Amphioxus Mitochondrial DNA , Chordate Phylogeny , and the Limits of Inference Based on Comparisons of Sequences

Ð Analyses of both the nucleotide and am ino acid sequences derived from all 13 m itochondr ial protein-encoding genes (12,234 bp) of 19 metazoan species, including that of the lancelet Branchiostom a ̄ oridae (``amphioxus’ ’ ), fail to yield the widely accepted phylogeny for chordates and, w ithin chordates, for vertebrates. G iven the breadth and the compelling nature of the data supporting that phylogeny, relationships supported by the m itochondrial sequence comparisons are almost certainly incorrect, despite their being supported by equally weighted parsimony, distance, and m aximum -likelihood analyses. The incorrect groupings probably result in part from convergent base-com positional similarities among some of the taxa, similarities that are strong enough to overwhelm the historical signal. Comparisons among very distantly related taxa are likely to be particularly susceptible to such artifacts, because the historical signal is already greatly attenuated. Empirical results underscore the need for approaches to phylogenetic inference that go beyond simple site-by-site comparison of aligned sequences . This study and others indicate that, once a sequence sample of reasonable size has been obtained, accurate phylogenetic estim ation may be better served by incorporating know ledge of molecu lar structures and processes into inference models and by seeking additional higher order characters embedded in those sequences , than by gathering ever larger sequence samples from the same organisms in the hope that the historical signal will eventually prevail. [Am phioxus; chordate phylogeny; homoplasy; m tDNA; molecu lar systematics; phylogenetic inference. ] The practice of inferring evolutionary trees from DNA sequences has ̄ ourished in recent years, its credibility bolstered by the observation that phylogenies of wellstudied groups are usually supported by sequence data. W hen the sequence of a particular gene or other well-de ® ned DNA segment yields an inference congruent w ith an accepted relationship for a particular group, there is a tendency to regard that segment as reliable for phylogenetic inference and to use it to determ ine phylogenies for taxa whose relationships are unknown (Graybeal, 1994; Cho et al., 1995). However, from the beginning of such studies it was recognized that any DNA segment can only be useful over a limited divergence range; outside that range the historical signal would be either too undeveloped or too attenuated to be reliable. Furtherm ore, with an increase in the num ber of such studies it also became apparent that the useful range varied among different taxa. Thus, there are instances in which sequence data provide accurate assessm ents for some relationships, and erroneous ones for others (Felsenstein, 1978; H illis, 1991; Kim , 1996; Philippe et al., 1994). The latter occur whenever the embedded historical signal is overturned by a stronger, homoplasious signal among the DNA sequences. Various methods are used for phylogenetic reconstruction, each implying a different model of evolutionary change and emphasizing different aspects of the observed character-state covariation among taxa. It is common practice to regard a phylogeny that is supported by several different methods as correct, and especially so when statistical methods for evaluating the streng th of support [e.g., bootstrapping (Felsenstein, 1985) and decay indices (Bremer, 1988; Donoghue et al., 1992)] are compelling. This stem s from a tacit assumption that an incorrect phylogeny, even if it is the best ® t to the available data, w ill not receive signi® cant statistical support when the result itself is evaluated. That assumption is incorrect. Statistical evaluations merely assess the streng th of the signal used to order the data hierarby gest on N ovem er 4, 2016 http://sysbfordjournals.org/ D ow nladed from 62 VOL. 47 SYSTEMATIC BIOLOGY chically (Swofford et al., 1996). Thus, if there is a hierarchical signal in the data that arises from a nonhistorical source and if that signal is suf® ciently strong, it can overw helm not only a weaker historical signal, but also a statistical evaluation of the resu lt. The ``total evidence’ ’ approach to phylogenetic reconstruction (Eernisse and Kluge, 1993) is currently among the most w idely applied. It ``uses character congruence to ® nd the best ® tting hypothesis for an unpartitioned set of synapomorphies, which is ideally all of the relevant available data’ ’ (Eernisse and Kluge, 1993). In its purest implementation the approach weights all characters equally in order to dispense with any need to identify different classes of information. Proponents m aintain that partitioning evidence into classes is arti® cial, ``because there is little reason to believe such categories are m ind-independent categories w ith d iscoverab le boundaries’ ’ (Eernisse and Kluge, 1993). This somew hat narrow perspective has gained a following because it obviates a need to specify explicit (and often poorly known) processes about the way in which traits evolve. Advocates of the method espouse a view that homoplasy (characterstate covariation among taxa due to in ̄ uences other than shared history) w ill be random ly distributed with respect to taxa, and that hierarchically structured historica l s ign al (ch arac ter-state covariat ion among taxa due to shared history) w ill overshadow the homoplasy if enough data are collected (see Farris, 1983). In keep ing with this view, it is assumed that any incorrect inferences will be due to stochastic error associated w ith an in su f ® c ient ly large sample size of characters, and that they will disappear as more data are collected. The sample size of sites required to ensure that the historical signal overturns the homoplasy depends to a large extent on the streng th of the historical signal in a data set and on the grain size of the homoplasy. If the homoplasy is dispersed in a ® ne-grained fashion Ð that is, is distributed in sm all ``packets,’ ’ each of which suggests a different nonhistorical associationÐ it w ill likely appear random ly distributed at relatively sm all sample sizes. If hom oplasy is d ispersed in a coarsegrained fashion, so that several sites suggest the same nonhistorical grouping, then larger sample sizes (i.e., more sequence) w ill be required before patterns of homoplasy appear randomly distributed. In essence, the sample size of sites at which the random ness of homoplasy becomes apparent is dictated by the gra in size of the homoplasy. Thus, even if homoplasy were random ly distributed among taxa at the level of the entire genome (an assumption that has not been empirically tested), it would appear to be highly nonrandomly distributed within a particular sample of sites if its grain size were coarse and the sample of sites insuf ® ciently large. Note, in the current context, that the term ``grain size’ ’ has no spatial connotation. W hen we refer to homoplasy as ``coarse-grained,’ ’ we mean only that several sites within a fragment imply the same nonhistorical grouping; the m isleading sites that collectively constitute a ``packet’ ’ need not be spatially contiguous along the sequence. The prem ise that hom oplasy is random ly distributed or unstructured with in data sets underlies the phylogenetically meaningful interpretations of bootstrapping , decay ind ices, and successive weigh ting (Farris, 1969). Groupings assessed as unreliable (i.e., those with little character support) are assumed to be due to chance, while those assessed as reliable are assumed to be so due to shared history. Unfortunately, if homoplasy is nonrandom ly distributed or if it shows ``system atic error’ ’ (Swofford et al., 1996), then analyses will not only yield erroneous phylogenetic inferences, but m any of the tests designed to evaluate the reliability of their constituent nodes will lead to falsely con ® den t assessments. Given the appeal of the ``total evidence,’ ’ equally weighted parsim ony approach, it would be useful to evaluate the extent to which its required assumption for random distribution of homoplasy is actually met by molecular data sets. When phylogeny is by gest on N ovem er 4, 2016 http://sysbfordjournals.org/ D ow nladed from 1998 63 NAYLOR AN D BROW NÐ LIMITS OF INFEREN CE TA B L E 1. Species used and their Genbank acces-