David M. Hillis

Increased taxon sampling greatly reduces phylogenetic error.

Several authors have argued recently that extensive taxon sampling has a positive and important effect on the accuracy of phylogenetic estimates. However, other authors have argued that there is little benefit of extensive taxon sampling, and so phylogenetic problems can or should be reduced to a few exemplar taxa as a means of reducing the computational complexity of the phylogenetic analysis. In this paper we examined five aspects of study design that may have led to these different perspectives. First, we considered the measurement of phylogenetic error across a wide range of taxon sample sizes, and conclude that the expected error based on randomly selecting trees (which varies by taxon sample size) must be considered in evaluating error in studies of the effects of taxon sampling. Second, we addressed the scope of the phylogenetic problems defined by different samples of taxa, and argue that phylogenetic scope needs to be considered in evaluating the importance of taxon-sampling strategies. Third, we examined the claim that fast and simple tree searches are as effective as more thorough searches at finding near-optimal trees that minimize error. We show that a more complete search of tree space reduces phylogenetic error, especially as the taxon sample size increases. Fourth, we examined the effects of simple versus complex simulation models on taxonomic sampling studies. Although benefits of taxon sampling are apparent for all models, data generated under more complex models of evolution produce higher overall levels of error and show greater positive effects of increased taxon sampling. Fifth, we asked if different phylogenetic optimality criteria show different effects of taxon sampling. Although we found strong differences in effectiveness of different optimality criteria as a function of taxon sample size, increased taxon sampling improved the results from all the common optimality criteria. Nonetheless, the method that showed the lowest overall performance (minimum evolution) also showed the least improvement from increased taxon sampling. Taking each of these results into account re-enforces the conclusion that increased sampling of taxa is one of the most important ways to increase overall phylogenetic accuracy.

Phylogenetic relationships of the dwarf boas and a comparison of Bayesian and bootstrap measures of phylogenetic support

Four New World genera of dwarf boas (Exiliboa, Trachyboa, Tropidophis, and Ungaliophis) have been placed by many systematists in a single group (traditionally called Tropidophiidae). However, the monophyly of this group has been questioned in several studies. Moreover, the overall relationships among basal snake lineages, including the placement of the dwarf boas, are poorly understood. We obtained mtDNA sequence data for 12S, 16S, and intervening tRNA-val genes from 23 species of snakes representing most major snake lineages, including all four genera of New World dwarf boas. We then examined the phylogenetic position of these species by estimating the phylogeny of the basal snakes. Our phylogenetic analysis suggests that New World dwarf boas are not monophyletic. Instead, we find Exiliboa and Ungaliophis to be most closely related to sand boas (Erycinae), boas (Boinae), and advanced snakes (Caenophidea), whereas Tropidophis and Trachyboa form an independent clade that separated relatively early in snake radiation. Our estimate of snake phylogeny differs significantly in other ways from some previous estimates of snake phylogeny. For instance, pythons do not cluster with boas and sand boas, but instead show a strong relationship with Loxocemus and Xenopeltis. Additionally, uropeltids cluster strongly with Cylindrophis, and together are embedded in what has previously been considered the macrostomatan radiation. These relationships are supported by both bootstrapping (parametric and nonparametric approaches) and Bayesian analysis, although Bayesian support values are consistently higher than those obtained from nonparametric bootstrapping. Simulations show that Bayesian support values represent much better estimates of phylogenetic accuracy than do nonparametric bootstrap support values, at least under the conditions of our study.

Evidence from 18S ribosomal DNA that the lophophorates are protostome animals

The suspension-feeding metazoan subkingdom Lophophorata exhibits characteristics of both deuterostomes and protostomes. Because the morphology and embryology of lophophorates are phylogenetically ambiguous, their origin is a major unsolved problem of metazoan phylogenetics. The complete 18S ribosomal DNA sequences of all three lophophorate phyla were obtained and analyzed to clarify the phylogenetic relationships of this subkingdom. Sequence analyses show that lophophorates are protostomes closely related to mollusks and annelids. This conclusion deviates from the commonly held view of deuterostome affinity.

The ascomycota tree of life: A phylum-wide phylogeny clarifies the origin and evolution of fundamental reproductive and ecological traits

We present a 6-gene, 420-species maximum-likelihood phylogeny of Ascomycota, the largest phylum of Fungi. This analysis is the most taxonomically complete to date with species sampled from all 15 currently circumscribed classes. A number of superclass-level nodes that have previously evaded resolution and were unnamed in classifications of the Fungi are resolved for the first time. Based on the 6-gene phylogeny we conducted a phylogenetic informativeness analysis of all 6 genes and a series of ancestral character state reconstructions that focused on morphology of sporocarps, ascus dehiscence, and evolution of nutritional modes and ecologies. A gene-by-gene assessment of phylogenetic informativeness yielded higher levels of informativeness for protein genes (RPB1, RPB2, and TEF1) as compared with the ribosomal genes, which have been the standard bearer in fungal systematics. Our reconstruction of sporocarp characters is consistent with 2 origins for multicellular sexual reproductive structures in Ascomycota, once in the common ancestor of Pezizomycotina and once in the common ancestor of Neolectomycetes. This first report of dual origins of ascomycete sporocarps highlights the complicated nature of assessing homology of morphological traits across Fungi. Furthermore, ancestral reconstruction supports an open sporocarp with an exposed hymenium (apothecium) as the primitive morphology for Pezizomycotina with multiple derivations of the partially (perithecia) or completely enclosed (cleistothecia) sporocarps. Ascus dehiscence is most informative at the class level within Pezizomycotina with most superclass nodes reconstructed equivocally. Character-state reconstructions support a terrestrial, saprobic ecology as ancestral. In contrast to previous studies, these analyses support multiple origins of lichenization events with the loss of lichenization as less frequent and limited to terminal, closely related species.

Increased Taxon Sampling Is Advantageous for Phylogenetic Inference

Until recently, it was believed that complex phylogenies might be extremely difficult to reconstruct due to the phenomenal rate of increase in the number of possible phylogenies as the number of taxa increases. However, Hillis (1996) showed through simulation that, for at least one complex phylogeny of angiosperms with 228 taxa, reconstruction was far more accurate than expected, even with relatively modest amounts of DNA sequence data. This led to a flurry of papers on the subject of taxon sampling and phylogenetic reconstruction, with focus quickly shifting from the question of whether complex phylogenies can be reconstructed to whether and how much an existing phylogeny can be improved through increased taxon sampling (Hillis, 1998; Kim, 1998; Poe, 1998; Poe and Swofford, 1999; Pollock and Bruno, 2000; Rannala et al., 1998; Yang, 1998). Although a statistician might intuitively believe that it is generally better (or at least no worse) to increase the amount of data to resolve a question in statistical inference, the benefits of taxon addition for phylogenetic inference remain controversial. Some researchers have argued that taxon addition can decrease accuracy (Kim, 1996,1998), while others believe that increased sampling improves accuracy (Graybeal, 1998; Hillis, 1996, 1998; Murphy et al., 2001; Poe, 1998; Pollock and Bruno, 2000; Pollock et al., 2000; Soltis et al., 1999). The reasons that different papers come to apparently contradictory conclusions deserve careful consideration. An often cited factor affecting the benefits of taxon addition is the phenomenon of long-branch attraction (LBA). Some phylogenetic methods have a bias toward preferential clustering of long branches, leading to erroneous results when those long branches do not actually represent a monophyletic assemblage (Felsenstein, 1978; Hendy and Penny, 1989). This phenomenon has been cited in favor of increased taxon sampling, since sampling can be designed to break up long branches (Hillis, 1998). However, increased sampling has also been implicated as a potential cause of LBA because addition of a new long branch may wrongly attract a pre-existing long branch that had previously been inferred correctly (Poe and Swofford, 1999; Rannala et al., 1998). LBA may also explain some simulations that have found problems in phylogeny estimation when sampling outside the taxonomic group of interest (but see Pollock and Bruno [2000] for an alternative explanation). Outside sampling in these simulations tended to add long branches, which tended to attract the longest unbroken branch in the group of interest (Hillis, 1998; Rannala et al., 1998). The degree to which LBA is a problem depends greatly on the method of analysis, and LBA is much less of a problem for maximum likelihood (ML) than for parsimony or distance methods (Bruno and Halpern, 1999). A recent paper on the subject of taxon addition (Rosenberg and Kumar, 2001) concludes that increased taxon sampling is of little benefit to phylogenetic inference when compared to increasing sequence length. We disagree with their interpretation and believe that their data support the importance of increased taxon sampling. In addition, some of their data were simulated under extreme conditions (i.e., substitution rates that were very high or low, or sequences that were unreasonably short). Large error values and nonlinear relationships at these extremes make it difficult to interpret effects for the majority of the range, and averaging across the entire range is inappropriate. Moreover, we do not believe that Rosenberg and Kumar (2001) used the most appropriate metric to measure the relative effect of taxon addition. Our reanalysis of their simulated data indicates that increased taxon sampling is highly beneficial for phylogenetic inference.

Is sparse taxon sampling a problem for phylogenetic inference

Rosenberg and Kumar (2001) addressed the importance of taxon sampling in phylogenetic analysis and concluded that phylogenetic error is “largely independent of taxon sample size” (2001:10756) and that their “results do not provide evidence in favor of adding taxa to problematic phylogenies” (2001:10756). In response to these conclusions, Zwickl and Hillis (2002) and Pollock et al. (2002) conducted additional simulations and reanalyzed the data presented by Rosenberg and Kumar (2001). Zwickl and Hillis and Pollock et al. showed that these conclusions of Rosenberg and Kumar could not be supported either by analyses of their original data or by new simulations that corrected a number of deficiencies in Rosenberg and Kumar’s original experimental design. Both Zwickl and Hillis and Pollock et al. found that increased taxon sampling resulted in greatly reduced phylogenetic estimation error, and Pollock et al. showed that the benefits of increased taxon sampling were similar to adding an equivalent amount of sequence length for the same taxa (in the ranges simulated by Rosenberg and Kumar). In their response, Rosenberg and Kumar (2002) focused on a slightly different conclusion from that in their original paper, which was that “longer sequences, rather than extensive sampling, will better improve the accuracy of phylogenetic inference” (2001:10751). In 2001, Rosenberg and Kumar argued that the beneficial effect of increasing taxa was 10-fold lower than the beneficial effect of increasing sequence length and that the effects of increased taxon sampling for the same genes were negligible (“largely independently” of phylogenetic error). Rosenberg and Kumar (2002) have now concluded that the beneficial effect of increasing taxon sample size is not small, but they suggested that the benefit comes simply from the overall increase in size of the data matrix (the total number of characters × taxa). Furthermore, they maintained that there is a greater benefit to increasing the total sequence length for few taxa than can be obtained by increasing taxon sampling for the same genes. Here, we discuss the two sets of conclusions reached by Rosenberg and Kumar (2001, 2002).

Resolution of Phylogenetic Conflict in Large Data Sets by Increased Taxon Sampling

The debate about whether phylogenetic accuracy is most efficiently increased by sampling more charac ters or more taxa is certainly not new (e.g., Kim, 1996; Graybeal, 1998; Poe, 1998a,b; Rannala et al., 1998; Poe and Swofford, 1999; Pollock and Bruno, 2000; Rosenburg and Kumar, 2001; Pollock et al., 2002; Zwickl and Hillis, 2002; Rosenberg and Kumar, 2003; Hillis et al., 2003). However, the recent increase of whole genomic sequences available from an assortment of distantly related taxa makes this debate highly relevant to researchers across fields of bi ology. Recently, Rokas et al. (2003) argued that the true species tree can be recovered despite conflicting phylo genetic signal between genes if enough genes are used in the analysis. Using the bootstrap proportion (BP) as a measure of phylogenetic accuracy, they concluded that approximately 20 genes are needed to ensure a robustly supported tree (>95% BP) for their study group of eight yeast taxa. From these empirical results, they generalized that most molecular phylogenetic studies have probably included insufficient numbers of genes to confidently re solve relationships within their respective focal groups. This approach to measuring accuracy can be sensitive to method inconsistency, or the failure to converge on the correct tree as the data set becomes infinitely large. When a method is inconsistent, measures of support such as nonparametric bootstrapping can increase as more se quence data are added?but in support of the wrong phy logeny (Phillips et al., 2004; Collins et al., 2005; Delsuc et al., 2005). Although most methods perform well over most of tree space (Huelsenbeck, 1995; Poe, 2003), regions of inconsistency have been identified in the literature for all of the most commonly used phylogenetic meth ods. For example, compositional bias can affect the accu racy of minimum evolution (Phillips et al., 2004), model misspecification may affect parametric methods such as maximum likelihood (ML) (Poe, 2003; Philippe et al, 2005; Collins et al., 2005), and branch-length asymme try can lead to inconsistency in maximum parsimony (Felsenstein, 1978; Hendy and Penny, 1989). Parsimony is particularly prone to long-branch attraction (LBA), an analytical artifact in which two taxa on long branches are incorrectly placed as sister taxa (Felsenstein, 1978; Hendy and Penny, 1989; Huelsenbeck and Hillis, 1993). Although there are many reasons for conflicting phylo genetic signal between genes, one relevant reason could be related to method inconsistency: differing rates of evo lution between genes could cause a particular method to be inconsistent for some genes and not for others. We argue that by addressing this source of conflict between genes, fewer genes may be needed to return an accu rate phylogeny. One source of conflict in the Rokas et al. (2003) data set may be nonstationarity: taxa that differ from the others in their base compositional bias may be erroneously drawn together as sister taxa (Collins et al., 2005). Here, we show that an additional source of conflict between the 106 genes in the Rokas et al. data set may be branch-length asymmetry. Using simulations of 106 genes from the Rokas et al. data set on a 79-taxon yeast phylogeny, we additionally show that when genes are added to a data set, support for the wrong reconstruc tion can increase when there is LBA. However, when taxa are added to the analysis, support for the correct reconstruction increases, and fewer genes are needed to achieve accuracy.

EVOLUTION OF RIBOSOMAL DNA: FIFTY MILLION YEARS OF RECORDED HISTORY IN THE FROG GENUS RANA

Evolution of nuclear ribosomal DNA (rDNA) arrays of frogs of the genus Rana was examined among 32 species that last shared a common ancestor approximately 50 million years ago. Extensive variation in restriction sites exists within the transcribed and nontranscribed rDNA spacer regions among the species, whereas rDNA coding regions exhibit comparatively little interspecific variation in restriction sites. The most parsimonious phylogenetic hypothesis for the evolution of the group was constructed based on variation in restriction sites and internal spacer lengths among the 32 species of Rana and one species of Pyxicephalus (examined for outgroup comparison). This analysis suggests that R. sylvatica of North America is more closely related to the R. temporaria group of Eurasia than to other North American Rana. The hypothesized phylogeny also supports the monophyly of the R. boylii group, the R. catesbeiana group, the R. palmipes group, the R. tarahumarae group, and the R. pipiens complex. Furthermore, the restriction site data provide information about the evolution within and among these species groups. This demonstrates that restriction site mapping of rDNA arrays provides a useful molecular technique for the examination of historical evolutionary questions across considerable periods of time.

Phylogeny and Biogeography of a Cosmopolitan Frog Radiation: Late Cretaceous Diversification Resulted in Continent-Scale Endemism in the Family Ranidae

Ranidae is a large anuran group with a nearly cosmopolitan distribution. We investigated the phylogenetic relationships and early biogeographic history of ranid frogs, using 104 representatives of all subfamilies and families, sampled from throughout their distribution. Analyses of approximately 1570 bp of nuclear gene fragments (Rag-1, rhod, Tyr) and approximately 2100 bp of the mitochondrial genome (12S rRNA, tRNAVAL, 16S rRNA) indicate that the monophyly of several taxa can be rejected with high confidence. Our tree is characterized by a clear historical association of each major clade with one Gondwanan plate. This prevalence of continent-scale endemism suggests that plate tectonics has played a major role in the distribution of ranid frogs. We performed dispersal-vicariance analyses, as well as analyses constrained by paleogeographic data, to estimate ancestral distributions during early ranid diversification. Additionally, we used molecular clock analyses to evaluate whether these scenarios fit the temporal framework of continental breakup. Our analyses suggest that a scenario in which the ancestors of several clades (Rhacophorinae, Dicroglossinae, Raninae) reached Eurasia via the Indian subcontinent, and the ancestor of Ceratobatrachinae entered via the Australia-New Guinea plate, best fits the paleogeographic models and requires the fewest number of dispersal/vicariance events. However, several alternatives, in which part of the ranid fauna colonized Laurasia from Africa, are not significantly worse. Most importantly, all hypotheses make clear predictions as to where to expect key fossils and where to sample other living ranids, and thus constitute a strong basis for further research.

Molecules and morphology in evolution : conflict or compromise?

Preface 1. Introduction Colin Patterson 2. Aspects of hominoid phylogeny Peter Andrews 3. Molecular and morphological analysis of high-level mammalian interrelationships Malcolm C. McKenna 4. Avian phylogeny reconstructed from comparisons of the genetic material, DNA Charles G. Sibley and Jon E. Ahlquist 5. Tetrapod relationships: the molecular evidence M. J. Bishop and A. E. Friday 6. Pattern and process in vertebrate phylogeny revealed by coevolution of molecules and morphologies Morris Goodman, Michael M. Miyamoto and John Czelusniak 7. Macroevolution in the microscopic world C. R. Woese 8. Divergence in inbred strains of mice: a comparison of three different types of data Walter M. Fitch and William R. Atchley Index.