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Response to Comment on "Hexapod Origins: Monophyletic or Paraphyletic?"
Assessing the relationships among arthropod taxa is an intenselydebated issue in metazoan phylogeny, with various studies testingdifferent character sets, phylogenetic methods, and strategiesfor analyzing molecular data (1–3). Delsuc et al. (4)criticize our recent hypothesis that Hexapoda is not monophyletic(3) on methodological grounds, including use of a suboptimalsubstitution matrix, lack of correction for among-site ratevariation (ASRV), and biased taxon selection. They further introducea method (5) that recodes nucleotide sequences into only twocategories— purines (R) and pyrimidines (Y)—anduse a maximum-likelihood approach to reanalyze our data recodedin this fashion. They claim that this method corrects for artefactualclustering of taxa that results from compositional bias of nucleotidecontent; this view is bolstered by the correct placement ofthe honeybee and louse, which were anomalously placed in ourtree and others (1, 6, 7), within Insecta. Their reanalysisplaces collembolans at the base of Hexapoda, although with moderateto low support, and thus questions the main conclusion of ourstudy.
Despite the correct placement of the honeybee and louse, itis not clear that the Delsuc et al. method performs better generally.In fact, one might expect that reducing nucleotide sequencedata set to only two states (R and Y) might exacerbate saturationat many sites, and be more susceptible to problems of nonstationarityof substitutions than an amino acid data set (8). In the latter,the model of substitution reflects the probability of a replacementto be fixed, and the use of 20-state characters decreases thepossibility of overlooking convergence. The inference of phylogeniesbased on amino acid sequences is common practice and is generallyaccepted as among the most reliable of methods (8–10).Although the matrix of amino acid change used in the first ofour two analyses was based on vertebrate sequences, it has beenextensively used to study relationships among invertebrateswith no reports of significant flaws (1, 6).
In our previous study (3), we used an alignment of amino acidsequences and two likelihood-based methods of analysis: A classicallikelihood reconstruction using a fixed-parameter model of aminoacid substitution, and a Bayesian analysis based on a generaltime reversible (GTR) model of substitution and modeling ratevariation across sites using an invariant + distribution. Bothanalyses converged on the same topology, which supports theplacement of Collembola outside of the Insecta + Crustacea assemblage.
The correction for rate variation among sites has been shownto potentially affect estimates of branch lengths and divergencetimes, but it seems to have only a limited effect on topology(11). To further address this issue, we calculated the likelihoodof competing trees under the same matrix, but modeled ASRV usinga distribution (12). Table 1 shows that the trees we produce(with Collembola outside Insecta + Crustacea) give higher likelihoodscores, although with different degrees of significance, regardlessof the use of correction.
Table 1. Comparison of tree topologies under the amino acid substitution model mtREV24 implemented in PAML (12, 19) and incorporating a -correction for ASRV [pKH = P value of the Kishino-Hasegawa test (17); pSH = P value of the Shimodaira-Hasegawa test (18); pRELL = P value of the RELL bootstrap (17)]. In the 15-taxon data set, the topology derived in Nardi et al. (3) is compared with a topology derived in Delsuc et al. (4), after pruning extra taxa and exchanging Anopheles gambiae with A. quadrimaculatus. In the 25-taxon data set, the topology from Fig. 1 is compared with the topology derived in (4). In the 35-taxon data set, the two topologies derived in (3) and (4) are compared.
To investigate the possible effects of taxon exclusion on theanalysis, we repeated the analysis described in (3) on the 25-taxondata set of Delsuc et al. (4). To rule out the possibility thatthe analysis is negatively affected by use of a suboptimal substitutionmatrix and lack of ASRV correction, we used the Bayesian methodoutlined in (3) (Fig. 1). Again, Collembola fall outside theInsecta + Crustacea clade, although with only moderate support.
Fig. 1. Maximum likelihood tree obtained applying the method outlined in (3) as implemented in MrBayes ver. 2.1 (20) (aamodel = gtr; rates = invgamma) to the 25-taxon data set of Delsuc et al. (4). The analysis was run for 570,000 generations and sampled every 100 generations. The first 150,000 generations were excluded from the analysis as the burn-in of the search. Numbers at each node indicate posterior probabilities. Branch lengths are drawn according to estimates obtained with PAML.
[View Larger Version of this Image (24K GIF file)]
Our past (3) and present analyses, the analysis of Delsuc etal. (4), as well as other molecular studies (13) demonstratethat a reliable reconstruction of the phylogeny of Arthropoda—andthe assessment of the mono- or paraphyly of Hexapoda, specifically—arestill disputable. Results differ when subjecting the same dataset to different methods of analysis or when using differentsubsets of data with the same methods. This leaves the impressionthat none of the competing hypotheses can yet be rejected withcertainty. However, we believe that the theory of hexapod nonmonophylyproposed by several studies (3, 7, 14, 15) must be considered.In this context, the recent discovery of a marine hexapod fromthe Lower Devonian (16) undermines the traditional associationbetween terrestrialization and the evolution of hexapods, leavingroom for alternative hypotheses concerning hexapod origins.
Francesco Nardi Giacomo Spinsanti
Department of Evolutionary Biology University of Siena via Aldo Moro 2 53100 Siena, Italy E-mail: nardifra{at}unisi.it
Jeffrey L. Boore
U.S. Department of Energy Joint Genome Institute and Lawrence Berkeley Laboratory Walnut Creek, CA 94598, USA
Antonio Carapelli Romano Dallai Francesco Frati
Department of Evolutionary Biology University of Siena
References and Notes
1. U. W. Hwang, M. Friedrich, D. Tautz, C. J. Park, W. Kim, Nature413, 154 (2001). [CrossRef] [Medline]
18. H. Shimodaira, M. Hasegawa, Mol. Biol. Evol.45, 1114 (1999).
19. This methodology, as implemented in PAML (12), is unfortunately not suitable to conduct a full likelihood search. However, it is efficient for comparing a limited number of trees.
20. The amino acid substitution model "gtr," present as an option in MrBayes 2.1, has not been implemented, in its original form, in the latest release MrBayes 3. It is not clear to us if the method is still available under a different set of commands or if it has been removed altogether.
21. We thank P. Liò for useful discussion on this topic.
Received for publication 4 June 2003. Accepted for publication 18 August 2003.
The editors suggest the following Related Resources on Science sites:
In Science Magazine
TECHNICAL COMMENTS
Frédéric Delsuc, Matthew J. Phillips, and David Penny (12 September 2003) Science301 (5639), 1482d.
[DOI: 10.1126/science.1086558] |Full Text »|PDF »
REPORTS
Francesco Nardi, Giacomo Spinsanti, Jeffrey L. Boore, Antonio Carapelli, Romano Dallai, and Francesco Frati (21 March 2003) Science299 (5614), 1887.
[DOI: 10.1126/science.1078607] |Abstract »|Full Text »|PDF »|Supporting Online Material »
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distribution. Both analyses converged on the same topology, which supports the placement of Collembola outside of the Insecta + Crustacea assemblage. 
