Phylogenetic Relationship of Muscle Tissues Deduced from Superimpositionof Gene Trees
Satoshi OOta and Naruya Saitou
Laboratory of Evolutionary Genetics, National Institute of Genetics, Mishima, Japan; and Department of Genetics, School ofLife Science, Graduate University for Advanced Studies, Mishima, Japan
Muscle tissues can be divided into six classes; smooth, fast skeletal, slow skeletal and cardiac muscle tissues forvertebrates, and striated and smooth muscle tissues for invertebrates. We reconstructed phylogenetic trees of six
protein genes that are expressed in muscle tissues and, using a newly developed program, inferred the phylogenyof muscle tissues by superimposition of five of those gene trees. The proteins used are troponin C, myosin essential
light chain, myosin regulatory light chain, myosin heavy chain, actin, and muscle regulatory factor (MRF) families.
Our results suggest that the emergence of skeletal-cardiac muscle type tissues preceded the vertebrate/arthropod
divergence (ca. 700 MYA), while vertebrate smooth muscle seemed to evolve independent of other muscles. In
addition, skeletal muscle is not monophyletic, but cardiac and slow skeletal muscles make a cluster. Furthermore,arthropod striated muscle, urochordate smooth muscle, and vertebrate muscles except for smooth muscle share acommon ancestor. On the other hand, arthropod nonmuscle and vertebrate smooth muscle and nonmuscle share acommon ancestor.

Introduction
How did tissues evolve? The most effective method
to infer the evolution of tissues is to use molecular phylogenetic trees of regulatory regions of tissue-specificgenes; however, this is virtually impossible because of
the lack of data. An alternative method is to use structural genes expressed in various tissues. Of course, evolution of regulatory regions does not necessarily correspond to evolution of deduced tissue distribution fromthe structural gene trees. There are three possibilities forthe relationship between regulatory and structural regions in terms of gene duplications:
1. A gene is duplicated, but its regulatory region is intact (fig. 1a).
2. A gene and its regulatory region are both duplicated(fig. 1b).
3. A gene is intact, but its regulatory region is duplicated (fig. 1c).
Depending on the case, the evolutionary differentiation
pattern may change. Phenomena corresponding to thesecases should be observed as expression patterns of isoforms in different tissues. The relationships between tissue differentiations and gene duplications can be categorized into the following three types:
1. Homologous genes are expressed in the same tissueclasses (fig. 1d ). This situation corresponds to figure
1a. It does not contribute to the inference of tissue
evolution, since the path of gene evolution does notreflect the path of tissue evolution.
2. Homologous genes are expressed in different tissueclasses (fig. 1e). This situation corresponds to figure
1b. Because the path of gene evolution is expected
Key words: muscle, phylogeny, tree superimposition, regulatory
region.
Address for correspondence and reprints: Naruya Saitou, Laboratory of Evolutionary Genetics, National Institute of Genetics, Mishima, 411-8540 Japan. E-mail: nsaitou@genes.nig.ac.jp.
Mol. Biol. Evol. 16(6):856–867. 1999
q 1999 by the Society for Molecular Biology and Evolution. ISSN: 0737-4038

856

to reflect the path of the tissue evolution, it does contribute to the inference of tissue evolution.
3. The same gene is expressed in more than one tissueclass (fig. 1f ). This situation corresponds to figure
1c. There is no duplicating event, and gene A is expressed in tissue classes a and b. This can be interpreted as the situation that the two tissue classes haveclose relationships.
The second and third types of gene duplications makepossible the inference of deduced tissue trees from structural gene trees. A single structural gene may not giveenough information to infer tissue evolution. Therefore,
superimpositions of the deduced tissue trees are expected to provide valid information.
We focus on the evolution of the developmental
pattern of muscle tissues in this study, because muscle
is the best understood example of actin-based motility,
and it is highly specialized compared with typical animal cells (e.g., Alberts et al. 1994). Furthermore, many
sequence data are available for muscles. The following
examples correspond to the above categories, respectively: case 1 (fig. 1d )—duplicated a and b human myosin heavy chain genes are both expressed in cardiac
muscle (Jaenicke et al. 1990; Matsuoka et al. 1991); case
2 (fig. 1e)—duplicated human actin genes are expressedin smooth and cardiac muscles (Hamada, Petrino, andKakunaga 1982; Taylor et al. 1988); case 3 (fig. 1f )—the human troponin C gene is expressed in both slow
skeletal and cardiac muscles (Schreier, Kedes, and Gahlmann 1990).
In vertebrates, there are four muscle tissue classes;
fast skeletal, slow skeletal, cardiac, and smooth muscles.
The fast and slow skeletal muscles are different in termsof twitching speed (Fitts 1994), and they are believed to
be derived from distinct myogenic precursors (Stockdale1992). Therefore, vertebrate tissues are classified asshown in table 1 with regard to muscles. Similarly, there
are three classes in invertebrate tissues: striated muscle(corresponding to vertebrate skeletal muscle), smoothmuscle, and nonmuscle, as shown in table 1.

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FIG. 1.—Three possible schemes of gene differentiations (top) and the corresponding patterns in tissue expression (bottom). a, A structuralgene was duplicated, but its regulatory region is intact. b, A structural gene and its regulatory region were duplicated. c, A structural gene is
intact, but its regulatory region was duplicated. Hatched boxes represent regulatory regions, and open boxes represent structural genes. d, A
gene duplication (a filled circle) produced two homologous genes, A and B, which are expressed in the same tissue class. e, Duplicated genes(A and B) are expressed in different tissue classes (1 and 2). f, the same gene (A) is expressed in different tissue classes (1 and 2).

There are 135,135 possible rooted phylogenetic relationships for these eight classes (five vertebrate tissueclasses and three invertebrate ones), even if each ofthem is monophyletic. Which topology is the most probable in terms of tissue evolution? Histologically, smoothmuscle is called the most ‘‘primitive’’ muscle, in thesense of being the most similar to nonmuscle cells (e.g.,
Alberts et al. 1994). Since ascidian adult muscle actin
is more similar to vertebrate skeletal muscle actin than
it is to other types of muscle actin, and the larval muscle
actin is more similar to cardiac actin than it is to othertypes of muscle actin, the divergence of the skeletal andcardiac isoforms has been suggested to have occurred
before the emergence of urochordates (Kovilur et al.
1993). Are such inferences true? According to a phylogenetic tree of myosin heavy chain, smooth muscle
and skeletal muscle myosins were independently derived
from nonmuscle myosin, and the tree suggests that thesimilarities between these types of muscle are the resultof convergent evolution (Goodson and Spudich 1993).
According to a phylogenetic tree of actin, however,smooth muscle and skeletal muscle actins were derived
from the same ancestor (Mounier et al. 1992). Whichevolution reflects the evolution of muscle tissues? Furthermore, the relationship of vertebrate and invertebratemuscle tissues is not clear; e.g., did vertebrate muscle
and arthropod muscle evolve independently or not? The
objective of this study is, therefore, to elucidate the phylogenetic relationship of muscle tissues using six genefamilies expressed in muscle and/or nonmuscle tissues.

These proteins are troponin C, myosin essential lightchain, myosin regulatory light chain, myosin heavy
chain, actin, and muscle regulatory factor (MRF).
Materials and Methods
Proteins Used in this Study
Troponin C, Myosin Essential Light Chain, andMyosin Regulatory Light Chain
Troponin C, myosin essential light chain, and myosin regulatory light chain belong to the EF-hand superfamily as well as calmodulin (Moncrief, Kretsinger,
and Goodman 1990). Ca21-binding protein troponin C
is one subunit of the ternary troponin complex in vertebrate skeletal and cardiac muscles. Through its association with actin and tropomyosin on the thin filament,
troponin C inhibits the actomyosin interaction at submicromolar Ca21 concentrations and stimulates the interaction at micromolar Ca21 concentrations (Farah andReinach 1995). Myosin essential light chain is essential
for the phosphorylation-dependent regulation of actinactivated ATPase activity (Katoh and Morita 1996). Covalent modification of myosin by phosphorylation of the
myosin regulatory light chains plays a significant role
in regulation of contractile activity in vertebrate smoothmuscle (Horowitz et al. 1996). We reconstructed a treeof the EF-hand superfamily (not shown) to root eachsubfamily.
Myosin Heavy Chain
Myosin heavy chain is a ubiquitous protein foundin all eukaryotic cells, where it provides the motor func-

Table 1Vertebrate and Invertebrate Tissue Classes






muscle
Invertebrate tissue (In) muscleVertebrate tissue (Ve)

cardiac muscle (C)skeletal muscle (K)smooth muscle (S)striated muscle (T)smooth muscle (S)nonmuscle (N)nonmuscle (N)slow skeletal muscle (Ks)fast skeletal muscle (Kf)858

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Table 2
Queries to Determine Each Gene Family
FamilyMyosin light chains and related
proteins. . . . . . . . . . . . . . . . . . . . . .
Troponin C . . . . . . . . . . . . . . . . . . . .
Myosin essential light chain . . . . . .
Myosin regulatory light chain . . . . .
Myosin heavy chain. . . . . . . . . . . . . . .
Actin . . . . . . . . . . . . . . . . . . . . . . . . . . .
MRF family . . . . . . . . . . . . . . . . . . . . .

Entry of
PIR

Database Description (species)

A25183

Myosin essential light chain,striated muscle (Patinopectensp.)

A23662ATAX
S20086

Myosin I, high molecular weight(Acanthamoeba sp.)Actin (Acanthamoeba castellanii)
MyoD1 protein (Ovis aries)N
Homa

N siteb

26
34
40

148
145
145

110
158
32

247
357
86

a Number of homologous entries found.
b Number of compared amino acid sites. We used only sites which do not contain any gaps.

tion for diverse movements such as cytokinesis, phagocytosis, and muscle contraction (Weiss and Leinwand
1996). There is no homology between light and heavy
chains in myosin molecules. Due to the extensive number of different molecules, myosin heavy chains havebeen divided into 7–13 distinct classes based on theproperties of the head domain (Cheney, Riley, and Mooseker 1993; Cope et al. 1996; Weiss and Leinwand
1996). We used class II myosin heavy chain to reconstruct a gene tree, and we used other classes to root thissubfamily.
Actin
Myosin interacts with actin fibers as actomyosin to
generate force (Huxley and Simmons 1971). Muscle
contraction is essentially the actomyosin ATPase reaction in solution (Sugi 1993). We reconstructed a phylogenetic tree of the whole actin family (not shown) andextracted the animal cluster that was monophyletic. A
cluster of nonanimal actins was used as an outgroup toroot the animal actin tree.
MRF Family
The MRF family regulates skeletal muscle determination and differentiation in vertebrates. The MRF
family consists of MyoD, myf5, myogenin and MRF4
subfamilies (Molkentin and Olson 1996; Yun and Wold
1996). We used the MASH family as an outgroup toroot the MRF tree.
Reconstruction of Gene Trees
All data used in this study were retrieved from the
PIR-International protein sequence database release52.00. To identify each gene family, we chose a sequence which obviously belongs to the family and carried out homology searches using BLAST (Altschul etal. 1990) and FASTA (Lipman and Pearson 1985) for
the whole database. The query sequences are listed intable 2. We conducted multiple alignments of each protein family using CLUSTAL W (Thompson, Higgins,
and Gibson 1994) and removed sites at which any gaps
existed. We used the neighbor-joining method (Saitou
and Nei 1987) to reconstruct phylogenetic trees using
CLUSTAL W (1,000 replications for bootstrap probabilities in every tree). Kimura’s (1983) method was used

to estimate evolutionary distances in terms of aminoacid substitutions. The maximum-likelihood method(Felsenstein 1981) was also used for some cases using
go/0, a parallel executable program (OOta, Saitou, andKunifuji 1995), and ProtML (Adachi and Hasegawa
1996). All sequences except one, which consisted of putative alternative splicing products, were eliminated.
TREEVIEW (Page 1996) was used to visualize trees.
Reconstruction of Tissue Trees from Gene Trees
Each gene was mapped to one or more tissue classes according to its expression pattern as describedabove. The basic criteria to map a gene tree to a tissue
tree are as follows:
1. A cluster whose genes were mapped to the same tissue class was considered a new OTU of a tissue tree.
The cluster is defined as a set of more than one genewhich share a certain ancestor and an expression pattern.
2. When a cluster of genes was mapped to more than
one tissue class, those tissue classes were considered
to make a cluster.
3. Since expression patterns often vary depending on
developmental stages, those of adults were used tomap genes to tissue classes.
4. When tissue classes in a tree were not monophyletic,
the tree was divided such that every tissue class wasmonophyletic. In such cases, all possible superimpositions were performed for each set of genes.
5. When an expression pattern of a gene was not available from the current literature, we did not map thegene to any tissue class.
6. For any case that was not described here, we omitted
any correspondence between a gene tree and a tissue
tree from mapping.
Since a part of the available data was not sufficient forour objectives, we provided exceptions:
1. When genes that shared the same ancestor could bemapped to different tissue classes which had a hierarchical relationship, the most specific tissue class
was chosen as long as there was no inconsistency.
For example, when the different tissue classes were
K and Kf, Kf was chosen (see table 1).

Phylogeny of Muscle Tissues

2. For the scallop, the sea squirt, and the fruit fly, thedefinition of a cluster was loosened: the cluster was
defined as a set of genes which share a certain ancestor and an expression pattern. In other words, acluster comprising only one gene is possible.
3. For the sea squirt, striated muscle of tadpole larvae
was included in the tissue classes because of evolutionary interest.
We thus reconstructed phylogenetic trees having tissueclasses as their OTUs. The expression patterns were retrieved from TITLE field of the PIR database and/or
original papers described in the database. In the case of
the fruit fly, FlyBase (Ashburner and Drysdale 1994)
was also used.
We have prepared a web site to reveal the expression patterns of genes that appeared in our gene trees.
At this site, every reference, with part of its abstract, isalso available. In addition, direct evidence of tissue
specificity of some genes can be retrieved. The URL is:
http://thinker.lab.nig.ac.jp/express/express.html.
Superimposition of Tissue Trees
Tree comparison methods have been described bymany authors (e.g., Foulds, Penny, and Hendy 1979;
Robinson and Foulds 1981; Zhang and Shasha 1989;
Shasha et al. 1994). Our intention is, however, not pairwise tree matching, but superimposition of multiple
trees. Furthermore, internal nodes are unlabeled in our
problem, since it is virtually impossible to know eachcorrespondence among them. We thus developed an algorithm to superimpose multiple tissue trees. In our algorithm, topological distances (Foulds, Penny, and Hendy 1979) between a given topology and each tissue tree
are computed, and their sum is assigned to the topology
as a ‘‘negative’’ score. This computation is iterated forall possible topologies. Finally, the topology (or topologies) having the smallest score is chosen as the mostprobable superimposed tree. Pruning costs and weights
for tree editing according to depth are introduced in general tree matching algorithms (Zhang and Shasha 1989;
Shasha et al. 1994). To reduce computational cost, they
were omitted in our method; that is, all the pruning costs
5 0 and all the weights 5 1.
The algorithm is summarized as follows:
1. A list of all tissue classes that appeared in the genetrees was generated.
2. Tissue classes that appeared just once were removed
from the list, because such classes do not affectscores when the pruning costs are omitted.
3. All possible topologies were generated for the tissueclasses.
4. Topological distances between each of the generated
topologies and tissue trees were computed and weresummed up as a score.
5. The former operation was iterated for all the possible
topologies.
6. Obtained scores were sorted.
A program named super was developed to perform theabove algorithm. Super is available as part of Deep-

859

Forest. For details, see http://thinker.lab.nig.ac.jp/
DeepForest/deepforest.html.
RESULTS
Multiple Alignment
Multiple alignments are not shown in this paper,
but they can be found at the following web site: http://
thinker.lab.nig.ac.jp/paper/data/data.html. Records of the
PIR database appearing in the phylogenetic trees canalso be found at the same site.
Phylogenetic Trees of Troponin C, Myosin Essential
Light Chain, and Myosin Regulatory Light Chain
A Global Tree
We first reconstructed a phylogenetic tree of myosin light chains and related proteins which included calmodulin, troponin C, myosin essential light chain, andmyosin regulatory light chain (tree not shown). The result was inconsistent with the phylogenetic tree produced by Moncrief, Kretsinger, and Goodman (1990).
We therefore used the maximum-likelihood method as
follows. Since the number of sequences is too large forapplication of our go/0 program, 10 sequences were extracted to generate a simplified tree. The topology of atree was first obtained by using the neighbor-joining
method, then the maximum-likelihood value was computed for its topology (log likelihood is 22,728). Ten
topologies were obtained by exchanging branches locally where the bootstrap values were low. A tree withthe highest likelihood value (log likelihood is 22,718)
among the 10 topologies was selected as the most probable tree. This tree was compatible with Moncrief, Kretsinger, and Goodman’s (1990) tree. Therefore, the topology that we consider to be reasonable can be represented in the Newick format as follows: ((vertebrate troponin C, invertebrate troponin C), (myosin essential
light chain, myosin regulatory light chain), calmodulin).
Troponin C
Figure 2 shows a phylogenetic tree of troponin C.
The fast skeletal muscle class and the cardiac and slow
skeletal muscle class are distinctly divided after a gene
duplication which occurred before the frog/mammal divergence (about 350 MYA). In vertebrates, there is only
one gene duplication leading to tissue classes in thistree.
In the fast skeletal muscle cluster (Kf in fig. 2), therelationship among the organisms is the same as Moncrief, Kretsinger, and Goodman’s (1990) tree. Although
this pattern is different from the established vertebrate
phylogeny, this inconsistency may be caused by shortbranches. We added 12 new invertebrate sequences to
Moncrief, Kretsinger, and Goodman’s (1990) tree andshowed their relationships. In comparison with vertebrate troponin C’s, their evolutionary rates are high (approximately 1.4 times). This may reflect the differenceof function; the Ca21-binding capacity of invertebrate
troponin C’s seems to be only one Ca21 ion per molecule, while vertebrate troponin C’s bind more than one
Ca21 ions per molecule (Shima et al. 1984).

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FIG. 2.—A phylogenetic tree of troponin C. Closed rhombuses denote gene duplications. Ve and In denote vertebrates and invertebrates,
respectively. T, S. and N in shaded boxes denote invertebrate striated muscle, smooth muscle, and nonmuscle, respectively. Kf, Ks, C, S, andN in open boxes denote vertebrate fast skeletal muscle, slow skeletal muscle, cardiac muscle, smooth muscle, and nonmuscle, respectively. a,b, and c indicate the upper limits of the vertebrate/invertebrate, bird/mammal, and frog/mammal divergences, respectively. Bootstrap values areless that 90% in gray branches. The scale bar stands for the number of substitutions per amino acid site. A broken line stands for an omitted
correspondence from the mapping according to the criteria (see text).

Myosin essential light chains

A Phylogenetic Tree of Myosin Heavy Chain

Figure 3 shows the phylogenetic tree of myosin
essential light chain. The features of this tree are similar
to those of the troponin C tree; gene duplications leading
to isoforms in the vertebrate tissue classes probably occurred after the vertebrate/invertebrate divergence. In
the vertebrate tissue classes, there are cardiac (C), slow
skeletal (Ks), and fast skeletal (Kf) muscle clusters. The
three classes emerged after the two gene duplications,which both occurred at least before the bird/mammal
divergence. On the other hand, there is the vertebratesmooth (S) muscle and nonmuscle (N) cluster in the
myosin essential light chain family, in contrast to thetroponin C family. In invertebrates, the differentiation
between smooth muscle (S) and striated muscle (T) myosin essential light chains occurred before the vertebrate/
invertebrate divergence.

Figure 5 shows a phylogenetic tree of class II myosin heavy chains. Since A24922, S04090, I38055, andA29320 in this tree are embryonic or perinatal, they
were excluded for reconstruction of a tissue tree. The
features of the tree are different from those of the three
genes we discussed before. There are two gene duplications which led to three major clusters: (1) nonmuscle
and smooth muscle, (2) invertebrate striated muscle, and(3) vertebrate cardiac and skeletal muscles.
It is characteristic that the coalescence point (a infig. 5) of the muscle tissues except for the vertebratesmooth muscle (T, Ks, Kf, and C in fig. 5) precedes thevertebrate/invertebrate divergence (a in fig. 5), and thiscluster contains nematode, arthropod, and mollusk myosin heavy chain. This suggests that vertebrates, nematodes, arthropods, and the mollusks may share the striated muscle tissue origin in terms of the myosin heavy
chain evolution.
Our result is consistent with Goodson and Spudich’s (1993) tree and Cheney, Riley, and Mooseker’s(1993) tree. Moore et al.’s (1993) tree, however, does
not agree with ours. They used only the light meromyosin (LMM) region, which is one of the components ofmyosin heavy chain rod. The reason for this inconsistency is currently not known.
The b myosin heavy chain gene of the mouse isexpressed in Ca and Ks (Rindt, Knotts, and Robbins

Myosin Regulatory Light Chains
The tree of myosin regulatory light chain (fig. 4)shows considerably different aspects than does that of
the essential light chain. For instance, gene duplications
which led to isoforms occurred before the vertebrate/
invertebrate divergence. In addition, there are several
isoforms in the same tissue class. For example, the cardiac muscle class has two isoform clusters (C-1 and C2 in fig. 4), and their divergence time is earlier than thatof the vertebrate/invertebrate divergence.

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FIG. 3.—A phylogenetic tree of myosin essential light chain. All other notations are the same as those in figure 2.

1995). Although these sequence data are not available
in PIR, we used this information in mapping for Ks infigure 5.
Actin of Animals
Our actin tree (fig. 6) is inconsistent with Kovilur
et al.’s (1993) tree, in which nonmuscle classes of sea
squirt and sea urchin are not monophyletic. They used
the maximum-parsimony method to construct their tree.
Since multiple alignments of actin sequences are quite
stable, the difference in topologies is probably caused
by a difference in tree-making methods. Although we
also applied the maximum-likelihood method using program go/0, there was no significant difference between
our tree and Kovilur et al.’s (1993) tree. In terms oforganismal relationship, however, nonmuscle classes ofsea squirt (S33386) and sea urchin (S07288) should bemonophyletic. This suggests that our tree is more probable than Kovilur et al.’s (1993) tree.
The cluster (T2) of the striated muscle actins ofarthropods is close to invertebrate and vertebrate nonmuscle clusters (see fig. 6). It suggests that arthropods
evolved muscle independently of vertebrate muscle.
However, this inference is inconsistent with that fromthe myosin heavy chain tree (fig. 5). The vertebrate muscle cluster, which includes smooth, skeletal, and cardiac
muscles, is monophyletic, and this is further clusteredwith sea squirt actins to form a chordate lineage. Mounier et al. (1992) proposed that muscle-specific actin
genes have appeared independently at least twice during
the evolution of animals; insect muscle actin genes haveemerged from an ancestral cytoplasmic actin gene within the arthropod phylum, whereas vertebrate muscle actin genes evolved within the chordate lineage. Our result

confirms their model, while it is inconsistent with Kovilur et al.’s (1993) conclusion that ‘‘the divergence ofthe skeletal and cardiac (actin) isoforms occurred before
the emergence of urochordates.’’
A Phylogenetic Tree of the MRF Family
A phylogenetic tree of the MRF family is shownin figure 7. Since the MRF family is so diverged, Atchley, Fitch, and Bronner-Fraser (1994) reconstructed thewhole MyoD family tree using only the 59-amino-acidbasic helix loop helix (bHLH) region. We compared not
only the bHLH region but also other conserved regions,and a total of 86 amino acids. Although Atchley, Fitch,
and Bronner-Fraser did not indicate the root of their tree
explicitly, our reconstructed tree of the MRF family is
consistent with theirs.
According to our result, a gene duplication after theupper limit of the vertebrate/invertebrate divergence (ain fig. 7) produced the vertebrate MRF family. The relationship among the invertebrate and vertebrate MRF
family is (Caenorhabditis elegans CeMyoD, (sea urchin
SUM-1, (fruit fly nautilus, vertebrate MRF family)) in
the Newick format. It is noteworthy that sea urchin
SUM-1 is an example of an invertebrate myogenic factor that is capable of functioning in mammalian cells(Venuti et al. 1991). This suggests that fruit fly nautilus
is also capable of functioning in mammalian cells.
Therefore, it is possible to speculate that metazoans
share the same ancestral muscle form, as suggested by
the result for the myosin heavy chain.
As shown in figure 7, the branching pattern of myogenin, MRF4, myf-5, and MyoD, ((myogenin, MRF4),(myf-5, MyoD)), is consistent with their putative functions estimated by gene knockout experiments; Myf-5

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FIG. 4.—A phylogenetic tree of myosin regulatory light chain. All notations are the same as those in figure 2.

and MyoD act jointly as determination genes, and double knockouts fail to either produce or sustain a significant population of myoblasts. Myf-5 and MyoD are epistatic to myogenin, which acts as a differentiator and is
epistatic to MRF4 (Yun and Wold 1996).
Unfortunately, each member of the MRF family
does not have isoforms and does not contribute to theinference of muscle tissue trees. We excluded MRF fromsuperimposition of tissue trees.
Comparison of Five Genes in Vertebrates andSuperimposition of Trees
Since superimposition of whole tissue trees is too
expensive to perform in terms of computational cost, weFIG. 5.—A phylogenetic tree of conventional myosin heavy chain. All notations are the same as those in figure 2.

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863

FIG. 6.—A phylogenetic tree of actin. Bootstrap values are less than 50% in gray branches. Other notations are the same as those in figure 2.

divided the superimposition into two steps. In this section, we estimate the phylogenetic relationship of vertebrate tissues (table 3) from the phylogenetic trees offive genes (figs. 2–6). Since we categorized vertebratemuscle tissues into five classes, there are 105 possiblerooted topologies. Our program super computed topological distances between a given topology and tissue
trees of table 3 and took summation of the topologicaldistances for each topology. Since the cardiac muscle
class is not monophyletic in myosin regulatory lightchain, two kinds of superimpositions were performed.
In the first case, myosin regulatory light chain (1) intable 3 was used. In the second case, myosin regulatory
light chain (2) in table 3 was used.
The best 12 topologies are shown in table 4. When
myosin regulatory light chain (1) was used, the bestscore was 4; however, when myosin regulatory lightchain (2) was used, the best score was 6. Correspondingscores and topological distances for myosin regulatory
light chain (2) appear in parentheses. Therefore, themost compatible topology for vertebrate muscle tissue
evolution is represented in the Newick format as follows: ((root, ((cardiac muscle, slow skeletal muscle),
fast skeletal muscle)), nonmuscle, smooth muscle). A
tree corresponding to this topology is shown in figure 8a.
The following aspects should be noted. First,
smooth muscle evolved independent of the other muscles. Second, skeletal muscle is not monophyletic, but
cardiac and slow skeletal muscles make a cluster.

Superimposition of Trees Including Both Vertebrate
and Invertebrate Tissue Classes
If each invertebrate tissue class were monophyletic,
the number of all tissue classes would be eight. By definition, however, invertebrates contain all organisms
which are not vertebrates, and each invertebrate tissueclass (T, S, and N) may not be monophyletic. For example, actin invertebrate striated muscle and nonmuscle
classes are not monophyletic (T1, T2, and N in fig. 6).
Therefore, each class for each invertebrate phylum isneeded to distinguish one from another.
There are at least 14 classes for invertebrate tissues
in the represented gene trees. The number of classes is
too large for our method, because the program super
performs an exhaustive topology search. From theseclasses, we chose arthropods for superimposition, because the relationships between vertebrates and arthropods (especially the fruit fly) are important in terms ofevolutionary development. We also used urochordates,
because the origin of vertebrate tissue classes can beclarified by including this group.
In the previous section, we superimposed the treesof the five vertebrate genes. For every gene tree, vertebrate skeletal and cardiac muscle classes make a single
cluster; that is, they are monophyletic. Therefore, we
refer to these tissue classes as a single tissue class, abbreviated as Vck. Including this new class, we haveeight classes for both vertebrate and invertebrate tissues.

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FIG. 7.—A phylogenetic tree for the MRF family and related protein genes. The branch with symbol SS is not proportional to the numberof substitutions in length. Other notations are the same as those in figure 2.

Superimposition was carried out in the same way
as that for vertebrate tissue classes. In this section, we
estimate the phylogenetic relationship of vertebrate andinvertebrate tissues (table 5) from the phylogenetic treesof five genes (figs. 2–6). Since arthropod nonmuscle
class is not monophyletic in actin, two topologies (1 and
2) are shown in table 5. The program super eliminatedclasses Ut and Un because they appeared just once
among the tissue trees. Therefore, these two tissue classes were excluded from the evaluation.
Results of superimposition are shown in table 6.
The best 15 topologies in which actin topology (1) wasused are presented. Corresponding scores and topological distances for actin topology (2) appear in parentheses. These two results happened to be identical for the
Table 3Vertebrate Tissue Trees for Each Gene
Gene

Tissue Tree

Troponin C . . . . . . . . . . . . . . . . . . . . . . .
Myosin essential light chain . . . . . . . . .
Myosin regulatory light chain (1) . . . .
Myosin regulatory light chain (2) . . . .
Myosin heavy chain. . . . . . . . . . . . . . . .
Actin . . . . . . . . . . . . . . . . . . . . . . . . . . . .

(0, Kf, (Ks, C))(0, (S, N), (Kf, (Ks, C)))
(0, S, N, (Kf, (Ks, C)))(0, (S, N), (C, (Kf, Ks)))(0, (S, N), (Ks, C))(0, (S, C, (Kf, Ks)), N)

NOTE.—Tissue class abbreviations are as follows: Kf, first skeletal muscle;
Ks, slow skeletal muscle; C, cardiac muscle; S, smooth muscle; N, nonmuscle;
0, root. Since the cardiac muscle class is not monophyletic in myosin regulatory
light chain, two topologies (1 and 2) are shown in the table.

best four topologies, with score of eight. All these topologies suggest that arthropod nonmuscle (An), vertebrate nonmuscle (Vn), and vertebrate smooth muscle(Vs) are clustered. But which topology is most probable? According to the relationships between appearing
phyla (arthropods and chordates), the topology shownin figure 8b (topology (0, (1, (3, 4)), (2, (5, 6))) in table
Table 4
The 12 Most Probable Topologies Representing
Vertebrate Tissue Evolution
Score

Topology

4 (6) . . .
8 (10) . .
8 (6) . . .
10 (12) . .
10 (10) . .
10 (12) . .
10 (12) . .
12 (10) . .
14 (14) . .
14 (16) . .
14 (16) . .
14 (12) . .

((0, ((3, 4), 5)), 1, 2)(0, 1, (2, ((3, 4), 5)))((0, (3, (4, 5))), 1, 2)(0, (1, ((3, 4), 5)), 2)((0, ((3, 5), 4)), 1, 2)(((0, 5), (3, 4)), 1, 2)(((0, (3, 4)), 5), 1, 2)(0, 1, (2, (3, (4, 5))))(0, 1, (2, ((3, 5), 4)))(0, 1, ((2, 5), (3, 4)))(0, 1, ((2, (3, 4)), 5))(0, (1, (3, (4, 5))), 2)

TROC MEL
0
0
2
0
2
0
0
2
2
0
0
2

0
2
2
2
2
2
2
4
4
4
4
4

MRL
0 (2)
2 (2)
2 (0)
2 (2)
2 (0)
2 (0)
2 (0)
4 (2)
4 (2)
4 (2)
4 (2)
4 (2)

MHC ACT
0
2
0
2
0
0
0
2
2
2
2
2

4
2
2
4
4
6
6
0
2
4
4
2

NOTE.—TROC, MEL, MRL, MHC, and ACT denote troponin C, myosin
essential light chain, myosin regulatory light chain, myosin heavy chain, andactin, respectively. Numbers in topologies indicate root or tissue classes: 0 5
root, 1 5 N, 2 5 S, 3 5 C, 4 5 Ks, 5 5 Kf. See table 3 for other abbreviations.
Since cardiac muscle class is not monophyletic in myosin regulatory light chain,
two sets of topological distances and scores are shown. Those for topology (2)are in parentheses.

Phylogeny of Muscle Tissues

865

the phylogenetic relationship of tissues and (2) that thegenes we chose here are such structural genes. It may
be possible to say that no correlation exists between
gene trees and tissue divergence. If this premise were
not true, however, we could hardly explain the following
observations: (1) Each gene tree makes clear clusters interms of expression patterns in various tissue classes.
(2) Several tissue divergences deduced from the genetrees can be superimposed without large inconsistencies.
In other words, they are surprisingly similar to each other.
The above observations suggest a high correlation
between the evolution of regulatory regions and the evolution of structural genes. We agree that the observations
contain some ‘‘noise’’ in terms of tissue divergence. We
believe, however, that superimposition of several tissue
trees reduces the noise and makes it possible to extract
significant information.
Incongruence in Actin
FIG. 8.—The proposed evolutionary relationship of muscle tissues. Only the topology is shown, and the branch lengths are not proportional to evolutionary time. a, The most probable relationship for
vertebrate muscle tissues. b, The most probable relationship for vertebrate and invertebrate muscle tissues.

6) is most probable. This result implies the following
views in terms of evolutionary differentiation: (1) Arthropod striated muscle and vertebrate skeletal and cardiac muscles share a common ancestor. In other words,
they did not evolve independently, although actin genes
suggest they did. (2) Urochordate smooth muscle shares
an ancestor with vertebrate skeletal and cardiac muscles.
(3) Urochordate smooth muscle evolved independent ofvertebrate smooth muscle. (4) Arthropod nonmuscle andvertebrate smooth muscle and nonmuscle are clustered.
(5) The divergence of vertebrate skeletal and cardiac
muscles/vertebrate smooth muscle and nonmuscle is atleast before that of vertebrates/arthropods. In otherwords, emergence of skeletal and cardiac muscle typetissues preceded the vertebrate/arthropod divergence (ca.
700 MYA).
DISCUSSION
The Concept of Building a Tissue Tree from a Gene
Tree
The most important premises in this study are (1)that phylogenetic trees of some structural genes reflect

According to tables 4 and 6, actin has the mostincongruent topologies to the superimposed trees having
the best scores. Why did actin genes give such incongruent topologies for the other genes? The simplest interpretation is that the obtained actin tree was wrong. If
the actin tree is appropriate, however, we have to discuss
the premises of the method used in this study.
We used an assumption to infer tissue evolution;phylogenetic trees of genes reflect the phylogenetic relationship of tissues. However, this may not be true forsome genes. The assumption implies that a gene wasexpressed in a certain tissue class and that this lineage
has been kept in the same tissue class. For instance, if
a mutation caused change in the expression pattern of agene, its lineage might disappear for the tissue. In thiscase, the gene tree no longer reflects the tissue evolution.
The animal actin gene may present such a case.
There is another possibility. So far, we have considered superimposition among different gene families.
However, we could superimpose subtrees in the samegene family’s phylogenetic tree. This idea is similar tothat of the reconciled tree between gene and species
trees (Page and Charleston 1997). A gene tree could befolded at a gene duplication to make up for the lack of
information. In other words, a gene tree is divided into
two subtrees, and we may be able to superimpose twotissue trees constructed from the two gene subtrees as if
they were different gene trees.

Table 5
Vertebrate and Invertebrate Tissue Trees for Each Gene
Gene

Tissue Tree

Troponin C. . . . . . . . . . . . . . . . . . . . . . . . . . . .
Myosin essential light chain . . . . . . . . . . . . . .
Myosin regulatory light chain . . . . . . . . . . . .
Myosin heavy chain . . . . . . . . . . . . . . . . . . . .
Actin (1). . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Actin (2). . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

(0, Vck, At)(0, Us, ((Vs, Vn), Vck))(0, (Vck, Us), ((Vs, Vn), An))(0, (Vck, At), ((Vs, Vn), An))(0, (Ut, (Vs, Vck)), (Un, ((An, At), Vn)))(0, (Ut, (Vs, Vck)), ((Un, An), (At, Vn)))

NOTE.—Tissue class abbreviations are as follows: Vck, vertebrate skeletal and cardiac muscles; Vn, vertebrate nonmuscle; Vs, vertebrate smooth muscle; Ut, urochordate striated muscle; Un, urochordate nonmuscle; Us, urochordate smoothmuscle; An, arthropod nonmuscle; At, arthropod striated muscle; 0, root. Since the arthropod nonmuscle class is notmonophyletic in actin, two topologies (1 and 2) are shown in the table.

866

OOta and Saitou

Table 6The 15 most Probable Topologies Representing Tissue
Evolution
Score

Topology

8 (8) . . . ((0, 5), (1, (3, 4)), (2, 6))
8 (8) . . . (0, (1, (3, 4)), ((2, 5), 6))
8 (8) . . . (0, (1, (3, 4)), ((2, 6), 5))
8 (8) . . . (0, (1, (3, 4)), (2, (5, 6)))10 (12) . . ((((0, 5), 6), (3, 4)), 1, 2)
10 (12) . . (((0, (5, 6)), (3, 4)), 1, 2)
10 (10) . . (((0, 5), 6), (1, (3, 4)), 2)
10 (10) . . ((0, (5, 6)), (1, (3, 4)), 2)
10 (10) . . ((0, 5), ((1, (3, 4)), 6), 2)
10 (10) . . (0, (((1, (3, 4)), 6), 5), 2)
10 (10) . . (0, ((1, (3, 4)), (5, 6)), 2)
10 (10) . . (0, ((1, (3, 4)), 5), (2, 6))
10 (10) . . (0, ((1, (3, 4)), 6), (2, 5))
12 (14) . . (((((0, 5), 6), 4), 3), 1, 2)
12 (14) . . ((((0, (5, 6)), 4), 3), 1, 2)

TROC MEL MRL MHC
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0

0
2
2
2
0
2
0
2
0
0
2
2
0
0
2

2
0
0
0
2
0
2
0
2
2
0
2
2
4
2

0
0
0
0
4
4
2
2
2
2
2
0
2
6
6

ACT
6 (6)
6 (6)
6 (6)
6 (6)
4 (6)
4 (6)
6 (6)
6 (6)
6 (6)
6 (6)
6 (6)
6 (6)
6 (6)
2 (4)
2 (4)

NOTE.—Numbers in topologies indicate root or tissue classes: 0 5 root, 1
5 An, 2 5 At, 3 5 Vn, 4 5 Vs, 5 5 Us, 6 5 Vck. See tables 4 and 5 for otherabbreviations. Since arthropod nonmuscle is not monophyletic in actin, two setsof topological distances and scores are shown. Those for topology (2) are inparentheses.

According to this idea, the actin gene tree can bedivided as follows (there are two possible tissue trees
because the arthropod nonmuscle class is not monophyletic in actin): (0, Ut, (Vs, Vck)) and (0, Un, ((An, At),Vn)), or (0, Ut, (Vs, Vck)) and (0, (An, Un), (At, Vn)).
Using them, we superimposed tissue trees. The same
four topologies in table 6 were also shown to be thebest. However, scores for the new definitions were all
4. This suggests that the actin phylogenetic tree consistsof two kinds of tissue evolution subtrees and that they
were divided at a gene duplication that occurred before
the vertebrate/arthropod divergence.
Evolution of Muscle Tissues
According to the Haeckel’s (1866) biogenetic law,smooth muscle and heart may be closer to each otherthan to other types of muscles. This is because our general knowledge of the lineage of the amniote tissues suggests that they differentiate from splanchnic mesoderm(e.g., Gilbert 1997). Our results, however, contradict thisdescription as shown in fig. 8a; smooth muscle is not
an intermediate tissue between nonmuscle and othermuscles.
Interestingly, arthropods and vertebrates share anancestor of striated muscle although they diverged morethan 700 MYA. This inference fits the fact that arthropods and vertebrates have strong similarities in the myogenic network (Yun and Wold 1996). In addition, arthropod nonmuscle and vertebrate smooth muscle andnonmuscle share a common ancestor.
Superimposition of Multiple Gene Trees
To superimpose trees, precisely speaking, it is necessary to align trees according to their internal nodes.
However, their accurate emergence times cannot be estimated. Therefore, we are unable to label internal nodes
and to align them. Thus, this inability to align the trees

is a limitation of our method. If the correspondencesamong nodes are incorrect in a superimposed tree, wewill be led to erroneous inference. Outside of such extreme cases, however, our method has significant potential in various fields. For example, when we obtain orthologous relationships from several gene trees, they
may be incongruent. In such cases, our method provides
the most probable relationship with a certain score. The
number of OTUs is limited to up to around 9 in ourcurrent computer program, because an exhaustive topology search is carried out. On the other hand, thenumber of gene trees to be superimposed is virtuallyunlimited (computational cost is an order of the numberof genes). It is noteworthy that even if the number of
OTUs is small, our intuition is not powerful enough toobtain a superimposed tree from the moderate numberof gene trees. This new method can be applied to arbitrary tissue classes and opens a new field of molecular
evolution.
Acknowledgments
Dr. Takashi Chikayama kindly advised S.O. about
parallel logic programming. Ms. Chie Takahashi patiently cooperated in checking our program. Ms. Tatsuko Kawamoto, Ms. Yumiko Yoshida, and Ms. Kumi Shimada
helped us to gather a huge literature. Ms. Rose Chapman
carefully checked this manuscript. Dr. Kenta Sumiyama,
Dr. Takashi Kitano, and Mr. Petur Henry Petersen commented on an early version of the manuscript. We also
thank an anonymous reviewer for useful comments toimprove this manuscript. This work was supported by
grants from Institute for Research Institute for Advanced
Information Technology, Japan, and a Grant-in-Aid for
Scientific Research on Priority Areas ‘‘Genome Science’’ from the Ministry of Education, Science, Sports
and Culture, Japan, to N.S.
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DAN GRAUR, reviewing editor
Accepted March 4, 1999