Testing the New Animal Phylogeny: First Use of Combined Large-Subunit
and Small-Subunit rRNA Gene Sequences to Classify the Protostomes
Jon Mallatt and Christopher J. Winchell
School of Biological Sciences, Washington State University
Although the small-subunit ribosomal RNA (SSU rRNA) gene is widely used in the molecular systematics, few
large-subunit (LSU) rRNA gene sequences are known from protostome animals, and the value of the LSU gene for
invertebrate systematics has not been explored. The goal of this study is to test whether combined LSU and SSU
rRNA gene sequences support the division of protostomes into Ecdysozoa (molting forms) and Lophotrochozoa, as
was proposed by Aguinaldo et al. (1997) (Nature 387:489) based on SSU rRNA sequences alone. Nearly complete
LSU gene sequences were obtained, and combined LSU 1 SSU sequences were assembled, for 15 distantly related
protostome taxa plus five deuterostome outgroups. When the aligned LSU 1 SSU sequences were analyzed by
tree-building methods (minimum evolution analysis of LogDet-transformed distances, maximum likelihood, and
maximum parsimony) and by spectral analysis of LogDet distances, both Ecdysozoa and Lophotrochozoa were
indeed strongly supported (e.g., bootstrap values .90%), with higher support than from the SSU sequences alone.
Furthermore, with the LogDet-based methods, the LSU 1 SSU sequences resolved some accepted subgroups within
Ecdysozoa and Lophotrochozoa (e.g., the polychaete sequence grouped with the echiuran, and the annelid sequences
grouped with the mollusc and lophophorates)—subgroups that SSU-based studies do not reveal. Also, the mollusc
sequence grouped with the sequences from lophophorates (brachiopod and phoronid). Like SSU sequences, our
LSU 1 SSU sequences contradict older hypotheses that grouped annelids with arthropods as Articulata, that said
flatworms and nematodes were basal bilateralians, and considered lophophorates, nemerteans, and chaetognaths to
be deuterostomes. The position of chaetognaths within protostomes remains uncertain: our chaetognath sequence
associated with that of an onychophoran, but this was unstable and probably artifactual. Finally, the benefits of
combining LSU with SSU sequences for phylogenetic analyses are discussed: LSU adds signal, it can be used at
lower taxonomic levels, and its core region is easy to align across distant taxa—but its base frequencies tend to be
nonstationary across such taxa. We conclude that molecular systematists should use combined LSU 1 SSU rRNA
genes rather than SSU alone.
Introduction
The small-subunit ribosomal RNA (SSU rRNA or
18S) gene is the gene used most widely for molecular
phylogenetic analysis, especially for evaluating deeplevel relationships among organisms (Adoutte et al.
2000; Van de Peer et al. 2000). A key study using this
gene (Aguinaldo et al. 1997) provided evidence that living triploblastic or bilateralian animals fall into three
groups: the classically recognized (1) deuterostomes and
protostomes; and a division of protostomes into (2) Ecdysozoa, and (3) Lophotrochozoa. The Ecdysozoa, all
of which molt and lack motile locomotory cilia, include
priapulids, kinorhynchs, nematodes, nematomorphs, tardigrades, onychophorans, and arthropods. The Lophotrochozoa, originally identified by Halanych et al.
(1995) and named for those members that have a trochophore larva or a feeding structure called a lophophore,
include the annelids, molluscs, brachiopods, phoronids,
and bryozoans, plus flatworms and related minor
groups, such as rotifers, gastrotrichs, and gnathostomulids (Garey and Schmidt-Rhaesa 1998; Giribet et al.
2000; also see Balavoine 1998). The Ecdysozoa and Lophotrochozoa hypotheses challenge traditional thinking.
Besides proposing a previously unanticipated relation
among the molting animals, they contradict the old
Key words: large-subunit ribosomal RNA, 28S rRNA, small-subunit ribosomal RNA, phylogeny, protostome, Ecdysozoa.
Address for correspondence and reprints: Jon Mallatt, Box
644236, School of Biological Sciences, Washington State University,
Pullman, Washington 99164-4236. E-mail: [email protected].
Mol. Biol. Evol. 19(3):289–301. 2002
q 2002 by the Society for Molecular Biology and Evolution. ISSN: 0737-4038
American-textbook view that flatworms (which lack a
coelom) branched from the base of the bilateralian tree,
followed by nematodes and other pseudocoelomate phyla (Barnes 1980, chapt. 3).
In the past few years, the Ecdysozoa and Lophotrochozoa hypotheses have been accepted by many molecular systematists, anatomists, developmental biologists, and paleontologists (Schmidt-Rhaesa et al. 1998;
Adoutte et al. 2000; Conway Morris 2000; Giribet et al.
2000; Peterson, Cameron, and Davidson 2000; Peterson
and Eernisse 2001). Although challenged by a few researchers (Foster and Hickey 1999; Wagele et al. 1999;
Hausdorf 2000), these hypotheses have accumulated
support from other gene sequences, namely from Hox
genes (de Rosa et al. 1999), b-thymosin (Manuel et al.
2000), and from expanded sets of SSU rRNA sequences
(Garey and Schmidt-Rhaesa 1998; Giribet et al. 2000;
Peterson and Eernisse 2001).
Despite the impact the Ecdysozoa and Lophotrochozoa hypotheses have had, they were founded on incomplete rRNA information. That is, the SSU gene is
just the smaller part of a gene family that also contains
the large-subunit rRNA genes (LSU: 28S, 5.8S, and the
little used 5S genes), which also are valuable for deeplevel phylogenetic analysis (Hillis and Dixon 1991).
Given these facts, LSU rRNA genes should be a powerful tool with which to test the Ecdysozoa and Lophotrochozoa hypotheses—for if the larger rRNA genes fail
to support them, these hypotheses will be severely compromised. In this study, we sequenced the LSU rRNA
genes from 15 distantly related taxa of protostomes and
used the combined LSU and SSU sequences to test the
289
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Mallatt and Winchell
Ecdysozoa and Lophotrochozoa hypotheses. Furthermore, because complete LSU sequences are almost unknown for protostomes (De Rijk et al. 1995), our new
sequences should provide a foundation for other complete rRNA gene studies in the future.
Materials and Methods
This is the fourth in a series of studies using nearly
complete LSU rRNA gene sequences for animal phylogeny, and most of the methodology is discussed in the
three previous papers (Mallatt and Sullivan 1998; Mallatt, Sullivan, and Winchell 2001; Winchell et al. 2002).
Specimens and Sequences
Information on the sources of the animals, voucher specimen numbers, precise parts of the genes used,
the new GenBank accession numbers (AF342785–
AF342805), and sequences taken from the literature, are
all available as supplementary material at the Molecular
Biology and Evolution website (http://www.smbe.org).
The 15 protostome species used were Amphiporus sp.
(nemertean), Chordodes morgani (nematomorph), Eisenia fetida (oligochaete), Halicryptus spinulosus (priapulan), Limulus polyphemus (arthropod), Peripatoides
novaezealandiae (onychophoran), Phascolopsis gouldii
(sipunculan), Phoronis ijimae (5vancouverensis) (phoronid), Placopecten magellanicus (mollusc), Proceraea
cornuta (polychaete), Sagitta elegans (chaetognath),
Stylochus zebra (platyhelminth), Terebratalia transversa (brachiopod), Trichinella spiralis (nematode), and
Urechis caupo (echiuran), and the five deuterostome
outgroups were Branchiostoma floridae (cephalochordate), Ciona intestinalis (tunicate), Florometra serratissima (echinoderm), Hydrolagus colliei (vertebrate), and
Ptychodera flava (hemichordate). In general, we used
taxa whose SSU rRNA genes are slowly evolving, as
did Aguinaldo et al. (1997). However, not all their taxa
were available to us, and only two of the protostome
species were the same in both studies (T. transversa and
T. spiralis). In using deuterostomes as the outgroup, we
assume that deuterostomes are the sister group of all
protostomes, an assumption supported by many recent
studies (Balavoine and Adoutte 1998; Giribet et al.
2000; Peterson and Eernisse 2001). Following the recommendation of Giribet et al. (2000), no diploblast outgroups were used because diploblast LSU and SSU sequences are very different from those of triploblasts
(Medina et al. 2001) and seem too dissimilar to assure
accuracy of the triploblast tree. The five outgroup taxa—
Branchiostoma, Ciona, Florometra, Hydrolagus, and
Ptychodera (taken from Winchell et al. 2002)—were
chosen primarily to yield uniform sampling across the
deuterostomes (one or two representatives per phylum)
and secondarily as the least divergent and most complete
sequences available. Previously sequenced LSU and
SSU genes from the protostomes Caenorhabditis elegans, Drosophila melanogaster, and Aedes albopictus
were not used because of high divergence (long branches: De Rijk et al. 1995). By contrast, LSU from the
chaetognath Sagitta was sequenced and used here, de-
spite the likelihood of high divergence because taxonomists have requested more gene sequences from this
enigmatic phylum (Garey and Schmidt-Rhaesa 1998;
Giribet et al. 2000). To assure that the divergent Sagitta
would not disrupt the phylogenetic analyses, such analyses were performed twice, first without Sagitta and
then with Sagitta included.
DNA extraction, primers, PCR amplification procedures, purification, sequencing, and fragment assembly were as described by Mallatt and Sullivan (1998)
and Winchell et al. (2002). The gene sequences were
imported into Seqlab, a Macintosh X Window application (Smith et al. 1994). The concatenated 28S, 5.8S,
and 18S rRNA genes were aligned by eye, with the
alignment rigidly based on secondary structure using
the LSU rRNA model of Xenopus laevis (Schnare et
al. 1996) and the SSU models of X. laevis and Strongylocentrotus purpuratus (Gutell 1994). Our alignments are available from EMBL (ftp://ftp.ebi.ac.uk/
pub/databases/embl/align) under the accession numbers
ALIGNp000087 and ALIGNp000088.
As in our past studies of deep-level phylogeny, the
entire conserved core of the 28S gene was readily alignable across taxa and was used; the divergent domains,
comprising over one-third of the 28S gene, were excluded (see table 7.1 in Mallatt, Sullivan, and Winchell
2001; Hassouna, Michot, and Bachellerie 1984). In the
SSU genes, about 15% of the sites could not be aligned
across taxa and were excluded. Overall, we used 2,348,
149, and 1,517 aligned sites from the 28S, 5.8S, and
18S genes, respectively, for a total of about 4,000 sites
in the analysis.
Phylogenetic Analyses
Three data sets were analyzed: (1) combined LSU
1 SSU genes; (2) SSU only; and (3) LSU only. We
combined the genes because the incongruence length
difference test yielded a value of P , 0.27, thereby suggesting that the LSU and SSU sequences were not significantly more incongruent than random partitions (but
see Sullivan [1996] for a criticism of that test as an
arbiter of data combination).
The search strategies for inferring optimal phylogenetic trees included equally weighted maximum parsimony (MP), maximum likelihood (ML), and minimum
evolution (ME) using LogDet-Paralinear distances (Lake
1994; Lockhart et al. 1994; Swofford et al. 1996). We
executed each of these optimality criteria with PAUP*
version 4.0 beta 4a (Swofford 2000). To obtain the best
tree using ML, we followed an iterative search strategy
(Swofford et al. 1996; Mallatt and Sullivan 1998), in
which the GTR 1 I 1 G model was found to fit the data
best.
Of the three tree-building methods used, ME analysis with LogDet distances is emphasized over ML and
MP because it is designed to give the best results when
the gene sequences differ in base composition across
taxa—as our sequences do (see Results). This LogDet
method only crudely accounts for heterogeneity in rates
of evolution across nucleotide sites, although it has been
Phylogeny of Protostomes
shown to yield results similar to more complex models
of rate heterogeneity (Waddell, Penny, and Moore 1997;
J. Sullivan, personal communication). The way it accounts for rate heterogeneity is by using a value called
Pinv, the proportion of invariable sites in the genes. Because Pinv cannot be measured directly, we used the ML
method of calculating this parameter, even though ML
slightly overestimates Pinv (see Winchell et al. 2002); if
the ML-derived value for Pinv led to many trees with
undefined values in 1,000 ME bootstrap replicates, it
was lowered slightly until it led to fewer than 10 such
undefined trees. To determine how sensitive the ME results were to variation in estimates of Pinv, we also used
the following as Pinv values; namely, the proportion of
sites that were constant in our set of protostome and
deuterostome sequences (Pc) and the arbitrarily decreasing range of Pinv values: 0.55, 0.45, 0.35, 0.25, and 0.15.
To measure nodal support for our ME, ML, and
MP trees, nonparametric bootstrap analyses (Felsenstein
1985) were performed with 1,000 replicates for the ME
and MP searches and 100 replicates for the ML
searches.
Spectral Analysis
Spectral analysis, a method that quantifies both
support (S) and conflict (C) within gene sequences for
groups of taxa (or splits), is an alternative to tree-based
analyses (Lento et al. 1995; Penny et al. 1999). In this
part of the study, the LogDet-transformed distances were
analyzed by the Spectrum program (Charleston 1998,
http://taxonomy.zoology.gla.ac.uk/;mac/spectrum/spectrum), with Pinv set at the same ML-based value as in
the ME-tree calculation. (Again, using Pinv is a crude
way to account for heterogeneous rates of evolution
across nucleotide sites.) This Spectrum program must
be supplied with a threshold support value, which must
be chosen according to several criteria: It must be low
enough to yield many splits but not so many as to overload the display buffer and crash the program; also, this
threshold value must yield standardized conflict values
(see subsequently) in roughly the same size range as the
support values so that both support and conflict are interpretable when plotted together on a histogram (as in
fig. 4). A threshold of 0.0003 substitutions per site was
chosen because it fit these criteria best. Conflict values
were standardized according to Lento et al. (1995).
Spectral analysis is not yet a statistical method, so
it does not provide an absolute basis for accepting one
split and rejecting another. To allow better comparisons
between such competing splits, we developed a ‘‘relative support-versus-conflict index’’ (I). This index was
calculated as the ratio, (S1/S2)/(C1/C2), where S1 and
C1 are the support and conflict values for the split being
evaluated, and S2 and C2 are the corresponding values
for the competing (mutually exclusive) split that had the
highest level of support. Although it is not statistical,
this index has a clear meaning: a split with an index of
n (say, 2) has n times more conflict-adjusted support
than does the nearest alternative split. To provide a standard, we identified many splits within deuterostomes
291
(Winchell et al. 2002) that are supported by morphological or independent molecular evidence and found all
such splits to have I-values over 4 (amphioxus plus vertebrates, hemichordates plus echinoderms, lamprey plus
hagfish, holothuroideans plus echinoideans); based on
this precedent from deuterostomes, we called protostome splits valid if they likewise had I-values over 4.
Results
Nucleotide Frequencies
Table 1 shows the proportions of A, C, G, and T
nucleotides in the combined LSU 1 SSU genes of the
taxa used in this study. Note that the Peripatoides and
Sagitta sequences are richest in C and G, whereas Trichinella is rich in A and T. As shown at the bottom of the
table, for the basic set of 19 taxa—that is, with Sagitta
left out—the chi-square test of homogeneity of base frequencies across taxa indicates strong nonstationarity (P
K 0.01), and this becomes even more extreme when
Sagitta is included. On the other hand, when the CGrich Sagitta and Peripatoides sequences are both omitted, the 18 remaining sequences are stationary.
Most of the nonstationarity of base frequencies in
the rRNA genes must be in the LSU gene because the
SSU sequences alone were always stationary (test results not shown).
Phylogenetic Trees
Figure 1 shows the trees from the basic set of 19
taxa (i.e., without Sagitta) as calculated from the combined LSU 1 SSU, the SSU-only, and the LSU-only
gene sequences by the ME, ML, and MP methods. Brief
examination of these trees reveals that SSU and LSU
produce similar phylogenies, and that relative branch
lengths are roughly similar in both the SSU and LSU
trees. That is, taxa with rapidly evolving SSU sequences
(longest branches) usually have rapidly evolving LSU
sequences, and slowly evolving (short-branched) SSU
sequences go with slowly evolving LSU sequences. This
suggests that the SSU and LSU rRNA genes evolve by
a similar set of rules.
In the combined-gene tree of figure 1A, the protostome, Lophotrochozoa, and Ecdysozoa clades are
strongly supported, with bootstrap values of 92% or
higher. Support is also evident (ME bootstrap values
over 60%) for the following seven groups within Lophotrochozoa and Ecdysozoa: (1) polychaete Proceraea
1 echiuran Urechis; (2) brachiopod Terebratalia 1 phoronid Phoronis 1 mollusc Placopecten; (3) Phoronis 1
Placopecten; (4) oligochaete Eisenia 1 Proceraea 1
Urechis 1 Terebratalia 1 Phoronis 1 Placopecten; (5)
all Ecdysozoa other than the priapulan Halicryptus; (6)
Peripatoides 1 arthropod Limulus; and (7) nematomorph Chordodes 1 nematode Trichinella. Comparison
of figure 1B and C shows that the SSU gene seems to
contain more signal for the higher-order groups, Lophotrochozoa and Ecdysozoa (which have SSU-ME bootstrap values of 73% and 81%, respectively, compared
with only 42% and 32% in the LSU tree), whereas the
LSU gene contains more signal for the seven lower-
292
Mallatt and Winchell
Table 1
Proportions of Nucleotide Types in the SSU and LSU rRNA Gene Alignments Used in the Analysis (28S divergent
domains and other nonalignable sites are excluded). The Number of Sites Used (28S 1 18S 1 5.8S) is Listed at Far
Right
Taxon
A
C
G
T
Number of
Sites Used
Amphiporus . . . . . . .
Chordodes . . . . . . . .
Eisenia . . . . . . . . . . .
Halicryptus. . . . . . . .
Limulus . . . . . . . . . .
Peripatoides . . . . . . .
Phascolopsis. . . . . . .
Phoronis . . . . . . . . . .
Placopecten . . . . . . .
Proceraea . . . . . . . . .
Sagitta. . . . . . . . . . . .
Stylochus . . . . . . . . .
Terebratalia. . . . . . . .
Trichinella . . . . . . . .
Urechis . . . . . . . . . . .
Branchiostoma . . . . .
Ciona . . . . . . . . . . . .
Florometra . . . . . . . .
Hydrolagus. . . . . . . .
Ptychodera . . . . . . . .
Mean. . . . . . . . . . . . .
0.2656
0.2740
0.2687
0.2714
0.2627
0.2441
0.2616
0.2681
0.2718
0.2619
0.2445
0.2720
0.2658
0.2858
0.2685
0.2568
0.2668
0.2596
0.2574
0.2582
0.2643
0.2189
0.2115
0.2163
0.2134
0.2250
0.2451
0.2259
0.2161
0.2159
0.2213
0.2499
0.2154
0.2165
0.2080
0.2089
0.2283
0.2111
0.2219
0.2267
0.2296
0.2213
0.2826
0.2778
0.2829
0.2815
0.2871
0.3045
0.2890
0.2826
0.2760
0.2846
0.3060
0.2785
0.2828
0.2679
0.2817
0.2958
0.2903
0.2890
0.2945
0.2942
0.2865
0.2328
0.2366
0.2321
0.2337
0.2253
0.2063
0.2234
0.2332
0.2363
0.2323
0.1996
0.2342
0.2350
0.2383
0.2409
0.2191
0.2318
0.2295
0.2213
0.2181
0.2280
3,855
3,981
3,818
3,887
4,013
3,795
3,910
3,860
3,863
4,013
3,975
3,993
3,996
3,860
3,993
3,899
3,800
3,927
4,003
3,912
3,918
NOTE.—x2 test of homogeneity of base frequencies: For 19 of the above taxa, Sagitta omitted: x2 5 84.2, P 5 0.0053. For all 20 of the above taxa: x2 5
126.3, P 5 0.00000037. For 18 taxa, Sagitta and Peripatoides omitted: x2 5 51.3, P 5 0.46.
order groups within Lophotrochozoa and Ecdysozoa—
all of which have ,50% bootstrap support in the SSU
tree.
To check the validity of the ME bootstrap values
in figure 1, ME bootstrap analyses were rerun upon the
combined LSU 1 SSU sequences over a wide range of
Pinv values (table 2). As seen in this table, support varied
with Pinv for only a few groups, and the high bootstrap
values for the main groups—protostomes, Lophotrochozoa, Ecdysozoa—seemed unaffected by Pinv. At or just
below the ML estimate of Pinv 5 0.62, there is bootstrap
support .60% for every group listed in the table.
Figure 2 shows the LSU 1 SSU tree calculated
from the taxa used in figure 1, except with the chaetognath Sagitta added. The highly divergent Sagitta sequence, represented by the longest branch in the tree,
groups with that of Peripatoides, which is also divergent
and CG rich. The Peripatoides 1 Sagitta group has an
85% ME bootstrap value in this combined gene tree,
and it also occurs in the SSU-only and LSU-only trees
(not illustrated) with bootstrap values of 88% and 62%,
respectively. The other groups in figure 2 resemble those
in figure 1A, except that there are slightly lower bootstrap values for the protostome, Lophotrochozoa, and
Ecdysozoa clades. Also, within Ecdysozoa, Limulus no
longer groups strongly with Peripatoides, and Halicryptus no longer branches off first. The relationships within
Lophotrochozoa, by contrast, seem unaffected by the
presence of Sagitta (cf. fig. 1).
Figure 3 shows the LSU 1 SSU tree that results
when both the Sagitta and Peripatoides sequences are
excluded. With the data set now exhibiting stationarity
of base frequencies (see previously), this tree should
have the most accurate likelihood and parsimony bootstrap values. Thus, it is noteworthy that both ML and
MP strongly support the protostome, Lophotrochozoa,
and Ecdysozoa groups. Within Lophotrochozoa, the relations are essentially the same as in the nonstationary
trees of figures 1A and 2. So few ecdysozoans remain,
however, that the relationships among crown ecdysozoans (Chordodes, Trichinella, and Limulus) are not resolved with this abbreviated set of taxa.
When this manuscript was in preparation, James
Garey analyzed our combined LSU 1 SSU sequences
with Bootstrapper’s Gambit using LogDet-Paralinear
distances and corrections for site to site variation (Lake
1995; Cameron, Garey, and Swalla 2000)—a method
that can better account for heterogeneous rates of evolution across nucleotide sites than can methods that use
Pinv. The Gambit program could only accommodate 15
sequences, so Peripatoides, Sagitta, Trichinella, Hydrolagus, and Ciona were omitted. Within Lophotrochozoa,
the following groups and bootstrap values (1,000 replicates) were attained: Eisenia 1 Proceraea 1 Urechis 1
Terebratalia 1 Phoronis 1 Placopecten: 75%; Terebratalia 1 Phoronis 1 Placopecten: 68%; Eisenia 1 Proceraea 1 Urechis: 50%. These results are similar to
those generated by the ME analysis using Pinv in figure
1A.
Spectral Analysis
Without Sagitta
The results reported in this section were derived
from the 19 core taxa without Sagitta (although adding
Sagitta did not change the following findings). Figure 4
Phylogeny of Protostomes
293
FIG. 1.—Phylogenetic trees for 14 protostomes (chaetognath Sagitta not included) and five deuterostome outgroups calculated from rRNA
gene sequences under the ME criterion using LogDet-Paralinear distances and estimated values for the proportion of invariable sites (Pinv: see
Materials and Methods). The percentages of bootstrap replicates supporting each node are shown and represent, from top to bottom (or right to
left): minimum evolution (ME), maximum likelihood (ML), and maximum parsimony (MP)-based values. Bootstrap values are not shown if the
ME values were ,50%, except for Lophotrochozoa and Ecdysozoa where all bootstrap values are shown. A, Combined LSU 1 SSU rRNA
gene tree: Pinv 5 0.62. B, SSU tree: Pinv 5 0.61. C, LSU tree: Pinv 5 0.59.
98
98
98
99
98
98
97
Pinv
0.15 . . . . . . . . .
0.25 . . . . . . . . .
0.35 . . . . . . . . .
0.45 . . . . . . . . .
0.55 . . . . . . . . .
0.62a . . . . . . . . .
0.64b . . . . . . . . .
93
95
95
95
95
92
87
Lophotrochozoa
(%)
91
93
94
95
94
93
87
Ecdysozoa
(%)
43
41
46
51
59
65
67
NOTE.—The Pinv values of 0.62, and a bit less than 0.62, are most likely to be correct (see text).
aP
inv estimated with the GTR 1 I maximum likelihood model (see fig. 1A).
b The actual proportion of constant sites in the 19 sequences used to calculate P .
inv
Protostomes
(%)
Proceraea,
Urechis
(%)
81
81
79
81
76
76
71
Phoronis,
Terebratalia,
Placopecten
(%)
Proceraea, Urechis,
Eisenia, Phoronis,
Terebratalia,
Placopecten (%)
72
68
70
70
67
61
51
Phoronis,
Placopecten
(%)
83
81
81
85
83
81
78
85
88
88
88
85
77
59
Peripatoides,
Chordodes,
Trichinella,
Limulus (%)
88
90
87
82
75
65
55
Peripatoides,
Limulus
(%)
Table 2
Bootstrap Values for 10 Key Groups in Figure 1, Computed Using LogDet Distances of LSU 1 SSU rRNA Sequences over a Range of Invariable Sites (Pinv)
90
91
89
82
74
64
52
Chordodes,
Trichinella
(%)
294
Mallatt and Winchell
FIG. 2.—Phylogenetic tree calculated from combined LSU 1 SSU
rRNA gene sequences for 15 protostomes. Same as in figure 1A, except
with the chaetognath Sagitta included. Pinv 5 0.59.
FIG. 3.—Phylogenetic tree calculated from combined LSU 1 SSU
rRNA gene sequences for 13 protostomes. Same as in figure 1A, except with both Sagitta and Peripatoides omitted. Pinv 5 0.64.
Phylogeny of Protostomes
295
FIG. 4.—Lento plot of support and conflict values for the 20 most highly supported splits (taxonomic groups) that were computed by
spectral analysis from LSU 1 SSU LogDet distances among the 19 taxa listed in figure 1 (Sagitta not included: Pinv 5 0.62). Units on the yaxis are substitutions per site, and the precise numerical values are listed in column 1 of table 3. Black bars represent splits that are present in
the ME tree (fig. 1A), whereas gray bars represent splits not in that tree: A 5 Chordodes 1 Trichinella, B 5 Peripatoides 1 Limulus, C 5
Lophotrochozoa, D 5 Trichinella 1 Limulus, E 5 protostomes, F 5 Peripatoides 1 Chordodes 1 Trichinella 1 Limulus, G 5 Peripatoides 1
Chordodes, H 5 Ecdysozoa, I 5 Proceraea 1 Urechis, J 5 Terebratalia 1 Phoronis 1 Placopecten, K 5 Eisenia 1 Proceraea 1 Urechis 1
Terebratalia 1 Phoronis 1 Placopecten, L 5 Chordodes 1 Trichinella 1 Limulus, M 5 Phoronis 1 Placopecten, N 5 deuterostomes 1
Peripatoides 1 Halicryptus 1 Chordodes 1 Limulus, O 5 Phascolopsis 1 Stylochus, P 5 Peripatoides 1 Halicryptus 1 Limulus, Q 5
Proceraea 1 Urechis 1 Eisenia, R 5 Stylochus 1 Amphiporus, S 5 Peripatoides 1 Halicryptus 1 Chordodes 1 Limulus, T 5 Peripatoides
1 Halicryptus 1 Trichinella 1 Limulus.
is a Lento plot of support and conflict values for various
splits calculated by spectral analysis of the LogDet distances from the combined LSU 1 SSU sequences. The
precise support and conflict values are listed in column
1 of table 3, as S1 and C1 values, respectively. Spectral
analysis showed most support for the groups that were
also supported by bootstrap analysis, although it should
be noted that most splits containing ecdysozoans have
high conflict values because of the several highly divergent sequences in this superphylum.
Because ranking splits by raw support values can
be misleading, we used a relative support-versus-conflict
index, I, that also takes conflict into account when comparing splits (see Materials and Methods). As shown in
column 1 of table 3, this index exceeded four for the
following eight splits, which are thus considered most
likely to be valid: Lophotrochozoa; Protostomes; Peripatoides 1 Chordodes 1 Trichinella 1 Limulus; Ecdysozoa; Proceraea 1 Urechis; Terebratalia 1 Phoronis
1 Placopecten; Eisenia 1 Proceraea 1 Urechis 1 Terebratalia 1 Phoronis 1 Placopecten; Phoronis 1 Placopecten. Note that all these groups were also identified
by .60% bootstrap values in figure 1. Also note that
the Chordodes 1 Trichinella and Peripatoides 1 Limulus splits had low indices (I # 1.2, table 3), despite
their high support values; their low indices resulted from
a relatively high support for the alternative split of
Trichinella 1 Limulus.
To assess which of the gene components—LSU or
SSU—contributed more to the splits, these two components were analyzed separately (compare columns 2
and 3 in table 3). On the basis of the relative sizes of
their support-versus-conflict indices, the SSU gene contributes more than LSU to the higher-order splits of Lophotrochozoa, protostomes, and Ecdysozoa (that is, the
SSU index exceeds the LSU index for each of these
groups). By contrast, LSU contributes more to the other,
lower-order, taxonomic splits (F, I–K, and M in the table) because the LSU index exceeds the SSU index for
these splits. Recall this is the same pattern exhibited by
bootstrap analysis (fig. 1A–C).
With Sagitta
When the sequence from the chaetognath Sagitta
was included, spectral analysis of the combined LSU 1
SSU data favored a Peripatoides 1 Sagitta pairing. The
support value for this pairing was greater (S 5 11.4 3
1023 cf. 2.6 3 1023) and the conflict value less (C 5
5.2 3 1023 cf. 11.6 3 1023) than for the best alternative
split of Peripatoides 1 Limulus, yielding a relative support-versus-conflict index of 10.0.
The main findings of this study are summarized in
figure 5.
296
Mallatt and Winchell
Table 3
Support and Conflict Values for the Various Groups (splits) of Protostomes Shown in Figures 4 and 1, Calculated by
Spectral Analysis
Split in Figure 4
A. Chordodes 1 Trichinella. . . . . . . . . .
B. Peripatoides 1 Limulus . . . . . . . . . .
C. Lophotrochozoa . . . . . . . . . . . . . . . . .
E. Protostomes . . . . . . . . . . . . . . . . . . . .
F. Peripatoides 1 Chordodes 1
Trichinella 1 Limulus . . . . . . . . . . . .
H. Ecdysozoa . . . . . . . . . . . . . . . . . . . . .
I. Proceraea 1 Urechis. . . . . . . . . . . . . .
J. Terebratalia 1 Phoronis 1
Placopecten . . . . . . . . . . . . . . . . . . . . .
K. Eisenia 1 Proceraea 1 Urechis 1
Terebratalia 1 Phoronis 1
Placopecten . . . . . . . . . . . . . . . . . . . . .
M. Phoronis 1 Placopecten . . . . . . . . . .
O. Phascolopsis 1 Stylochus. . . . . . . . .
Q. Proceraea 1 Urechis 1 Eisenia . . . .
Column 1: LSU 1 SSU
S1/S2@C1/C2 5 I
Column 2: SSU only
S1/S2@C1/C2 5 I
Column 3: LSU only
S1/S2@C1/C2 5 I
S1/S2: 7.26/5.89a 5 1.2
C1/C2: 6.71/6.76a 5 1.0
I 5 1.2
S1/S2: 6.51/5.89a 5 1.1
C1/C2: 6.53/6.76a 5 1.0
I 5 1.1
S1/S2: 6.22/0.85c 5 7.3
C1/C2: 3.06/12.7c 5 0.24
I 5 30.4
S1/S2: 5.8/1.73c 5 3.4
C1/C2: 4.1/11.5c 5 0.36
I 5 9.4
S1/S2: 3.91/6.82a 5 0.6
C1/C2: 5.3/4.0a 5 1.3
I 5 0.4
S1/S2: 1.98/6.82a 5 0.3
C1/C2: 7.0/4.0a 5 1.8
I 5 0.2
S1/S2: 4.79/1.01d 5 4.8
C1/C2: 4.2/11.5d 5 0.37
I 5 13
S1/S2: 6.37/1.15f 5 6.1
C1/C2: 2.5/11.1f 5 0.23
I 5 26.5
S1/S2: 6.97/5.08b 5 1.4
C1/C2: 5.6/6.5b 5 0.86
I 5 1.6
S1/S2: 8.08/5.08b 5 1.6
C1/C2: 4.4/6.5b 5 0.7
I 5 2.3
S1/S2: 3.66/3.66c 5 1.0
C1/C2: 4.01/6.0c 5 0.67
I 5 1.5
S1/S2: 3.35/2.42c 5 1.4
C1/C2: 4.1/7.12c 5 0.58
I 5 2.4
S1/S2: 4.54/1.73c 5 2.6
C1/C2: 6.82/11.5c 5 0.59
I 5 4.4
S1/S2: 4.22/1.73c 5 2.4
C1/C2: 6.25/11.5c 5 0.54
I 5 4.4
S1/S2: 2.92/0.81h 5 3.6
C1/C2: 0.72/2.57h 5 0.28
I 5 12.9
S1/S2: 0.3/1.63g 5 0.2
C1/C2: 5.4/7.0g 5 0.77
I 5 0.3
S1/S2: 6.28/1.0d 5 6.3
C1/C2: 3.9/11.5d 5 0.34
I 5 18.6
S1/S2: 1.41/1.33i 5 1.1
C1/C2: 6.8/2.6i 5 2.6
I 5 0.4
S1/S2: 4.21/3.66c 5 1.2
C1/C2: 7.9/6.0c 5 1.3
I 5 0.9
S1/S2: 2.43/3.66c 5 0.7
C1/C2: 7.7/6.0c 5 1.3
I 5 0.6
S1/S2: 2.5/0.74h 5 3.4
C1/C2: 0.4/1.3h 5 0.3
I 5 11.1
S1/S2: 2.0/0.47j 5 4.3
C1/C2: 0.41/2.49j 5 0.16
I 5 26.1
S1/S2: 0.6/1.33i 5 0.5
C1/C2: 2.7/2.6i 5 1.04
I 5 0.5
S1/S2: 1.69/1.17k 5 1.4
C1/C2: 0.86/0.92k 5 0.93
I 5 1.5
S1/S2: 1.96/0.55l 5 3.6
C1/C2: 1.4/5.4l 5 0.26
I 5 13.9
S1/S2: 1.82/0.37o 5 4.9
C1/C2: 0.1/0.9o 5 0.11
I 5 45
S1/S2: 1.65/1.23r 5 1.3
C1/C2: 3.0/3.25r 5 0.9
I 5 1.5
S1/S2: 1.45/0.81h 5 1.8
C1/C2: 1.53/2.57h 5 0.595
I 5 3.03
S1/S2: 0.35/3.39m 5 0.1
C1/C2: 9.6/2.2m 5 4.3
I 5 0.2
S1/S2: 1.49/0.61p 5 2.4
C1/C2: 1.0/3.2p 5 0.3
I58
S1/S2: ,0.3/3.4m 5 ,0.1
C1/C2: —t
I 5 low
S1/S2: ,0.3/2.4s 5 ,0.13
C1/C2: —t
I 5 low
S1/S2: 2.33/0.46n 5 5.1
C1/C2: 0.34/2.0n 5 0.17
I 5 30
S1/S2: 2.34/0.51q 5 4.6
C1/C2: 0.36/1.65q 5 0.22
I 5 21
S1/S2: 2.05/3.66c 5 0.6
C1/C2: 3.6/6.0c 5 0.6
I51
S1/S2: 2.71/1.17k 5 2.3
C1/C2: 0.8/0.9k 5 0.9
I 5 2.9
NOTE.—Splits with the highest I values in column 1 (I . 4) are the most likely to be valid (I 5 relative support-versus-conflict index).
NOTE.—S1 and C1 are support and conflict values (in units of substitutions per site, 3 1023) for the split listed, whereas S2 and C2 are the corresponding
values for the most highly supported contradictory split. Such second-best splits, as indicated by the symbols for footnotes, are:
a Trichinella 1 Limulus; b Peripatoides 1 Chordodes; c Stylochus 1 Trichinella; d Proceraea 1 Peripatoides; e Deuterostomes 1 Peripatoides 1 Halicryptus 1
Chordodes 1 Limulus; f Ecdysozoans 1 Branchiostoma; g Halicryptus 1 Chordodes 1 Trichinella; h Urechis 1 Terebratalia 1 Phoronis 1 Placopecten 1 Eisenia;
i Urechis 1 Terebratalia; j Proceraea 1 Urechis 1 Phoronis 1 Placopecten; k Eisenia 1 Terebratalia; l Phascolopsis 1 Stylochus 1 Eisenia; m Phascolopsis 1
Eisenia; n Terebratalia 1 Phoronis 1 Placopecten 1 Amphiporus; o Terebratalia 1 Phoronis; p Urechis 1 Placopecten; q Terebratalia 1 Placopecten 1 Eisenia;
r Stylochus 1 Amphiporus; s All lophotrochozoans except Proceraea. t Conflict values could not be calculated when support values were less than 0.3 3 1023,
although the conflicts for such weakly supported groups can be assumed to be high.
Discussion
Ecdysozoa and Lophotrochozoa
The main goal of this study was to test whether
combined LSU 1 SSU rRNA gene sequences support
the division of protostomes into Lophotrochozoa and
Ecdysozoa, as Aguinaldo et al. (1997) proposed based
on SSU sequences alone. The answer is an emphatic
yes: Adding the LSU to the SSU data increased the ME
bootstrap support for these two clades markedly, from
73% and 81% in our SSU-only tree (fig. 1B) to 92%
and 93%, respectively, in the combined LSU 1 SSU
tree (fig. 1A). It is noteworthy that although we used
taxa whose SSU genes contained less bootstrap support
for Lophotrochozoa (73%) and for Ecdysozoa (81%)
than did the taxa used by Aguinaldo et al. (.90%), adding the LSU sequences raised our bootstrap values to
.90%, as seen in that previous study. Furthermore, our
combined LSU 1 SSU sequences continued to support
the existence of Ecdysozoa and Lophotrochozoa when
the disruptive sequence of the chaetognath Sagitta was
included (fig. 2), when the taxa were limited to those
yielding stationary base frequencies (fig. 3), and when
the sequences were analyzed by spectral analysis (fig. 4
and table 3). Thus, this study not only supports the di-
Phylogeny of Protostomes
FIG. 5.—Summary tree showing the findings of this study, derived
from combined LSU 1 SSU rRNA gene sequences. Numbers represent
ME bootstrap percentages (from fig. 1A) over I values (relative support-versus-conflict indices from column 1 of table 3); and key morphological characters are mapped onto some important nodes. Note
that the position of chaetognaths within protostomes is treated as uncertain (?). As for sipunculans, evidence from mitochondrial genes
suggests they are akin to annelids (Boore and Staton 2002), although
our rRNA data do not reveal this. For documentation of the morphological characters see Nielsen (1995, spiral cleavage and protostomy);
Schmidt-Rhaesa (1998, aflagellate sperm); Schmidt-Rhaesa et al.
(1998, dorsal heart); Giribet et al. (2000, haemal system); McHugh
(2000, chaetae), and Peterson and Eernisse (2001, protrusile chaetae).
vision of protostomes into Ecdysozoa and Lophotrochozoa but also strengthens it. We also found that SSU is
best at resolving deeper nodes in the protostome tree,
whereas LSU is best at revealing more apical nodes.
Subgroups of Taxa
Together, the LSU 1 SSU sequences seemed to resolve some subgroups within Ecdysozoa and Lophotrochozoa. Identifying such subgroups is a major goal of
invertebrate taxonomy, toward which SSU alone has
made only a small contribution, despite the availability
of hundreds of SSU sequences from a wide range of
taxa (Garey and Schmidt-Rhaesa 1998; Adoutte et al.
2000; Giribet et al. 2000). For example, SSU-based phylogenies show extensive paraphyly of molluscs, annelids, and some other phyla within the Lophotrochozoa
(Aguinaldo et al. 1997; Halanych 1998; Winnepenninckx, Van de Peer, and Backeljau 1998; Giribet et al.
2000). Given the small number of taxa used in our study
and the fact that our bootstrap values were only in the
60% and 70% range, our proposal that LSU 1 SSU
sequences can resolve subgroups within Ecdysozoa and
Lophotrochozoa is made with caution. Nonetheless,
297
these subgroups were also supported by our spectral
analysis, and most of them have been proposed previously based on more extensive morphological and molecular evidence. To be specific, such subgroups include:
(1) echiurans 1 polychaetes (evidenced by morphology
and elongation factor 1a genes; McHugh 1997, 2000),
(2) a lophophorate 1 mollusc 1 annelid clade (from
fossil evidence; Conway Morris and Peel 1995), which
is independent of flatworms (Garey and Schmidt-Rhaesa
1998), and (3) priapulids as basal ecdysozoans (based
on extensive and conserved SSU sequences; Peterson
and Eernisse 2001; Garey 2001; but see Schmidt-Rhaesa
et al. 1998).
Despite these apparent successes, the sipunculan
(Phascolopsis) sequence suggests a failure. Morphology
places sipunculans with annelids and molluscs, and their
mitochondrial genes relate them to annelids (Boore and
Staton 2002). Our data missed this relation, probably
because sipunculan rRNA sequences are divergent
(Winnepenninckx, Van de Peer, and Backeljau 1998; Giribet et al. 2000) and attracted artifactually to the longbranched flatworm sequence (fig. 1A). This sipunculan
example shows that adding LSU to SSU does not yield
a perfect phylogeny—and adding sequences from more
protostomes will undoubtedly yield more anomalies and
other examples of paraphyly. Even so, the value of combining LSU and SSU is demonstrated by the fact that
our LSU 1 SSU data resolved known subgroups that
our SSU data did not (compare fig. 1A and B).
Our combined-gene analysis joins the lophophorate
and mollusc sequences (Terebratalia, Phoronis, and Placopecten). This echoes an old view that allied brachiopods with molluscs, a view not held by modern experts
(Hyman 1959; p. 516), who either consider brachiopods
and molluscs closer to some polychaete than to each
other (Conway Morris 1998, p. 188) or else consider
lophophorates basal to all other lophotrochozoans, including molluscs (Valentine 1997; Peterson and Eernisse
2001). In one sense our evidence is questionable, in that
it places the phoronid closer to the mollusc than to the
brachiopod. This placement is unconvincing, considering that phoronids and brachiopods are similar animals
united by a lophophore and similar coeloms (Brusca and
Brusca 1990, chapt. 21). In another sense, however, our
evidence does support a lophophorate 1 mollusc clade,
in that it places the sequences of both lophophorates
closer to that of the mollusc than to any other protostome (fig. 5). If the lophophorate 1 mollusc clade
should prove to be real, then brachiopod and mollusc
shells could be homologous, rather than just analogous
as is now believed. Shell structure and pores, shell muscles, and shell formation from a mantle are similar in
both phyla, although these shells have different relations
to the body axis (Barnes 1980, p. 914; Pechenik 1985,
p. 378; Meglitsch and Schram 1991, pp. 212, 514; Nielsen 1995, p. 352; Reindl, Salvenmoser, and Haszprunar
1995).
Results Explained
Despite our modest taxon sampling, there are three
reasons why our results should contain true signal and
298
Mallatt and Winchell
not be the result of sampling error. First, there seems to
be a real benefit in adding LSU sequences to the traditionally used SSU sequences. The evidence for this, as
mentioned previously, is that LSU added resolution
within the Lophotrochozoa and Ecdysozoa, whereas no
such resolution existed in the SSU tree (compare fig. 1B
with A). This is the ‘‘adding more characters’’ approach
to increasing phylogenetic resolution (Mitchell, Mitter,
and Regier 2000), and its apparent success in this study
suggests that the characters in the LSU genes are indeed
valuable additions.
The second reason for the resolution attained must
be our use of the LogDet-based distance method because
the ML and MP methods did not identify the subgroups
of Ecdysozoa and Lophotrochozoa that ME did (compare the ME, ML, and MP bootstrap values in fig. 1A).
The effectiveness of this LogDet method for retrieving
well-known clades from rRNA data has been noted before (Aguinaldo et al. 1997; Mallatt, Sullivan, and Winchell 2001; Garey 2001). Its effectiveness may stem not
only from the fact that this method performs best when
nucleotide frequencies are nonstationary across taxa but
also because all 12 types of nucleotide substitutions are
free to occur at different rates, unlike in the ML or MP
methods (Swofford et al. 1996, pp. 459–461). Spectral
analysis of the LogDet distances also yielded high taxonomic resolution. Spectral analysis has a theoretical advantage over tree-building methods because in the treebuilding process support values from characters that favor contradictory trees cancel one another so that information is lost, whereas in spectral analysis all these
values are retained to produce total support values; that
is, in spectral analysis, no support signal is lost. As mentioned previously, another advantage of spectral analysis
is that it allows conflict values to be taken into account,
whereas tree-building methods do not.
The third reason for the apparent resolving power
of this study is that most of the taxa used had slowly
evolving sequences. There is much debate in molecular
phylogenetics over whether the best way to resolve difficult phylogenies is to use just the most slowly evolving
sequences (the approach of Aguinaldo et al. 1997 and
Garey and Schmidt-Rhaesa 1998) or to add more and
more sequences to split up the long branches (the dominant approach used with SSU gene sequences; Eernisse
1997; Giribet et al. 2000). We favor the former approach
because we believe that long-branch attraction (Felsenstein 1978) remains a major confounding problem in
SSU-based studies, that using slowly evolving sequences minimizes this problem, and that the currently dominant approach of adding taxa for the sake of thoroughness has not improved resolution much, perhaps because
new long branches have been added as often as the older
branches were broken up. Even so, our philosophy is to
avoid using the most divergent sequences rather than to
use only the most conserved ones, and we certainly believe that many more taxa than the 15 used here are
needed to resolve the relationships within Ecdysozoa
and Lophotrochozoa. This idea that the judicious omission and addition of taxa improves phylogenetic estimation was championed, in more depth, by Kim (1998).
Position of Chaetognaths
The phylogenetic position of chaetognath worms is
a longstanding problem. Their anatomical characters are
confusing (Bone, Kapp, and Pierrot-Bults 1991, p. 2),
and their rRNA sequences—the only molecular information available from this phylum—are highly divergent. The SSU sequences of chaetognaths have always
grouped with other rapidly evolving protostome sequences—rhabditid nematodes, gastrotrichs, gnathostomulids, and recently, with onychophorans (Halanych
1996, 1998; Eernisse 1997; Littlewood et al. 1998; Zrzavy et al. 1998; Giribet et al. 2000)—with a lack of consistency that suggests artifactual long-branch attraction.
Both our phylogenetic tree (fig. 2) and spectral analysis
placed Sagitta with Peripatoides, which we suspect is
another artifact of long-branch attraction, here between
two highly divergent and CG-rich sequences (note that
even LogDet cannot correct for the most extreme cases
of nucleotide nonstationarity; Foster and Hickey 1999).
As evidence supporting this suspicion, when we omitted
the Peripatoides sequence and reran the LSU 1 SSU
bootstrap analysis, Sagitta no longer joined the Ecdysozoa but appeared as an independent protostome lineage (in ME and MP trees: not shown). Thus, the position of chaetognaths is left unresolved in our summary
figure 5.
Testing Some Other Hypotheses
The Ecdysozoa and Lophotrochozoa hypotheses
contradict several classical, morphology-based hypotheses of triploblast relationships: that annelids and arthropods are interrelated as Articulata (based on similarities in body segmentation; Schmidt-Rhaesa et al.
1998) and that the flatworm line, then the nematode line,
branched from the base of the triploblast tree before the
appearance of deuterostomes and protostomes (Barnes
1980, chapt. 3). In parts 1–3 of table 4, these three classical hypotheses are tested by spectral analysis of our
combined LSU 1 SSU-gene data. None of these hypotheses has even one-twentieth as much support as the
Ecdysozoa and Lophotrochozoa hypotheses that oppose
it. Further evidence against these classical hypotheses
has been provided by many recent molecular and anatomical studies (Ruppert 1991; Eernisse, Albert, and
Anderson 1992; Balavoine 1998; Boore and Brown
2000; Peterson and Eernisse 2001).
The findings of this study can also test two hypotheses about deuterostomes. The first is that nemerteans are related to chordates (Jensen 1988). SSU-based
evidence speaks against a nemertean-deuterostome link
(Winnepenninckx, Backeljau, and De Wachter 1995;
Sundberg, Turbeville, and Harlin 1998), as does our
combined LSU 1 SSU evidence. In addition to the high
bootstrap support for Amphiporus as a protostome and
lophotrochozoan (fig. 1A), spectral analysis indicates
that the support for Amphiporus 1 deuterostomes is
only one-ninth as high as for Amphiporus-as-lophotrochozoan, and the conflict is three times greater (see part
4 in table 4).
Phylogeny of Protostomes
299
Table 4
Testing Older Hypotheses of Protostome Relationships Based on Spectral Analysis of
LogDet Distances from LSU 1 SSU Genes (taxa of figs. 1 and 4 were used)
Classical Hypothesis
Preferred Alternate Split, This Study
1. Articulata (Annelids 1 Panarthropoda: Eisenia 1
Proceraea 1 Urechis 1 Peripatoides 1 Limulus): S
5 0.08
2. Flatworm (Stylochus) not a Protostome: S 5 20.5
3. Nematode (Trichinella) not a Protostome: S 5 0.2
4. Nemertean (Amphiporus) 1 Deuterostomes: S 5
0.7; C 5 210.1
5. Lophophorates (Terebratalia, Phoronis) 1 Deuterostomes: S 5 20.2
1. Lophotrochozoa: S 5 6.2
Ecdysozoa: S 5 4.2
2. Stylochus in Lophotrochozoa: S 5 6.2
3. Trichinella in Ecdysozoa: S 5 4.2
4. Amphiporus in Lophotrochozoa: S 5
6.2; C 5 23.1
5. Terebratalia and Phoronis in Lophotrochozoa: S 5 6.2
NOTE.—Support (S) and conflict (C) values are substitutions per site (31023). Conflict values could not be calculated
when levels of support were below the threshold of 0.3 3 1023 (see Materials and Methods).
The second hypothesis about deuterostomes is that
they are related to lophophorates (brachiopods, phoronids, bryozoans). Lophophorates have been classified as
deuterostomes based on their development and the fact
that their lophophore resembles a similar feeding structure in pterobranch hemichordates (Hyman 1959; Brusca
and Brusca 1990, p. 797). However, molecular evidence
from many genes now indicates that lophophorates are
protostomes (Halanych et al. 1995; de Rosa et al. 1999;
Saito, Kojima, and Endo 2000). Furthermore, as seen in
part 5 of table 4, spectral analysis gives the lophophorate
1 deuterostome hypothesis negative support.
Relative Value of the LSU Versus the SSU Gene in
Molecular Phylogeny
Over 60 LSU rRNA sequences are now known
from animals (listed in Mallatt and Sullivan 1998; Medina et al. 2001; Winchell et al. 2002). On the basis of
our experience with these sequences, it is possible to
compare the relative strengths and weaknesses of the
LSU and SSU genes for phylogenetic analysis. Overall,
the LSU gene seems to contain less phylogenetic signal,
at least for resolving the deepest branches of the animal
tree (compare fig. 1C to B; also see Winchell et al.
2002). This means that LSU should not be used alone
for deep-level phylogeny but should always be combined with SSU, and indeed, in our experience, the combined LSU 1 SSU sequences always give better resolution than do SSU sequences alone. Although the LSU
gene does add signal, it requires about three times more
work to amplify and sequence than the SSU gene. However, the fact that the conserved core region of the 28S
gene is both well defined and informative about deeplevel animal taxonomy makes aligning LSU genes simpler and more objective than aligning SSU genes.
The divergent domains of the 28S gene, although
not used in this higher-level phylogenetic analysis, can
be included for lower-level analyses all the way down
to the species or even subspecies level (Littlewood
1994; Mallatt and Sullivan 1998; Jarman et al. 2000;
Litvaitis et al. 2000; Winchell 2001). The SSU gene, by
contrast, has few rapidly evolving regions and provides
little resolution at lower taxonomic levels.
Finally, as already mentioned, a weakness of protostome and deuterostome LSU gene sequences is that
they are susceptible to nonstationarity of base frequencies. By contrast, all the SSU data sets with which we
have dealt were stationary.
Conclusions
The combined LSU 1 SSU rRNA gene sequences
used in this study uphold and strengthen the hypotheses
of Aguinaldo et al. (1997), based on SSU sequences
alone, that protostome animals consist of Ecdysozoa and
Lophotrochozoa (fig. 5). More intriguingly, our admittedly preliminary findings suggest that the LSU 1 SSU
sequences can help resolve the topology within Ecdysozoa and Lophotrochozoa, which is one of the main
goals of animal systematics. For example, the results
suggest an unexpected clade of lophophorates and molluscs. More bilateralian LSU sequences are greatly
needed.
Although additional, independent genes must also
be used, LSU rRNA gene sequences could help to free
systematists who now use SSU from the frustrating cycle that many are experiencing, i.e., a cycle of adding
progressively more SSU sequences without much improvement in taxonomic resolution. Widespread taxonomic sampling of protostome LSU sequences should
begin, and if the findings of the present study are true
indicators, the new studies should emphasize slowly
evolving over rapidly evolving sequences and should
include LogDet-based distance methods for maximum
efficiency. We foresee a time when no rDNA-based phylogenetic study will be considered complete unless it
includes both the LSU and SSU genes.
Acknowledgments
Thanks are extended to James Garey for critiquing
the manuscript and running the Gambit analysis, to Jack
Sullivan for methodological discussions, to Gary Thorgaard for sharing lab facilities, to Kevin Pullen of the
Charles R. Conner Museum of Zoology at Washington
State University for help with the voucher specimens,
to D. T. J. Littlewood for advice on which flatworm to
sequence, to J. M. Turbeville for the Amphiporus SSU
sequence, and to all those who provided specimens.
300
Mallatt and Winchell
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Accepted November 21, 2001