Syst. Biol. 54(4):651–659, 2005
c Society of Systematic Biologists
Copyright ISSN: 1063-5157 print / 1076-836X online
DOI: 10.1080/10635150500221044
Poriferan mtDNA and Animal Phylogeny Based on Mitochondrial Gene Arrangements
D ENNIS V. LAVROV,1,3 AND B. FRANZ LANG 1,2
1
Département de Biochimie, Université de Montréal, Succursale Centre-Ville, Montreal, Que H3C 3J7, Canada
Robert Cedergren Centre, Program in Evolutionary Biology, Canadian Institute of Advanced Research, Montreal, Canada
3
Current Address: Department of Ecology, Evolution and Organismal Biology, Iowa State University, 343A Bessey Hall, Ames, IA 50011, USA;
E-mail: [email protected]
2
Abstract.—Phylogenetic relationships among the metazoan phyla are the subject of an ongoing controversy. Analysis of mitochondrial gene arrangements is a powerful tool to investigate these relationships; however, its previous application outside
of individual animal phyla has been hampered by the lack of informative out-group data. To address this shortcoming, we
determined complete mitochondrial DNA sequences for the demosponges Geodia neptuni and Tethya actinia, two representatives of the most basal animal phylum, the Porifera. With sponges as an outgroup, we investigated phylogenetic relationships
of nine bilaterian phyla using both breakpoint analysis of global mitochondrial gene arrangements and maximum parsimony
analysis of mitochondrial gene adjacencies. Our results provide strong support for a group that includes protostome (but not
deuterostome) coelomate, pseudocoelomate, and acoelomate animals, thus clearly rejecting the Coelomata hypothesis. Two
other groups of bilaterian animals, Lophotrochozoa and Ambulacraria, are also supported by our analyses. However, due
to the remarkable stability of mitochondrial gene arrangements in Deuterostomia and the Ecdysozoa, conclusions on their
evolutionary history cannot be drawn. [Coelomata hypothesis; metazoan phylogeny; mitochondrial DNA; phylogenetic
inference; Porifera.]
Metazoan phyla can be defined as “the largest groupings of taxa that can readily be seen to be more closely
related to each other than to any other groups” (Budd and
Jensen, 2000), and thus essentially representing the upper
limit in the resolving power of traditional morphological systematics. The interrelationships among different
phyla have been the subject of a century-long controversy, and no consensus on the global animal phylogeny
has been reached based on morphological and/or embryological data (Jenner and Schram, 1999). Despite this
lack of consensus, metazoan relationships have been traditionally depicted as a gradual progression in morphological complexity from simple to complex forms,
with a special emphasis on the type of body cavity and
the presence or absence of segmentation (Barnes, 1987;
Brusca and Brusca, 1990; Hyman, 1940). Accordingly,
“acoelomate” platyhelminthes were placed at the base of
the bilaterian tree, followed first by “pseudocoelomate”
worms, such as nematodes and priapulids, and then by
the assemblage of “coelomate” animals (the Coelomata),
consisting of the Protostomia (e.g., annelids, mollusks,
and arthropods) and the Deuterostomia (e.g., echinoderms, hemichordates, chordates, and often lophophorates) (Fig. 1A).
The advent of molecular studies, primarily based on
nuclear small subunit (SSU or 18S) ribosomal RNA sequences, has challenged this traditional view on animal
phylogeny. Unexpectedly, these studies have placed the
acoelomate and pseudocoelomate taxa within the Protostomia, thus refuting the Coelomata hypothesis and
shifting the split between the protostome and deuterostome animals to the base of bilaterian evolution (Fig. 1B).
Several other changes were proposed, including the relocation of the lophophorate taxa from deuterostome to
protostome lineage (Halanych et al., 1995; Mackey et al.,
1996); the recognition of two large protostome clades, the
Lophotrochozoa (e.g., platyhelminthes, annelids, mollusks, and lophophorates) and Ecdysozoa (e.g., arthro-
pods, priapulids, and nematodes) (Aguinaldo et al., 1997;
Carranza et al., 1997; Halanych et al., 1995); and the
grouping of echinoderms and hemichordates in the Ambulacraria, to the exclusion of chordates (Halanych, 1995;
Turbeville et al., 1994; Wada and Satoh, 1994). For a more
detailed review of molecular studies related to animal
phylogenetics, see Halanych (2004).
Although often referred to as the “new animal phylogeny,” the metazoan tree based on 18S sequences has
found rather limited support by the analyses of independent molecular datasets (Copley et al., 2004; Telford,
2004; Wagele and Misof, 2001). Some corroboratory evidence comes from the analysis of Hox genes (de Rosa
et al., 1999; but see Telford, 2000), Na/K ATPase gene
(Anderson et al., 2004), LSU rRNA (Mallatt and Winchell,
2002), and more recently, multiple protein-coding genes
(Philippe et al., 2005). By contrast, several other studies
based on multiple protein genes reject the rRNA-based
phylogeny and advocate the return to the traditional
Coelomata hypothesis (Blair et al., 2002; Mushegian et al.,
1998; Philip et al., 2005; Wolf et al., 2004). Additional independent data sets are clearly needed to resolve this
controversy.
Comparisons of mitochondrial gene arrangements is
a powerful tool for phylogenetic inference (Boore, 1999;
Boore and Brown, 1998). The large number of possible gene arrangements makes convergence in the gene
order unlikely, while the usually slow rate of gene
rearrangements in animal mtDNA preserves the phylogenetic signal from mutational oversaturation. Analyses
of mitochondrial gene orders in animals have provided
convincing results in several cases where all other data
were equivocal, including the relationships among major groups of echinoderms (Smith et al., 1993), arthropods
(Boore et al., 1995, Boore, Lavrov, and Brown, 1998), and
crustaceans (Lavrov et al., 2004; Morrison et al., 2002).
However, application of this data set to the study of
metazoan phylogeny outside of individual phyla has
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the Porifera. Here we compare poriferan mitochondrial
gene arrangements to those of other animals and use
them as an outgroup for the analysis of bilaterian phylogeny based on mitochondrial gene order.
M ATERIALS AND M ETHODS
Specimen Collection, DNA Extraction, Amplification,
Cloning, and Sequencing
FIGURE 1. Old and new views of animal phylogeny. (A) A traditional (pre-1995) animal phylogeny showing the increase in animal
complexity throughout evolution. Simple (acoelomate and pseudocoelomate) animals are placed at the base of the tree. (B) rRNA-based
metazoan phylogeny with the acoelomate and pseudocoelomate taxa
distributed among protostome animals. The relationships within the
Lophotrochozoa are according to Winchell and Mallatt (2002). Broken
lines indicate major disagreements in published molecular phylogenies. Only the phyla analyzed in the present study are shown.
been hampered by the lack of informative data from accepted outgroups. The nonmetazoan mitochondrial gene
arrangements, even of the closest relatives of animals,
are too derived to be used in outgroup comparisons
(Burger et al., 2003), whereas studied diploblastic animals (cnidarians) lack most mitochondrial tRNA genes
(Beagley et al., 1998). In search for an appropriate outgroup for bilaterian animals, we determined complete
mtDNA sequences from the demosponges Geodia neptuni and Tethya actinia (Lavrov et al., 2005), two representatives of arguably the most basal animal phylum,
A specimen of Geodia neptuni (Sollas, 1886) (Class
Demospongiae: Order Astrophorida: Family Geodiidae) was collected from Tennessee Reef, Florida Keys,
at a depth of 15 m. A specimen of Tethya actinia
(de Laubenfels, 1950) (Class Demospongiae: Order
Hadromerida: Family Tethyidae) was collected from
mangrove roots in Zane Grey Creek, Long Key, Florida.
Portions of each sponge were stored frozen after addition
of an 8M guanidinium chloride solution (pH 8). Total
DNA was prepared from ∼1-cm3 piece of each specimen
by phenol-chloroform extraction following proteinase K
digestion. Portions of cox1 and rnl were amplified using primers HCO, LCO, 16S-ARL, and 16S-BRH (Folmer
et al., 1994; Hillis et al., 1996) and used to design specific
primers for these regions. Complete mtDNA from both
species was amplified in two overlapping fragments using the TaKaRa LA-PCR kit under recommended conditions. Random clone libraries were constructed from
the purified PCR products by shearing them into fragments 1–3 kbp in size (Okpodu et al., 1994) and by
cloning them into a modified pBluescript II KS+ vector
with a shortened multi-cloning site (pBFL). Clones were
sequenced on a Li-Cor automated sequencer (Lincoln,
NE) and assembled using the STADEN software suite
(Staden, 1996). Problematic and underrepresented regions were sequenced directly from PCR products by
primer-walking. tRNA genes were identified by the
tRNAscan-SE program (Lowe and Eddy, 1997); other
genes were identified by similarity searches in local
databases using the FASTA program (Pearson, 1994),
and in GenBank at the NCBI using BLAST network service (Benson et al., 2003). The sequence data from this
study have been deposited in GenBank (AY320032 and
AY320033).
Phylogenetic Analysis
Two different approaches to the encoding and analysis of gene arrangement data are possible. In the first approach, the ordering of the genes along the chromosome
is encoded as a single multistate character, and a distance measure is used to determine the transformational
cost among different character states. Since the true evolutionary distance between any two genomes is usually
unknown, it is approximated either by the edit distance
(the minimum number of permitted evolutionary events
that can transform one gene order into another), or by the
breakpoint distance (the number of adjacencies present
in one genome but not the other) (Watterson et al., 1982).
Although such pairwise distances can be used in distance matrix methods (e.g., Sankoff et al., 1992), a better
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LAVROV AND LANG—PORIFERAN mtDNA AND ANIMAL PHYLOGENY
alternative is to use them in direct optimization analyses that reconstruct genomes for internal nodes and
minimize the total length of the tree (Blanchette et al.,
1999; Tang and Moret, 2003). Direct optimization methods have been developed for breakpoint (Sankoff et al.,
1996) and inversion (Bader et al., 2001) distances and
have been named minimum breakpoint analysis (Sankoff
and Blanchette, 1998) and inversion phylogeny (Moret
et al., 2002), respectively. Given that inversions constitute only a small fraction of all rearrangements in animal mtDNA (Boore, 1999; Blanchette, 1999), we used
the minimum breakpoint analysis as implemented in the
GRAPPA 1.7 program (Moret et al., 2001) for our study.
Furthermore, due to the limit on the number of taxa that
can be simultaneously analyzed in GRAPPA (<15; Moret
et al., 2004), we constrained the evaluated trees to those
that preserve the monophyly of the 10 individual phyla
used (see below). These constrains reduce the number
of evaluated trees from ∼1.9 × 1017 to just over 2 million and allow us to complete the minimum breakpoint
analysis within 1 week on a 2.5-GHz G5 computer.
In the second approach to the analysis of gene order
data, the ordering of genes along the chromosome is
encoded as a set of characters, corresponding either to
observed gene adjacencies or to relative positions of individual genes. If gene adjacencies are used, the presence/absence status for every possible pair of signed
genes is recorded as a binary character (Cosner et al.,
2000), where a gene’s “sign” indicates its transcriptional
polarity. If relative gene positions are used, the signed
identities of upstream and downstream neighbors for
every gene are usually recorded as two multistate characters (Boore et al., 1995; Bryant, 2004; but see Gallut
and Barriel, 2002; Wang et al., 2002). In both cases resulting matrices are used directly in the maximum parsimony analysis, and the two methods have been named
maximum parsimony on binary encoding (MPMB) and
maximum parsimony on multistate encoding (MPME),
respectively (Wang et al., 2002). We used only the MPME
method for our analysis because it is known to outperform both the MPBE and distance-based methods (Wang
et al., 2002). To automate the encoding procedure, we
wrote a set of Perl scripts that convert a Genbank flatfile to a matrix of 74 characters corresponding to upstream and downstream position of 37 genes typical for
bilaterian mtDNA, as suggested by Boore et al. (1995).
This matrix was subsequently used in PAUP∗ 4.0 Beta10
(Swofford, 2002) for maximum parsimony and bootstrap
(1,000 replicates) analyses, and for Kishino-Hasegawa
and Templeton tests. Bremer support values were determined using the AutoDecay program by Torsten Eriksson; MacClade 3.04 (Maddison and Maddison, 1992) was
used for reconstruction of ancestral states. Both the Perl
scripts and the MPME matrix are available upon request.
R ESULTS
Mitochondrial Gene Arrangements of Demosponges
The mitochondrial genomes of G. neptuni and T. actinia (Fig. 2A) contain all 37 genes typical for animals,
653
plus genes for subunit 9 of ATP synthase (atp9), and for
two (G. neptuni) or three (T. actinia) additional tRNAs.
All genes are transcribed from the same DNA strand of
the molecule in both mtDNAs. The relative arrangement
of protein and RNA genes is conserved between the two
genomes, except for the position of nad6, whereas the locations of 13 tRNA genes differ between them (Fig. 2B).
Analysis of gene adjacencies in mtDNAs reveals that 18
identical gene boundaries are present in the two demosponges, and up to 12 between demosponges and bilaterian animals (Fig. 3). Our simulation studies show
that two randomly reshuffled genomes with all genes
transcribed from the same strand would share on average only one gene boundary. The probability for sharing more than three boundaries is less than 0.05, and
even smaller when two transcriptional polarities are allowed. Accordingly, poriferan mitochondrial gene arrangements contain strong, genuine phylogenetic signal
that can be used to root a bilaterian tree.
Phylogenetic Analysis of Animal Relationships Based
on Mitochondrial Gene Order Data
The mitochondrial gene orders from two demosponges and selected representatives of nine bilaterian
phyla were used for phylogenetic analyses (Table 1).
Within each phylum, we chose the phylogenetically
most distant species with the best-conserved gene arrangements (as estimated by the number of shared gene
boundaries with other phyla): two each from Annelida,
Arthropoda, Brachiopoda, Chordata, Echinodermata,
Mollusca, and Platyhelminthes, plus the only available
hemichordate, Balanoglossus carnosus, and the nematode
Trichinella spiralis (Table 1). Although complete mtDNA
sequences are available from several nematode species,
all but T. spiralis have highly derived gene arrangements
statistically indistinguishable from randomly shuffled
genomes with the same gene content (data not shown).
Furthermore, two other animal phyla for which complete
mtDNA sequences are available, the Cnidaria (Beagley
et al., 1995) and the Chaetognatha (Helfenbein et al.,
2004), have been excluded from this analysis because
their mtDNA lacks more than half of the genes found
in other animal phyla.
Minimum breakpoint analysis using GRAPPA 1.7 produced a single best tree with a score of 302 (Fig. 4A). The
maximum parsimony analysis of the gene adjacency matrix from the same dataset produced five most parsimonious trees each 578 steps long, consistency index, CI =
0.92 and retention index, RI = 0.86 (Fig. 4B). Both analyses support a monophyletic group including protostome
coelomate, pseudocoelomate and acoelomate animals
(= protostomia sensu Aguinaldo et al. [1997]). Within this
group, the lophotrochozoan clade (annelids, mollusks,
brachiopods, and platyhelminths) is supported, whereas
the relationship among Lophotrochozoa, Nematoda and
Arthropoda remains unresolved. The second large group
of bilaterian animals, the Deuterostomia (Chordata,
Hemichordata, and Echinodermata), is supported only
by the MP analysis (Fig. 4B). However, the only character
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FIGURE 2. Mitochondrial genomes of G. neptuni and T. actinia: gene maps (A) and gene rearrangements (B). Protein and ribosomal genes
(gray) are: atp 6-9, ATP synthase subunits; cob, cytochrome b apoprotein; cox1–3, cytochrome c oxidase subunits; nad1-6, NADH dehydrogenase
subunits; rnl, LSU rRNA; rns, SSU rRNA. tRNA genes (black) are identified by the one-letter code for their corresponding amino acid. Two
arginine, two isoleucine, two leucine, and two serine tRNA genes are differentiated by their anticodon sequence with trnR(ucg) marked as
R1, trnR(ucu)—as R2, trnI(gau)—as I1, trnI(cau) as I2, trnL(uag)—as L1, trnL(uaa)—as L2, trnS(gcu)—as S1, and trnS(uga)—as S2. All genes are
transcribed clockwise (A) or left to right (B). The innermost circle shows the locations of PCR amplification products (A). Translocations of tRNA
genes are marked with gray lines; rearrangements of larger mtDNA fragments are marked with black lines (B).
that supports the monophyly of the Deuterostomia in
all five MP trees (the presence of -trnS(uga) upstream of
trnD) does not appear to be synapomorphic. Rather, the
inferred ancestral state for the gene upstream of trnD
(+cox1 + trnD) found in one of the sponges and the mollusk Katharina tunicata likely results from an isolated
case of convergent evolution. In contrast, Ambulacraria
(Hemichordata plus Echinodermata) is recovered as a
monophyletic group both in minimum breakpoint and
maximum parsimony analyses.
Bremer support values and bootstrap percentages are
moderate for most internal nodes on the MPME tree, an
expected result given the small size of the dataset and
the relative stability of gene order. Interestingly, bootstrap values appear to be more homogeneous than Bremer support values, with both the Protostomia (Bremer
support = 3) and the Deuterostomia (Bremer support =
1) recovered in ∼75% of bootstrap replicates. To test
whether there is a statistically significant signal in the
gene order data, we conducted Kishino-Hasegawa and
Templeton tests on the MPME matrix for the two a priori
hypotheses of animal relationships shown in Figure 1.
Both tests showed strong preference for the new animal
phylogeny, rejecting the traditional tree with high statistical support (p < 0.002). As corroborative evidence,
the best tree that preserves the monophyly of Coelomata
in the minimum breakpoint analysis is 313 steps long,
11 steps (3.6%) longer than the best tree without such
constraint.
Mitochondrial Gene Arrangement of Ancestral Bilaterian
and Gene Order Evolution
One of the open questions in animal evolution is the
nature of the last common ancestor of bilaterian animals
(Erwin and Davidson, 2002). Both minimum breakpoint
and maximum parsimony analyses reconstruct character states for internal (ancestral) nodes, providing an
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655
FIGURE 3. Numbers of gene boundaries shared between bilaterian animals and demosponges Geodia neptuni (gray bars) and Tethya actinia
(black bars). Species names abbreviated below each bar can be found in Table 1. The broken horizontal line indicates the number of shared gene
boundaries expected to occur purely by chance in two randomly rearranged genomes.
opportunity to address a part of this issue related to mitochondrial gene order. Figure 5A presents the minimum
breakpoint reconstruction for the mitochondrial gene order of the common ancestor of bilaterian animals and
the MP support for individual gene boundaries in this
arrangement, shown by two dots above each of them.
As shown in Figure 5A, both analyses agree well on the
inferred ancestral bilaterian gene arrangement; only the
position of three tRNA genes remains uncertain (shown
in gray).
Figure 5B compares the inferred ancestral bilaterian
gene order with those for present-day deuterostome (H.
TABLE 1. Species, taxonomic classification, and accession numbers.
Species
Geodia neptuni
Tethya actinia
Homo sapiens
Branchiostoma floridae
Balanoglossus carnosus
Florometra serratissima
Arbacia lixula
Trichinella spiralis
Limulus polyphemus
Drosophila yakuba
Katharina tunicata
Loligo bleekeri
Lumbricus terrestris
Platynereis dumerilii
Terebratulina retusa
Laqueus rubellus
Fasciola hepatica
Taenia crassiceps
∗
New sequence.
Classification
Porifera, Demospongiae
Porifera, Demospongiae
Chordata, Craniata
Chordata, Cephalochordata
Hemichordata, Enteropneusta
Echinodermata, Crinoidea
Echinodermata, Echinozoa
Nematoda, Enoplea
Arthropoda, Chelicerata
Arthropoda, Insecta
Mollusca, Polyplacophora
Mollusca, Cephalopoda
Annelida, Oligochaeta
Annelida, Polychaeta
Brachiopoda, Rhynchonellata
Brachiopoda, Rhynchonellata
Platyhelminthes, Trematoda
Platyhelminthes, Cestoda
Accession
number
AY
AY
NC
NC
NC
NC
NC
NC
NC
NC
NC
NC
NC
NC
NC
NC
NC
NC
320032∗
320033∗
001807
000834
001887
001878
001770
002681
003057
001322
001636
002507
001673
000931
000941
002322
002546
002547
sapiens) and protostome (L. polyphemus) animals. This
comparison indicates that the differences previously
observed between vertebrate and arthropod genomes
(Fig. 4C, D; Clary and Wolstenholme, 1985) are due predominantly to gene rearrangements that occurred in the
protostome lineage. By contrast, the gene arrangement
typical for present-day vertebrates is almost identical to
that of the common ancestor of bilaterian animals. An
important implication of this finding is that mitochondrial gene arrangement cannot be used to support either
deuterostome monophyly or the affinity of other animals
with deuterostomes (despite some recent claims to the
contrary; Bourlat et al., 2003). Interestingly, a similar pattern in occurrence of gene rearrangements is repeated
in Protostomia. The reconstructed gene arrangement of
the protostome ancestor is identical to that of Limulus
polyphemus (not shown). Accordingly, the monophyly of
the Ecdysozoa or Arthropoda cannot be tested by mitochondrial gene rearrangements.
D ISCUSSION
Conservation of Mitochondrial Gene Arrangement Across
the Metazoa
One peculiar feature of animal mtDNA is the stability
of gene arrangements, which often remain unchanged
over long evolutionary periods. Identical or nearly identical mitochondrial gene orders have been found in animal taxa that diverged more than 500 MYA, and some
gene clusters are conserved across most animal phyla
(Boore, 1999). The present study suggests that the factors underlying this genome stability evolved in the common ancestors of multicellular animals because identical
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gene clusters are present in mtDNAs of demosponges
and bilaterian animals, but not between animals and
those of other eukaryotes, including the closest studied outgroup, the choanoflagellate Monosiga brevicollis
(Burger et al., 2003). The prevalence of tRNA translocations among gene rearrangements found in two demosponges is also typical of animal mtDNA and suggests
a common mechanism of mitochondrial gene rearrangements throughout the Metazoa.
Mitochondrial Gene Rearrangements and Animal Phylogeny
Adoutte et al. (2000) have aptly compared phylogenetics to cosmology, where experimental testing of
hypotheses is impossible, and where confidence in the
inference depends on the congruence between results obtained from independent data sets. The present analysis
of mitochondrial gene arrangements provides strong, independent support for two previously proposed groups
of bilaterian animals, Protostomia (sensu Aguinaldo et al.
(1997)) and Lophotrochozoa (Halanych et al., 1995). The
placement of protostome coelomate, pseudocoelomate
and acoelomate animals in Protostomia clearly rejects
the Coelomata hypothesis in its traditional form (e.g.,
Barnes, 1987) and also its recent reincarnation (Philip
et al., 2005; Wolf et al., 2004). Furthermore, we recover the
grouping of hemichordates and echinoderms (Ambulacraria) within Deuterostomia, although in maximum
parsimony analysis the support for this group is based
on a single shared derived boundary trnE+trnT. Finally,
the monophyly of either Deuterostomia or Ecdysozoa is
not supported in our analysis, due to the apparent lack
of synapomorphic rearrangements in these groups.
For comparison, maximum likelihood, maximum parsimony, and neighbor-joining analyses based on the
amino acid sequences of mitochondrial genes for the
same set of species produce a phylogeny, with nematodes
and platyhelminthes forming a monophyletic group
at the base of Bilateria (Fig. 6). This result is likely due to
the long branch attraction, a phylogenetic artifact that for
these two taxa persists even when many more genes and
many more species are included in the analysis (Philippe
et al., 2005).
FIGURE 4. Phylogeny of bilaterian relationships based on the analyses of mitochondrial gene arrangements. (A) The shortest minimum
breakpoint tree based on circular ordering of the 36 mitochondrial
genes present in all compared genomes. To reduce computational load,
topologies were constrained to those that preserve monophylies of individual phyla. The breakpoint scores were calculated using GRAPPA 1.7
with the adjacency-parsimony option for iteration (Moret et al., 2001).
(B) Strict consensus of the five most parsimonious trees (each 578 steps
long, CI = 0.92, RI = 0.86) based on a 74 character gene adjacency matrix for the 37 typical bilaterian mitochondrial genes. Numbers above
branches indicate Bremer’s support values (Bremer, 1994), numbers
below show bootstrap support (Felsenstein, 1985).
Gene Arrangements and Phylogenetics
Although sometimes considered to be a novel character (Dowton et al., 2002), gene order (pattern of chromosomal banding), have been used for phylogenetic inference, even before the role of DNA in inheritance was
known (Sturtevant and Dobzhansky, 1936). Later, the
gene order–based differences in organellar DNA restriction patterns have been used to study evolutionary relationships among plant species (Knox et al., 1993; Palmer
and Zamir, 1982; Timothy et al., 1979). The influx of complete genome sequences, both nuclear and organellar,
opens new possibilities for the use of gene order data in
phylogenetics. Although not all phylogenetic questions
can be studied based on the gene order data, we hope
that the present study will inspire further use of this data
set in animal phylogenetics, and the development of algorithms that use gene rearrangements for phylogenetic
inference.
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657
FIGURE 5. Mitochondrial gene arrangement for the common ancestor of bilaterian animals (A) and gene order evolution in the protostome
and the deuterostome lineages (B). Genes are abbreviated as in Figure 2 and are transcribed from left to right except when underlined; underlining
indicates the opposite transcriptional polarity. The reconstruction of the ancestral bilaterian gene order is based on the minimum breakpoint
analysis. The maximum parsimony support for individual gene boundaries in this arrangement is shown by two dots above them. Two dots
are needed because each gene boundary is reconstructed twice, based on the relative positions of two genes that form it. Open dots indicate
that the MP reconstruction of the character state is consistent with the gene boundary, and unambiguous; half-filled dots indicate that the
reconstruction is consistent with the gene boundary, but ambiguous; filled dots indicate that the reconstruction is inconsistent with the gene
boundary. The positions of genes shown in gray differ in minimum breakpoint and maximum parsimony reconstructions (A) or are not conserved
(B). Translocations of tRNA genes are marked with gray lines; rearrangements of large mtDNA fragments are marked with black lines.
ACKNOWLEDGEMENTS
We thank Michelle Kelly for help with specimen collection, Lise
Forget and Zhang Wang for assistance in cloning and sequencing, and
Bernard Moret for help with GRAPPA. We are grateful to the Editor Roderic Page, Associate Editor Marshal Hedin, two referees, Jon
Mallatt and Susan Masta, and to Henner Brinkmann, Wesley Brown,
Tim Collins, Karri Haen, Amy Hauth, Nicolas Lartillot, and Herve
Philippe for many valuable comments and suggestions. Salary and
interaction support from the Canadian Institutes of Health Research
(DVL, BFL), the Canadian Institute for Advanced Research (BFL),
and supply of laboratory equipment and informatics infrastructure by
Genome Quebec/Canada are also gratefully acknowledged.
R EFERENCES
FIGURE 6. Maximum likelihood (ML) phylogram based on inferred
amino acid sequences from four mitochondrial genes (cob, cox1, cox2,
cox3) (JTT matrix, +I). Identical positions of Nematodes and Platyhelminthes were obtained in maximum parsimony (MP) and neighbor joining (NJ) analyses. Bootstrap support values for “Coelomata”
and Nematoda + Platyhelminthes are based on NJ with gamma, MP,
ML without gamma, and ML with gamma analyses of 100 bootstrap
replicates.
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First submitted 6 May 2004; reviews returned 9 August 2004;
final acceptance 15 April 2005
Associate Editor: Marshal Hedin