

et al. 1998). Mouse CLOCK and BMAL1 form a hetero-

dimer through the PAS domains (Gekakis et al. 1998;

Hogenesch et al. 1998). The mouse CLOCK:BMAL1

heterodimer activates transcription of three Period (Per)

and two Cryptochome (Cry) genes by binding E-boxes in

the Per and Cry promoter regions with the bHLH motif

(Gekakis et al. 1998; Hogenesch et al. 1998). Tetrapods

including mouse also have the second Bmal gene, Bmal2

(Hogenesch et al. 2000; Okano et al. 2001), and BMAL2

also can form a heterodimer with CLOCK (Hogenesch

et al. 2000). In contrast, the fruit fly (Drosophila melano-

gater), an insect, has only one bmal1 or cycle gene (Rutila

et al. 1998) and one clock gene (Allada et al. 1998). In a

similar way, fly CLOCK and CYCLE/BMAL1 form the

CLOCK:CYCLE/BMAL1 heterodimer that drives rhyth-

mic expression of fly per and tim (timeless) (Allada et al.

1998; Darlington et al. 1998; Rutila et al. 1998).

Teleost fishes have the most species among all verte-

brate groups, show enormous phenotypic diversity in

morphology and behaviors, and live in diverse aquatic

habitats (Nelson 2006). Gene and genome duplication have

been thought to contribute to the magnificent teleost radi-

ation (Amores et al. 1998; Meyer and Schartl 1999;

Postlethwait 2007). While extra copies of many develop-

mentally important genes such as hox genes have been

suggested to promote the emergence of morphological

complexity of teleost fishes (Amores et al. 1998; Cresko

et al. 2003; Meyer and Schartl 1999; Postlethwait et al.

2004), very little is known about possible roles of gene and

genome duplication in the generation of diverse behaviors

including circadian behaviors of teleost fishes (Wang

2008a, b). Because many life history traits such as mating,

spawning or migration are temporally controlled, investi-

gation of the evolution of the teleost fish circadian clock

genes should shed light on the tremendous teleost explo-

sion. Comparative analysis of the genomes of the Japanese

pufferfish (fugu) (Takifugu rubripes) (Aparicio et al.

2002), the spotted green pufferfish (tetraodon) (Tetra-

odon nigroviridis) (Jaillon et al. 2004), the Japanese

medaka fish (Oryzias latipes) (Kasahara et al. 2007), the

three-spine stickleback (Gasterosteus aculeatus) and zeb-

rafish (Danio rerio) has already provided invaluable

information concerning the evolution of teleost fish circa-

dian clock genes (Wang 2008a, b). Zebrafish have

preserved the per1a/per1b ancient duplicate, whereas

medaka, fugu, tetraodon and stickleback have maintained

the per2a/per2b ancient duplicate (Wang 2008a). Further,

zebrafish, fugu and tetraodon also have retained the

clock1a/clock1b duplicate (Wang 2008b). Like period and

clock genes, zebrafish also have more bmal genes than

tetrapods (Cermakian et al. 2000; Ishikawa et al. 2002).

The first two zebrafish bmal genes were isolated also using

the yeast two-hybrid screen with zebrafish clock1a as bait

(Cermakian et al. 2000; Wang 2008b). The third zebrafish

bmal gene was isolated by degenerate reverse transcrip-

tion-polymerase chain reaction (RT-PCR) (Ishikawa et al.

2002). However, no other bmal genes from fugu, tetraodon,

medaka or stickleback have been reported to date.

Here interrogation of the five teleost fish genome

sequences in combination with phylogenetic, splice site

and genome neighborhood analyses was used to uncover

fish bmal genes, to reconstruct the evolutionary relation-

ships of these bmal genes and to determine the number of

ancient duplicate bmal genes preserved in each of the five

fishes. Modes of selection then were estimated for these

ancient bmal duplicate pairs, and the Tajima relative rate

test was conducted to determine whether asymmetric

evolutionary rates occurred between each of these ancient

bmal duplicates following the duplication. Similar to tele-

ost fish period and clock genes (Wang 2008a, b), the extra

teleost fish bmal genes also were derived from the fish-

specific genome duplication (FSGD), and differential gene

loss after the duplication resulted in retention of different

ancient bmal duplicates in different teleost fishes, which

could have contributed to the evolution of the complex

teleost fish timekeeping mechanisms (Wang 2008a, b).

Materials and methods

Phylogenetic analysis

The 14 teleost fish bmal genes (Table 1) were obtained

from Ensembl (http://www.ensembl.org/index.html) as of

September, 2008. Multiple sequence alignments of Bmal

coding sequences (cds) were generated using ClustalX

(Thompson et al. 1997). The phylogenetic tree of Bmal

coding sequences (Fig. 1) was constructed using the

neighbor-joining method with MEGA4 (Tamura et al.

2007).

Splice site analysis

Exon boundaries of the coding regions of these 14 teleost

fish bmal genes as well as human, mouse, chicken Bmal

genes, and fly bmal1/cyc gene were determined according

to Ensembl (Curwen et al. 2004). The numbers of nucle-

otides (nt) for each exon as well as the phase of each

splicing site also were determined, and shown in Figs. 2

and 3, respectively.

150 Genetica (2009) 136:149–161

123

Syntenic analysis

Using human BMAL1 (11p15, 13.26 mb) and BMAL2

(12p12.2-p11.2, 27.38 mb) (NCBI Build 36.2) as anchor

sites, the orthologous comparisons of the genes in the

regions of approximately 20–100 mb flanking the human

BMAL loci with the mouse genome (NCBI Build 37.1),

the chicken genome (WASHUC2), the zebrafish genome

(Ensembl Zv7), the fugu genome (FUGU 4.0), the tet-

raodon genome (TETRAODON 7), the medaka genome

(MEDAKA1) or stickleback genome (BROAD S1) were

conducted with the BioMart mode (Kasprzyk et al.

2004) in Ensembl, respectively. From the BioMart out-

put tables, only genes that show 1 to 2 (human/mouse/

chicken to zebrafish/fugu/tetraodon/medaka/stickleback)

orthology that was supported by phylogenetic analysis

(data not shown) were chosen. The Ensembl gene IDs

and the genomic locations of these human, mouse,

chicken, zebrafish, fugu, tetraodon, medaka and stickle-

back genes are showed in Supplementary Tables 1

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Fig. 1 The phylogenetic analysis of Bmal genes using their coding

sequences (cds) with the fly cycle/bmal1 cds as an outgroup. The tree

was constructed by the neighbor-joining method with the maximum

composite likelihood model and 1,000 bootstraps, and executed with

MEGA4 (Tamura et al. 2007). Shown is the bootstrap consensus tree.

The numbers indicate the percentage bootstrap support. Dm,

Drosophila melanogaster; Dr, Danio rerio; Tr, Takifugu rubripes;

Tn, Tetraodon nigroviridis; Ol, Oryzias latipes; Ga, Gasteros-teus aculeatus; Gg, Gallus gallus; Mm, Mus musculus; and Hs,

Homo sapiens. The Ensembl gene IDs of these genes are listed in

Table 1 and Supplementary Table 3

Genetica (2009) 136:149–161 151

123

Ratio of nonsynonymous to synonymous substitutions

(dN/dS)

The DNA sequences of the fish and tetrapod Bmal genes

were aligned following the amino acid alignment by

ClustalX (Thompson et al. 1997). The numbers of

nonsynonymous nucleotide substitutions per nonsynony-

mous site (dN) and the numbers of synonymous nucleotide

substitutions per synonymous site (dS) were calculated

with the maximum likelihood method using PAML4 (Yang

2007).

Relative evolutionary rate test

Tajima relative rate tests (Tajima 1993) were conducted

with cDNA sequences for the four fish bmal duplicate pairs

using MEGA4 (Tamura et al. 2007).

Results

Phylogenetic analysis of teleost fish bmal genes

Interrogation of the five teleost fish genomes revealed 14

fish bmal genes (Table 1). Phylogenetic analysis indicated

that six fish bmal1 genes form a sister clade with tetrapod

Bmal1 genes (Fig. 1). Zebrafish are the only fish that have

two bmal1 genes, bmal1a, which was previously named as

‘‘zfbmal1’’ (Cermakian et al. 2000; Ishikawa et al. 2002),

and bmal1b, which was previously called as ‘‘zfbmal3’’

(Ishikawa et al. 2002). In contrast, the remaining four tel-

eost fishes, fugu, tetraodon, medaka and stickleback each

have only bmal1a (Fig. 1). Further, eight fish bmal2 genes

form a monophyletic group with tetrapod Bmal2 genes

(Fig. 1). Interestingly, fugu, tetraodon and medaka each

have two bmal2 genes, bmal2a and bmal2b, while zebrafish

have only bmal2a and stickleback have only bmal2b

(Fig. 1).

Splice site analysis

The conserved properties such as exons with the same

numbers of nucleotides and the conserved intron phases

can be determined by examining exonic structures of the

genes under comparison (von Schantz et al. 2006; Wang

2008a; b). The majority of the 20 vertebrate Bmal genes

studied here, i.e., 14 teleost bmal genes and 6 tetrapod

Bmal genes, share four linked exons with the same num-

bers of nucleotides (nt), i.e., a 158-nt exon, a 80-nt exon, a

93-nt exon and a 118-nt exon, near the 50 end or the middle

of the transcripts (Figs. 2, 3). In addition, most of these 20

vertebrate Bmal genes also share two linked exons each

Fig. 2 Exonic structures of fly cycle and 11 vertebrate Bmal1 genes.

The splice sites crossing or flanking the exons are shown on the top of

each exon boundary. Dm, Drosophila melanogaster; Dr, Danio rerio;

Tr, Takifugu rubripes; Tn, Tetraodon nigroviridis; Ol, Oryzias latipes;Ga, Gasterosteus aculeatus; Gg, Gallus gallus; Mm, Mus musculus;

and Hs, Homo sapiens. The size of each exon is not drawn to scale

152 Genetica (2009) 136:149–161

123

with 107 nt and 151 nt, respectively, near the 30 end of the

transcripts (Figs. 2, 3).

Besides the commonly shared six linked exons with the

same numbers of nt described above, most of the nine

vertebrate Bmal1 genes share the two more linked exons

each with 150 nt and 247 nt, respectively, at the 30 end of

the four linked exons; and two additional linked exons each

with 109 nt and 94 nt, respectively, near the 30 end of the

transcripts (Fig. 2). All the six teleost bmal1 genes also

share a 94-nt exon near the 30 end of the transcripts

(Fig. 2). The three tetrapod Bmal1 genes share additional

three linked exons each with 21 nt, 39 nt and 42 nt,

respectively, adjacent to the 50 end of the 158-nt exon,

except that human BMAL1 has a 148-nt exon in stead of the

21-nt exon; and the 21-nt and 39-nt exons are also present

in zebrafish bmal1a and bmal1b (Fig. 2). Furthermore,

while three of the four teleost bmal2a genes also share a

97-nt exon near the 30 end of the transcripts, three of the

four teleost bmal2b genes also share a 112-nt nt exon near

the 30 end of the transcript (Fig. 3). Chicken, mouse and

human Bmal2 share additional three linked exons each with

109 nt, 86 nt and 109 nt, respectively, near the 30 end of the

transcripts (Fig. 3). Mouse and human Bmal2 genes also

share two more exons, a 102-nt exon near the 50 end of the

transcripts, and a 241-nt exon adjacent the 50 end of the

107-nt exon (Fig. 3).

For these exons with the same numbers of nt, the con-

served intron phases are also observed (Figs. 2, 3).

Therefore, it is apparent that a number of conserved intron

phases are shared by all 20 vertebrate Bmal genes, by all

nine vertebrate bmal1 genes, and shared by all 11 verte-

brate Bmal2 genes, respectively (Figs. 2, 3). In addition,

the six fish bmal1 genes or 8 fish bmal2 genes also share a

number of conserved phases, respectively (Figs. 2, 3).

Finally, in comparison with vertebrate bmal genes, the fly

bmal1/cycle gene appears to have much fewer numbers of

Fig. 3 Exonic structures of seven vertebrate Bmal2 genes. The splice

sites crossing or flanking the exons are shown on the top of each exon

boundary. Numbers in boxes are nucleotide numbers. Dr, Danio

rerio; Tr, Takifugu rubripes; Tn, Tetraodon nigroviridis; Ol, Oryziaslatipes; Ga, Gasterosteus aculeatus; Gg, Gallus gallus; Mm, Mus mus-culus; and Hs, Homo sapiens. The size of each exon is not drawn to scale

Genetica (2009) 136:149–161 153

123

exons than the vertebrate Bmal genes. Even so, the verte-

brate bmal genes and the fly bmal1/cycle gene share a few

conserved intron phases (Figs. 2, 3).

Genomic neighborhood analysis

Conserved syntenic regions defined by closely linked two

or more orthologous genes on a single chromosome or a

chromosomal fragment in each of two or more different

species provide critical information concerning how genes

and genomes evolve (Ehrlich et al. 1997; Nadeau 1989;

Postlethwait 2007; Wang 2008a; b; Woods et al. 2005).

Using the BioMart function in Ensembl, the orthologs or

co-orthologs of mouse, chicken, zebrafish, fugu, tetra-

odon, medaka and stickleback to human genes in the

chromosomal regions approximately 20 to 100 mb

flanking BMAL1 or BMAL2 were determined. The chro-

mosomal locations, Ensembl Gene ID as well as the

peptide lengths of these genes in human, mouse, chicken,

zebrafish, fugu, tetraodon, medaka and stickleback were

also obtained (Supplementary Tables 1, 2). Comparison of

the human chromosome 11 region harboring human

BMAL1 with the mouse chromosome 7 region containing

mouse Bmal1 and the chicken chromosome 5 region

possessing chicken Bmal1 reveals a highly conserved

synteny. In particular, mouse and human maintain the

same gene order for eight genes including SOX6 (Sex

determining region Y box 6), PTH (Parathyroid hor-

mone), BTBD10 (BTB/POZ domain-containing protein

10), BMAL1, TEAD1 (TEA domain family member 1),

PARVA (Parvin, alpha), GALNT4 (Polypeptide N-acety-

lgalactosaminyltransferase-like protein 4) and LMO1

(LIM domain only 1). However, inversions are observed

for the remaining eight genes in the mouse chromosome 7

region as well as almost all 15 genes flanking Bmal1 in

the chicken chromosome 5 region (Fig. 4). Further, two

paralogons each harboring one of the two co-orthologs of

the genes flanking BMAL1 on human chromosome 11 are

observed in zebrafish (chromosome 25 vs. chromosome 7),

tetraodon (chromosome 13 vs. chromosome 5) and stick-

leback (group XIX vs. group GII) (Fig. 4). Indeed, each of

the two co-orthologs of GAS2 (Growth-arrest-specific

protein 2), SLC17A6 (Solute carrier family 17, member 6),

TMEM16E (Transmembrane protein 16E), TPH1 (Tryp-

tophan 5-hydroxylase 1), NAP1L4 (Nucleosome assembly

protein 1 like 4), IGF2 (Insulin-like growth factor II),

RASSF7 (Ras association domain-containing protein 7),

PTH, BTBD10, TEAD1, PARVA and GALNTL4 that are

closely linked to BMAL1 in human chromosome 11, are

located in zebrafish chromosome 25 and chromosome 7,

respectively (Fig. 4). Numerous co-ortholog pairs of these

genes are also observed in two different chromosomes,

linkage groups or scaffolds of tetraodon, stickleback

(Fig. 4), fugu and medaka (Supplementary Table 1),

respectively. Further, presumably due to differential gene

loss following the duplication (Postlethwait et al. 2004;

Taylor et al. 2001; Wang 2008a; b), only one of the two

co-orthologs of some these tetrapod genes, for instance,

tetraodon tph1a and rassf7a, and zebrafish lmo1b and

sox6b, are preserved in these fish genomes (Fig. 4 and

Supplementary Table 1). Together, these fish co-orthologs

or single orthologs define both the fish bmal1a paralogon

and the fish bmal1b paralogon that are co-orthologous to

the tetrapod Bmal1 paralogon (Fig. 4 and Supplementary

Table 1).

Likewise, a highly conserved synteny is also observed

among the human chromosome 12 region harboring human

BMAL2, the mouse chromosome 6 region containing

mouse Bmal2 and the chicken chromosome 1 region pos-

sessing chicken Bmal2, even though Tph2 (Tryptophan

5-hydroxylase 2), Nap1l1 (Nucleosome assembly protein

1-like 1) and Gas2l3 (GAS2-like protein 3) appear to be

translocated to mouse chromosome 10 (Fig. 5). Two

medaka, zebrafish, or stickleback paralogons defined by

fish bmal2 genes and co-orthologs or single orthologs of

the tetrapod genes in chromosomal regions containing

tetrapod Bmal2 are also observed (Fig. 5). For instance,

each of the two co-orthologs of ERGIC2 (Endoplasmic

reticulum-Golgi intermediate compartment protein 2),

RASSF8 (Ras association domain-containing protein 8),

CD9 (CD9 antigen), GALNT8 (Polypeptide N-acetylga-

lactosaminyltransferase 8), FGF6 (Fibroblast growth

factor 6), C12ORF5 (Chromosome 12 open reading frame

5), CCND2 (Cyclin D2), PRMT8 (Protein arginine

N-methyltransferase 8), and TSPAN9 (Tetraspanin 9) that

are closely linked to BMAL2 in human chromosome 12, are

distributed in medaka chromosome 6 and chromosome 23,

respectively (Fig. 5). A number of co-ortholog pairs or

single orthologs of these genes are also founded in two

different chromosomes, linkage groups, scaffolds of

zebrafish, stickleback (Fig. 5), fugu and medaka (Supple-

mentary Table 1), respectively. Interestingly, zebrafish

bmal2a with four closely linked genes, tph2a, prmt8a,

tspan9a and cd9a appear to be translocated chromosome

18 (Fig. 5). Together, these fish co-orthologs or single

orthologs define both the fish bmal2a paralogon and the

fish bmal2b paralogon that are co-orthologous to the tet-

rapod Bmal2 paralogon (Fig. 5 and Supplementary

Table 1).

Finally, comparison of the human, mouse and chicken

Bmal1 paralogons with the human, mouse and chicken

Bmal2 paralogons reveals that Bmal1/Bmal2, Gas2/Gas2l3,

Tph1/Tph2, Sox6/Sox5 (Sex determining region Y box 5),

Galntl4/Galntl8, Lmo1/Lmo3 (LIM domain only 3),

Nap1l4/Nap1l1, and Rassf7/Rassf8 are paralog pairs,

respectively (Figs. 4, 5).

154 Genetica (2009) 136:149–161

123

Evolution of the coding sequences of the four fish bmal

duplicates

The ratio of the numbers of nonsynonymous substitutions

per nonsynonymous site (dN) to the numbers of synony-

mous substitutions per synonymous site (dS) can be used to

estimate the modes of selection. Specifically, dN/dS [ 1

indicates positive selection, dN/dS \ 1 purifying selection,

and dN/dS = 1 neutral selection (Yang and Nielsen 2000).

The phylogenetic, splice site and syntenic analyses dis-

cussed-above discovered four pairs of fish bmal ancient

duplicates, i.e., zebrafish bmal1a/bmal1b, fugu, tetraodon

and medaka bmal2a/bmal2b. The dN/dS values for these

four bmal duplicate pairs are all less than one (Table 2),

implicating that these ancient duplicates have been under

purifying selection. Further, Tajima relative rate tests

(Tajima 1993) were performed to investigate whether one

of the fish bmal duplicates has evolved at an accelerated

rate following the duplication. A statistically significant

increase of evolutionary rate has occurred in one of the

duplicates for fugu, tetraodon and medaka bmal2a/bmal2b,

whereas no statistically significant increase of evolutionary

rate is observed for zebrafish bmal1a/bmal1b (Table 3).

Discussion

Extra copies of fish bmal genes were generated from the

FSGD

Four of the five teleost fishes studied have three bmal genes

(Table 1), whereas the fruit fly has one cycle/bmal1 gene

Fig. 4 Comparison of gene orders surrounding Bmal1 in chromo-

somes of human, mouse, chicken, zebrafish, tetraodon and

stickleback. Orthologous genes flanking Bmal1 define a synteny with

high degree of conservation among human (chromosome 11), mouse

(chromosome 7) and chicken (chromosome 5) and the tetrapod Bmal1paralogon. For the tetrapod Bmal1 paralogon, two zebrafish, tetraodon

and stickleback paralogons, i.e., the fish bmal1a paralogon and the

fish bmal1b paralogon each bearing one of the two co-orthologs of the

genes flanking Bmal1 on tetrapod chromosomes are shown. See the

text for detailed description. Dr, Danio rerio; Tn, Tetraodon nigro-viridis; Ga, Gasterosteus aculeatus; Gg, Gallus gallus; Mm,

Mus musculus; and Hs, Homo sapiens. The Ensembl IDs of these

genes are listed in Supplementary Table 1. m, Million base pairs from

one end of the chromosome, linkage group, or scaffold where the

gene is located. The positions of genes on chromosomes are not

drawn to scale

Genetica (2009) 136:149–161 155

123

and the three tetrapods, chicken, mouse and human, each

have two Bmal genes. Phylogenetic analysis showed that

zebrafish have two bmal1 genes, bmal1a and bmal1b, and

bmal2a; fugu, tetraodon and medaka each have two bmal2

genes, bmal2a and bmal2b, and bmal1a; and stickleback

have bmal1a and bmal2b (Fig. 1 and Table 1). In support

of the phylogenetic analysis, splice site analysis showed

that the majority of the 20 vertebrate Bmal genes studied

here share the six linked conserved exons and a number of

conserved intron phases, thereby providing evidence for

their common ancestry as does the phylogenetic analysis

(Figs. 1–3); and that most of the 9 vertebrate Bmal1 genes

share four more conserved exons and a number of con-

served intron phases (Fig. 2), whereas most of the 11

vertebrate Bmal2 genes share conserved exons and a

number of conserved intron phases (Fig. 3); which is

consistent with that vertebrate Bmal1 genes and vertebrate

Bmal2 genes form closely related but separate clades in the

phylogenetic analysis (Fig. 1). Splice site analysis also

showed a number of conserved exons and conserved intron

Fig. 5 Comparison of gene orders surrounding Bmal2 in chromo-

somes of human, mouse, chicken, zebrafish, medaka and stickleback.

Shown is a conserved synteny surrounding tetrapod Bmal2 among

human (chromosome 12), mouse (chromosome 6) and chicken

(chromosome 1). Translocation seemed to break part of the synteny

and move the chromosomal fragments containing orthologous genes

of human TPH2, NAP1L1 and GAS2L3 into to chromosome 10 in

mouse. For the tetrapod Bmal2 paralogon, two zebrafish, medaka and

stickleback paralogons defined by fish bmal2 and co-orthologs or

orthologs of the tetrapod genes are also shown. See the text for

detailed description. Dr, Danio rerio; Ol, Oryzias latipes; Ga, Gast-erosteus aculeatus; Gg, Gallus gallus; Mm, Mus musculus; and Hs,

Homo sapiens. The Ensembl IDs of these genes are listed in

Supplementary Table 2. m, Million base pairs from one end of the

chromosome, linkage group, or scaffold where the gene is located.

The positions of genes on chromosomes are not drawn to scale

Table 2 The ratios of nonsynonymous to synonymous substitutions

(dN/dS) for the fish bmal duplicate pairs

Duplicate paira dNb dSc dN/dS

bma1a_Dr versus bma1b_Dr 0.0141 1.8973 0.0074

bmal2a_Tr versus bmal2b_Tr 0.1820 10.1881 0.0179

bmal2a_Tn versus bmal2b_Tn 0.2021 8.3368 0.0242

bmal2a_Ol versus bmal2b_Ol 0.1789 3.2932 0.0543

a Dr: Danio rerio, Tr: Takifugu rubripes, Tn: Tetraodon nigroviridis,

Ol: Oryzias latipesb dN, number of nonsynonymous nucleotide substitutions per

nonsynonymous sitesc dS, number of synonymous nucleotide substitutions per synony-

mous sites

156 Genetica (2009) 136:149–161

123

phases for the three tetrapod Bmal1 genes, the three tet-

rapod Bmal2 genes, the four teleost bmal2a genes and the

four teleost bmal2b genes, respectively (Figs. 2, 3).

Comparison of the tetrapod chromosomal regions con-

taining Bmal1 showed a highly conserved synteny,

supported by as many as 16 sets of orthologous genes,

among human chromosome 11, mouse chromosome 7 and

chicken chromosome 5, which defined the tetrapod Bmal1

paralogon (Fig. 4). Similarly, the tetrapod Bmal2 paralo-

gon is supported by a highly conserved synteny, defined by

as many as 14 sets of orthologous genes, among human

chromosome 12, mouse chromosome 6 and chicken

chromosome 1 (Fig. 5 and Supplementary Table 2). Inter-

estingly, comparison of the tetrapod Bmal1 paralogon with

the tetrapod Bmal2 paralogon reveals the human chromo-

somal regions (Ch.11/Ch.12), the mouse chromosomal

regions (Ch.7/Ch.6), and the chicken chromosomal regions

(Ch.5/Ch.1) are paralogon pairs, respectively, as evidenced

by Gas2/Gas2l3, Tph1/Tph2, Sox6/Sox5, Galntl4/Galntl8,

Lmo1/Lmo3, Nap1l4/Nap1l1, and Rassf7/Rassf8 paralo-

gous pairs which are closely linked to Bma11 or Bmal2,

respectively (Figs. 4, 5). Thus, this syntenic analysis sup-

ports that tetrapod Bmal1/Bmal2 is a paralog pair resulting

from genome or chromosomal duplication (Figs. 1, 4, 5)

(Hogenesch et al. 2000; Okano et al. 2001).

Zebrafish, tetraodon and stickleback each have two

paralogons defined by as many as 12 co-ortholog pairs and

a number of single orthologs of the tetrapod genes flanking

Bmal1, i.e., both the fish bmal1a paralogon and the fish

bmal1b paralogon are co-orthologous to the tetrapod Bmal1

paralogon (Fig. 4). The two fish bmal1 paralogons also are

clearly present in medaka (chromosome 6 vs. chromosome

3) and fugu (for instance, scaffold 14 vs. scaffold 1) (See

Supplementary Table 1). Likewise, two paralogons that are

defined by fish bmal2 genes and as many as 9 co-ortholog

pairs or a number of single orthologs of the tetrapod genes

flanking Bmal2 also exist in zebrafish (Ch. 25/Ch. 4),

medaka (Ch. 6/Ch. 23), and stickleback (group XIX/Group

IV) (Fig. 5), tetraodon (Ch. 13/CH. 19) and fugu (for

instance, scaffold 105/scaffold 8) (see Supplementary

Table 2). Both the fish bmal2a paralogon and the fish

bmal2b paralogon are co-orthologous to the tetrapod Bmal2

paralogon (Fig. 5). The most parsimonious evolutionary

path for the evolution of teleost fish bmal genes is through

the ancient FSGD event that occurred in the teleost lineage

after its divergence from the tetrapod lineage but before its

radiation (Amores et al. 1998; Christoffels et al. 2004;

Meyer and Schartl 1999; Meyer and Van de Peer 2005;

Naruse et al. 2000; Postlethwait et al. 1998; Taylor et al.

2003; Vandepoele et al. 2004; Woods et al. 2005)

Primarily based upon allozyme and genome size data

almost forty years ago, Susumo Ohno proposed that two

successive rounds of genome duplications (2R hypothesis)

that occurred during the origin of early vertebrates might

have contributed to increasingly complex vertebrate gen-

ome structures by providing genetic materials for natural

selection to act upon (Ohno 1970). The third round of fish-

specific genome duplication (FSGD or 3R hypothesis)

occurred in the teleost lineage after its divergence from the

tetrapod lineage but before its radiation (Amores et al.

1998; Christoffels et al. 2004; Meyer and Van de Peer

2005; Postlethwait et al. 1998; Taylor et al. 2003; Woods

et al. 2005). This FSGD hypothesis also was initially

suggested by Ohno (Ohno 1970), and recent mounting

evidence such as the discoveries of the 7 hox clusters

located in seven separate chromosomes in zebrafish and

medaka (Amores et al. 1998; Naruse et al. 2000) and the

whole genome analyses of fugu (Christoffels et al. 2004;

Vandepoele et al. 2004) and tetraodon (Jaillon et al. 2004)

appears to confirm it. The FSGD hypothesis has been

successfully used to account for the evolution of fish genes

and gene families (Taylor et al. 2003; Wang et al. 2007;

Wang et al. 2008; Wang 2008a; b). The previous analyses

of teleost fish period and clock evolution were consistent

with the FSGD hypothesis (Wang 2008a; b). Similarly, the

current study of teleost fish bmal genes also supports the

FSGD hypothesis.

Preservation of different ancient bmal duplicates

in different fishes

The genome neighborhood analysis also supports the

notion that the ancestral Bmal1 gene was duplicated during

the third round of genome duplication and gave rise to

Table 3 Results of Tajima relative rate tests of fish bmal duplicate

pairs with their human orthologsa

Testing group Mtb M1c M2d v2 Pe

bma1a_Dr/bmal1b_Dr with

BMAL1_Hs1324 140 157 0.97 0.3239

bmal2a_Tr/bmal2b_Tr with

BMAL2_Hs852 125 266 50.85 0.0000

bmal2a_Tn/bmal2b_Tn with

BMAL2_Hs639 69 173 44.69 0.0000

bmal2a_Ol/bmal2b_Ol with

BMAL2_Hs943 173 310 38.86 0.0000

a The equality of evolutionary rate between a fish duplicate pair is

tested using their human ortholog as an outgroup using Tajima rela-

tive rate test with cDNA sequencesb Mt is the sum of the identical sites and the divergent sites in all

three sequences testedc M1 is the number of unique differences in the first fish paralogd M2 is the number of unique differences in the second fish paraloge If P \ 0.05, the test rejects the equal substitution rates between the

two fish duplicates and infers that one of the two duplicates has an

accelerated evolutionary rate

Genetica (2009) 136:149–161 157

123

bmal1a and bmal1b, both of which have been preserved

only in zebrafish but only bmal1a is found in fugu, tetra-

odon, medaka and stickleback (Fig. 6). Likewise, the

ancestral Bmal2 gene was duplicated and gave rise to

bmal2a and bmal2b during the same round of the genome

duplication, both of which have been preserved in fugu,

tetraodon and medaka but only bmal2a in zebrafish and

only bmal2b in stickleback (Fig. 6).

The three zebrafish bmal genes were named as zfbmal1,

zfbmal2 and zfbmal3 without considering the evolutionary

histories of these genes (Ishikawa et al. 2002; Whitmore

et al. 1998). Based upon the current comparative genomic

analysis, zebrafish zfbmal1is renamed as ‘‘bmal1a,’’ and

zfbmal3 as ‘‘bmal1b.’’ These new names are intended to

reflect the evolutionary context and enhance our under-

standing of the genome connectedness as well as the

functions of these Bmal genes.

Among these five fishes studied here, zebrafish have

retained the bmal1a/ bmal1b ancient duplicate, while fugu,

tetraodon and medaka have preserved the bmal2a/bmal2b

ancient duplicate. It also is intriguing that reciprocal gene

loss (RGL) (Semon and Wolfe 2007) has occurred for

bmal2 duplicates in zebrafish and stickleback where the

former has preserved only bmal2a and the latter only

bmal2b (Fig. 1 and Table 1). Previous studies showed that

zebrafish have preserved the per1a/per1b ancient duplicate,

while medaka, fugu, tetraodon and stickleback have

maintained the per2a/per2b ancient duplicate (Wang

2008a); and zebrafish, fugu and tetraodon have retained the

clock1a/clock1b ancient duplicates (Wang 2008b). The

differential losses of duplicates in different teleost lineages

following the whole genome-wide genome duplication

resulted in preservation of different ancient circadian clock

gene duplicates in different fishes as well as reciprocal

gene loss of certain duplicates in different fishes, which

likely have shaped the evolution of distinct timekeeping

mechanisms in these teleost fishes (Looby and Loudon

2005; Lynch and Conery 2000; Postlethwait 2007; Semon

and Wolfe 2007; Taylor et al. 2001; Wang 2008a; b). It

appears that most of the preserved ancient circadian clock

duplicates have evolved significantly from each other

(Wang 2008a; b) (also see Table 3 and discussion below).

Thus, it is possible that like their magnificent morpholog-

ical diversity, teleost fishes likely have evolved diverse

timekeeping mechanisms that in turn have played a role in

their tremendous explosion.

Evolution of the fish bmal duplicates

The fates of duplicate genes can be nonfunctional, neo-

functional or subfunctional (Force et al. 1999; He and

Zhang 2005; Hughes 1994; Stoltzfus 1999). Classic

Fig. 6 A model of the teleost fish bmal gene evolution. The common

ancestor of the teleost fishes and tetrapods was assumed to have two

Bmal genes, Bmal1 and Bmal2, which can be found in chicken, mouse

and human. During the third round of genome duplication in teleost

fishes, bmal1 gave rise to bmal1a and bmal1b, both of which have

been preserved in zebrafish but only bmal1a in fugu, tetraodon,

medaka and stickleback; bmal2 gave rise to bmal2a and bmal2b, both

of which have been retained in fugu, tetraodon and medaka but only

bmal2a in zebrafish and bmal2b in stickleback. Here, tetrapod Bmal1and Bmal2 are paralogs; fish bmal1a and bmal1b are paralogs and co-

orthologs of tetrapod Bmal1; and fish bmal2a and bmal2a are paralogs

and co-orthologs of tetrapod Bmal2. For simplicity, only two linked

genes with Bmal1 or Bmal2 are shown

158 Genetica (2009) 136:149–161

123

population genetics models proposed that one of the two

duplicate genes should become a pseudogene resulting

from accumulation of predominant deleterious mutations

over time, i.e., nonfunctionalization or it can evolve into a

novel gene due to accumulation of rare beneficial muta-

tions, i.e., neofunctionalization (Force et al. 1999; Hughes

1994; Stoltzfus 1999). Neofunctionalization as well as

higher gene dosage constraints would result in the pres-

ervation of gene duplicates. However, both models cannot

be responsible for the much higher rate of the preserva-

tion of gene duplicates observed (Force et al. 1999). New

models that account for co-existence of both ancient

duplicates presumed that the functions of the ancestral

gene were subdivided between the two duplicates, i.e.,

subfunctionalization, and both the duplicates were

required to function as an equivalent of the ancestral gene

(Force et al. 1999; Hughes 1994; Postlethwait et al. 2004;

Stoltzfus 1999). Subfunctionalization of the ancestral gene

can occur in distinct motifs of coding regions (Hughes

1994); which may exhibit different nucleotide substitution

rates for the duplicate genes (Steinke et al. 2006; Van de

Peer et al. 2001); or in cis- regulatory elements of non-

coding regions that may elicit distinct spatiotemporal

expression patterns (Force et al. 1999; Postlethwait et al.

2004). In either case or both, testable predictions can be

examined to determine mechanisms underpinning

subfunctionalization.

The nucleotide substitution rates in coding regions of

the duplicate genes have been used to examine these

competing hypotheses of the fates of the ancient dupli-

cates (Kellis et al. 2004; Kondrashov et al. 2002; Steinke

et al. 2006; Van de Peer et al. 2001). The dN/dS ratios for

the four fish bmal duplicate pairs are all smaller than one

(Table 2), implicating that they have been subject to

purifying selection; and pairwise comparisons of dN/dS

ratios among all 14 fish bmal genes, between fish bmal

genes and tetrapod Bmal genes, and among tetrapod Bmal

genes indicated that these Bmal genes also have been

subject to purifying selection (Supplementary Fig. 1),

which are in line with the recent genome-wide analyses of

duplicate genes (Kondrashov et al. 2002; Lynch and

Conery 2000; Steinke et al. 2006; Van de Peer et al.

2001). The dN/dS ratio of\1 between duplicate pairs was

used to reject the neofunctionalization model (Kondra-

shov et al. 2002). However, some genes undergoing

neofunctionalization likely also are subject to a certain

level of functional constraints (He and Zhang 2005). In

addition, due to the conservative nature of the dN/dS

estimation, several genes with a dN/dS value of \1

indeed have been subject to positive selection throughout

evolution (Dorus et al. 2004).

Conversely, Tajima relative rate test detected acceler-

ated evolutionary rates in one of the duplicates for three of

the fish bmal duplicate pairs (Table 3). Specifically, fugu,

tetraodon and medaka bmal2b have significantly higher

numbers of unique sites than fugu, tetraodon and medaka

bmal2b, respectively (Table 3), inferring that the former

have been under positive selection or relaxed functional

constraint. Asymmetric evolutionary rates between dupli-

cates were used to support neofunctionalization (Kellis

et al. 2004). However, asymmetric evolutionary rates also

should be expected between the two duplicate genes that

have been under subfunctionalization because they might

have retained different ancestral functions that might have

evolved at different rates (He and Zhang 2005).

Studies of fly, zebrafish and mouse Bmal genes showed

that the three model organisms differ not only in numbers

of Bmal genes they have but also in expression patterns of

Bmal genes. Fly bmal1/cycle is constitutively expressed

(Rutila et al. 1998), mouse Bmal1 is expressed rhythmi-

cally and synchronously in most tissues (Oishi et al. 2000),

and mouse Bmal2 mRNA levels remain constant (Okano

et al. 2001); while all three zebrafish bmal genes are

expressed rhythmically but asynchronously in various tis-

sues (Cermakian et al. 2000; Ishikawa et al. 2002).

Rhythmic expression of mouse Bmal1 is positively and

negatively regulated by retinoic acid-related orphan

nuclear receptors, Rora and Rev-erba, whose expression is

regulated directly by the CLOCK:BMAL1 heterodimer

(Preitner et al. 2002; Sato et al. 2004). Rora and Rev-erbacompete to bind ROREs (retinoic acid-related orphan

nuclear receptor response elements) in the Bmal1 promoter

region, and then RORa activates the Bmal1 expression,

while REV-ERBa inhibits the transcription (Guillaumond

et al. 2005; Preitner et al. 2002; Sato et al. 2004). Thus, it

is not uncommon that distinct molecular mechanisms

underlying expression of Bmal genes have evolved in fly,

zebrafish and mouse. Further studies of expression and

their regulatory mechanisms for the remaining 11 teleost

fish bmal genes should shed light on the evolution of bmal

genes, in particular whether the fish bmal duplicate pairs

have gone subfunctionalization or subneofunctionalization

(He and Zhang 2005).

In conclusion, extra copies of fish bmal genes were

derived from the third-round FSGD, divergent resolution

following the duplication resulted in retaining a subset of

bmal duplicates as well as reciprocal gene loss in dif-

ferent fishes, which might have shaped the evolution of

the intricate and complex teleost fish timekeeping mech-

anisms. These results should add to the growing body of

knowledge of the evolution of teleost genes and genomes,

set the stage for studying the functions of these extra

copies of fish bmal genes, and help gain a better under-

standing of the evolution of the teleost fish circadian

regulation system that might have contributed to the

explosive burst of teleost fishes.

Genetica (2009) 136:149–161 159

123

Acknowledgments This study was supported by grants from the

Whitehall foundation (2002-12-103) and the Oklahoma Health

Research Program (HR04-140S).

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