Point Counter Point What Can 18 S rDNA Do for Bivalve Phylogeny ?

In the July (1996) issue of Journal of Molecular Evolution, Steiner and Mu ̈ller (1996) discuss the limitations of 18S rDNA sequences to achieve a ‘‘reasonable’’ phylogeny of bivalve mollusks. After a nice introduction to the subject and thorough references to molecular techniques applied to Bivalvia, they collect 21 18S rDNA sequences published to date—2 Polyplacophora, 3 Gastropoda, and 16 Bivalvia belonging to the Filibranchia (4Pteriomorphia) and Eulamelibranchia ( 4Heterodonta)—plus 2 new sequences (both also Filibranchia). Eight of these sequences, however, were excluded from the main analyses. Two more partial sequences were not included: one Cephalopoda and one Polyplacophora (Wheeler et al. 1993). Different tree-constructing algorithms, based on parsimony, neighbor-joining, and maximum-likelihood, as well as other methods, to test the reliability of the clades, such as the PRN and spectral analysis were used. The results obtained led them to propose a ‘‘preferred tree (their Fig. 9) combining the results from the 18S rDNA data and morphological characters compiled by Waller (1978, 1990). ’’ In their words, this tree ‘‘is not a computer-constructed tree but an evaluation of hypotheses on bivalve phylogeny from the given morphological and molecular trees. ’’ Moreover ‘‘the failure of neighbor-joining and parsimony to resolve this clade [Bivalvia]’’ is probably due to the different substitution rates among species. We have reanalyzed the data set presented by Steiner and Müller (1996), to which we added 12 more molluscan sequences available via GenBank. Sequence alignments were generated using ClustalW (Thompson et al. 1996), with subsequent manual correction based on secondary structure constraints. Two alignments were analyzed, one with the whole data set and another in which four ambiguous-alignment zones corresponding to loops were removed. Alignments are available at the ftp site porthos.bio. ub.es/users/ftp/pub/teu/18Sphylogeny. Parsimony analyses were performed with PAUP 3.1. 1 (Swofford 1993). Analyses were run for both alignments, using unweighted and weighted parsimony at different transition/transversion rates (3 and 5), as well as using transversion parsimony (transitions downweighted to 0). Gaps were treated in two ways, as missing data or as a fifth state. Three independent data sets were analyzed: the 13 taxa analyzed in the original paper (data set 1), the 21 taxa reported in their Table 2 (data set 2), and all molluscan sequences published to date (data set 3). All the analyses run with data sets 2 and 3 rendered Bivalvia monophyletic (Figs. 1B and C) with an internal unstable topology, varying depending on the analysis condition. The following clades above family level are always monophyletic: Pectinidae, Arca+ Pectinidae, Ostreidae, Mytilidae, Mactridae, and the two represented Eulamelibranchia groups ( Mya + Mactridae). The paraphyletic Bivalvia have only been recovered in some cases when data set 1 was used for the analyses (Fig. 1A). Although the internal topology differs slightly from their Fig. 4, the two Gastropoda also cluster with the two Mactridae, which are the longest branches of the tree. These results clearly indicate that the paraphyletic Bivalvia is an artifact, most likely due to the low number of taxa used in the analyses, combined with the long branch attraction effect among the Gastropoda and the Mactridae. Simulations have demonstrated that adding large numbers of additional taxa to phylogenetic analyses may increase the accuracy of the estimated trees, reduce the long branch attraction effect, and, at the same time, reduce the need for computationally complex methods of analysis (Hillis 1996). In our case, solely increasing the umber of taxa from 13 to 21 is enough to obtain a robust monophyletic Bivalvia clade. From these results we suggest that computational efforts in phylogenetic analysis should focus on analyzing the biggest available data sets instead of performing more costly computational methods (e.g., the PRN and spectral analyses) with fewer taxa. The failure of recovering a reliable internal phylogeny J Mol Evol (1999) 48:256–261