Hexapod Origins: Monophyletic or Paraphyletic?

Abstract

Recent morphological and molecular evidence has changed interpretations of arthropod phylogeny and evolution. Here we compare
complete mitochondrial genomes to show that Collembola, a wingless group traditionally considered as basal to all insects,
appears instead to constitute a separate evolutionary lineage that branched much earlier than the separation of many crustaceans
and insects and independently adapted to life on land. Therefore, the taxon Hexapoda, as commonly defined to include all six-legged
arthropods, is not monophyletic.

Full text

DOI: 10.1126/science.1078607
, 1887 (2003); 299Science
et al.Francesco Nardi,
Hexapod Origins: Monophyletic or Paraphyletic?
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energetics models without acceleration-
based transport predicted the offshore mi-
gration (1, 2), they had limited skill pre-
dicting the total change to the beach over
45 days because they failed to predict on-
shore migration between storms (2). The
energetics model that was extended to in-
clude acceleration better predicted the
change in the sea-floor both onshore and
offshore of the bar crest (Fig. 4), and the
overall evolution of the cross-shore depth
profile (Fig. 5).
References and Notes
1. E. Thornton, R. Humiston, W. Birkemeier, J. Geophys.
Res. 101, 12097 (1996).
2. E. Gallagher, S. Elgar, R. Guza, J. Geophys. Res. 103,
3203 (1998).
3. D. Aubrey, J. Geophys. Res. 84, 6347 (1979).
4. L. Wright, A. Short, Mar. Geol. 56, 93 (1984).
5. J. Roelvink, M. Stive, J. Geophys. Res. 94, 4185
(1989).
6. L. Wright, J. Boon, S. Kim, J. List, Mar. Geol. 96,19
(1991).
7. A. Bowen, in The Coastline of Canada, S. McCann, Ed.
(Geol. Surv. Canada Pap. 10-80, Ottawa, Canada,
1980), pp. 1–11.
8. J. Bailard, J. Geophys. Res. 86, 10938 (1981).
9. J. Trowbridge, D. Young, J. Geophys. Res. 94, 10971
(1989).
10. S. Elgar, E. Gallagher, R. Guza, J. Geophys. Res. 106,
11623 (2001).
11. S. Elgar, R. Guza, M. Freilich, J. Geophys. Res. 93, 9261
(1988).
12. T. G. Drake, J. Calantoni, J. Geophys. Res. 106, 19859
(2001).
13. O. Madsen, in Proc. 14th Int. Conf. Coastal Eng.,
Copenhagen, Denmark (American Society of Civil
Engineers, Reston, VA, 1974), p. 776 –794.
14. P. Nielsen, Coastal Eng. 45, 53 (2002).
15. R. Hallermeier, Cont. Shelf Res. 1, 159 (1982).
16. D. Hanes, D. Huntley, Cont. Shelf Res. 6, 585 (1986).
17. E. Gallagher, S. Elgar, E. Thornton, Nature 394, 165
(1998).
18. R. Bagnold, U.S. Geol. Surv. Prof. Pap. 422-I (1966).
19. Support was provided by the Army Research Office,
the Office of Naval Research, NSF, and a fellowship
from Conselho Nacional de Desenvolvimento
Cientı´fico e Tecnolo´gico (CNPq), Brazil. E. Gal-
lagher, R. Guza, T. Herbers, and B. Raubenheimer
made valuable comments and helped obtain the
field observations. The staff of the Field Research
Facility and the Center for Coastal Studies provid-
ed excellent logistical support during arduous field
conditions.
12 December 2002; accepted 12 February 2003
Hexapod Origins: Monophyletic
or Paraphyletic?
Francesco Nardi,
1
* Giacomo Spinsanti,
1
Jeffrey L. Boore,
2
Antonio Carapelli,
1
Romano Dallai,
1
Francesco Frati
1
Recent morphological and molecular evidence has changed interpretations of ar-
thropod phylogeny and evolution. Here we compare complete mitochondrial ge-
nomes to show that Collembola, a wingless group traditionally considered as basal
to all insects, appears instead to constitute a separate evolutionary lineage that
branched much earlier than the separation of many crustaceans and insects and
independently adapted to life on land. Therefore, the taxon Hexapoda, as com-
monly defined to include all six-legged arthropods, is not monophyletic.
The phylum Arthropoda comprises the major
groups Hexapoda (insects and presumed allies),
Myriapoda (e.g., centipedes and millipedes),
Chelicerata (e.g., spiders and horseshoe crabs),
and Crustacea (e.g., crabs and lobsters). Many
studies have attempted to reconstruct the evolu-
tionary relationships among arthropods using
various approaches such as paleontology (1),
comparative morphology (2), comparative devel-
opmental biology (3, 4), and molecular phyloge-
netics (5, 6).
It has long been held that hexapods (7) con-
stitute a monophyletic taxon (8, 9) and that their
closest relatives are to be found in myriapods
(10). More recently, molecular and developmen-
tal studies have rejected this relationship (3–5,
11, 12) in favor of a closer affinity between
Hexapoda and Crustacea (Pancrustacea or Tetra-
conata). In this context, special attention must be
given to the apterygotes (springtails, silverfish,
and their allies), the wingless hexapods thought
to branch at the base of Hexapoda. The phyloge-
netic position of these groups is still unclear
(13–16), casting doubt even on the monophyly of
the Hexapoda (17).
A potentially powerful technique for resolv-
ing deep relationships is to compare whole mito-
chondrial genomes (5, 17, 18). Phylogenetic
analysis of the only complete mitochondrial
sequence available for an apterygotan species
(17 ) suggested the possibility that Collem-
bola might not be included within Hexapoda,
contrasting with the classic view of a mono-
phyletic taxon that includes all six-legged
arthropods. Collembola have been clustered
within crustaceans in other molecular and/or
combined data sets (15, 16), but the possible
paraphyly of Hexapoda has not been given
specific attention and the deserved consider-
ation. We have now sequenced the complete
mitochondrial genomes of two additional
species (19) specifically chosen to address
this problem: Tricholepidion gertschi, repre-
senting one of the most basal lineages of the
Insecta (Zygentoma), and Gomphiocephalus
hodgsoni, another collembolan, to test sup-
port for the two competing hypotheses of a
monophyletic versus paraphyletic Hexapoda.
An initial phylogenetic analysis performed on
the 35-taxon data set (19) produced the tree
shown in Fig. 1. The tree has high support at most
nodes, with support decreasing toward deeper
relationships. This analysis strongly supports the
Pancrustacea hypothesis, with the exception of
the position of Apis and Heterodoxus. T. gertschi
is basal to all the pterygotan insects, supporting
the monophyly of the Insecta. The four crusta-
cean sequences are divided into two well-defined
groups (representing Malacostraca and Bran-
chiopoda), but their reciprocal relationships and
position relative to the Insecta are not resolved.
The Crustacea ⫹ Insecta node is well supported,
and it excludes the two collembolans, which
cluster together as the basal lineage of the Pan-
crustacea. A second group unites the Cheli-
cerata ⫹ Myriapoda [as in (20)] but also includes
the insects Apis and Heterodoxus, presumably as
an artefact.
Although this tree shows many interesting
outcomes, it also contains some evidently unten-
able relationships, which nevertheless have
strong statistical support. This indicates the pres-
ence of anomalies in the evolution of these se-
quences that introduce strong systematic errors
in the analysis. The most likely factors that can
cause these anomalies are unequal base compo-
sition [which can bias amino acid composition
(21)] and uneven rates of evolution among
different lineages. This problem might be
especially acute, because some taxa share an
extremely high AT bias—Apis (84.8%), Rh-
ipicephalus (78.0%), and Heterodoxus
(79.3%)—and different rates of evolution,
1
Department of Evolutionary Biology, University of
Siena, via Aldo Moro 2, 53100 Siena, Italy.
2
U.S.
Department of Energy Joint Genome Institute and
Lawrence Berkeley National Laboratory, 2800 Mitch-
ell Drive, Walnut Creek, CA 94598, USA.
*To whom correspondence should be addressed. E-
mail: nardifra@unisi.it
Fig. 5. Observed and predicted cross-shore bot-
tom elevation profiles spanning a 45-day period.
Sea-floor elevation relative to mean sea level
observed 1 September 1994, 1900 hours (solid
black curve), observed 15 October 1994, 2200
hours (dashed black), and predicted for 15 Octo-
ber 1994, 2200 hours by the energetics (blue) and
energetics plus acceleration (red) models versus
cross-shore position.
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which could potentially cause artefactual at-
traction (22) in this analysis. Such sequenc-
es are usually removed from phylogenetic
analyses owing to their evidently incorrect
placement and disturbance to the reconstruc-
tion. To recognize and exclude from the
analysis those sequences whose placement
in the phylogenetic tree could be influenced
by such anomalies in the mechanism of evo-
lution, rather than by the true historical pro-
cess, we performed a detailed statistical test
(19) to select a subset of sequences with
homogeneous modes of evolution and whose
rate of evolution is compatible with that of
Gomphiocephalus and Tricholepidion. The
placement of these two taxa is key to assess-
ing the monophyly of the Hexapoda, so it is
especially important that the taxa compared
are compatible with them. The methods of
analysis outlined above, applied to this re-
duced data set, produced the two trees
shown in Fig. 2, which differ only in the
placement of Ostrinia with respect to the
remaining Holometabola. Again, strong sup-
port is obtained for the Pancrustacea, with
Tricholepidion basal to the remaining ptery-
gotan insects, and the two collembolans
placed outside the Crustacea ⫹ Insecta
clade. The trees also show monophyly of
Crustacea, although with a lower level of
support. Limulus is recovered as the sister
group of the Pancrustacea, in contrast with
the analysis based on the 35-taxon data set,
but again with very low support. The result-
ing trees do not seem to be sensitive to the
taxa included (fig. S2).
The most interesting result produced by
this study is certainly the nonmonophyly of
Hexapoda—that is the position of the two
collembolans outside the Crustacea ⫹ Insecta
clade, agreed upon by all analyses and with
high levels of support. To test the relative
positioning of Crustacea, Collembola, and
Insecta in more detail, we compared two
alternative topologies using analytical tests.
The hypothesis of Crustacea external to a
monophyletic Hexapoda (here, Insecta ⫹
Collembola) is strongly rejected (Table 1) in
favor of the proposed nonmonophyly of
Hexapoda. We also applied the same tests to
the problem of the basal trichotomy between
Chelicerata, Myriapoda, and Pancrustacea. A
sister group relationship between Pancrusta-
cea and Myriapoda (⫽Mandibulata) is
strongly rejected (Table 1), and no significant
difference in support was found for the other
two possible hypotheses. This accords with
the low levels of support found in all trees at
this node.
It has been generally accepted that the taxon
Hexapoda, including the basal apterygotan or-
ders, is monophyletic. This conclusion is
strongly supported by similarities in their body
organization (composed of head, thorax, and
abdomen), as well as other morphological char-
acters including eye and leg structure and the
absence of limbs in one of the cephalic seg-
ments (9). On the other hand, the interpretation
of such characters also depends on which is the
closest relative of the Hexapoda, and even on
the basal splitting of the latter taxon (9). Nev-
ertheless, apterygotan taxa, including Collem-
bola, show a number of peculiar features that at
least complicate the analysis of their affinities
with the Insecta sensu stricto (9, 23) and leave
some room to question these affinities altogeth-
er. The acceptance of nonmonophyly of
Hexapoda implies that the tripartite and six-
legged body plan typical of Hexapoda would be
a convergent acquisition of collembolans and
the “true insects.”
Fig. 1. Maximum-likelihood
[ProtML (24)] phylogenetic re-
construction, complete data set.
Numerals at each node show lo-
cal bootstrap probability values.
Branch lengths are drawn pro-
portionally to maximum-likeli-
hood estimates.
Fig. 2. Maximum-likeli-
hood [ProtML (24) and
MrBayes (25)] phyloge-
netic reconstructions, re-
duced data set. Al-
ternative placement of
Ostrinia follows MrBayes
reconstruction. Numerals
above each node show
local bootstrap probabili-
ty values (ProtML), and
numerals below each
node indicate posterior
probabilities (MrBayes).
Branch lengths are pro-
portionate to maximum-
likelihood estimates pro-
duced by ProtML.
Table 1. Tests of significance for competing hypotheses. Statistical tests of significance were conducted
for different competing phylogenetic hypotheses within Pancrustacea and within arthropod classes. au,
approximately unbiased test; kh, Kishino-Hasegawa test; sh, Shimodaira-Hasegawa test (26).
Tree ⫺ln L (ProtML) au kh sh
(Collembola, (Crustacea, Insecta)) 19723.73 0.991 0.979 0.979 Best
(Crustacea, (Collembola, Insecta)) 19744.96 0.009 0.021 0.021
(Myriapoda, (Chelicerata, Pancrustacea)) 19723.73 0.509 0.496 0.649 Best
((Myriapoda, Chelicerata), Pancrustacea) 19723.97 0.509 0.504 0.626
(Chelicerata, (Myriapoda, Pancrustacea)) 19739.90 0.006 0.032 0.084
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Our analysis, based on a large, specifical-
ly targeted data set and modern statistical
tools, strongly supports the view that
Hexapoda is not monophyletic, that at least
some apterygotes have adapted to life on land
independently from insects, and that those
features shared between some apterygotes
and insects might have originated indepen-
dently in these lineages.
References and Notes
1. D. E. G. Briggs, R. A. Fortey, Science 246, 241 (1989).
2. M. J. Emerson, F. R. Schram, in Arthropod Relation-
ships, R. A. Fortey, R. H. Thomas, Eds. (Chapman &
Hall, London, 1997), pp. 67– 86.
3. C. E. Cook, M. L. Smith, M. J. Telford, A. Bastianello, M.
Akam, Curr. Biol. 11, 759 (2001).
4. M. Duman-Scheel, N. H. Patel, Development 126,
2327 (1999).
5. J. L. Boore, D. V. Lavrov, W. M. Brown, Nature 392,
667 (1998).
6. J. W. Shultz, J. C. Regier, Proc. R. Soc. London Ser. B
267, 1011 (2000).
7. Hexapoda is defined as composed of the apterygotan
orders Protura, Collembola, Diplura, Microcoryphia,
Zygentoma, and all pterygotan orders (Pterygota).
Microcoryphia ⫹ Zygentoma ⫹ Pterygota constitute
the Insecta sensu stricto.
8. W. Hennig, Insect Phylogeny (Wiley, New York,
1981).
9. K.-D. Klass, N. P. Kristensen, Ann. Soc. Entomol. Fr.
37, 265 (2001).
10. O. Kraus, in Arthropod Relationships, R. A. Fortey,
R. H. Thomas, Eds. (Chapman & Hall, London, 1997),
pp. 295–303.
11. W. Dohle, Ann. Soc. Entomol. Fr. 37, 85 (2001).
12. M. Friedrich, D. Tautz, Nature 376, 165 (1995).
13. C. Bitsch, J. Bitsch, Zool. Scr. 29, 131 (2000).
14. A. Carapelli, F. Frati, F. Nardi, R. Dallai, C. Simon,
Pedobiologia 44, 361 (2000).
15. G. Giribet, C. Ribera, Cladistics 16, 204 (2000).
16. T. Spears, G. Abele, in Arthropod Relationships,R.A.
Fortey, R. H. Thomas, Eds. (Chapman & Hall, London,
1997), pp. 169 –188.
17. F. Nardi, A. Carapelli, P. P. Fanciulli, R. Dallai, F. Frati,
Mol. Biol. Evol. 18, 1293 (2001).
18. E. Garcia-Machado et al., J. Mol. Evol. 49, 142 (1999).
19. Materials and methods are available as supporting
online material on Science Online.
20. U. W. Hwang, M. Friedrich, D. Tautz, C. J. Park, W.
Kim, Nature 413, 154 (2001).
21. K. G. Helfenbein, W. M. Brown, J. L. Boore, Mol. Biol.
Evol. 18, 1734 (2001).
22.
D. L. Swofford, G. J. Olsen, P. J. Waddell, D. M. Hillis, in
Molecular Systematics, D. M. Hillis, C. Moritz, B. K. Mable,
Eds. (Sinauer, Sunderland, MA, 1996), pp. 407–514.
23. R. Dallai, B. A. Afzelius, in The Male Gamete, from
Basic Science to Clinical Applications, C. Gagnon, Ed.
(Cache River, Vienna, 1999), pp. 333–350.
24. J. Adachi, M. Hasegawa, Comput. Sci. Monogr. 28,1
(1996).
25. J. P. Huelsenbeck, F. Ronquist, Bioinformatics 17, 754
(2001).
26. H. Shimodaira, Syst. Biol. 51, 492 (2002).
27. This study was supported by grants from the Minis-
tero dell’Istruzione, dell’Universita` e della Ricerca,
from the Progetto Nazionale di Ricerca in Antartide,
and from the University of Siena. Part of this work
was performed under the auspices of the U.S. Depart-
ment of Energy, Office of Biological and Environmen-
tal Research, and by the University of California,
Lawrence Berkeley National Laboratory, under con-
tract DE-AC03-76SF00098.
Supporting Online Material
www.sciencemag.org/cgi/content/full/299/5614/1887/
DC1
Materials and Methods
Figs. S1 and S2
References
20 September 2002; accepted 7 February 2003
Role of EphA4 and EphrinB3 in
Local Neuronal Circuits That
Control Walking
Klas Kullander,
1,2
* Simon J. B. Butt,
3
James M. Lebret,
3
Line Lundfald,
3
Carlos E. Restrepo,
3
Anna Rydstro¨m,
2
Ru¨diger Klein,
4
† Ole Kiehn
3
*†
Local circuits in the spinal cord that generate locomotion are termed central
pattern generators (CPGs). These provide coordinated bilateral control over the
normal limb alternation that underlies walking. The molecules that organize the
mammalian CPG are unknown. Isolated spinal cords from mice lacking either
the EphA4 receptor or its ligand ephrinB3 have lost left-right limb alternation
and instead exhibit synchrony. We identified EphA4-positive neurons as an
excitatory component of the locomotor CPG. Our study shows that dramatic
locomotor changes can occur as a consequence of local genetic rewiring and
identifies genes required for the development of normal locomotor behavior.
Rhythmic movements such as locomotion and
swimming require that muscles contract and
relax in a complex repetitive pattern. Central
pattern generators, or CPGs, are local spinal
neuronal networks that generate and coordinate
these rhythmic muscle activities (1, 2). In the
fruit fly, it was recently shown that the CPG for
peristaltic crawling develops in the complete
absence of sensory input (3). In two nonmam-
malian vertebrate species, the lamprey and the
Xenopus tadpole, the critical neuronal compo-
nents of the locomotor CPG have been identi-
fied (4, 5). In mammals, the CPGs controlling
limb movements are located in the ventromedial
part of the spinal cord (6). However, the neuro-
nal organization is still poorly understood (2),
and no molecules that contribute to CPG devel-
opment have been identified. Because CPGs are
important for spinal control of walking in hu-
mans (7), understanding their neuronal organi-
zation and molecular determination is essential
in the ongoing effort to reestablish locomotor
1
Department of Medical Biochemistry, Gothenburg
University, Medicinaregatan 9 A, 405 30 Gothenburg,
Sweden.
2
AstraZeneca Transgenics and Comparative
Genomics, AstraZeneca, 431 83 Mo¨lndal, Sweden.
3
Mammalian Locomotor Laboratory, Department of
Neuroscience, The Karolinska Institute, Retzius vag 8,
171 77 Stockholm, Sweden.
4
Max-Planck Institute of
Neurobiology, Am Klopferspitz 18A, D-82152 Martin-
sried, Germany.
*To whom correspondence should be addressed. E-
mail: klas.kullander@medkem.gu.se (K.K.); ole.kiehn@
neuro.ki.se (O.K.)
†These authors contributed equally to this work.
Fig. 1. The lumbar seg-
ments of the spinal
cords of neonatal
ephA4- and ephrinB3-
null mice exhibit syn-
chronous left-right
ventral root activity.
(A to C) Images of WT
mice displaying nor-
mal locomotor activity
(A) or ephA4-null mice
(B) and ephrinB3-null
mice (C) displaying a
rabbitlike gait. (D to F)
Recorded activity after
application of NMDA
and serotonin to the
isolated spinal cord
(a 4-␮M solution of
each drug) of WT mice
(D), ephA4-null mice
(E), and ephrinB3-null
mice (F) in flexor (L2)
and extensor ventral
(L5) roots. r, right; l,
left. (G to I) Circular
phase diagrams derived from 20 locomotor cycles for the WT (G), ephA4-null (H), and ephrinB3-null (I)
mice shown in (A) to (C), respectively, (J to L) Plots show the vector points of L2 pairings for all
experiments conducted on WT mice (n ⫽ 5) ( J, green squares); ephA4 heterozygotes (n ⫽ 13) (K, black
triangles) and homozygotes (n ⫽ 14) (K, blue circles); and ephrinB3 homozygotes (n ⫽ 9) (L).
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