Molecular Phylogenetics and Evolution 43 (2007) 1005–1022
www.elsevier.com/locate/ympev
Ribosomal RNA genes and deuterostome phylogeny revisited:
More cyclostomes, elasmobranchs, reptiles, and a brittle star
Jon Mallatt a,¤, Christopher J. Winchell b
a
School of Biological Sciences, Washington State University, Pullman, WA 99164-4236, USA
b
Department of Ecology and Evolutionary Biology, University of California, Los Angeles,
Box 951606, 621 Charles E. Young Dr. South, Los Angeles, CA 90095-1606, USA
Received 2 June 2006; revised 27 October 2006; accepted 22 November 2006
Available online 9 December 2006
Abstract
This is an expanded study of the relationships among the deuterostome animals based on combined, nearly complete 28S and 18S
rRNA genes (>3925 nt.). It adds sequences from 20 more taxa to the »45 sequences used in past studies. Seven of the new taxa were
sequenced here (brittle star Ophiomyxa, lizard Anolis, turtle Chrysemys, sixgill shark Hexanchus, electric ray Narcine, Southern Hemisphere lamprey Geotria, and Atlantic hagWsh Myxine for 28S), and the other 13 were from GenBank and the literature (from a chicken,
dog, rat, human, three lungWshes, and several ray-Wnned Wshes, or Actinopterygii). As before, our alignments were based on secondary
structure but did not account for base pairing in the stems of rRNA. The new Wndings, derived from likelihood-based tree-reconstruction
methods and by testing hypotheses with parametric bootstrapping, include: (1) brittle star joins with sea star in the echinoderm clade,
Asterozoa; (2) with two hagWshes and two lampreys now available, the cyclostome (jawless) Wshes remain monophyletic; (3) Hexanchiform sharks are monophyletic, as Hexanchus groups with the frilled shark, Chlamydoselachus; (4) turtle is the sister taxon of all other
amniotes; (5) bird is closer to the lizard than to the mammals; (6) the bichir Polypterus is in a monophyletic Actinopterygii; (7) ZebraWsh
Danio is the sister taxon of the other two teleosts we examined (trout and perch); (8) the South American and African lungWshes group
together to the exclusion of the Australian lungWsh. Other Wndings either upheld those of the previous rRNA-based studies (e.g., echinoderms and hemichordates group as Ambulacraria; orbitostylic sharks; batoids are not derived from any living lineage of sharks) or were
obvious (monophyly of mammals, gnathostomes, vertebrates, echinoderms, etc.). Despite all these Wndings, the rRNA data still fail to
resolve the relations among the major groups of deuterostomes (tunicates, Ambulacraria, cephalochordates and vertebrates) and of
gnathostomes (chondrichthyans, lungWshes, coelacanth, actinopterygians, amphibians, and amniotes), partly because tunicates and
lungWshes are rogue taxa that disrupt the tree. Nonetheless, parametric bootstrapping showed our RNA-gene data are only consistent
with these dominant hypotheses: (1) deuterostomes consist of Ambulacraria plus Chordata, with Chordata consisting of tunicates and
‘vertebrates plus cephalochordates’; and (2) lungWshes are the closest living relatives of tetrapods.
© 2006 Elsevier Inc. All rights reserved.
Keywords: 28S rRNA; Ribosomal RNA genes; Molecular phylogeny; Ambulacraria; Asterozoa; Chordata; Vertebrata; Hexanchformes; Parametric
bootstrapping
1. Introduction
Deuterostomes are a large group of animals, consisting
of the vertebrates and other chordates, the echinoderms,
hemichordates, and the rare worm-like Xenoturbella (Bour-
*
Corresponding author. Fax: +1 509 335 3184.
E-mail address: [email protected] (J. Mallatt).
1055-7903/$ - see front matter © 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.ympev.2006.11.023
lat et al., 2003; Holland, 2005; Kardong, 2006; Rowe, 2004).
The relations among these phyla have long been studied,
and can throw light on the origin of the chordates and of
vertebrates (Cameron et al., 2000; Gee, 1996, 2006; Mallatt
and Chen, 2003; Northcutt, 2005; Winchell et al., 2002).
Many investigations of these relations used traditional systematics with morphological characters (e.g., Maisey,
1986a; SchaeVer, 1987), but molecular-phylogenetic studies
are advancing to the point where giant, multi-gene analyses
1006
J. Mallatt, C.J. Winchell / Molecular Phylogenetics and Evolution 43 (2007) 1005–1022
are now revealing the relationships among major deuterostome groups (Delsuc et al., 2006; Philippe et al., 2005;
Takezaki et al., 2003, 2004; also see Blair and Hedges,
2005). However, these big phylogenomic studies, which are
based on hundreds of genes, require great sequencing eVort,
so their progress is slow and the number of sequenced taxa
is still small. In the meantime, nearly complete sequences of
ribosomal RNA genes (28S and 18S rRNA) can be generated more easily, from a wide range of deuterostomes. At
over 5000 nt. in length, the rRNA-gene family is known to
contain phylogenetic information at many taxonomic levels
(Giribet, 2002; Hillis and Dixon, 1991), and several laboratories are working to sequence 28S genes from more animals to combine with the thousands of 18S sequences that
are available (Collins et al., 2006; Giribet et al., 2005; Lockyer et al., 2003; Mallatt and Giribet, 2006; Mallatt and
Winchell, 2002; Mallatt et al., 2004; Passamaneck and
Halanych, 2006; Passamaneck et al., 2004; Telford et al.,
2003).
The present study builds on previous rRNA-based studies of deuterostome relationships: those of Winchell et al.
(2002, 2004) and Mallatt and Sullivan (1998). To provide a
more thorough and even representation of taxa within the
deuterostomes, we added 20 more sequences, 7 of which are
from key taxa sequenced here, and 13 of which we retrieved
from GenBank and the literature. The original goal of this
study was to get sequences from another hagWsh and lamprey to revisit the question of whether these jawless Wshes
group together as Cyclostomata (Mallatt and Sullivan,
1998). Next, we sequenced genes from two more elasmobranchs, to help deWne the relationships of sharks and
batoids (skates and rays) (after Winchell et al., 2004). Then,
we wanted to add some amniotes, and because no 28S genes
of reptiles had ever been sequenced, we sequenced these
genes from a turtle and a lizard. Finally, we sequenced the
rRNA genes of an ophiuroid (brittle star), the only major
clade of echinoderms that was not included in our previous
study (Winchell et al., 2002). The sequences we retrieved
from GenBank and the literature were from a bird, various
bony Wshes, and some mammals.
In bringing the number of complete deuterostomerRNA sequences to about 65, we seek to approach the level
of sophistication of a recent, comparable study of the relations among arthropods and their relatives (ecdysozoans),
in which over 80 taxa were included (Mallatt and Giribet,
2006). As in that ecdysozoan study, our methods are
upgraded over those of the earlier investigation of basaldeuterostome relationships (Winchell et al., 2002), especially by extensive statistical testing with parametric bootstrapping.
2. Materials and methods
Table 1 lists the 20 species added by this study, with the
GenBank numbers of their rRNA sequences. Also listed are
the other deuterostomes and the protostome outgroups,
whose sequences were taken from our previous studies.
Table S1 in the Supplementary material gives more information about the seven taxa whose rRNA genes we newly
sequenced.
Routine procedures were as described previously: specimen preservation, DNA extraction using the Qiagen
(Valencia, CA) DNeasy® Tissue Kit, gene ampliWcation by
PCR, primers, sequencing methods, and the assembly and
alignment of sequences with GCG computer programs:
Wisconsin Package, Version 10.3, Accelrys Inc., San Diego
CA (see Mallatt and Giribet, 2006; Mallatt and Sullivan,
1998; Mallatt et al., 2004; Winchell et al., 2004). All the
rRNA-gene sequences are over 86% complete, at least for
the alignable regions used in our phylogenetic analyses.
Sequences were aligned by eye, rigidly following secondary-structure models of rRNA (but not accounting for the
pairing of nucleotides in the stems: see Mallatt and Giribet,
2006, for a detailed explanation). First, when aligning
across all deuterostomes (Fig. 1), we included only the conserved core of the 28S genes (Mallatt et al., 2001) plus those
sites in the 18S genes that showed identical nucleotides in at
least 70% of the taxa. This led to a data set with 3925
aligned-nucleotide sites. Second, to obtain additional characters for investigating relationships within deuterostome
clades (in echinoderms, vertebrates, amniotes, elasmobranchs: Figs. 2–5), we expanded the 28S part of the alignment to include all sites in the 28S gene that had identical
nucleotides in 770% of taxa. All the alignments are presented in Supplementary material, S2.
For phylogenetic reconstruction, both likelihood-based
and classical maximum-parsimony (MP) methods were
used. We favor the likelihood methods because they use
explicit models of nucleotide substitution during gene
evolution, have good statistical properties, and are relatively resistant to long-branch-attraction artifacts caused
by divergent gene sequences (Buckley and Cunningham,
2002; Felsenstein, 2004; Springer et al., 2004; SwoVord
et al., 1996; Whelan et al., 2001). The likelihood-based
methods we used were (1) Bayesian inference, and (2)
maximum-likelihood (ML). Although these two methods
usually produce similar results in our studies (Mallatt and
Giribet, 2006; Winchell et al., 2004), they diVer in some
ways. Bayesian inference (Huelsenbeck et al., 2001) calculates support for clades in the form of posterior probabilities (PP), which are unambiguous statements of statistical
probability (Jow et al., 2002); however, the PP values
often seem too high for clades with short edge lengths
(Lewis et al., 2005), which has caused concern (Kjer et al.,
2006). This apparent “overconWdence” is not due to any
Xaws in the Bayesian method, but to the weaknesses of
current models of nucleotide substitution, upon which all
likelihood-based methods rely (J. Huelsenbeck, University
of California, Berkeley, personal communication). The
ML method, on the other hand, supports clades with nonparametric-bootstrapping values, whose meaning and signiWcance are not clear, but which are more conservative
estimates of conWdence. In this study, we accept clades as
signiWcant if they have Bayesian PP values >95% and ML
J. Mallatt, C.J. Winchell / Molecular Phylogenetics and Evolution 43 (2007) 1005–1022
1007
Table 1
Information on species used in this study
ClassiWcation
Chordata
Craniata (vertebrates)
Cyclostomata
Myxiniformes
Petromyzontiformes
Gnathostomata
Chondrichthyes
Holocephali
Elasmobranchii
( D Neoselachii)
“Sharks”
Batoidea
Species
GenBank numbers
28S rDNA
18S rDNA
Myxine glutinosa¤¤
Eptatretus stouti
Geotria australis¤¤
Petromyzon marinus
AY859635-39
AF061797
AY859628-31
AF061798
M97574
M97572
AY859632
M97575
Hydrolagus colliei
AF061799
Stock (1992)
Hexanchus griseus¤¤
Triakis semifasciata
Narcine brasiliensis¤¤
Raja schmidti
AY859633
AF212182
AY859640-41
AF278683
AY859634
AF212180
AY859642
AF278682
Plus: 26 more Chondrichthyes sequenced, mostly AY049805-61, from Winchell et al. (2004)
Osteichthyes
Sarcopterygii
Coelacanthiformes
Dipnoi
Latimeria chalumnae
Neoceratodus forsteri¤
Protopterus aethiopicus and P. sp.¤
Lepidosiren paradoxica¤
U34336
U34338
U34339
U34337
L11288
Stock (1992)
AF188370
Stock (1992)
AF212178
M59384
Testudines
Lepidosauria
Ambystoma macrodactylum and A.
mexicanum
Rana nigromaculata and R. chensinensis¤
Xenopus laevis
Chrysemys sp.¤¤
Anolis carolinensis¤¤
AB099628
X59734
AY859625-26
AY859621-23
AY145522, AB099628
X59734
AY859627
AY859624
Archosauria (Aves)
Gallus gallus¤
AADN01030883.1,a AADN01055467.1,
AADN01055478.1, AADN01030819.1,
AADN01108159.1, AADN01093675.1,
AADN01055474.1, AADN01030820.1
AF173612
Mammalia
Homo sapiens¤
Rattus norvegicus¤
Canis familiaris¤
M11167
V01270
NW_878945, NW_876295
U13369
V01270
NW_878945
Polypterus ornatipinnis and P. delhezi¤
Acipenser brevirostrum and A.
transmontanus
Oncorhynchus mykiss and O. kisutch¤
Danio rerio¤
Siniperca chuatsi¤
AF154052
U34340
AF198114
(Stock, 1992)
U34341, AF061801
BX296557, BX537263
AY452491-94
AF030250
BX296557, BX537263
AY452489, AY452490, AY452495
Branchiostoma Xoridae
AF061796
M97571
Ciona intestinalis
Styela plicata
Thalia democratica
Oikopleura sp.
AF212177
AF158724
AF158725
AF158726
AB013017
M97577
D14366
D14360
Florometra serratissima and Antedon
serrata
Asterias forbesi and A. amurensis
Strongylocentrotus purpuratus
AF212168
D14357
AF212169
AF212171
D14358
L28056
Tetrapoda
Amphibia
Urodela
Anura
Actinopterygii
Polypteriformes
Chondrostei
Teleostei
Cephalochordata
Tunicata (Urochordata)
Ascideacea
Thaliacea
Appendicularia
Other deuterostomes
Echinodermata
Crinoidea
Asteroidea
Echinoidea
(continued on next page)
1008
J. Mallatt, C.J. Winchell / Molecular Phylogenetics and Evolution 43 (2007) 1005–1022
Table 1 (continued)
ClassiWcation
Holothuroidea
Ophiuroidea
Hemichordata
Protostomes (outgroups)
Ecdysozoa
Lophotrochozoa
Species
GenBank numbers
Arbacia punctulata and A. lixula¤
Cucumeria salva and C. sykion
Ophiomyxa australis¤¤
28S rDNA
AY026367
AF212170
AY859643-44
18S rDNA
Z37514
Z80950
AY859645
Cephalodiscus gracilis
Saccoglossus pusillus
Harrimania planktophilus
Ptychodera Xava
Ptychodera sp. WCJ2000
AF212172
AF212174
AF212173
AF212176
AF278684
AF236798
AF236800
AF236799
AF278681
AF278685
Priapulus caudatus
Limulus polylphemus
Proceraea cornuta
Placopecten magellanicus
AY210840
AF212167
AF212165
AF342798
Z38009
U91490
AF212179
X53899
Note. The 7 species indicated by double asterisks (**) were newly sequenced in the present study for 28S (and for 18S when needed). The sequences of the
13 species indicated by single asterisks (*) were newly obtained from GenBank and the literature. The others were used in the phylogenetic analyses of
Winchell et al. (2002, 2004) or Mallatt and Sullivan (1998).
a
Plus other contigs in the Chicken (Gallus) Genome Project that overlap these eight contigs.
bootstrap values over 60–70% (Mallatt and Giribet, 2006),
although we rely more on the ML values, to err on the
side of caution.
All our ML-based analyses used the GTR + I + model
of nucleotide substitution (with eight gamma categories)
because this model was universally favored by the AIC procedure in Modeltest (Posada and Buckley, 2004; Posada
and Crandall, 1998). The ML and MP searches were run
with PAUP¤ 4.0 beta 10 (SwoVord, 2002), with 100 (ML)
and 1000 (MP) nonparametric-bootstrap replicates. Partitioned Bayesian inference was performed with MrBayes 3.0.
The command was ‘nst D 6 invgamma’ for both the 28S and
18S partitions (Huelsenbeck and Ronquist, 2003). Separate
Bayesian analyses were performed, with and without the
covarion model of nucleotide evolution at sites through
time (Galtier, 2001; Huelsenbeck, 2002; Penny et al., 2001).
The Bayesian analyses ran for 1.5 million generations. All
parameters stabilized by 200,000 generations, so these were
discarded as burn-in.
Parametric bootstrapping (pboot), a rigorous way of
testing phylogenetic hypotheses (Huelsenbeck et al., 1996b),
was used to determine whether the rRNA genes support
alternate hypotheses not indicated by the optimal genetrees. The steps in pboot are as follows (Mallatt and Sullivan, 1998; Wilcox et al., 2002). (1) From the actual, aligned
data set, PAUP¤ calculates the best likelihood tree that is
constrained to the alternate hypothesis. (2) The likelihoodmodel parameters of that tree are then entered into SeqGen
1.2.5 (Rambaut and Grassly, 2001), to simulate 100 replicate alignments. (3) From each of the simulated alignments,
PAUP¤ calculates the likelihood value of the unconstrained
best tree (Ln Lbest) and of the tree constrained to the alternate hypothesis (Ln Lhyp); then, the distribution of the 100diVerence values, Ln Lbest ¡ Ln Lhyp, is recorded; this provides a 99% conWdence limit for the actual diVerence. (4)
Finally, the likelihood value of the best model-constrained
tree from step 1 is subtracted from the likelihood value of
the actual optimal tree (both values from the real, not simulated, data sets). This diVerence, , is compared to the 100
Ln Lbest ¡ Ln Lhyp values of step 3, and if lies outside the
range of these values, the alternate hypothesis is rejected at
P < 0.01.
3. Results
Fig. 1 shows the ML tree calculated from the rRNA genes
of 46 taxa that span the full range of deuterostomes, and thus
shows the relationships among the major subclades. Support
values are indicated at the nodes of the tree.
This 46-taxon data set has a compositional bias in its
nucleotide frequencies (2 test of nucleotide homogeneity
across taxa: 2 D 223.7; P D 0.0000024; see Supplementary
material, S2), and such bias can cause errors in tree reconstruction (Jermiin et al., 2004). So, next we eliminated the
two most extreme sequences, the AT-rich sequence of tunicate Oikopleura and the CG-rich sequence of hemichordate
Cephalodiscus, and this made the data set stationary
(2 D 151.2; P D 0.089). The resulting 44-taxon tree had an
almost identical topology to the original 46-taxon tree, and
the support values were similar; these values from the stationary set are also indicated at the nodes in Fig. 1, in
parentheses.
Those taxa that were used previously (by Winchell et al.,
2002 in their Fig. 1C) came out in the same places here in
Fig. 1 as in the previous tree, with similar support values.
Monophyly of the Tunicata and of Ambulacraria (Echinodermata plus Hemichordata) remain strongly supported.
The position of tunicates within Deuterostomia remains
unresolved. The only apparent diVerence from the previous
results is lower ML-bootstrap support for a “Euchordata”
clade uniting the cephalochordate Branchiostoma with
vertebrates: 42–50% here compared to 97% previously.
J. Mallatt, C.J. Winchell / Molecular Phylogenetics and Evolution 43 (2007) 1005–1022
Chordata
Ambulacraria
Chordata
Tunicata
Hemichordata
Asterozoa
Protostomia
(outgroup)
Vertebrata
Petromyzon (lamprey)
Geotria ** (lamprey)
81/100/100/95
Ambystoma (salamander)
(82/100/100/95)
Xenopus (frog)
83/100/100/100
Rana * (frog)
(85/100/100/100)
Hexanchus ** (shark)
86/100/100/72
(87/100/100/74)
Triakis (shark)
Hydrolagus (ratfish)
All 100
Raja (ray)
<50/91/96/<50
Latimeria (coelacanth)
(<50/89/97/<50)
Acipenser (sturgeon)
<50/100/100/<50
(<50/100/100/<50)
Polypterus * (bichir)
64/100/100/<50
Danio * (zebrafish)
(55/100/100/<50)
Oncorhynchus * (trout)
95/87/95/93
Siniperca * (perch)
(97/83/93/91)
Chrysemys ** (turtle)
67/100/100/98
(83/100/100/97)
Homo * (human)
All 100
Canis * (dog)
All 100
<50/99/100/90
Rattus * (rat)
(52/98/100/90)
Gallus * (chicken)
93/100/100/71
(98/100/100/70)
Anolis ** (lizard)
Lepidosiren * (lungfish)
91/100/
Protopterus * (lungfish)
97/98/100/54
100/99
All 100
(91/95/99/56)
(89/100/
Neoceratodus
*
(lungfish)
100/100)
Florometra (crinoid)
Cucumeria
All 100
(sea cucumber)
All 100
Arbacia * (sea urchin)
88/100/100/55
All 100
(94/100/100/60)
Strongylocentrotus (sea urchin)
All 100
Ophiomyxa ** (brittle star)
82/70/92/<50
(81/85/96/<50)
Asterias (sea star)
Cephalodiscus (pterobranch)
87/100/100/98
All 100
Saccoglossus (acorn worm)
(---/---/---/---)
Harrimania (acorn worm)
99/100/
Ptychodera flava (acorn worm)
96/100/100/100
100/99
(94/100/100/100)
(100/100/
Ptychodera sp. (acorn worm)
100/100)
Oikopleura (larvacean)
92/100/100/63
Styela (ascidian)
(100/100/100/100)
Ciona
(ascidian)
85/100/100/54
100/100/100/100
(---/---/---/---)
(94/100/100/96)
Thalia (thaliacean)
Priapulus (penis worm)
92/100/100/97
Limulus (horseshoe crab)
(97/100/100/98)
Placopecten (scallop)
90/100/100/96
(86/100/100/97)
Proceraea (polychaete)
50/100/100/90
(42/100/100/90)
Echinodermata
Eptatretus (hagfish)
Myxine ** (hagfish)
Cyclostomata
All 100%
99/100/100/90
(97/100/100/91)
Gnathostomata
Branchiostoma
(cephalochordate)
1009
0.1 substitutions per site
Fig. 1. Deuterostome tree. ML tree calculated from the broadest data set, consisting of concatenated 28S and 18S rRNA genes from 46 deuterostomes and
outgroups, with 3925 aligned nt. The species that were added since the study of Winchell et al. (2002) are indicated by asterisks (when from GenBank or
the literature) and by double asterisks (when newly sequenced here). The Ln L value of this optimal tree is ¡31,242.68. The four support values at each
node are, in order: ML bootstrap percentage, Bayesian-covarion and Bayesian non-covarion posterior probabilities, and MP bootstrap percentage. The
second sets of values at each node, in parentheses, are from the 44-taxon tree, whose sequences were made stationary for nucleotide composition by
removing the hemichordate Cephalodiscus and the tunicate Oikopleura. Nodes with no numbers had ML-bootstrap support <60 and posterior probabilities <95, meaning they are unsupported. For more-accurate and expanded relations within various clades, see Figs. 2–5.
However, this could merely derive from the fact that eight
gamma-rate categories are used in the present likelihood
model, whereas four categories were used previously. Supporting this interpretation, when we reanalyzed the present
data with the fast-ML program, GARLI (D. Zwickl: http://
www.bio.utexas.edu/faculty/antisense/garli/Garli.html), which
only allows four gamma categories, high support for
cephalochordate-with-vertebrates was again achieved
(82%).
Findings that involve the newly added taxa will be presented next.
Echinoderms. Within echinoderms, the brittle star Ophiomyxa grouped with sea star Asterias, as the clade Asterozoa (Janies, 2001), with 82% and 81% ML-bootstrap
support in Fig. 1. Upholding this clade, when we limited the
taxa to just echinoderms, hemichordates, and the cephalochordate in order to increase the number of alignable sites
(to 4275 nt., from 3925), the resulting data set also
Strongylocentrotus (sea urchin)
Cucumaria
(sea cucumber)
100/99
Ophiomyxa (brittle star)
100/100
Asterias (sea star)
69/<50
Echinodermata
88/66
Eleutherozoa
Arbacia (sea urchin)
Asterozoa
100/100
Echinozoa
J. Mallatt, C.J. Winchell / Molecular Phylogenetics and Evolution 43 (2007) 1005–1022
Echinoidea
1010
Florometra (crinoid)
Saccoglossus
(acorn worm)
Harrimania
(acorn worm)
Ptychodera flava
(acorn worm)
100/
100
Ptychodera sp.
(acorn worm)
Outgroup
100/
100
Hemichordata
100/100
Branchiostoma (cephalochordate)
0.1 substitutions per site
Fig. 2. Echinoderm tree. ML tree calculated from the echinoderms and the
nearest outgroups, hemichordates and Branchiostoma; 28S and 18S rRNA
genes with more aligned nucleotides than in the all-deuterostome analysis
of Fig. 1 (4275, cf. 3925, nt.). One hemichordate, Cephalodiscus, was left
out of the outgroup, to make the data set stationary in nucleotide composition (2 D 20; P D 0.91), although the same relations among the echinoderms were obtained when Cephalodiscus was included (tree not shown).
The Ln L value of this optimal tree is ¡14,606.15. The support values at
the nodes are ML and MP bootstrap percentages. ML explores tree space
eVectively when so few taxa are used, so no Bayesian analyses were run on
this small data set.
recovered Ophiomyxa + Asterias (Fig. 2), although with
slightly lower ML-bootstrap support (69%).
Cyclostomes. The newly sequenced rRNA genes from
the Southern Hemisphere lamprey Geotria were remarkably similar to those of the Northern Hemisphere lamprey
Petromyzon, with 5367 of the 5678 shared sites (or 95% of
the entire genes) having identical nucleotides. The new
sequence from hagWsh Myxine was not as similar to that of
hagWsh Eptatretus, as only 5165 of 6235 alignable sites (or
83%) were identical. However, hagWsh rRNA genes have
very long variable regions (Mallatt and Sullivan, 1998), and
the similarity between the conserved cores of the Myxine
and Eptatretus genes was much higher, at least 97.8%. With
such strong similarities, it is not surprising that the lampreys and the hagWshes each formed clades with 100% MLbootstrap support (Fig. 1). Also, lampreys united with
hagWshes as Cyclostomata, with ML-bootstrap values of
99% and 97%.
Gnathostomes (jawed vertebrates). To investigate the
relationships among gnathostomes more closely than in
Fig. 1, we limited the taxa to just vertebrates and the Branchiostoma outgroup (Fig. 3), which provided more data by
increasing the number of alignable sites from 3925 to 4881.
As in the previous study (Winchell et al., 2002), the present
tree (Fig. 3) shows no resolution among the basic clades of
gnathostomes, even though we have now added Dipnoi
(lungWshes) and more actinopterygians (ray-Wnned Wshes).
This 24-taxon data set had a biased nucleotide composition
(2 D 99.0; P D 0.01, see Supplementary material, S2), so we
made it stationary by eliminating the CG-rich sequence of
the hagWsh (2 D 76.7; P D 0.17). However, the resulting 23taxon tree did not resolve the basic gnathostomes either
(see the values in parentheses in Fig. 3).
These trees calculated from 24 and 23 vertebrate taxa
showed monophyly of the Actinopterygii (ML: 77% and
78% in Fig. 3). Tetrapoda (amphibians plus amniotes) was
present in the optimal ML tree, but without support (<50%
ML). This more-complete data set united Protopterus and
Lepidosiren within Dipnoi, unlike in Fig. 1, which was
based on 20% fewer nucleotides. Amniota (reptiles, birds,
and mammals) was well supported (ML: 100% and 99%),
and the turtle Chrysemys was separate from all other
amniotes (ML: 87% and 85%). The sequence of the lizard
Anolis joined to that of the bird Gallus (ML: 79% and 80%),
to the exclusion of mammals. This bird-lizard clade was
also supported when the analysis was conWned to amniotes
(Fig. 4), which increased the number of alignable sites to
5074 nt.
Chondrichthyes (cartilaginous Wshes). Fig. 5 shows the
tree that was calculated after adding sequences from the
sixgill shark Hexanchus and electric ray Narcine to the
chondrichthyans used by Winchell et al. (2004). In
batoids, Narcine joined to a clade consisting of the guitarWsh Rhinobatos and the stingrays Potamotrygon and
Urobatis (96% ML-bootstrap support), to the exclusion
of the skate Raja. Hexanchus grouped strongly with the
other hexanchiform shark, Chlamydoselachus. Otherwise,
the new tree of Fig. 5 is the same as the previous tree
(Fig. 2A in Winchell et al., 2004), with two exceptions: (1)
it has higher ML support for a clade of orbitostylic
sharks (72%, cf. 59%), which consists of the squaliforms
(spiny dogWshes), Squatina (angel shark), pristiophoriforms (saw sharks), and hexanchiforms; and (2) higher
support for locating Squatina in this orbitostylic clade as
the sister taxon to the dogWshes (82%, cf. 63%
previously).
Pboot tests used our rRNA data to evaluate 28
alternate hypotheses about deuterostome relationships, as
presented in Table 2. To improve accuracy, only the
stationary data sets without nucleotide-compositional
bias were used for these tests. These pboot results will be
considered in Section 4.
4. Discussion
4.1. General performance
This study expanded our previous analyses of deuterostome relationships based on nearly complete rRNA genes
(Mallatt and Sullivan, 1998; Winchell et al., 2002, 2004) by
adding 20 taxa and by evaluating the Wndings more thoroughly with the statistical, pboot test. How well did this
approach succeed at reconstructing deuterostome phylogeny? Before answering this question, we must admit that
the »65 taxa used here are still only a small sample of the
deuterostome superphylum, and that rRNA has the
98/100/100/80
(96/100/100/81)
Raja (ray)
69/100/100/87
(76/100/100/85)
Sarcopterygii
Triakis (shark)
100/100/100/99
(100/100/100/97)
Hexanchus** (shark)
<50/<50/84/50
(<50/58/95/<50)
Hydrolagus (ratfish)
1011
Chondrichthyes
J. Mallatt, C.J. Winchell / Molecular Phylogenetics and Evolution 43 (2007) 1005–1022
Oncorhynchus* (trout)
Danio* (zebrafish)
Polypterus* (bichir)
77/100/100/<50
(78/100/100/62)
Acipenser (sturgeon)
All 100%
Chrysemys** (turtle)
<50/88/99/<50
(<50/54/94/<50)
Xenopus (frog)
Rana* (frog)
Amphibia
All
100%
100/100/100/98
(100/100/100/99)
Ambystoma (salamander)
Osteichthyes
79/99/100/76
(80/99/100/73)
Anolis** (lizard)
All 100%
87/99/100/85
(85/99/100/87)
Sarcopterygii
Gallus* (chicken)
Amniota
Rattus* (rat)
100/99/100/91
(99/100/100/96)
Tetrapoda
Homo* (human)
Gnathostomata
96/100/100/99
(97/100/100/99)
Actinopterygii
Siniperca* (perch)
100/100/100/<50
(100/100/100/51)
Teleostei
Latimeria (coelacanth)
Lepidosiren* (lungfish)
79/98/100/85
(78/99/100/83)
Dipnoi
Protopterus* (lungfish)
All 100%
Eptatretus
(hagfish)
Petromyzon
(lamprey)
Branchiostoma
(cephalochordate)
Outgroup
Neoceratodus* (lungfish)
91/99/100/<50
(--- /--- /--- /---)
0.1 substitutions per site
Fig. 3. Vertebrate tree. ML tree calculated from 24 vertebrates and their nearest outgroup, Branchiostoma; 28S and 18S rRNA genes with more aligned
nucleotides than in the all-deuterostome analysis of Fig. 1 (4881, cf. 3925, nt.). Note that neither Osteichthyes (bony Wshes and their descendents) nor Sarcopterygii (lobe-Wnned Wshes and their descendents) appears as monophyletic. The Ln L value of this optimal tree is ¡23,973.8. Asterisks mean the same as
in Fig. 1, as do the support values at the nodes. The second sets of support values, in parentheses, are from the 23-taxon tree, whose sequences were made
stationary for nucleotide composition by removing the hagWsh Eptatretus. For more-accurate relations within the Chondrichthyes, see Fig. 5.
limitation of being just one gene family (albeit an informative one: Giribet, 2002). To answer the question, all seven of
the newly sequenced taxa contributed well-resolved Wndings, as did the chicken and most of the Wsh sequences from
GenBank; these Wndings are summarized in Table 3. Overall, however, the performance of rRNA genes on deuterostomes was merely “good,” rather than excellent. That is,
although our trees successfully recovered some independently established clades (Ambulacraria, Vertebrata,
Cyclostomata, Actinopterygii, Amniota, Batoidea), they
have two major “trouble spots” with poor resolution: Wrst,
at the base of the deuterostomes and chordates (Fig. 1); and
second, at the base of the gnathostomes (Fig. 3). Also unresolved is whether sharks are monophyletic (Fig. 5).
By contrast, nearly complete rRNA performs better on
some other major clades of animals than on deuterostomes.
In studies of the basal lineages of Metazoa (Collins et al.,
2006; Medina et al., 2001; also see Telford et al., 2003), the
relations of ctenophores, cnidarians, and bilaterians were
clearly resolved, and the only trouble spot was in Porifera,
where it was unclear whether the sponges are monophyletic
or diphyletic. In Ecdysozoa, rRNA genes provide strong
1012
J. Mallatt, C.J. Winchell / Molecular Phylogenetics and Evolution 43 (2007) 1005–1022
Homo (human)
Rattus (rat)
100/100
Mammalia
46/48
Canis (dog)
Gallus (chicken)
Anolis (lizard)
Diapsida
95/
86
Chrysemys (turtle)
0.01 substitutions per site
Fig. 4. Amniote tree. ML tree from amniotes, with more aligned nucleotides than for the vertebrate analysis in Fig. 3 (5074, cf. 4881, nt.). The data
set was stationary for nucleotide frequency (2 D 7.3; P D 0.95). The Ln L
value of this optimal tree is ¡9265.1. Support values at the nodes are ML
and MP bootstrap percentages, as in Fig. 2. Relations among the three
mammals are unresolved, but both mammalian monophyly and
‘bird + lizard’ are upheld, as in Fig. 3.
resolution at many diVerent taxonomic levels, with just one
trouble spot at the base of the arthropods (Mallatt and
Giribet, 2006). Additionally, rRNA cleanly resolves relations among the Xatworms of the large Platyhelminthes
phylum (Lockyer et al., 2003).
Nevertheless, rRNA genes perform much better on deuterostomes than they do on the remaining major group of
animals, the Lophotrochozoa (annelids, mollusks, brachiopods, etc.: Halanych et al., 1995). There, very few relationships among the lophotrochozoan phyla are recoverable
with existing tree-building methods (Passamaneck and
Halanych, 2006; also see Passamaneck et al., 2004).
Despite any current diYculties in rRNA-based phylogenetics, we expect rRNA genes to perform much better on all
animal groups in the future. Better ways of aligning by secondary structure, along with new models of base-pair evolution in the rRNA stems, have already improved the
phylogenetic resolution obtained with 18S genes (Hudelot
et al., 2003; Kjer, 2004; Savill et al., 2001; Telford et al.,
2005), and these improvements will soon be available for
28S genes as well (Gillespie et al., 2005).
4.2. Basic deuterostome groups, and a consideration of
parametric bootstrapping
This is the Wrst rRNA gene-based study to test basicdeuterostome relations with parametric bootstrapping
(Table 2, Hypotheses 1–5).
The classical, morphology-based hypothesis of deuterostome interrelationships, favored through most of the 20th
century, said echinoderms are the sister group of all other
deuterostomes, hemichordates group with chordates, and
in chordates the cephalochordates are closest to vertebrates
(Maisey, 1986a; Romer, 1970; SchaeVer, 1987). In this
hypothesis, hemichordates and chordates were united by
the shared characters of gill slits and perceived homologies
of a dorsal, hollow nerve cord and a notochord (see Ruppert, 2005). Part of this hypothesis toppled in the 1990s
under a load of genetic and developmental evidence that
showed hemichordates do not group with chordates but
with echinoderms, as Ambulacraria (Bronham and
Degnan, 1999; Cameron et al., 2000; Castresana et al., 1998;
Furlong and Holland, 2002; Halanych, 1995; MetschnikoV,
1881; Peterson, 2004; Turbeville et al., 1994; Zeng and
Swalla, 2005). Anatomically, the Ambulacraria are united
by the larval characters of similar bands of circum-oral cilia
and an asymmetric heart–kidney (Ruppert, 2005).
The currently dominant hypothesis of deuterostome
relationships (Kardong, 2006; Zeng and Swalla, 2005)
accepts Ambulacraria and Chordata but it still joins cephalochordates with vertebrates as Euchordata, based on such
characters as cardinal veins, hepatic-portal veins, and myomere segments—which tunicates lack (Mallatt and Chen,
2003). Now, however, this Euchordata concept faces a challenge from phylogenomic studies that recover tunicates as
the sister group of vertebrates (Delsuc et al., 2006; also see
Blair and Hedges, 2005; Philippe et al., 2005). These studies
used large numbers of concatenated, nuclear proteinencoding genes, up to 34,000 amino acids in total length.
In the present study, the rRNA genes recovered Ambulacraria with strong support, but they did not provide any
other resolution among the basal-deuterostome groups, as
they failed to recover a monophyletic Chordata (Fig. 1).
Suspicion as to the source of the problem falls on the tunicates, whose entire genomes are highly divergent (Delsuc
et al., 2006; Gissi et al., 2004; Holland and Gibson-Brown,
2003; Yokobori et al., 1999). Tunicate rRNA genes are ATrich, are shortened as part of a genome-wide simpliWcation
(Delsuc et al., 2006), and, attesting to their rapid evolution,
are diYcult to align with the rRNA genes of other bilaterian animals (personal observation). In our rRNA tree
(Fig. 1), tunicates were at the base of the deuterostomes.
This position had no ML-bootstrap or Bayesian support,
however, and given the peculiarities of tunicate genes, it
might be interpreted as an artifact of long-branch attraction toward the protostome outgroup (Bergsten, 2005). To
test this interpretation we used pboot to evaluate the dominant, alternate hypothesis that tunicates are in Chordata
(Table 2, Hypothesis 1: D 3.6). Parametric bootstrapping
accepted this hypothesis, whose best-supported constraint
tree had a Chordata consisting of tunicates plus euchordates (personal observation). Therefore, tunicates could be
chordates but act as rogue taxa in Fig. 1.
All the other hypotheses of deuterostome relationships
were rejected by parametric bootstrapping (Table 2,
Hypotheses 2–5). These hypotheses are, from the least- to
the most-strongly rejected:
Hypothesis 2: Tunicates, not cephalochordates, are the
sister lineage of vertebrates ( D 13.4). Thus, the rRNA
J. Mallatt, C.J. Winchell / Molecular Phylogenetics and Evolution 43 (2007) 1005–1022
88/99/100/91
1013
Centroscymnus (roughskin dogfish)
Squal.
92/100/100/90
Dalatias (kitefin shark)
82/99/100/75
Squalus (piked dogfish)
92/100/100/89
Squatina (Pacific angel shark)
Prist.
Pristiophorus (longnose saw shark)
72/90/96/88
Hexanchus** (bluntnose sixgill shark)
Hexan.
92/100/92/100
<50/84/96/71
Orbitostylic sharks
Centroscyllium (black dogfish)
Deania (birdbeak dogfish)
Sharks
Chlamydoselachus (frilled shark)
Heterodontus (horn shark)
93/100/100/90
97/100/100/92
Lamn.
Carcharias (sand tiger shark)
All 100%
Hemiscyllium (epaulette shark)
Orect.
Orectolobus (ornate wobbegong shark)
51/93/100/<50
Triakis (leopard shark)
100/100/100/89
Galeocerdo (tiger shark)
97/100/100/100
Galeomorphs
Mitsukurina (goblin shark)
69/99/100/<50
Apristurus (deepwater cat shark)
96/100/
100/94
Carch.
Scyliorhinus (cloudy cat shark)
All 100%
All 100%
All
100%
All 100%
Urobatis (Carribean yellow stingray)
Rhinobatos (shovelnose guitarfish)
Narcine** (lesser
electric ray)
Raja (browneye skate)
Hydrolagus (ratfish)
Callorhynchus (Southern elephantfish)
Acipenser (sturgeon)
Latimeria (coelacanth)
Osteichthyes
(outgroup)
Holocephali
96/99/
100/65
Potamotrygon (porcupine river stingray)
Batoidea
88/100/100/72
All 100%
Neoselachii
Alopias (pelagic thresher shark)
46/82/95/51
0.1 substitutions per site
Fig. 5. Chondrichthyes tree. ML tree from cartilaginous Wshes, based on the 28S, 5.8S, and 18S-gene data set of Winchell et al. (2004), to which sequences
from Hexanchus and Narcine (marked with asterisks **) have been added; 5320 aligned nt. The data set was stationary for nucleotide frequency (2 D 13.3;
P D 1.0). The Ln L value of this optimal tree is ¡18,338.9. Support values listed at the nodes are as in Fig. 1. Abbreviations: Squal., Squaliformes; Prist.,
Pristiophoriformes; Hexan., Hexanchiformes; Lamn., Lamniformes; Orect., Orectolobiformes; Carch., Carcharhiniformes.
data rejected the new ‘tunicates-plus-vertebrates’
hypothesis that was based on phylogenomic studies
(Delsuc et al., 2006; Philippe et al., 2005).
Hypothesis 3: Cephalochordate Branchiostoma plus
Ambulacraria ( D 15.1). The rRNA data reject a linking of cephalochordates with echinoderms, a link that
was tentatively suggested by the phylogenomic study of
Delsuc et al. (2006). Those authors were careful to note,
however, that their statistical support for this clade was
non-signiWcant, and their study did not include any
sequence from the key, hemichordate, phylum. When a
hemichordate was recently added to a comparable
multi-gene data set (Bourlat et al., 2006), the cephalochordate-echinoderm link collapsed in favor of chordate monophyly.
Hypothesis 4: Tunicates plus cephalochordate Branchiostoma ( D 24.7). Jollie (1973) united these two clades
because both have an atrium around their pharynx; they
also share similarities of their notochords (Ruppert,
2005). Jollie (1973) was an early advocate of the Ambulacraria concept, so his placement of tunicates with cephalochordates is the only way in which his hypothesis
diVers from the currently dominant one. Even so, the value of 24.7 seems high enough to indicate that rRNA
strongly resists joining tunicates and cephalochordates
as sister taxa.
Hypothesis 5: Hemichordates plus chordates ( D 58.0).
This is the classical hypothesis, and it is the most
strongly rejected. Its only distinguishing feature is that it
separates hemichordates from echinoderms, so its
1014
J. Mallatt, C.J. Winchell / Molecular Phylogenetics and Evolution 43 (2007) 1005–1022
Table 2
Parametric bootstrapping: results of tests of alternate hypotheses of deuterostome relationships Hypotheses 1–28, listed as constraint trees; rejection is at P<0.01
Hypothesis
h
Range of 100
simulated-diVerence
values (conWdence
interval)
Reject or
Accept?
A. Basic deuterostome groupsa: Best tree (Fig. 1): ((Tunicates, T; ((Hemichordates, H; Echinoderms, E),
(Branchiostoma, B; Vertebrates, V))), outgroup, protostomes)
1. Chordata: (outgroup, E, H, (T, B, V))
2. Tunicates plus vertebrates: (outgroup, E, H, B, (T, V))
3. Branchiostoma plus Ambulacraria: (outgroup, T, V, (E, H, B))
4. Tunicates plus Branchiostoma: (outgroup, ((E, H), ((T, B), (V)))): Jollie (1973)
5. Hemichordates plus chordates: (outgroup, E, (H, (T, (A, V))))
3.6
13.4
15.1
24.7
58.0
0–6.4
0–3.9
0–5.0
0–7.5
0–3.5
Accept
Reject
Reject
Reject
Reject
B. Echinodermsb: Best tree (Fig. 2): (Crinoid, C; ((Ophiuroid, O; Asteroid, A), (Echinoids, E; Holothuroid, H))),
outgroup, enteropneusts and Branchiostoma)
6. Cryptosyringida: (C, (A, (O, (E, H))))
7. Asteroidea with Echinozoa: (outgroup, O, (A, (E, H)))
8. Ophiuroidea with Echinoidea: (outgroup, A, H, (O, E))
2.6
3.9
14.1
0–1.9
0–2.3
0–4.7
Reject
Reject
Reject
C. Cyclostomatac: Best tree (Fig. 1): same tree as in Part A above, with the vertebrates subdivided as: ((Lampreys,
L; HagWshes, H), Gnathostomes, G)
9. Lampreys plus gnathostomes: (all others, H, (L, G))
21.7
0–2.7
Reject
2.1
5.7
6.1
7.2
9.1
9.4
9.4
13.5
0–3.9
0–3.2
0–4.8
0–4.5
0–5.5
0–3.9
0–3.9
0–3.2
Accept
Reject
Reject
Reject
Reject
Reject
Reject
Reject
4.8
6.3
0–5.2
0–4.8
Accept
Reject
3.2
6.5
0–3.7
0–3.1
Accept
Reject
7.5
7.7
10.2
29.6
50.7
75.1
0–2.0
0–3.1
0–3.1
0–4.6
0–3.3
0–5.5
Reject
Reject
Reject
Reject
Reject
Reject
94.9
0–8.1
Reject
d
D. Basic gnathostome groups : Best tree (Fig. 3): ((LungWshes, L; ((Chondrichthyes, Ch; Coelacanth, Co),
(Actinopterygii, A; Tetrapoda, T))), outgroup, lamprey and Branchiostoma)
10. LungWshes plus tetrapods: (outgroup, Ch, A, Co, (L, T))
11. LungWshes plus coelacanth: (outgroup, Ch, A, T, (L, Co))
12. Coelacanth plus tetrapods: (outgroup, Ch, A, L, (Co, T))
13. Bird plus mammals: (outgroup, Ch, A, L, Co, Amphibians, Turtle, Lizard, (Bird, Mammals))
14. Sarcopterygii: (outgroup, Ch, A, (Co, L, T))
15. Osteichthyes: (outgroup, Ch, (A, Co, L, T))
16. Accepted gnathostome phylogeny: (outgroup, (Ch, (A, (Co, (L, T)))))
17. Turtles as diapsids: (outgroup, Ch, A, L, Co, Amphibians, Mammals, (Turtle, Lizard, Bird))
E. EVects of lungWshes on the gnathostome tree: Best tree after lungWshes removede: ((((Co, Ch), A), T),
outgroupDlamprey and Branchiostoma), and best tree after coelacanth removedf: ((L, (Ch, (A, T))),
outgroupDlamprey and Branchiostoma). (Same taxon abbreviations as in Part D above.)
18. Osteichthyes (after lungWshes removed from the data set): (outgroup, Ch, (A, (Co, T)))e
19. Osteichthyes (after coelacanth removed from the data set): (outgroup, Ch, (A, (L, T)))f
F. Elasmobranchsg : Best tree (Fig. 5): (((Batoids, B; ((Carcharhiniformes, C; (Lamniformes, L; Orectolobiformes,
O)), (Heterodontus, Ht; (Hexanchiformes, Hx; (Squaliformes with Pristiophorus and Squatina, S)))))),
Holocephali, H; outgroup, Latimeria and Acipenser)
20. Hexanchiformes as sister group of all other elasmobranchs: (outgroup, H, ((Hx), (other sharks and batoids)))
21. Narcine as sister group of other batoids: (outgroup, H, sharks, Narcine, (Raja, Rhinobatos, Potamotrygon,
Urobatis))
22. Lamniformes with Carcharhiniformes: ((L, C), (other elasmobranchs), H, outgroup)
23. Heterodontus as a galeomorph shark: ((Ht, C, L, O), other elasmobranchs, H, outgroup)
24. Heterodontus as sister group of all other elasmobranchs: (outgroup, ((H), (Ht, (other sharks and batoids))))
25. Hexanchiformes paraphyletic: (outgroup, H, other elasmobranchs, (Chlamydoselachus, (Hexanchus, (S))))
26. Hypnosqualea: ((B, Squatina, Pristiophorus), other elasmobranchs, H, outgroup)
27. Maisey (1980): (outgroup, H, ((B), ((C, L, O, Ht), (Squatina, (Chlamydoselachus, (Hexanchus,
(Squaliformes)))))))
28. Squalea: ((Hx, Squatina, Pristiophorus, B, Squaliformes), other elasmobranchs, H, outgroup)
Note. In each set of hypotheses, A–E, hypotheses are listed in order from best-supported to worst-supported, as indicated by the sizes of their values.
a
The 41 taxa used for these basic-deuterostome tests were the same as in Fig. 1, except Oikopleura and Cephalodiscus were omitted in order to yield stationary
nucleotide frequencies, and Siniperca, Danio, and Canis were omitted because several other ray-Wnned Wshes and mammals were already present. LnL of the best tree
was ¡27,961.01.
b
The 11 taxa used for these echinoderm tests were the same as in Fig. 2. LnL of the best tree was ¡14,606.15.
c
The 41 taxa used for this lamprey/hagWsh test were the same as for the basic-deuterostome tests above. LnL of the best tree was ¡27,961.01.
d
The 21 taxa used for these basic-gnathostome tests were the same as in Fig. 3, except Eptatretus, Danio and Siniperca were left out. LnL of the best tree was
¡20,686.4.
e
The 18 taxa used for this no-lungWsh test were the same as in Fig. 3, except Neoceratodus, Lepidosiren, Protopterus, Eptatretus, Danio and Siniperca were left out.
LnL of the best tree was ¡17,837.05.
f
The 20 taxa used for this no-coelacanth test were the same as in Fig. 3, except Latimeria, Eptatretus, Danio and Siniperca were left out. LnL of the best tree was
¡20,235.48.
g
The 28 taxa used for these elasmobranch tests were the same as in Fig. 5. LnL of the best tree was ¡18,338.9.
h
is the diVerence between the likelihood scores (LnL) of (1) the absolute optimal tree and (2) the best tree constrained to Wt the alternate hypothesis (both these
trees having been calculated from the actual data, not from the simulated data). A hypothesis is rejected when is larger than the range of 100 simulated-diVerence
values.
J. Mallatt, C.J. Winchell / Molecular Phylogenetics and Evolution 43 (2007) 1005–1022
1015
Table 3
Summary of results contributed by the 20 taxa added in this study
Taxa
Result contributed
Taxa sequenced in this study, for rRNA genes
1. Ophiomyxa (brittle star)
2. Myxine (hagWsh)
3. Geotria (lamprey)
4. Hexanchus (sixgill shark)
5. Narcine (electric ray)
6. Chrysemys (turtle)
7. Anolis (lizard)
1. Brittle star is sister taxon of sea star Asterias
2. Upholds Cyclostomata; low diversity within hagWshes
3. Upholds Cyclostomata; low diversity within lampreys
4. Extremely similar to frilled shark, Chlamydoselachus; monophyletic Hexanchiformes
5. Raja (skate), not Narcine, is sister taxon of other batoids
6. Turtle sister to the other amniotes (Anapsida concept)
7. Bird (Gallus) with lizard as diapsid reptiles
Taxa whose sequences were taken from GenBank and the literature
8. Arbacia (sea urchin)
8. (As expected, Arbacia grouped with the other sea urchin, Strongylocentrotus)a
9. Neoceratodus (Australian lungWsh)
9–11. Although position of lungWshes within gnathostomes was unresolved,
10. Lepidosiren (South American lungWsh)
‘lungWshes-plus- tetrapods’ is the only grouping that was not rejected; Lepidosiren plus
11. Protopterus (African lungWsh)
Protopterus form Lepidosireniformes
12. Polypterus (bichir)
12. Polypterus in a monophyletic Actinopterygii
13. Oncorhynchus (trout)
13–15. Monophyletic Teleostei, in which
14. Danio (zebraWsh)
trout grouped with perch to the exclusion
15. Siniperca (perch)
of zebraWsh
16. Rana (frog)
16. (As expected, Rana grouped with frog Xenopus in Anura)a
17. Gallus (chicken)
17. Bird with lizard (Anolis) as diapsid reptiles
18. Rattus (rat)
18–20. (Strong monophyly of these three mammals)a
19. Homo (human)
20. Canis (dog)
a
These results in parentheses are minor, or obvious, contributions.
rejection means our rRNA data strongly support Ambulacraria. Indeed, in Fig. 1, Ambulacraria has high MLbootstrap values of 91% and 89%.
Parametric bootstrapping. A careful consideration of the
pboot method suggests that some of the above hypotheses
should not be rejected out of hand. That is, pboot (and
other statistical methods that simulate gene sequences
based on evolutionary models of nucleotide substitution)
tend to reject alternate hypotheses too readily (Antezana,
2003; Buckley, 2002). This is because the methods are sensitive to model misspeciWcation, and current models are still
too crude (Jermiin et al., 2005). Additionally, most of the
models—including the GTR + I + model used here—
assume that the processes of rRNA evolution were the
same across all taxonomic lines, and this is demonstrably
false for the tunicates, with their shortened and exceptionally AT-rich rRNA genes. Violation of model assumptions
causes the simulated sequences to be too homogeneous,
producing conWdence intervals that are too small (F. Delsuc, University of Montpellier: personal communication).
This means that hypotheses involving the peculiar tunicates
should be rejected only if their likelihood scores diVer
greatly from the score of the optimal hypothesis—so, for
example, it is not clear whether a of 13.4 is large enough
to reject the ‘tunicates-plus-vertebrates’ hypothesis
(Hypothesis 2). In summary, until better models of nucleotide substitution become available, it is safest to consider
tunicates as essentially unclassiWable within chordates, by
rRNA genes.
Despite these uncertainties about using parametric
bootstrapping for determining absolute support levels, it
is still valuable to compare the relative sizes of the Ln L
and values from diVerent hypotheses, because such
comparison shows which hypotheses are better
supported than others.1
4.3. SpeciWc subclades
4.3.1. Echinoderms
For the Wve major clades of echinoderms, most studies
have found that Crinoidea is the sister lineage of the other
four, which form the Eleutherozoa; and that in Eleutherozoa, the Echinoidea (sea urchins and sand dollars) join with
Holothuroidea (sea cucumbers) to form the Echinozoa
(Janies, 2001; Littlewood et al., 1997; Ruppert et al., 2004;
Wray, 1998). However, the relative positions of Ophiuroidea (brittle stars) and Asteroidea (sea stars) within
Eleutherozoa are debated. The most extensive morphological and molecular-phylogenetic studies have had diYculty
distinguishing between two preferred arrangements: the
Asterozoa hypothesis that joins the two stars ((Ophi,
Aster), (Echinoidea, Holo)) (Littlewood et al., 1997; Smith,
1988; Wray, 1998), and the Cryptosyringida hypothesis that
joins the brittle stars with Echinozoa (Aster, (Ophi,
(Echinoidea, Holo))). Additionally, the analyses of
Littlewood et al. (1997) favored a third arrangement, of the
1
Along with parametric bootstrapping, we also used the SH test (Shimodaira and Hasegawa, 1999) to evaluate the hypotheses in Table 2. However, the SH test proved so conservative that it rejected almost nothing,
which seems to happen whenever this type of test is applied to this sort of
sequence data (see Inoue et al., 2003; Zardoya et al., 1998). The SH test is
more vulnerable to criticism than is parametric bootstrapping: It makes
many simplifying assumptions and is not as rigorously model-based (see
Goldman et al., 2000).
1016
J. Mallatt, C.J. Winchell / Molecular Phylogenetics and Evolution 43 (2007) 1005–1022
asteroid-sea stars with Echinozoa (Ophi, (Aster, (Echinoidea, Holo))); and a fourth possibility joins Ophiuroidea
with Echinoidea because these two clades share some larval
traits, such as a pluteus stage (see p. 416 in Littlewood et al.,
1997).
Our rRNA-gene trees support the Asterozoa hypothesis
uniting brittle star with sea star, while also recovering all
the accepted relations among echinoderms, namely
Eleutherozoa and Echinozoa (Figs. 1 and 2). Furthermore,
our pboot tests reject all the alternate hypotheses (Table 2:
Hypotheses 6–8) in the following order, from least-to moststrongly rejected: Cryptosyringida < (Ophi, (Aster, (Echinoidea, Holo))) < Ophiuroidea + Echinoidea. Ours is the
strongest support for Asterozoa ever obtained, because the
18S and mitochondrial-genes used in past-studies did not
contain enough signal to provide a well-supported decision
(Janies, 2001; Littlewood et al., 1997; Scouras and Smith,
2006). Proposed synapomorphies of Asterozoa are a saccate gut and undiVerentiated ambulacral ossicles (Janies,
2001).
4.3.2. Cyclostomes
Morphological studies tend to unite lampreys with
gnathostomes and to place hagWshes as the sister taxon to
all the other vertebrates (Donoghue et al., 2000; Hardisty,
1982; Janvier, 1996; Mallatt, 1984). HagWshes, however,
have so many autopomorphic traits that their “primitive”
characters could instead be secondary reversions to a primitive state, artifactually causing hagWshes to be displaced
basally in morphology-based phylogenies (also see Mallatt,
1997). Molecular-phylogenetic studies paint a diVerent picture of lamprey–hagWsh relationships. Starting with 18S
rRNA (Stock and Whitt, 1992), many genes have joined
lampreys with hagWshes in a monophyletic Cyclostomata,
which is the sister group of gnathostomes (Blair and
Hedges, 2005; Cotton and Page, 2002; Delsuc et al., 2006;
Furlong and Holland, 2002; Kuraku et al., 1999; Takezaki
et al., 2003).
Mallatt and Sullivan (1998) added 28S to 18S rRNA
genes and also recovered Cyclostomata, but their and most
other molecular studies used just one species of lamprey
and hagWsh, raising the possibility that adding more species
might change the result. To address this, we here sequenced
the rRNA genes of another hagWsh and lamprey that are,
taxonomically, as distant as possible from those used previously (Fernholm, 1998; Hardisty, 1979; Hubbs and Potter,
1971). Myxine represents the hagWshes with a single, external gill-opening (Myxininae), whereas Eptatretus represents
the hagWshes with multiple gill-openings (Eptatretinae).
Geotria represents the Southern Hemisphere lampreys,
whereas Northern Hemisphere lampreys (e.g., Petromyzon)
were used in every previous molecular-phylogenetic study.
We found that the rRNA genes of the two hagWshes are
quite similar, and so are the genes of the two lampreys (as
indicated by the short branches to the cyclostome genera in
Fig. 1). Our new tree upholds our original Wnding of a
monophyletic Cyclostomata (Mallatt and Sullivan, 1998;
Winchell et al., 2002), still with high support (>97%). Also,
pboot continues to reject the alternate hypothesis of ‘lampreys plus gnathostomes’ (Table 2, Hypothesis 9). In fact,
the rejection is much stronger now, with a value of 21.7
here compared to 5.2 in the previous study (Mallatt and
Sullivan, 1998).
4.3.3. Base of the gnathostomes
Our rRNA trees provide no resolution among the major
groups of gnathostomes: the chondrichthyans, actinopterygians, lungWshes, coelacanth, and tetrapods (Figs. 1 and 3).
By contrast, comprehensive studies based on many genes
(Kikugawa et al., 2004; Takezaki et al., 2003, 2004) support
the widely accepted scheme of chondrichthyans versus osteichthyans (bony Wshes), with the latter divided into actinopterygians (the ray-Wnned Wshes) and sarcopterygians (the
lobe-Wnned Wshes: lungWshes, coelacanth, and tetrapods)
(Janvier, 1996; Maisey, 1996; Meyer and Zardoya, 2003).
The lack of resolution from rRNA genes might be dismissed as nothing more than a locally poor performance in a
region of the vertebrate tree where all other phylogenetic
analyses that were based on a small number of genes also
had diYculty—a diYculty that has been attributed to a rapid
radiation of the gnathostomes right after they Wrst appeared
over 400 million years ago (Meyer and Zardoya, 2003; Takezaki et al., 2004; Zardoya et al., 1998). However, our rRNAbased Wndings cannot be dismissed so easily because, suspiciously, they reject the monophyly of Osteichthyes and of
Sarcopterygii (Table 2; Hypotheses 14–16). This challenges
the fundamental value of rRNA genes as a phylogenetic
marker within the vertebrates, because although markers
may sometimes fail to resolve relationships, they are not supposed to reject valid clades. Nonetheless, we hesitate to
accept the pboot tests’ rejections of Osteichthyes and Sarcopterygii, because of the utter lack of support against or for
these clades in the phylogenetic trees (Figs. 1 and 3).
Paraphyly of Osteichthyes and of Sarcopterygii would
match the Wndings of Arnason et al. (2001, 2004), but those
Wndings were from mitochondrial genomes, which evolve
faster and lose signal at deep-phylogenetic levels (Cotton
and Page, 2002; Hassanin, 2006; Iwabe et al., 2005; Kikugawa et al., 2004; Naylor and Brown, 1998; Yokobori et al.,
1999). This means that the lamprey, the only outgroup used
by Arnason et al. (2001, 2004), could be too distant an outgroup to allow resolution among the basal gnathostomes in
a mitochondrial-genome study.
If Osteichthyes and Sarcopterygii are valid clades after
all, then their rejection in the present study must be
explained. It might have resulted from the tendency of the
pboot test to reject too readily (see above), and more
importantly, from the nature of the rRNA genes of
lungWshes. LungWshes2 have some of the most AT-biased
2
The following discussion of lungWshes assumes their 28S sequences in
GenBank are accurate, the possibility of sequencing error being raised by
the fact that the two independently obtained sequences from Lepidosiren
paradoxica (U34337 and AJ306594) are only 92% identical.
J. Mallatt, C.J. Winchell / Molecular Phylogenetics and Evolution 43 (2007) 1005–1022
and divergent rRNA genes among gnathostomes (see S2 in
Supplementary material and the long branches to the
lungWshes in Fig. 3), so they might be rogue taxa that disrupt gnathostome relations through long-branch attraction
toward the base of the gnathostomes in the tree.
To test this interpretation, we removed the lungWsh
sequences from the vertebrate data set, then ran a pboot
test, and found that pboot now accepted the monophyly of
Osteichthyes and of Sarcopterygii (Table 2, Hypothesis 18).
As a control step to check this Wnding, we re-inserted the
lungWsh sequences and then removed the coelacanth
(Latimeria)—another sarcopterygian of uncertain placement on the rRNA trees in Figs. 1 and 3—and re-ran the
pboot test. Now, with the lungWshes back in the data set,
pboot rejected both Osteichthyes and Sarcopterygii again
(Table 2, Hypothesis 19), supporting our interpretation that
the lungWshes are rogue taxa that disrupted these two
clades in the Wrst place.
The relationships among the three subgroups of sarcopterygians—lungWshes, coelacanth, and tetrapods—have
generated much debate (reviewed by Meyer and Zardoya,
2003). Obviously, we cannot resolve this debate, because we
did not even recover a monophyletic Sarcopterygii. However, it is worth reporting that our pboot tests accept the
dominant hypothesis (Brinkmann et al., 2004b; Zardoya
et al., 2003) that lungWshes group with tetrapods (Table 2,
Hypothesis 10), while rejecting the two alternate hypotheses of lungWshes with coelacanth (Hypothesis 11) and of
coelacanth with tetrapods (Hypothesis 12).
4.3.4. Within lungWshes and Actinopterygii
LungWshes. Our most-complete vertebrate data set
(Fig. 3) grouped the South American Lepidosiren with the
African Protopterus with 79% and 78% ML-bootstrap support, separate from the Australian Neoceratodus. This
matches the classical division of lungWshes into Lepidosireniformes and Ceratodontiformes, which is also supported
by nuclear and mitochondrial protein-coding genes (Brinkmann et al., 2004a,b).
Actinopterygii. The monophyletic Actinopterygii recovered here (Fig. 3) contained the bichir Polypterus, whose
inclusion in this clade has been debated (Janvier, 1996;
Gardiner et al., 2005). Within the monophyletic teleosts,
perch Siniperca joined with trout Oncorhynchus to the
exclusion of zebraWsh Danio. All these Wndings were previously attained with mitochondrial genes, which are good at
resolving relations within the actinopterygians (Inoue et al.,
2003; Miya et al., 2005).
4.3.5. Amniotes
Among the amniotes, bony and soft structures indicate
that birds are most closely related to crocodilians as archosaur reptiles, and that lizards and snakes are the next closest to the archosaurs, all within the Diapsida (Currie et al.,
2004; Gauthier et al., 1988; Kardong, 2006). The position of
turtles in amniotes, however, is debated (http://tolweb.org/
tree/phylogeny.html). Although we lack rRNA sequences
1017
from the key crocodilian clade, our Wndings bear on these
questions.
Birds. Even though some recent molecular-phylogenetic
studies do recover birds as reptiles (Meyer and Zardoya,
2003), the pioneering studies with 18S rRNA genes joined
birds with mammals instead (Hedges et al., 1990; Hedges
and Poling, 1999; Huelsenbeck et al., 1996a; Marshall,
1992). A few workers truly believed in this unorthodox
‘bird + mammal’ view (e.g., Gardiner, 1982), but others felt
that the 18S rRNA gene, once touted as the “universal
yardstick of molecular phylogenetic reconstruction” (Xia
et al., 2003), was failing seriously by supporting such an
unlikely clade (Huelsenbeck et al., 1996a).
Xia et al. (2003) re-examined this question by improving
the 18S-based analysis; that is, they upgraded the 18S data
set, alignments, and evolutionary models. They found that
18S genes need not support a grouping of birds with mammals after all. However, they mostly used distance-based
phylogenetic methods and when they instead used the preferable, character-based method of ML (with a GTR + I + model), the ML tree contained questionable clades: that is,
although the bird and crocodile did go together, their nextnearest neighbor was the mammal, to the exclusion of all
other reptiles.
Suspecting that the 18S gene evolves too slowly to reveal
such relations among the amniotes, we added the 28S genesequences, 28S being longer and containing more variable
sites. The resulting trees supported the orthodox hypothesis
of ‘bird + lizard’ as diapsids, with ML-bootstrap values
>79% (Figs. 1, 3, 4), and pboot rejected the ‘bird + mammal’
hypothesis (Table 2, Hypothesis 13). However, our rRNA
evidence for ‘bird + lizard’ is not entirely conclusive, for
three reasons: The value for rejecting ‘bird + mammal,’ at
7.2, is not large; the lizard Anolis sequence was not quite
complete (789%); and a crocodilian sequence is needed.
Turtles. There has been much debate over whether turtles are primitively ‘anapsids’ (with no temporal opening in
the skull) or whether their anapsid condition was secondarily derived from a diapsid ancestor (with two temporal
openings: Meyer and Zardoya, 2003; Zardoya and Meyer,
2001). Some of the morphological and most of the molecular-phylogenetic evidence places turtles in Diapsida (Cao
et al., 2000; de Braga and Rieppel, 1997; Hill, 2005; Iwabe
et al., 2005; Rieppel and Reisz, 1999; Zardoya and Meyer,
1998). By contrast, our present Wndings support the original
form of the anapsid hypothesis, with turtles as the sister
group of all the other amniotes (Lee, 1997, 2001; Romer,
1956, 1966; Willinston, 1917). In fact, the pboot test rejected
the hypothesis of turtles as diapsids (Table 2, Hypothesis
17) more soundly than any other hypothesis we tested with
the vertebrate data set; that is, its value of 13.5 was the
highest. Our Wnding is unprecedented among molecularphylogenetic studies, and it certainly must be re-examined
by adding rRNA sequences from more reptiles. Even so,
rRNA genes have been said to support turtles-as-diapsids
(Hedges and Poling, 1999), and our study, which added
complete 28S genes, shows that this need not be so.
1018
J. Mallatt, C.J. Winchell / Molecular Phylogenetics and Evolution 43 (2007) 1005–1022
4.3.6. Relations within elasmobranchs
Here, we added rRNA-gene sequences from electric ray
Narcine and sixgill shark Hexanchus to the 26 sequences
from our earlier study of elasmobranch relationships
(Winchell et al., 2004). That study used pboot to test several
hypotheses, which we re-tested here along with Wve new
hypotheses (Table 2: Hypotheses 20–28).
The only alternate hypothesis not rejected by pboot was
a very old view that the hexanchiform sharks are the sister
group of all other neoselachians (living elasmobranchs)
(Table 2, Hypothesis 20). This view arose because hexanchiforms seem to share some primitive characters with extinct
Paleozoic sharks, such as a notochordal spinal column
without vertebral centra and a post-orbital joint between
the upper jaw and the braincase (Compagno, 1977; Gudger
and Smith, 1933). However, no modern morphological-taxonomic study has supported this view, and our rRNA-gene
tree does not either (Fig. 5). Instead, this optimal tree shows
ML-bootstrap support (72%) for the modern view that
places the hexanchiforms with squaliforms, pristiophoriforms and Squatina (de Carvalho, 1996).
Among the most strongly rejected hypotheses, with values of 50.7 and 94.9, are the concepts of Hypnosqualea
and Squalea (Shirai, 1992, 1996). These hypotheses (#26
and #28 in Table 2) say batoids are derived versions of
squaliform sharks, akin to pristiophoriforms, Squatina, and
the dogWsh Squalus. They were based on morphological
characters, and no molecular-phylogenetic study has ever
supported them (Douady et al., 2003; Human et al., 2006;
Fig. 3 in Winchell et al., 2004).
Less strongly rejected by pboot are the traditional
hypotheses that unite the highly predaceous lamniform and
carcharhiniform sharks ( D 7.5; Hypothesis 22); (see de
Carvalho, 1996) and that place the horn shark Heterodontus in Galeomorpha ( D 7.7; Hypothesis 23); (Compagno,
1973). Given the tendency of pboot tests to reject too readily, these two questions should probably be considered
unresolved. It should be noted, however, that the Heterodontus-as-galeomorph hypothesis went from ‘accept’ in
our previous study (Winchell et al., 2004) to ‘reject’ here.
Morphological studies also have diYculty positioning Heterodontus within the elasmobranchs (Compagno, 1973,
1977; Maisey, 1983). In any case, our data more-Wrmly
rejected a diVerent hypothesis about Heterodontus (Maisey,
1982, 1989), which claimed it is a basal neoselachian and
the sister taxon of all other living elasmobranchs ( D 10.2;
Hypothesis 24).
The hypothesis of Maisey (1980) united squaliforms,
pristiophoriforms, hexanchiforms, and Squatina into a
clade of ‘orbitostylic sharks’ (Hypothesis 27 in Table 2),
because all these sharks have a projection into the orbit
from the upper-jaw cartilage. At Wrst glance, our data seem
to reject this hypothesis and even more strongly than
before, with a of 75.1 here compared to the 18.0 found by
Winchell et al. (2004). However, the only reason the rejection is stronger now is because Maisey (1980) originally
advocated paraphyly of the hexanchiforms, which grossly
contradicts our new data (see below). Later, Maisey
accepted hexanchiform monophyly (Maisey and Wolfram,
1984; Maisey, 1986b), and when his 1980 hypothesis is
updated to include this, it becomes less inconsistent with
our rRNA data, with the now reduced to 29.9. The continued rejection of Maisey’s updated hypothesis must be
due to only minor diVerences between that hypothesis and
our optimal tree, such as the precise position of Squatina,
because our tree does support Maisey’s orbitostylic clade
(Fig. 5), which was the heart of his hypothesis anyway. In
fact, our ML-bootstrap support for orbitostylic sharks
increased in the present study, to a respectable 72% (Fig. 5)
from a weak 59% in our previous study (Winchell et al.,
2004). Thus, ‘orbitostylic sharks’ is upheld and strengthened.
ML bootstrapping supported the skate Raja as the sister
lineage of the other four batoids (Fig. 5). This diVers from
morphology-based Wndings, which place the torpedinoid
rays (Narcine here) or sawWshes (pristiforms, not sampled
here) in this basal position and join Raja with rhinobatids
(see McEachran and Aschliman, 2004; McEachran et al.,
1996; and Shirai, 1996). Our pboot test rejected the orthodox hypothesis of Narcine as the sister taxon of all other
batoids (Table 2, Hypothesis 21) but the value was only
6.5, perhaps lower than one might predict from the high
support for Raja in this position instead (ML: 96% in
Fig. 5). Narcine’s rRNA genes are the most divergent of all
those in the elasmobranch study, with by far the longest
branch in Fig. 5. Thus, Narcine may be artifactually misplaced on the gene tree.
Our tree strongly supports a monophyletic Hexanchiformes consisting of Hexanchus and Chlamydoselachus (ML
bootstrap: 92%, Fig. 5). In fact, the rRNA genes of these two
genera are almost identical, with 5224 of 5246 sites (99.6%)
sharing the same nucleotide. By contrast, Chamydoselachus is
distinct enough in its teeth, vertebral column, and jaw attachment that some morphologists have placed it outside a clade
consisting of the other hexanchiforms plus all the squaliforms (e.g., Bigelow and Schroeder, 1948; Herman et al.,
1993, 1994). This claim for hexanchiform paraphyly was reexamined by de Carvalho (1996), who reaYrmed the monophyly of this taxon, based on such synapomorphies as more
than Wve gills and just a single dorsal Wn. Our pboot analysis
of rRNA genes likewise rejects hexanchiform paraphyly
(Table 2; Hypothesis 25), and the value of 29.6 is quite high
considering that this hypothesis involved moving just one
taxon (Chlamydoselachus) just one step lower than in the
optimal tree. Clearly, the rRNA-gene sequences speak
strongly against splitting the hexanchiforms.
5. Conclusions
This study added and retrieved nearly complete rRNAgene sequences from 20 more deuterostomes, to increase
the number of sequences from this clade to »65. These new
taxa contributed the taxonomic results listed in Table 3.
They provided evidence for Asterozoa (sea star plus brittle
J. Mallatt, C.J. Winchell / Molecular Phylogenetics and Evolution 43 (2007) 1005–1022
star), for a monophyletic Cyclostomata (lampreys and
hagWshes), for monophyly of hexanchiform and of orbitostylic sharks, for the bichir Polypterus in a monophyletic
Actinopterygii, for a clade of trout and perch exclusive of
zebraWsh, for birds as diapsid reptiles, and for turtles as
anapsids. Parametric bootstrapping tested many alternate
hypotheses about deuterostome relationships, and the
results are shown in Table 2. Although this pboot method is
susceptible to the statistical error of rejecting hypotheses
too readily, it accepted the currently dominant hypotheses
of basic-deuterostome relationships (Ambulacraria and
Chordata, with the latter consisting of tunicates and
euchordates) and of lungWshes as the nearest relative of tetrapods. Complete rRNA genes should be sequenced from
more deuterostomes, especially crocodilians, paleognathid
birds, monotreme and marsupial mammals, gymnophiona
(caecilian) amphibians, more non-teleost actinopterygians,
pristiform rays, Xenoturbella worms, and Wnally, from a
wide variety of tunicates, to further clarify the relationships
within the diYcult tunicate clade (Swalla et al., 2000).
Acknowledgments
Thanks are extended to Gary Thorgaard, Joe Brunelli,
Stacia Sower, Birte Kalveram, Richard Johnson, Kelly Cassidy, Derek Pouchnik, and Frederic Delsuc for help with
various aspects of this study. Special thanks go to Germán
Pequeño for supplying the diYcult-to-obtain Geotria, a key
species in this study.
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.ympev.
2006.11.023.
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